The evolution over time of the peptic ulcer epidemiology reflects a complex and multifactorial etiology. Peptic ulcers were rare before 1800, so the gastric ulcer pathology was not described until 1835 (J. 1835 Cruveilhier maladies de l'estomac In Anatomy of Human Bailliere Pathologique du Corps, Paris ...). During the nineteenth century, the predominant form was gastric ulcer in young women, while duodenal ulcer was rare until the twentieth century. However, duodenal ulcer was gradually more prevalent until become the more frequent condition in the middle of the century. Moreover, in developed countries the mortality from peptic ulcer has been drastically reduced for the cohorts born after the start of the twentieth century (Sonnenberg, 2007).

It is now clear that the epidemiology of the peptic ulcer is mainly due to external environmental factors, among which are include *H. pylori* infection, the use of NSAIDs and smoking. However, these factors do not explain the whole story of the evolution in time and the birth-cohort effect for peptic ulcer. Specifically, *H. pylori* was a prevalent human infection before 1800, so that infection per se cannot explain the increased prevalence of the ulcer after this time nor the shift from gastric ulcer to duodenal ulcer (Graham, 2003). Interest in understanding the role of *H. pylori* in gastroduodenal diseases was started in the 1970s with the visualization of bacteria in the stomach of gastric ulcer patients, although the more important researches about it were carry out by the Australian pathologist Berry Marshal in 1979 in collaboration with J Robin Warren from 1981 (Warren JR, 1983). Almost by accident, successful culture of *H. pylori* in 1982 occurred. Marshall frustrated by not being able to get a good animal model of infection, ingested these bacteria and he became ill, developed inflammation and ulcer of stomach, and he was able to culture the bacteria from his own ulcer, proving *H. pylori* to be the cause of ulcers. They published the results of selfinduced infection in 1985 (Marshall et al., 1985) and initially, the isolated bacteria was termed *Campylobacter pyloridis* but it was re-named *Helicobacter pylori* (*H. pylori*) when biochemical and genetic characterization of the organism showed that it was not a member of the *Campylobacter* genus (Tan & Wong, 2011).

#### **4.2 Morphological and biochemical characteristics**

*H. pylori* is a spiral-shaped, flagellated, microaerophilic Gram-negative bacillus. It inhabits various areas of stomach and duodenum. Stomach is normally a hostile environment to the survival of viruses, bacteria and other micro-organisms due to its low pH. However *H. pylori* has evolved to be uniquely suited to thrive in the harsh stomach environment (Fig. 1).

show that ROS levels are directly correlated with the infective load of *H. pylori* (Zhang et al., 2007) and lipid peroxidation has been also shown to be increased in *H. pylori*-positive gastric

But although it has been established a clear association between bacteria infection and impaired synthesis of ROS, many other clinical data suggest that other factors inherent to host conditions (stress, diet, tobacco, hygiene, genetics...) contribute to the pathogenesis of this infection (Oh et al., 2005). It should be noted that some of these factors, including ingested food and tobacco smoke, directly influence in mucosal oxidative status, since they expose the gastric epithelium to the ROS that they generate within the gastric lumen in a

The evolution over time of the peptic ulcer epidemiology reflects a complex and multifactorial etiology. Peptic ulcers were rare before 1800, so the gastric ulcer pathology was not described until 1835 (J. 1835 Cruveilhier maladies de l'estomac In Anatomy of Human Bailliere Pathologique du Corps, Paris ...). During the nineteenth century, the predominant form was gastric ulcer in young women, while duodenal ulcer was rare until the twentieth century. However, duodenal ulcer was gradually more prevalent until become the more frequent condition in the middle of the century. Moreover, in developed countries the mortality from peptic ulcer has been drastically reduced for the cohorts born after the

It is now clear that the epidemiology of the peptic ulcer is mainly due to external environmental factors, among which are include *H. pylori* infection, the use of NSAIDs and smoking. However, these factors do not explain the whole story of the evolution in time and the birth-cohort effect for peptic ulcer. Specifically, *H. pylori* was a prevalent human infection before 1800, so that infection per se cannot explain the increased prevalence of the ulcer after this time nor the shift from gastric ulcer to duodenal ulcer (Graham, 2003). Interest in understanding the role of *H. pylori* in gastroduodenal diseases was started in the 1970s with the visualization of bacteria in the stomach of gastric ulcer patients, although the more important researches about it were carry out by the Australian pathologist Berry Marshal in 1979 in collaboration with J Robin Warren from 1981 (Warren JR, 1983). Almost by accident, successful culture of *H. pylori* in 1982 occurred. Marshall frustrated by not being able to get a good animal model of infection, ingested these bacteria and he became ill, developed inflammation and ulcer of stomach, and he was able to culture the bacteria from his own ulcer, proving *H. pylori* to be the cause of ulcers. They published the results of selfinduced infection in 1985 (Marshall et al., 1985) and initially, the isolated bacteria was termed *Campylobacter pyloridis* but it was re-named *Helicobacter pylori* (*H. pylori*) when biochemical and genetic characterization of the organism showed that it was not a member

*H. pylori* is a spiral-shaped, flagellated, microaerophilic Gram-negative bacillus. It inhabits various areas of stomach and duodenum. Stomach is normally a hostile environment to the survival of viruses, bacteria and other micro-organisms due to its low pH. However *H. pylori* has evolved to be uniquely suited to thrive in the harsh stomach environment (Fig. 1).

**4.** *Helicobacter pylori* **and gastroduodenal diseases**

start of the twentieth century (Sonnenberg, 2007).

of the *Campylobacter* genus (Tan & Wong, 2011).

**4.2 Morphological and biochemical characteristics**

mucosa.

sustained manner.

**4.1 Short history**

The bacterium secretes urease, a special enzyme that converts urea to ammonia. Ammonia reduces the acidity of stomach, making it more hospitable home for *H. pylori* (Pandey et al., 2010)*.*

Fig. 1. *H. pylori* localization in the gastric mucosa and ammonia production.

*H. pylori* exerts a trophic influence on the gastric epithelium, but before it can attach to the epithelium surface it has first to cross the thick mucus layer by adhering to the mucosal surface. The presence of unipolar flagella helps to establish the *H.pylori* colonization of the stomach and its attachment to the epithelial surface of mucosa in the stomach despite the host attempts to rid itself of the bacterial infection. It is of interest that mutant *H. pylori* strains that are non-motile are unable to colonize the stomach of gnotobiotic piglets (Eaton et al., 1989).

### **4.3 Time, mode and geographic distribution of infection**

*H. pylori* colonizes the gastric mucosa of more than 50% of human population. Infection is usually acquired in childhood while natural acquisition in adults is rare. The major risk factor for *H. pylori* infection are the socio-sanitary conditions lived during childhood, particularly at home, being important factors the level of sanitation, hygiene, and number of people in the household (the overcrowding). Studies in Kazakhstan (Nurgalieva et al., 2002) and Peru (Klein et al., 1991), have confirmed that high *H. pylori* infection prevalence in children in these countries, is related to these factors besides with the use of contaminated water with bacteria. These data determine that the water could be a reservoir and a transmission route for the bacteria.

Moreover, although it has been demonstrated a family association for infection (Nam et al., 2011), the transmission mode of *H. pylori* between individuals and within families remains to be elucidated and several interesting myths related to oral-oral transmission have been

*Helicobacter pylori* and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis 171

Fig. 3. *H. pylori*, gastritis and disease. During infection with *H. pylori* a number of

antibodies. However, this vigorous response fails to eliminate infection, and the continued presence of *H. pylori* leads to the development of a second arm of the immune response more specifically aimed at preventing the damaging effects of intraluminal pathogens. The initial acute phase of infection is subclinical in the great majority of subjects. This phase is short lived, and histologically results in a neutrophilic gastritis followed by a gradual infiltration of the gastric mucosa by inflammatory cells, and is coupled with a transient

In a small minority of people, and particularly in childhood, the bacteria may be spontaneously cleared, the cellular infiltrate resolves, and the mucosa return to normal appearance. However, in the majority of subjects although *H. pylori* stimulates a robust inflammatory and immune response, the host fails to eliminate the infection and over the next 3 or 4 weeks there is a gradual accumulation of chronic inflammatory cells that come to dominate the histological picture. An acute neutrophilic gastritis gives way to an active chronic gastritis or "bening gastritis" characterized by mild pangastritis with little disruption of gastric acid secretion. This phenotype is exhibited by the asymptomatic subjects and those who do not develop serious gastrointestinal alterations (Amieva & El-Omar, 2008). In fact, it is commonly accepted that *H. pylori* infection, followed by the induction of inflammatory changes in gastric mucosa, may persist for decades without

Chronic gastritis is a common denominator linking peptic ulceration, gastric carcinoma, and lymphoma, and the histological picture encompassed chronic inflammation, atrophy, and intestinal metaplasia and finally adenocarcinoma (Dixon, 2001) (Fig. 3). Once chronic gastritis is established, it can progress mainly towards two topographic patterns that are

inflammation patterns are associated with different disease stages.

causing any gastric disturbances (Konturek et al., 2009).

hypochlorhydria (Egan et al., 2007).

debunked when a study of couples without children revealed a low concordance of *H.pylori* infection (Perez-Perez et al., 1991); currently favored mechanisms of transmission appear to be gastro-oral and faecal-oral routes (Xia & Talley, 1997). Genetic susceptibility also appears to be significant in the acquisition of *H.pylori* infection as well as its clearance (Malaty et al., 1994).

Fig. 2. *H. pylori* world distribution

Although generally speaking a rate of infection worldwide by about 50%, prevalences varying widely within countries (Fig. 2), and even between geographical areas of the same country (Krejs, 2010). Thus, in developing countries such as the eastern regions of Asia and in some parts of Latin America, the infection prevalence is characterized by a rapid rate of acquisition, usually in childhood, so that about 80% of the population is infected by the age of 20 (Graham et al., 1991).

In contrast, in developed countries such as Spain, USA, UK or Australia, the prevalence of *H. pylori* infection in children is low for ages below 10 years, and peaks of 40% aproximately, occur about to 30-40 years of age (Lehours & Yilmaz, 2007).

### **4.4 General features of** *Helicobacter pylori* **infection**

In all infected subjects by *H. pylori*, the basic process that mediates the mucosa damage is the development of gastritis, whose extent and distribution will determine the clinical outcome (Amieva & El-Omar, 2008).

The arrival of lymphocytes and plasma cells in the mucosa signals augmentation of the acute inflammatory response by the production of cytokines and specific anti-*H. pylori*

debunked when a study of couples without children revealed a low concordance of *H.pylori* infection (Perez-Perez et al., 1991); currently favored mechanisms of transmission appear to be gastro-oral and faecal-oral routes (Xia & Talley, 1997). Genetic susceptibility also appears to be significant in the acquisition of *H.pylori* infection as well as its clearance (Malaty et al.,

Although generally speaking a rate of infection worldwide by about 50%, prevalences varying widely within countries (Fig. 2), and even between geographical areas of the same country (Krejs, 2010). Thus, in developing countries such as the eastern regions of Asia and in some parts of Latin America, the infection prevalence is characterized by a rapid rate of acquisition, usually in childhood, so that about 80% of the population is infected by the age

In contrast, in developed countries such as Spain, USA, UK or Australia, the prevalence of *H. pylori* infection in children is low for ages below 10 years, and peaks of 40% aproximately,

In all infected subjects by *H. pylori*, the basic process that mediates the mucosa damage is the development of gastritis, whose extent and distribution will determine the clinical outcome

The arrival of lymphocytes and plasma cells in the mucosa signals augmentation of the acute inflammatory response by the production of cytokines and specific anti-*H. pylori*

occur about to 30-40 years of age (Lehours & Yilmaz, 2007).

**4.4 General features of** *Helicobacter pylori* **infection**

1994).

Fig. 2. *H. pylori* world distribution

of 20 (Graham et al., 1991).

(Amieva & El-Omar, 2008).

Fig. 3. *H. pylori*, gastritis and disease. During infection with *H. pylori* a number of inflammation patterns are associated with different disease stages.

antibodies. However, this vigorous response fails to eliminate infection, and the continued presence of *H. pylori* leads to the development of a second arm of the immune response more specifically aimed at preventing the damaging effects of intraluminal pathogens.

The initial acute phase of infection is subclinical in the great majority of subjects. This phase is short lived, and histologically results in a neutrophilic gastritis followed by a gradual infiltration of the gastric mucosa by inflammatory cells, and is coupled with a transient hypochlorhydria (Egan et al., 2007).

In a small minority of people, and particularly in childhood, the bacteria may be spontaneously cleared, the cellular infiltrate resolves, and the mucosa return to normal appearance. However, in the majority of subjects although *H. pylori* stimulates a robust inflammatory and immune response, the host fails to eliminate the infection and over the next 3 or 4 weeks there is a gradual accumulation of chronic inflammatory cells that come to dominate the histological picture. An acute neutrophilic gastritis gives way to an active chronic gastritis or "bening gastritis" characterized by mild pangastritis with little disruption of gastric acid secretion. This phenotype is exhibited by the asymptomatic subjects and those who do not develop serious gastrointestinal alterations (Amieva & El-Omar, 2008). In fact, it is commonly accepted that *H. pylori* infection, followed by the induction of inflammatory changes in gastric mucosa, may persist for decades without causing any gastric disturbances (Konturek et al., 2009).

Chronic gastritis is a common denominator linking peptic ulceration, gastric carcinoma, and lymphoma, and the histological picture encompassed chronic inflammation, atrophy, and intestinal metaplasia and finally adenocarcinoma (Dixon, 2001) (Fig. 3). Once chronic gastritis is established, it can progress mainly towards two topographic patterns that are

*Helicobacter pylori* and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis 173

isolates which induce a strong and rapid oxidative burst in neutrophils are associated with higher inflammation scores in gastric ulcer patients and with histological mucosal damage (Louw et al., 1993). Furthermore, IL-8 levels secreted by mucosa cells positively correlate with infiltration of PMN and mononuclear cells, and with higher production of ROS in *H.*

ROS production in *H. pylori* infection is catalyzed by nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase; Nox) on the cell membrane (Lambeth, 2004). It is also produced superoxide anion (O2-), a precursor of microbicidal oxidants. This O2-

converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) catalysis, or by nonenzymatic dismutation in the phagosome. H2O2 can passively permeate cell membranes and is converted to hypochlorous acid (HOCl), which is 100 times more toxic than H2O2. This conversion is mediated by myeloperoxidases (MPO) released by Azur granules in phagocytes in the presence of chloride ions (Cl-). H2O2 also reacts nonenzymatically with O2- to form hydroxyl radicals (OH) in the presence of ferrous (Fe2+) or cuprous (Cu+) ions. In general, these highly reactive ROS (i.e., HOCl and OH) are used by the phagocyte to kill

However, in *H. pylori*-infected gastric mucosa, these ROS cannot eradicate *H. pylori* and this excessive production is believed to be a major cause of gastric mucosal damage, inducing

Thus, the role of oxidative stress on gastric mucosa is multi-functional. For neutrophils, it is the result of excessive defense reactions of the body against *H. pylori* intrusion, and for *H.*

is

*pylori*-infected antral gastric mucosa (Danese et al., 2001).

oxidative stress to the gastric mucosa cells (Handa et al., 2010).

*pylori*, it is a convenient tool for invading the human gastric mucosa.

Prx

SodB

KatA

RocF

*NOS*

**Fe+3**

NapA

Fenton reaction

**RNH2**

**HO·**

Trx-TrxR

**LOH**

Fig. 4. Sources of ROS and the detoxification systems in *H. pylori*

**NOH**

**OONO-**

**O2 · NO .**

**H2O2**

**H2O+O2**

**Fe+2**

pathogenic bacteria (Fig.4).

**Q**

**QH2**

Lipid

MdaB

**LH LOO·**

peroxidation

related to different clinical outcomes. The first pattern named "duodenal ulcer" phenotype is an antral predominant gastritis, accounts for up 15% of infected subjects and is characterized by inflammation mostly limited to the antrum. Subjects with this phenotype have high antral inflammatory scores, high gastrin, relatively healthy corpus mucosa, and very high acid output (Graham & Yamaoka, 1998). These subjects also have defective inhibitory control of gastric acid secretion.

This combination of pathophysiologic abnormalities contributes to the development of peptic ulcers, and confers protection against gastric cancer. Conversely, gastric ulcers are thought to be initially associated with a chronic non-atrophic gastritis which progresses to chronic atrophic gastritis involving both corpus and, invariably, the antrum and decreased acid output.

The second pattern, termed "gastric cancer" phenotype, is one of progressive pan-gastritis or multifocal atrophic gastritis, and hypo- or achlorhydria, characterized by active infection of both the gastric corpus and antrum with progressive development of gastric atrophy and intestinal metaplasia. These abnormalities affect at 1% of *H. pylori*-infected individuals who presented an increased risk of development gastric carcinoma (Amieva & El-Omar, 2008).

It is clear that *H. pylori* infection can lead to several divergent clinical outcomes. Explaining this apparent paradox is essential for understanding the pathogenesis of *H. pylori*-related disease because the mechanisms underlying the differences in outcome of *H. pylori* infection are currently poorly understood. It appears that the host reaction to the infection is very complex and it has been hypothesized that many sequential events and several mechanisms of tissue injury participate in the process. Between these pathogenic mechanisms of *H. pylori* are identified the induction of gastric inflammation, the disruption of the gastric mucosal barrier and the altered gastrin-gastric acid homeostasis (Dunn et al., 1997). Also, these different clinical manifestations of the infection are influenced by the host characteristics, the environmental factors and the bacterial genetic. Host factors are mainly related to the recognition of *H. pylori* by the immune system, variations in the level of cytokine response, sex and hereditary influences. Environmental data include tobacco smoke, diet, stress and the action of drugs such as NSAIDs. Bacterial factors may increase the risk of more severe disease, causing increased proinflammatory cytokine release. Strains possessing the "cag pathogenicity island" are more likely to be associated with peptic ulceration or gastric adenocarcinoma than strains lacking it. Another genes and proteins, such as iceA, vacA, OipA, BabA, have been analyzed but different studies were unable to show an association between them and the pattern of gastritis (Egan et al., 2007).

### **4.5 Toxicity of** *Helicobacter pylori* **on gastric mucosa (ROS)**

In the early stages of infection, *H. pylori* induces secretion of chemokines (RANTES, GRO, MIP-1, ENA-78, MCP-1, and IL-8), as well as of proinflammatory cytokines (IL-1, IL-6 and TNF-, mainly) (Ibraghimov & Pappo, 2000). These molecules provoke recruitment of cells (macrophages, PMN, mast cells, T and B lymphocytes) to the infected gastric tissue, being neutrophils the initial inflammatory component of the response to the pathogen (Naito & Yoshikawa, 2002). Recruited PMNs, in turn, secrete more inflammatory mediators that amplify the primary signal and mediates directly the influx of more PMN to the gastric mucosa. Furthermore, soluble proteins of *H. pylori* can also function as chemoattractants for neutrophils. PMNs into gastric tissue induce oxidative burst responses in phagocytes being these activities typical in the development of gastric disease. It is demonstrated that *H. pylori*

related to different clinical outcomes. The first pattern named "duodenal ulcer" phenotype is an antral predominant gastritis, accounts for up 15% of infected subjects and is characterized by inflammation mostly limited to the antrum. Subjects with this phenotype have high antral inflammatory scores, high gastrin, relatively healthy corpus mucosa, and very high acid output (Graham & Yamaoka, 1998). These subjects also have defective

This combination of pathophysiologic abnormalities contributes to the development of peptic ulcers, and confers protection against gastric cancer. Conversely, gastric ulcers are thought to be initially associated with a chronic non-atrophic gastritis which progresses to chronic atrophic gastritis involving both corpus and, invariably, the antrum and decreased

The second pattern, termed "gastric cancer" phenotype, is one of progressive pan-gastritis or multifocal atrophic gastritis, and hypo- or achlorhydria, characterized by active infection of both the gastric corpus and antrum with progressive development of gastric atrophy and intestinal metaplasia. These abnormalities affect at 1% of *H. pylori*-infected individuals who presented an increased risk of development gastric carcinoma (Amieva & El-Omar, 2008). It is clear that *H. pylori* infection can lead to several divergent clinical outcomes. Explaining this apparent paradox is essential for understanding the pathogenesis of *H. pylori*-related disease because the mechanisms underlying the differences in outcome of *H. pylori* infection are currently poorly understood. It appears that the host reaction to the infection is very complex and it has been hypothesized that many sequential events and several mechanisms of tissue injury participate in the process. Between these pathogenic mechanisms of *H. pylori* are identified the induction of gastric inflammation, the disruption of the gastric mucosal barrier and the altered gastrin-gastric acid homeostasis (Dunn et al., 1997). Also, these different clinical manifestations of the infection are influenced by the host characteristics, the environmental factors and the bacterial genetic. Host factors are mainly related to the recognition of *H. pylori* by the immune system, variations in the level of cytokine response, sex and hereditary influences. Environmental data include tobacco smoke, diet, stress and the action of drugs such as NSAIDs. Bacterial factors may increase the risk of more severe disease, causing increased proinflammatory cytokine release. Strains possessing the "cag pathogenicity island" are more likely to be associated with peptic ulceration or gastric adenocarcinoma than strains lacking it. Another genes and proteins, such as iceA, vacA, OipA, BabA, have been analyzed but different studies were unable to show an association

In the early stages of infection, *H. pylori* induces secretion of chemokines (RANTES, GRO, MIP-1, ENA-78, MCP-1, and IL-8), as well as of proinflammatory cytokines (IL-1, IL-6 and TNF-, mainly) (Ibraghimov & Pappo, 2000). These molecules provoke recruitment of cells (macrophages, PMN, mast cells, T and B lymphocytes) to the infected gastric tissue, being neutrophils the initial inflammatory component of the response to the pathogen (Naito & Yoshikawa, 2002). Recruited PMNs, in turn, secrete more inflammatory mediators that amplify the primary signal and mediates directly the influx of more PMN to the gastric mucosa. Furthermore, soluble proteins of *H. pylori* can also function as chemoattractants for neutrophils. PMNs into gastric tissue induce oxidative burst responses in phagocytes being these activities typical in the development of gastric disease. It is demonstrated that *H. pylori*

inhibitory control of gastric acid secretion.

between them and the pattern of gastritis (Egan et al., 2007).

**4.5 Toxicity of** *Helicobacter pylori* **on gastric mucosa (ROS)**

acid output.

isolates which induce a strong and rapid oxidative burst in neutrophils are associated with higher inflammation scores in gastric ulcer patients and with histological mucosal damage (Louw et al., 1993). Furthermore, IL-8 levels secreted by mucosa cells positively correlate with infiltration of PMN and mononuclear cells, and with higher production of ROS in *H. pylori*-infected antral gastric mucosa (Danese et al., 2001).

ROS production in *H. pylori* infection is catalyzed by nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase; Nox) on the cell membrane (Lambeth, 2004). It is also produced superoxide anion (O2- ), a precursor of microbicidal oxidants. This O2 is converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) catalysis, or by nonenzymatic dismutation in the phagosome. H2O2 can passively permeate cell membranes and is converted to hypochlorous acid (HOCl), which is 100 times more toxic than H2O2. This conversion is mediated by myeloperoxidases (MPO) released by Azur granules in phagocytes in the presence of chloride ions (Cl-). H2O2 also reacts nonenzymatically with O2 - to form hydroxyl radicals (OH) in the presence of ferrous (Fe2+) or cuprous (Cu+) ions. In general, these highly reactive ROS (i.e., HOCl and OH) are used by the phagocyte to kill pathogenic bacteria (Fig.4).

However, in *H. pylori*-infected gastric mucosa, these ROS cannot eradicate *H. pylori* and this excessive production is believed to be a major cause of gastric mucosal damage, inducing oxidative stress to the gastric mucosa cells (Handa et al., 2010).

Thus, the role of oxidative stress on gastric mucosa is multi-functional. For neutrophils, it is the result of excessive defense reactions of the body against *H. pylori* intrusion, and for *H. pylori*, it is a convenient tool for invading the human gastric mucosa.

Fig. 4. Sources of ROS and the detoxification systems in *H. pylori*

*Helicobacter pylori* and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis 175

a decrease in cell numbers could be related to an increase in cellular apoptosis, whereas tissue proliferation may be associated with molecular inhibition of apoptosis; in some diseases, internal or external factors could trigger the proapoptotic machinery within the cell causing cell death and tissue destruction; or cellular malignant transformation may occur because of a failure to activate apoptosis and delete cells with genetic damage (i.e., oncogenic mutations). These concepts suggest the possibility that several treatment strategies are useful in certain clinical conditions. Thus, inhibition of apoptosis could facilitate tissue repair processes by promoting cellular proliferation, and tissue regeneration, and, moreover, induction of apoptosis could be proven useful in treating malignant

In relationship with the gastrointestinal tract, apoptosis plays an important role in the regulation of epithelial cell numbers, being the deregulation of the apoptotic pathway implicated in a number of disease processes in the gastrointestine. In *H. pylori*-induced chronic gastritis, cell loss by apoptosis is excessive compared with proliferation, suggesting that infection with the bacteria triggers the acceleration of apoptosis, fact that has been

Two distinct, but partially overlapping, pathways are known to lead to apoptosis (Fig.5):

ligands more studied are Fas, tumor necrosis factor receptor or TRAIL receptors.

The extrinsic pathway is activated by apoptotic stimuli comprising external signals such as the binding of death inducing ligands to cell surface receptors. Among death inducing

Death ligand stimulation results in oligomerization of the receptors and recruitment of the adaptor protein Fas-associated death domain (FADD) and caspase-8, forming a deathinducing signalling complex (DISC). Autoactivation of caspase-8 at the DISC is followed by activation of effector caspases, including caspase-3, -6 and -7, which function as downstream effectors of the cell death program (Ashkenazi & Dixit, 1998). Fas has a central role in the physiological regulation of apoptosis and has been implicated in the pathogenesis of various malignancies as well as in diseases of the immune system. Fas is involved in cytotoxic T-cell mediated killing of cells (for example, CTL-mediated killing of virus-infected cells), destruction of inflammatory and immune cells in immune-privileged sites, deletion of selfreacting B cells and activated T-cells at the end of an immune response (Jin & El-Deiry, 2005). Some *in vitro* studies reveal that *H. pylori* stimulates apoptosis of gastric epithelial cells in association with the enhanced expression of the Fas receptor, indicating a role for Fasmediated signaling in the programmed cell death that occurs in response to *H. pylori*  infection (Jones et al., 1999). Wang et al. (Wang et al., 2000) also demonstrated that local Th1 cells (cellular subtype that is mainly recruited to the gastric mucosa during the infection) may contribute to the pathogenesis of gastric disease during *H. pylori* infection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions. Moreover, *H. pylori* can sensitize human gastric epithelial cells and enhance

Intrinsic apoptotic pathway is initiated inside cells. The most important turning point in the course of the intrinsic apoptotic process occurs in the mitochondria. Their structure and compartmentalization are highly related to a perfect performance of their functions, being the most relevant, in eukaryotic cells, energy production in ATP form molecules (Chinnery

carcinomas (Que & Gores, 1996).

proven in *in vivo* experiments (Hall et al., 1994).

susceptibility to TRAIL-mediated apoptosis (Wu et al., 2004).

& Schon, 2003).

As noted above, some factors that may further aggravate the pathogenicity caused by *H. pylori* are the genetic characteristics of bacteria. In fact, the possession of "cytotoxinassociated gene (cagA) or pathogenicity island" is associated with a high prevalence of peptic ulcer disease. And also, according to the authors Danese et al (Danese et al., 2001) and others, the gastric mucosa of patients infected by CagA (+) strains are characterized by increased ROS generation and neutrophil counts greater than that observed in CagA (-) subjects.

### **4.6** *Helicobacter pylori***, ROS and cellular apoptosis**

Several studies have shown a relationship between *H. pylori* infection and an increased apoptosis rate in gastric lesions such as chronic gastritis, generally accompanied by glandular atrophy, gastric ulcer and intestinal metaplasia. Specifically, studies in patients with gastric ulcer determine a higher apoptotic index in this type of gastric lesion with *H. pylori* infection compared to *H. pylori*-negative normal gastric mucosa (Targa et al., 2007). Results of Satoh et al (Satoh et al., 2003) showed that apoptosis, not only of surface epithelial cells but also of glandular cells in the upper portion of fundic glands, is increased in *H. pylori*-positive patients with gastric ulcers and decreased to normal levels after eradication of *H. pylori*. Recent studies are intended to avoid triggering apoptotic processes caused by *H. pylori* (Hsu et al., 2010). In these experiments, authors use substances capable of inhibiting the activation of caspase cascade (extract solanum lyratum in this study). This could be a new approach for the treatment of infection with *H. pylori*.

It is known that some of the major stimuli that can induce apoptosis are: ionising radiation, viral infection, growth factor depletion, cytoplasmic stress, serum starvation, presence of hormones such as corticosteroids, the damage to genetic material and an excessive presence of free radical. Given that one of the factors that triggers cellular apoptosis is the excess of ROS, it is reasonable to think that the apoptosis observed in gastric mucosal cells infected by *H. pylori* may have this origin.

Apoptosis was first described in 1972 by Currie and colleagues (Kerr et al., 1972). It plays a pivotal part in many physiological settings, including the embryonic and post-embryonic development of multicellular organisms, tissue homeostasis and the removal of damaged and/or infected cells. It is delicately regulated and balanced in a physiological context. Failure of this regulation results in pathological conditions such as developmental defects, autoimmune diseases, neurodegeneration or cancer (Thompson, 1995). Apoptosis is by far the best-characterized mode of programmed cell death that is associated to morphological features that had been repeatedly observed in various tissues and cell types. Apoptotic cells display typical morphological features such as nuclear fragmentation, chromatin condensation and cell shrinkage, and eventually break down into apoptotic bodies. These morphological changes are accompanied by ATP-dependent biochemical changes that lead to the cells to various functional alterations. The results of these changes are reflected in an asymmetry of the cytoplasmic membranes by translocation of phosphatidylserine to the surface, breaking of DNA in multiples fragments (ladder pattern), release of pro-apoptotic proteins (cytochrome c (cyt c), AIF,…) from the mitochondria to cytosol, activation of caspases cascade, and finally, cell death.

Apoptotic cascade appears to have many regulatory "switch points'' between proapoptotic and antiapoptotic forces. The imbalance of these forces has led to several key concepts regarding the importance of apoptosis in health and disease. Thus, atrophy of an organ with a decrease in cell numbers could be related to an increase in cellular apoptosis, whereas tissue proliferation may be associated with molecular inhibition of apoptosis; in some diseases, internal or external factors could trigger the proapoptotic machinery within the cell causing cell death and tissue destruction; or cellular malignant transformation may occur because of a failure to activate apoptosis and delete cells with genetic damage (i.e., oncogenic mutations). These concepts suggest the possibility that several treatment strategies are useful in certain clinical conditions. Thus, inhibition of apoptosis could facilitate tissue repair processes by promoting cellular proliferation, and tissue regeneration, and, moreover, induction of apoptosis could be proven useful in treating malignant carcinomas (Que & Gores, 1996).

In relationship with the gastrointestinal tract, apoptosis plays an important role in the regulation of epithelial cell numbers, being the deregulation of the apoptotic pathway implicated in a number of disease processes in the gastrointestine. In *H. pylori*-induced chronic gastritis, cell loss by apoptosis is excessive compared with proliferation, suggesting that infection with the bacteria triggers the acceleration of apoptosis, fact that has been proven in *in vivo* experiments (Hall et al., 1994).

Two distinct, but partially overlapping, pathways are known to lead to apoptosis (Fig.5):

174 Peptic Ulcer Disease

As noted above, some factors that may further aggravate the pathogenicity caused by *H. pylori* are the genetic characteristics of bacteria. In fact, the possession of "cytotoxinassociated gene (cagA) or pathogenicity island" is associated with a high prevalence of peptic ulcer disease. And also, according to the authors Danese et al (Danese et al., 2001) and others, the gastric mucosa of patients infected by CagA (+) strains are characterized by increased ROS generation and neutrophil counts greater than that observed in CagA (-)

Several studies have shown a relationship between *H. pylori* infection and an increased apoptosis rate in gastric lesions such as chronic gastritis, generally accompanied by glandular atrophy, gastric ulcer and intestinal metaplasia. Specifically, studies in patients with gastric ulcer determine a higher apoptotic index in this type of gastric lesion with *H. pylori* infection compared to *H. pylori*-negative normal gastric mucosa (Targa et al., 2007). Results of Satoh et al (Satoh et al., 2003) showed that apoptosis, not only of surface epithelial cells but also of glandular cells in the upper portion of fundic glands, is increased in *H. pylori*-positive patients with gastric ulcers and decreased to normal levels after eradication of *H. pylori*. Recent studies are intended to avoid triggering apoptotic processes caused by *H. pylori* (Hsu et al., 2010). In these experiments, authors use substances capable of inhibiting the activation of caspase cascade (extract solanum lyratum in this study). This could be a

It is known that some of the major stimuli that can induce apoptosis are: ionising radiation, viral infection, growth factor depletion, cytoplasmic stress, serum starvation, presence of hormones such as corticosteroids, the damage to genetic material and an excessive presence of free radical. Given that one of the factors that triggers cellular apoptosis is the excess of ROS, it is reasonable to think that the apoptosis observed in gastric mucosal cells infected by

Apoptosis was first described in 1972 by Currie and colleagues (Kerr et al., 1972). It plays a pivotal part in many physiological settings, including the embryonic and post-embryonic development of multicellular organisms, tissue homeostasis and the removal of damaged and/or infected cells. It is delicately regulated and balanced in a physiological context. Failure of this regulation results in pathological conditions such as developmental defects, autoimmune diseases, neurodegeneration or cancer (Thompson, 1995). Apoptosis is by far the best-characterized mode of programmed cell death that is associated to morphological features that had been repeatedly observed in various tissues and cell types. Apoptotic cells display typical morphological features such as nuclear fragmentation, chromatin condensation and cell shrinkage, and eventually break down into apoptotic bodies. These morphological changes are accompanied by ATP-dependent biochemical changes that lead to the cells to various functional alterations. The results of these changes are reflected in an asymmetry of the cytoplasmic membranes by translocation of phosphatidylserine to the surface, breaking of DNA in multiples fragments (ladder pattern), release of pro-apoptotic proteins (cytochrome c (cyt c), AIF,…) from the mitochondria to cytosol, activation of

Apoptotic cascade appears to have many regulatory "switch points'' between proapoptotic and antiapoptotic forces. The imbalance of these forces has led to several key concepts regarding the importance of apoptosis in health and disease. Thus, atrophy of an organ with

**4.6** *Helicobacter pylori***, ROS and cellular apoptosis**

new approach for the treatment of infection with *H. pylori*.

*H. pylori* may have this origin.

caspases cascade, and finally, cell death.

subjects.

The extrinsic pathway is activated by apoptotic stimuli comprising external signals such as the binding of death inducing ligands to cell surface receptors. Among death inducing ligands more studied are Fas, tumor necrosis factor receptor or TRAIL receptors.

Death ligand stimulation results in oligomerization of the receptors and recruitment of the adaptor protein Fas-associated death domain (FADD) and caspase-8, forming a deathinducing signalling complex (DISC). Autoactivation of caspase-8 at the DISC is followed by activation of effector caspases, including caspase-3, -6 and -7, which function as downstream effectors of the cell death program (Ashkenazi & Dixit, 1998). Fas has a central role in the physiological regulation of apoptosis and has been implicated in the pathogenesis of various malignancies as well as in diseases of the immune system. Fas is involved in cytotoxic T-cell mediated killing of cells (for example, CTL-mediated killing of virus-infected cells), destruction of inflammatory and immune cells in immune-privileged sites, deletion of selfreacting B cells and activated T-cells at the end of an immune response (Jin & El-Deiry, 2005).

Some *in vitro* studies reveal that *H. pylori* stimulates apoptosis of gastric epithelial cells in association with the enhanced expression of the Fas receptor, indicating a role for Fasmediated signaling in the programmed cell death that occurs in response to *H. pylori*  infection (Jones et al., 1999). Wang et al. (Wang et al., 2000) also demonstrated that local Th1 cells (cellular subtype that is mainly recruited to the gastric mucosa during the infection) may contribute to the pathogenesis of gastric disease during *H. pylori* infection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions. Moreover, *H. pylori* can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis (Wu et al., 2004).

Intrinsic apoptotic pathway is initiated inside cells. The most important turning point in the course of the intrinsic apoptotic process occurs in the mitochondria. Their structure and compartmentalization are highly related to a perfect performance of their functions, being the most relevant, in eukaryotic cells, energy production in ATP form molecules (Chinnery & Schon, 2003).

*Helicobacter pylori* and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis 177

Fig. 6. Mitochondria: structure and function. **A)** Mitochondrial structure. **B)** Mitochondrial membranes: relationship between electron transport chain complexes, cytochrome c, and cardiolipin. **C**) Mitochondrial transition pore formation: ruptured links between protein complexes and cardiolipin, and release of cytochrome c into the cytosol . OMM: outer mitochondrial membrane; IMM: inner mitochondrial membrane; CL: cardiolipin; cyt c:

proteins play a prominent role in the regulation and execution of this process. As a result of this work, three different types of Bcl-2 family proteins have been identified: (a) the proapoptotic mediators, namely Bax and Bak; (b) the anti-apoptotic effectors, notably Bcl-2, Bcl-XL and Mcl-1; and (c) a host of Bcl-2 homology domain 3 (BH3-only) proteins which control either both the pro- and anti-apoptotic family members or only a specific member of one group (Youle & Strasser, 2008). The exact mechanisms of action and interplay of all these proteins are still a matter of vibrant debate. But there is general agreement that Bax and Bak are the terminal mediators of OMM permeabilization. As a result of Bax insertion /oligomerization into the OMM (promoted, i.e., by the cleavage of the BH3-only protein, Bid), pores are formed that mediate the release of pro-apoptotic proteins from the intermembrane space into the cytosol.  Thus, in this research line we can find papers that evidence how VacA, one of the major pathogenic products of *H. pylori* which induces large vacuoles within gastric epithelial cells, stimulates apoptosis via a mitochondria-dependent pathway (Chiozzi et al., 2009; Domanska et al., 2010; Galmiche et al., 2000; Kim et al., 2010). Some authors say that it interferes with mitochondrial permeability and reduces ΔΨm, followed by cyt c release. It is suggested that VacA may not act directly to induce cyt c release from mitochondria, instead Bax and Bcl-2 homologous antagonist/killer (Bak), were activated and relocated to mitochondria, promoting cyt c release (Calore et al., 2010; Matsumoto et al.,

they are proteinaceous or lipidic, is currently not known (Ott et al., 2009).

cytochrome c; MTP: mitochondrial transition pores.

2010; Yamasaki et al., 2006).

Fig. 5. Apoptotic pathways: the extrinsic pathway involves so-called death receptors, the intrinsic one involves mitochondrial alterations. Both pathways converge at caspase-3 activation, where classic biochemical and morphological changes in association with the apoptotic phenotype are originated

Every mitochondrion has a double lipid envelope that delimites the matrix, located within the inner mitochondrial membrane (IMM) and the intermembrane space located, in turn, between the IMM and the outer mitochondrial membrane (OMM). IMM cristae or invaginations increase the area where specific mitochondrial processes develop (electron transport and oxidative phosphorylation) (Fig. 6). The efficacy of these processes greatly depends on the bilayer's appropriate composition and structure, which relies on the role of the phospholipid cardiolipin (CL). CL is a specific component of IMM and the most abundant in it. On the CL are anchored many proteins such as respiratory chain I-IV complexes and cytochrome c (cyt c) (Calvino Fernandez & Parra Cid, 2010). Particularly relevant is the electrostatic binding between CL and cyt c in the IMM since this limits the amounts of cyt c that can be released during apoptosis (Iverson & Orrenius, 2004; Ott et al., 2002).

The crucial step in mitochondrion regulated apoptosis is the permeabilization of the OMM, often considered as the "point of no return" in apoptosis signalling (Orrenius et al., 2003), accompanied by the loss of mitochondrial membrane potential (ΔΨm) and mitochondrial transition pores (MTP) opening. OMM permeabilization is followed by the release of caspase activating proteins such as cyt c and second mitochondrion-derived activator of caspase (SMAC; also known as DIABLO) into the cytosol.

The past 10 years have seen considerable efforts to decipher the molecular pathways leading to permeabilization of the OMM, and it was early recognized that the Bcl-2 family of

Fig. 5. Apoptotic pathways: the extrinsic pathway involves so-called death receptors, the intrinsic one involves mitochondrial alterations. Both pathways converge at caspase-3 activation, where classic biochemical and morphological changes in association with the

Every mitochondrion has a double lipid envelope that delimites the matrix, located within the inner mitochondrial membrane (IMM) and the intermembrane space located, in turn, between the IMM and the outer mitochondrial membrane (OMM). IMM cristae or invaginations increase the area where specific mitochondrial processes develop (electron transport and oxidative phosphorylation) (Fig. 6). The efficacy of these processes greatly depends on the bilayer's appropriate composition and structure, which relies on the role of the phospholipid cardiolipin (CL). CL is a specific component of IMM and the most abundant in it. On the CL are anchored many proteins such as respiratory chain I-IV complexes and cytochrome c (cyt c) (Calvino Fernandez & Parra Cid, 2010). Particularly relevant is the electrostatic binding between CL and cyt c in the IMM since this limits the amounts of cyt c that can be released

The crucial step in mitochondrion regulated apoptosis is the permeabilization of the OMM, often considered as the "point of no return" in apoptosis signalling (Orrenius et al., 2003), accompanied by the loss of mitochondrial membrane potential (ΔΨm) and mitochondrial transition pores (MTP) opening. OMM permeabilization is followed by the release of caspase activating proteins such as cyt c and second mitochondrion-derived activator of

The past 10 years have seen considerable efforts to decipher the molecular pathways leading to permeabilization of the OMM, and it was early recognized that the Bcl-2 family of

apoptotic phenotype are originated

during apoptosis (Iverson & Orrenius, 2004; Ott et al., 2002).

caspase (SMAC; also known as DIABLO) into the cytosol.

Fig. 6. Mitochondria: structure and function. **A)** Mitochondrial structure. **B)** Mitochondrial membranes: relationship between electron transport chain complexes, cytochrome c, and cardiolipin. **C**) Mitochondrial transition pore formation: ruptured links between protein complexes and cardiolipin, and release of cytochrome c into the cytosol . OMM: outer mitochondrial membrane; IMM: inner mitochondrial membrane; CL: cardiolipin; cyt c: cytochrome c; MTP: mitochondrial transition pores.

proteins play a prominent role in the regulation and execution of this process. As a result of this work, three different types of Bcl-2 family proteins have been identified: (a) the proapoptotic mediators, namely Bax and Bak; (b) the anti-apoptotic effectors, notably Bcl-2, Bcl-XL and Mcl-1; and (c) a host of Bcl-2 homology domain 3 (BH3-only) proteins which control either both the pro- and anti-apoptotic family members or only a specific member of one group (Youle & Strasser, 2008). The exact mechanisms of action and interplay of all these proteins are still a matter of vibrant debate. But there is general agreement that Bax and Bak are the terminal mediators of OMM permeabilization. As a result of Bax insertion /oligomerization into the OMM (promoted, i.e., by the cleavage of the BH3-only protein, Bid), pores are formed that mediate the release of pro-apoptotic proteins from the intermembrane space into the cytosol. The molecular nature of such pores, that is whether they are proteinaceous or lipidic, is currently not known (Ott et al., 2009).

 Thus, in this research line we can find papers that evidence how VacA, one of the major pathogenic products of *H. pylori* which induces large vacuoles within gastric epithelial cells, stimulates apoptosis via a mitochondria-dependent pathway (Chiozzi et al., 2009; Domanska et al., 2010; Galmiche et al., 2000; Kim et al., 2010). Some authors say that it interferes with mitochondrial permeability and reduces ΔΨm, followed by cyt c release. It is suggested that VacA may not act directly to induce cyt c release from mitochondria, instead Bax and Bcl-2 homologous antagonist/killer (Bak), were activated and relocated to mitochondria, promoting cyt c release (Calore et al., 2010; Matsumoto et al., 2010; Yamasaki et al., 2006).

*Helicobacter pylori* and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis 179

detected. All these changes were eliminated by incubating the cells with Vit E during the

Fig. 7. Morphologic and functional alterations caused by *H. pylori* in epithelial gastric cells.

Solanum lyratum extract (SLE) was also able to suppress *H. pylori*-induced apoptosis (Hsu et al., 2010). SLE inhibited caspase-8 activation, thereby preventing the release of cyt c from mitochondria and activation of the subsequent downstream apoptotic pathway. Thus, SLE may offer a new approach for the treatment of *H. pylori* by down-regulation of apoptosis in the *H. pylori* infected gastric epithelium. As it does not directly target bacteria, SLE

General knowledge of scientific advances is necessary not only to read and interpret the literature but also to diagnose and treat diseases appropriately. The biological phenomena contributing to oxidative stress and cell death, fundamental processes in many gastrointestinal diseases, are important in the science and practice of gastroenterology. Currently a scientific revolution is ongoing in the understanding of cell death by a process referred to as "apoptosis or programmed cellular death", and the underlying causes of

*H. pylori* infection is associated with chronic gastritis, peptic ulcers, and gastric cancer. Extensive scientific evidence shows that in these alterations are involved oxidative stress

ROS can directly affect mitochondria and trigger apoptotic process.

treatment might not cause development of resistant strains.

apoptosis are being the subject of many studies.

**5. Conclusions**

period of infection. The antioxidant, also prevented that the cells died by apoptosis.

Not only VacA has been seen to be involved in *H. pylori*-induced mitochondria pathway apoptosis. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and *H pylori* (and ROS) induce APE-1 expression in human gastric epithelial cells, increasing intracellular calcium ion concentration of these cells which induces APE-1 acetylation. This *H. pylori*-mediated acetylation of APE-1 suppresses Bax expression, preventing p53-mediated apoptosis when *H. pylori* infects gastric epithelial cells (Bhattacharyya et al., 2009; Chattopadhyay et al., 2010).

*In vivo* studies indicate that *H. pylori*-induced apoptosis is associated with an increase in Bak expression in gastric biopsies from patients colonized by the bacterium. Konturek et al. reported induction of apoptosis with evidence of Bax up-regulation and Bcl-2 downregulation in duodenal ulcer patients with *H. pylori* infection (Konturek et al., 1999).

It is known that mitochondria are one of the possible targets and the major intracellular source of free radicals (ROS), since it is estimated that 1–2% of the oxygen consumed by mitochondria (and they consume 85% of all body oxygen) in the electron transport chain is converted to O2.- (Shigenaga et al., 1994). Elevated amounts of O2.- could have detrimental effects on nearby molecules, modifying several proteins of the mitochondrial membrane, lipids or even mitochondrial DNA (which has limited protection because of its lack of histones). In physiological conditions, mitochondria have several enzymes (manganesedependent superoxide dismutase, glutathione peroxidase) and non-enzimatic systems (NADPH, Vit C and E) that maintain O2.- concentrations at very low levels. But when some events cause an overproduction of free radicals, these systems are not able to eliminate the excess. Therefore, overproduction of ROS may also reduce the antioxidant defenses.

This oxidative stress may damage cellular components (Fig. 7), including polyunsaturated fatty acids, carbohydrates, structural and regulatory proteins, and DNA (Baik et al., 1996; Calvino-Fernandez et al., 2008; Jacobson, 1996).

It could well play a role in epithelial proliferation, apoptosis, and oxidative DNA damage.

Lipids of cellular membranes, such as CL, are particularly susceptible to oxidation due to the amount of double bonds in their structure, and it is expected that structural changes would be deleterious to normal mitochondrial function (Calvino-Fernandez et al., 2008; Calvino Fernandez & Parra Cid, 2010).

It is widely known that *H. pylori* infection increases epithelial apoptosis in gastric mucosa, which may play an important role in gastric carcinogenesis (Xia & Talley, 2001), and *H. pylori*-induced apoptosis may stimulate compensatory hyperproliferation which results in potential preneoplastic changes in chronic *H. pylori* infection (Moss et al., 1996). Besides, as noted above, higher levels of apoptosis have been detected in ulcer lesions (Satoh et al., 2003; Targa et al., 2007) in patients infected with bacteria, and also, this increased apoptosis disappears when *H. pylori* is eradicated. In both clinical alterations, ulcer and cancer, are also shown that there is an increase of oxidative stress.

With all these data, it is reasonable to assume that whether *H. pylori* causes oxidative stress with an increase of free radicals production, these ROS could directly affect mitochondria and trigger the apoptosis by a similar way shown in Fig.7 (Calvino Fernandez & Parra Cid, 2010).

An indisputable proof that these two processes (ROS and apoptosis) are related to, is the inhibition of apoptosis by treatment with antioxidants. In *in vitro* experiments (Calvino-Fernandez et al., 2008), *H. pylori* caused apoptosis in gastric epithelial cells. Simultaneously alterations in several structural and functional characteristics of mitochondria (high O2 . synthesis, decreased levels of antioxidant enzymes, cardiolipin oxidation, loss of membrane potential, large amounts of cytosolic cit c, higher levels of Bax and caspases,...) were detected. All these changes were eliminated by incubating the cells with Vit E during the period of infection. The antioxidant, also prevented that the cells died by apoptosis.

Fig. 7. Morphologic and functional alterations caused by *H. pylori* in epithelial gastric cells. ROS can directly affect mitochondria and trigger apoptotic process.

Solanum lyratum extract (SLE) was also able to suppress *H. pylori*-induced apoptosis (Hsu et al., 2010). SLE inhibited caspase-8 activation, thereby preventing the release of cyt c from mitochondria and activation of the subsequent downstream apoptotic pathway. Thus, SLE may offer a new approach for the treatment of *H. pylori* by down-regulation of apoptosis in the *H. pylori* infected gastric epithelium. As it does not directly target bacteria, SLE treatment might not cause development of resistant strains.

### **5. Conclusions**

178 Peptic Ulcer Disease

Not only VacA has been seen to be involved in *H. pylori*-induced mitochondria pathway apoptosis. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and *H pylori* (and ROS) induce APE-1 expression in human gastric epithelial cells, increasing intracellular calcium ion concentration of these cells which induces APE-1 acetylation. This *H. pylori*-mediated acetylation of APE-1 suppresses Bax expression, preventing p53-mediated apoptosis when *H. pylori* infects gastric epithelial cells

*In vivo* studies indicate that *H. pylori*-induced apoptosis is associated with an increase in Bak expression in gastric biopsies from patients colonized by the bacterium. Konturek et al. reported induction of apoptosis with evidence of Bax up-regulation and Bcl-2 down-

It is known that mitochondria are one of the possible targets and the major intracellular source of free radicals (ROS), since it is estimated that 1–2% of the oxygen consumed by mitochondria (and they consume 85% of all body oxygen) in the electron transport chain is converted to O2.- (Shigenaga et al., 1994). Elevated amounts of O2.- could have detrimental effects on nearby molecules, modifying several proteins of the mitochondrial membrane, lipids or even mitochondrial DNA (which has limited protection because of its lack of histones). In physiological conditions, mitochondria have several enzymes (manganesedependent superoxide dismutase, glutathione peroxidase) and non-enzimatic systems (NADPH, Vit C and E) that maintain O2.- concentrations at very low levels. But when some events cause an overproduction of free radicals, these systems are not able to eliminate the

regulation in duodenal ulcer patients with *H. pylori* infection (Konturek et al., 1999).

excess. Therefore, overproduction of ROS may also reduce the antioxidant defenses.

This oxidative stress may damage cellular components (Fig. 7), including polyunsaturated fatty acids, carbohydrates, structural and regulatory proteins, and DNA (Baik et al., 1996;

It could well play a role in epithelial proliferation, apoptosis, and oxidative DNA damage. Lipids of cellular membranes, such as CL, are particularly susceptible to oxidation due to the amount of double bonds in their structure, and it is expected that structural changes would be deleterious to normal mitochondrial function (Calvino-Fernandez et al., 2008;

It is widely known that *H. pylori* infection increases epithelial apoptosis in gastric mucosa, which may play an important role in gastric carcinogenesis (Xia & Talley, 2001), and *H. pylori*-induced apoptosis may stimulate compensatory hyperproliferation which results in potential preneoplastic changes in chronic *H. pylori* infection (Moss et al., 1996). Besides, as noted above, higher levels of apoptosis have been detected in ulcer lesions (Satoh et al., 2003; Targa et al., 2007) in patients infected with bacteria, and also, this increased apoptosis disappears when *H. pylori* is eradicated. In both clinical alterations, ulcer and cancer, are also

With all these data, it is reasonable to assume that whether *H. pylori* causes oxidative stress with an increase of free radicals production, these ROS could directly affect mitochondria and trigger the apoptosis by a similar way shown in Fig.7 (Calvino Fernandez & Parra Cid, 2010). An indisputable proof that these two processes (ROS and apoptosis) are related to, is the inhibition of apoptosis by treatment with antioxidants. In *in vitro* experiments (Calvino-Fernandez et al., 2008), *H. pylori* caused apoptosis in gastric epithelial cells. Simultaneously alterations in several structural and functional characteristics of mitochondria (high O2. synthesis, decreased levels of antioxidant enzymes, cardiolipin oxidation, loss of membrane potential, large amounts of cytosolic cit c, higher levels of Bax and caspases,...) were

(Bhattacharyya et al., 2009; Chattopadhyay et al., 2010).

Calvino-Fernandez et al., 2008; Jacobson, 1996).

shown that there is an increase of oxidative stress.

Calvino Fernandez & Parra Cid, 2010).

General knowledge of scientific advances is necessary not only to read and interpret the literature but also to diagnose and treat diseases appropriately. The biological phenomena contributing to oxidative stress and cell death, fundamental processes in many gastrointestinal diseases, are important in the science and practice of gastroenterology. Currently a scientific revolution is ongoing in the understanding of cell death by a process referred to as "apoptosis or programmed cellular death", and the underlying causes of apoptosis are being the subject of many studies.

*H. pylori* infection is associated with chronic gastritis, peptic ulcers, and gastric cancer. Extensive scientific evidence shows that in these alterations are involved oxidative stress

*Helicobacter pylori* and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis 181

Calore, F., Genisset, C., Casellato, A., Rossato, M., Codolo, G., Esposti, M. D., Scorrano, L., &

Calvino-Fernandez, M., Benito-Martinez, S., & Parra-Cid, T. (2008). Oxidative stress by

Calvino Fernandez, M., & Parra Cid, T. (2010). H. pylori and mitochondrial changes in

Chattopadhyay, R., Bhattacharyya, A., & Crowe, S. E. (2010). Dual regulation by

Chinnery, P. F., & Schon, E. A. (2003). Mitochondria. *J Neurol Neurosurg Psychiatry,* Vol.74,

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Das, D., Bandyopadhyay, D., Bhattacharjee, M., & Banerjee, R. K. (1997). Hydroxyl radical is

Davies, G. R., Simmonds, N. J., Stevens, T. R., Sheaff, M. T., Banatvala, N., Laurenson, I. F.,

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(exacerbated synthesis of ROS) caused by the bacteria by different mechanisms. In addition, a common feature of all these pathologies is the activation of apoptosis, either by extrinsic or, as it is more accepted today, for intrinsic pathway.

Novel therapeutic strategies that may be useful in the prevention or treatment of disorders of the stomach and duodenum caused by bacteria, should have as target the inhibition of the synthesis of ROS and/or apoptosis. Whereas the excess of free radicals is the origin of these alterations, the first step to take would be to restore the balance between oxidants and antioxidant systems. This would eliminate the oxidative stress of the gastric mucosa.

With respect to ROS, the actions may be aimed to the inhibition of its exacerbated synthesis, which serve to mitigate the toxic effects caused by the observed deficiencies in the antioxidant levels. Another strategy could be external supplementation of scavengers to remove this excess of free radicals. The antioxidant vitamins are the logical first choice as therapeutic agents, because of the large amount of data available on their role in *H. pylori* infection, gastroduodenal disease and gastric cancer.

Other new approach for the treatment of *H. pylori* could be the down-regulation of apoptosis in infected gastric epithelium. This might be possible by administering substances that inhibit the activation of caspases. Thus, it would prevent the release of cyt c from mitochondria and as a result, the subsequent downstream apoptotic pathway.

These actions are directed against the toxic manifestations caused by infection, while the traditional treatments with antibiotics have as target the bacterium itself. Consequently, these strategies would also prevent the development of resistant strains, an increasingly common problem due to the usual antibiotic treatments.

### **6. Acknowledgment**

This chapter was partly funded by a research grant PI-2008134 of Fundación para la Investigación Sanitaria en Castilla La-Mancha (FISCAM). The work of M. Calvino was funded by Instituto de Salud Carlos III through a "Contrato de Apoyo a la Investigación en el SNS" (CA07/00157).

### **7. References**

(exacerbated synthesis of ROS) caused by the bacteria by different mechanisms. In addition, a common feature of all these pathologies is the activation of apoptosis, either by extrinsic

Novel therapeutic strategies that may be useful in the prevention or treatment of disorders of the stomach and duodenum caused by bacteria, should have as target the inhibition of the synthesis of ROS and/or apoptosis. Whereas the excess of free radicals is the origin of these alterations, the first step to take would be to restore the balance between oxidants and

With respect to ROS, the actions may be aimed to the inhibition of its exacerbated synthesis, which serve to mitigate the toxic effects caused by the observed deficiencies in the antioxidant levels. Another strategy could be external supplementation of scavengers to remove this excess of free radicals. The antioxidant vitamins are the logical first choice as therapeutic agents, because of the large amount of data available on their role in *H. pylori*

Other new approach for the treatment of *H. pylori* could be the down-regulation of apoptosis in infected gastric epithelium. This might be possible by administering substances that inhibit the activation of caspases. Thus, it would prevent the release of cyt c from

These actions are directed against the toxic manifestations caused by infection, while the traditional treatments with antibiotics have as target the bacterium itself. Consequently, these strategies would also prevent the development of resistant strains, an increasingly

This chapter was partly funded by a research grant PI-2008134 of Fundación para la Investigación Sanitaria en Castilla La-Mancha (FISCAM). The work of M. Calvino was funded by Instituto de Salud Carlos III through a "Contrato de Apoyo a la Investigación en

Amieva, M. R., & El-Omar, E. M. (2008). Host-bacterial interactions in Helicobacter pylori

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Bhattacharyya, A., Chattopadhyay, R., Burnette, B. R., Cross, J. V., Mitra, S., Ernst, P. B.,

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Bhakat, K. K., & Crowe, S. E. (2009). Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis. *Gastroenterology,* Vol.136, No.7, pp. 2258-2269, ISSN 1528-0012

antioxidant systems. This would eliminate the oxidative stress of the gastric mucosa.

mitochondria and as a result, the subsequent downstream apoptotic pathway.

or, as it is more accepted today, for intrinsic pathway.

infection, gastroduodenal disease and gastric cancer.

common problem due to the usual antibiotic treatments.

**6. Acknowledgment**

el SNS" (CA07/00157).

0016-5085 (Linking)

5472 (Print) 0008-5472 (Linking)

(Electronic) 0016-5085 (Linking)

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**10**

*1Poland 2Germany*

**Role of New Appetite Hormones Ghrelin,**

**of Healing of Chronic Gastric Ulcers**

*2Thuringia-Clinic Saalfeld Georgiou's Agricola GmbH Teaching*

*Hospital of the University of Jena,*

**Orexin-A and Obestatin in the Mechanism**

Thomas Brzozowski Aleksandra Szlachcic1, Robert Pajdo1, Zbigniew Sliwowski1, Danuta Drozdowicz1, Jolanta Majka1, Wladyslaw Bielanski1, Peter. C. Konturek2, Stanislaw J. Konturek1 and Wieslaw W. Pawlik1 *1Department of Physiology, Jagiellonian University Medical College, Cracow*

The regulation of appetite is regulated by complex mechanism involving brain-gut axis and hormonal and non-hormonal factors acting through the activity of satiety and hunger centers located in the hypothalamus [Wren & Bloom, 2007]. Among hormones controlling appetite are gastrointestinal hormones such as cholecystokinin, PYY, pancreatic polypeptide, glucagon-like peptide 1, oxyntomedulin, the endocrine pancreatic hormones including insulin and glucagon and endocannabinoids [Wren &Bloom, 2007]. Recent discoveries of ghrelin and orexin-A gave a new insight into the understanding of mechanism of appetite control, satiety and obesity. Ghrelin is a novel 28-amino acid peptide that has recently been discovered in rat and human gastrointestinal tract, particularly in gastric mucosa, as an endogenous ligand for growth hormone (GH) secretagogue receptor (GHS-R) [Kojima et al., 1999]. Ghrelin stimulates food intake and body weight gain exerting a modulating effect on energy expenditure acting through afferent nerves and directly on hypothalamic feeding centers [Kojima et al., 1999; Tomasetto et al., 2000]. This peptide was also shown to enhance the gastric motility and gastric secretion [Tschop et al., 2000; Date et al., 2001]. Ghrelin is considered as an orexigenic peptide produced by the stomach, acting as a meal initiator [Masuda et al., 2000; Ariyasu et al., 2001]. Little is known about other factors that might affect ghrelin secretion in the stomach and its influence on gastrointestinal integrity has not been fully elucidated. A recent study in humans revealed that stomach is a major source of circulating ghrelin and that gastrectomy produced dramatic fall in the plasma ghrelin levels, whereas fasting and *anorexia nervosa* was

The orexins also called hypocretins, are neuropeptides novel originally discovered in a small group of neurons in the hypothalamic area (LHA) [Sakurai et al., 1998; de Lecea et al., 1998]. identified two peptides that were endogenous ligands to orphan receptor HFGAN72 [Sakurai et al., 1998]. They found that these peptides after i.c.v. injection stimulated food

accompanied by elevated plasma ghrelin levels [Ariyasu et al., 2001].

**1. Introduction**

### **Role of New Appetite Hormones Ghrelin, Orexin-A and Obestatin in the Mechanism of Healing of Chronic Gastric Ulcers**

Thomas Brzozowski Aleksandra Szlachcic1, Robert Pajdo1, Zbigniew Sliwowski1, Danuta Drozdowicz1, Jolanta Majka1, Wladyslaw Bielanski1, Peter. C. Konturek2, Stanislaw J. Konturek1 and Wieslaw W. Pawlik1 *1Department of Physiology, Jagiellonian University Medical College, Cracow 2Thuringia-Clinic Saalfeld Georgiou's Agricola GmbH Teaching Hospital of the University of Jena, 1Poland 2Germany*

### **1. Introduction**

186 Peptic Ulcer Disease

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apoptosis in gastric cancer cells through the mitochondrial pathway. J Gastroenterol Hepatol, Vol.22, No.7, pp. 1051-1056, ISSN 0815-9319 (Print) 0815The regulation of appetite is regulated by complex mechanism involving brain-gut axis and hormonal and non-hormonal factors acting through the activity of satiety and hunger centers located in the hypothalamus [Wren & Bloom, 2007]. Among hormones controlling appetite are gastrointestinal hormones such as cholecystokinin, PYY, pancreatic polypeptide, glucagon-like peptide 1, oxyntomedulin, the endocrine pancreatic hormones including insulin and glucagon and endocannabinoids [Wren &Bloom, 2007]. Recent discoveries of ghrelin and orexin-A gave a new insight into the understanding of mechanism of appetite control, satiety and obesity. Ghrelin is a novel 28-amino acid peptide that has recently been discovered in rat and human gastrointestinal tract, particularly in gastric mucosa, as an endogenous ligand for growth hormone (GH) secretagogue receptor (GHS-R) [Kojima et al., 1999]. Ghrelin stimulates food intake and body weight gain exerting a modulating effect on energy expenditure acting through afferent nerves and directly on hypothalamic feeding centers [Kojima et al., 1999; Tomasetto et al., 2000]. This peptide was also shown to enhance the gastric motility and gastric secretion [Tschop et al., 2000; Date et al., 2001]. Ghrelin is considered as an orexigenic peptide produced by the stomach, acting as a meal initiator [Masuda et al., 2000; Ariyasu et al., 2001]. Little is known about other factors that might affect ghrelin secretion in the stomach and its influence on gastrointestinal integrity has not been fully elucidated. A recent study in humans revealed that stomach is a major source of circulating ghrelin and that gastrectomy produced dramatic fall in the plasma ghrelin levels, whereas fasting and *anorexia nervosa* was accompanied by elevated plasma ghrelin levels [Ariyasu et al., 2001].

The orexins also called hypocretins, are neuropeptides novel originally discovered in a small group of neurons in the hypothalamic area (LHA) [Sakurai et al., 1998; de Lecea et al., 1998]. identified two peptides that were endogenous ligands to orphan receptor HFGAN72 [Sakurai et al., 1998]. They found that these peptides after i.c.v. injection stimulated food

Role of New Appetite Hormones Ghrelin, Orexin-A

hyperemic effects of ghrelin.

**2. Material and methods**

**2.1 Production of gastric ulcers**

and Obestatin in the Mechanism of Healing of Chronic Gastric Ulcers 189

GHS-R1a and OX-A1 receptors with D-lys3GHRP-6 and SB334867 [Peeters, 2005; Smart et al., 2001] respectively, on spontaneous ulcer healing and the alteration in ulcer size induced by ghrelin and orexin-A. We also determined the effects of PG inhibition with a nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (rofecoxib) inhibitors and blockade with L-NNA, the NO-synthase inhibitor on healing of these ulcers and accompanying changes in the GBF in rats without and with ghrelin or orexin-A administration. In addition, the expression of ghrelin mRNA at the ulcer margin and that in non-ulcerated gastric mucosa was assessed in rats with gastric ulcer. Finally, ghrelin rats were concomitantly treated with obestatin to check whether obestatin, recently considered as physiological opponent of ghrelin regarding food intake and appetite control [Zhang et al., 2005], could influence the ulcer healing itself and affect the healing and

Male Wistar rats, weighing 200-250 g and fasted for 24 h were used in all studies. All experimental procedures were run according to Helsinki Declaration and approved by the

Gastric ulcers were produced in rats using our modification (19) of acetic acid method originally proposed by Okabe et al. (1971) with our group modification [Konturek et al., 1987]. Animals were anesthetized with ether, the stomach was exposed and 75 l of acetic acid was poured through the plastic mold (6 mm diameter) onto serosal surface of anterior wall of the stomach just proximal to the antral gland area for 25 s. This produced an immediate necrosis of the entire mucosa and submucosa (but not serosa) within the area where the acetic acid was applied, i.e., about 28 mm2. The excess of acetic acid was then removed and the serosa was gently washed out with saline. Our previous studies documented that these ulcers became chronic within 2-3 days and healed completely within 2-3 weeks without perforation or penetration to the surrounding organs as described in original technique [Konturek et al., 1987; Okabe et al., 1971]. After the application of acetic acid the animals were allowed to recover from anesthesia and received only water at the day of operation (day 0). Then, they were divided into various groups and received normal

**2.2 Effect of orexin-A, ghrelin without and with the antagonists of ghrelin and orexin-A**

Several groups of rats with gastric ulcers, each consisting of 6-8 animals, were treated daily either with 1) vehicle (saline), 2) ghrelin or orexin-A (2.5-30 g/kg-d i.p.), 3) D-Lys3-GHRP-6 (200 g/kg i.p.), the ghrelin receptor antagonist (Peeters, 2005), or SB 334867 which is a selective non-peptide OX-R1 receptor antagonist [Smart, *et al.*, 2001; Holland, *et al.*, 2006] administered alone or in the combination with ghrelin and orexin-A (standard dose of 30 g/kg-d i.p.), respectively. SB 334867 was dissolved in DMSO and kept in a 20 mM solution as recommended by manufacturer [Tocris Cookson Ltd., Bristol, UK]. The dose of OX-R1 antagonist used in this study was selected on the basis of our earlier studies (unpublished

Jagiellonian University Institutional Animal Care and Use Committee.

chow and water *ad libitum* for the next 9 days and then were sacrificed.

**receptors on ulcer healing and the alterations in GBF at ulcer margin**

intake and their expression was upregulated upon fasting. Therefore, the group of these peptides has been named "orexins" after the Greek word "orexis" meaning appetite [Komaki et al., 2001]. The orexin group of peptides was originally identified as regulators of food intake and of sleep behavior and act at one or the other of 2 G protein-coupled receptors, the orexin-1 (OX-R1) and orexin-2 (OX-R2) receptors [Sakurai et al., 1998; Mondal et al., 1999]. OX-A is a 33-residue peptide possessing the identical sequence in humans and rodents, whereas human and rat OX-B differ by two residues. OX-A and OX-B activate two G protein-coupled receptors known as OX-1 and OX-2, the OX-1 receptor having a greater affinity for OX-A over OX-B, whereas the OX-2 receptor has similar affinity for both ligands [Sakurai, 2003]. Besides the presence in the brain, orexin-like immunoreactivity was found in the ENS of different species including guinea pig, rat, mouse and humans [Kirchgessner & Liu, 1999]. Interestingly, the prepro-orexin, orexin A and orexin receptor mRNA expression were demonstrated in the myenteric plexus and afferent nerves co-localizing with VIP-ergic nerves and in the gastrin-producing cells of the rat stomach [Kirchgessner & Liu, 1999]. Orexin-A has been linked with the peripheral energy balance and central nervous system mechanisms that coordinate sleep-wakefulness and motivated behaviors such as food seeking, especially in physiological state of fasting stress [Mondal et al., 1999; Sakurai, 2003]. Recent studies in humans revealed that the plasma orexin-A concentrations are increased during fasting in humans [Komaki et al., 2001].

The question remains as to whether these newly discovered peptides could play a role in the mechanism of gastric integrity and gastric mucosal defense has not been fully explored. Both, ghrelin and orexin-A were implicated in the mechanism of gastroprotection but their role in the healing process of chronic gastric ulcers has been little investigated. Previous studies by [Sibilia et al., 2003] and our group [Brzozowski et al., 2004; Brzozowski et al., 2006 b.; Konturek et al., 2004] revealed that that central and peripheral administration of ghrelin reduced the formation of acute gastric lesions induced by ethanol and cold stress. It was proposed that NO and sensory neuropeptides mediate these gastroprotective effects of ghrelin because the blockade of NO-synthase (NOS) activity with L-NAME and the functional ablation of sensory afferent nerves with capsaicin attenuated the protection and gastric hyperemia induced by ghrelin [Brzozowski et al., 2004; Brzozowski et al., 2006a.; Dimitrova et al., 2010]. In contrast to prevention of acute damage to the mucosal structure, the healing of chronic gastric ulcers lasts days and weeks and may involve restoration of gland architecture, angiogenesis and scar formation and it remains unknown whether appetite hormones may influence these processes.

Recently, endogenous prostaglandins (PG) have been implicated in the control of food intake and appetite [Lugarini et al., 2002; Scholz, 2003] but the possibility that these arachidonate metabolites may play any important role in the mechanism of ghrelin or orexin-A affecting ulcer healing has not been explored. This prompted our interest in endogenous PG because these arachidonate metabolites play an essential role in the mechanism of gastric defense and ulcer healing [Brzozowski et al., 2001] but the importance of PG derived from cyclooxygenase (COX-1) and COX-2 in the possible ulcer healing effects of orexigenic peptides has not been so far determined.

This study was designed to compare the effects of daily intraperitoneal (i.p.) administration of vehicle, ghrelin, orexin-A and obestatin on healing of preexisting gastric ulcers and accompanying changes in the gastric blood flow (GBF) at ulcer margin and the generation of PGE2 in the gastric mucosa. An attempt was made to examine the effect of antagonism of GHS-R1a and OX-A1 receptors with D-lys3GHRP-6 and SB334867 [Peeters, 2005; Smart et al., 2001] respectively, on spontaneous ulcer healing and the alteration in ulcer size induced by ghrelin and orexin-A. We also determined the effects of PG inhibition with a nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (rofecoxib) inhibitors and blockade with L-NNA, the NO-synthase inhibitor on healing of these ulcers and accompanying changes in the GBF in rats without and with ghrelin or orexin-A administration. In addition, the expression of ghrelin mRNA at the ulcer margin and that in non-ulcerated gastric mucosa was assessed in rats with gastric ulcer. Finally, ghrelin rats were concomitantly treated with obestatin to check whether obestatin, recently considered as physiological opponent of ghrelin regarding food intake and appetite control [Zhang et al., 2005], could influence the ulcer healing itself and affect the healing and hyperemic effects of ghrelin.

### **2. Material and methods**

188 Peptic Ulcer Disease

intake and their expression was upregulated upon fasting. Therefore, the group of these peptides has been named "orexins" after the Greek word "orexis" meaning appetite [Komaki et al., 2001]. The orexin group of peptides was originally identified as regulators of food intake and of sleep behavior and act at one or the other of 2 G protein-coupled receptors, the orexin-1 (OX-R1) and orexin-2 (OX-R2) receptors [Sakurai et al., 1998; Mondal et al., 1999]. OX-A is a 33-residue peptide possessing the identical sequence in humans and rodents, whereas human and rat OX-B differ by two residues. OX-A and OX-B activate two G protein-coupled receptors known as OX-1 and OX-2, the OX-1 receptor having a greater affinity for OX-A over OX-B, whereas the OX-2 receptor has similar affinity for both ligands [Sakurai, 2003]. Besides the presence in the brain, orexin-like immunoreactivity was found in the ENS of different species including guinea pig, rat, mouse and humans [Kirchgessner & Liu, 1999]. Interestingly, the prepro-orexin, orexin A and orexin receptor mRNA expression were demonstrated in the myenteric plexus and afferent nerves co-localizing with VIP-ergic nerves and in the gastrin-producing cells of the rat stomach [Kirchgessner & Liu, 1999]. Orexin-A has been linked with the peripheral energy balance and central nervous system mechanisms that coordinate sleep-wakefulness and motivated behaviors such as food seeking, especially in physiological state of fasting stress [Mondal et al., 1999; Sakurai, 2003]. Recent studies in humans revealed that the plasma orexin-A concentrations are

The question remains as to whether these newly discovered peptides could play a role in the mechanism of gastric integrity and gastric mucosal defense has not been fully explored. Both, ghrelin and orexin-A were implicated in the mechanism of gastroprotection but their role in the healing process of chronic gastric ulcers has been little investigated. Previous studies by [Sibilia et al., 2003] and our group [Brzozowski et al., 2004; Brzozowski et al., 2006 b.; Konturek et al., 2004] revealed that that central and peripheral administration of ghrelin reduced the formation of acute gastric lesions induced by ethanol and cold stress. It was proposed that NO and sensory neuropeptides mediate these gastroprotective effects of ghrelin because the blockade of NO-synthase (NOS) activity with L-NAME and the functional ablation of sensory afferent nerves with capsaicin attenuated the protection and gastric hyperemia induced by ghrelin [Brzozowski et al., 2004; Brzozowski et al., 2006a.; Dimitrova et al., 2010]. In contrast to prevention of acute damage to the mucosal structure, the healing of chronic gastric ulcers lasts days and weeks and may involve restoration of gland architecture, angiogenesis and scar formation and it remains unknown whether

Recently, endogenous prostaglandins (PG) have been implicated in the control of food intake and appetite [Lugarini et al., 2002; Scholz, 2003] but the possibility that these arachidonate metabolites may play any important role in the mechanism of ghrelin or orexin-A affecting ulcer healing has not been explored. This prompted our interest in endogenous PG because these arachidonate metabolites play an essential role in the mechanism of gastric defense and ulcer healing [Brzozowski et al., 2001] but the importance of PG derived from cyclooxygenase (COX-1) and COX-2 in the possible ulcer healing effects

This study was designed to compare the effects of daily intraperitoneal (i.p.) administration of vehicle, ghrelin, orexin-A and obestatin on healing of preexisting gastric ulcers and accompanying changes in the gastric blood flow (GBF) at ulcer margin and the generation of PGE2 in the gastric mucosa. An attempt was made to examine the effect of antagonism of

increased during fasting in humans [Komaki et al., 2001].

appetite hormones may influence these processes.

of orexigenic peptides has not been so far determined.

Male Wistar rats, weighing 200-250 g and fasted for 24 h were used in all studies. All experimental procedures were run according to Helsinki Declaration and approved by the Jagiellonian University Institutional Animal Care and Use Committee.

### **2.1 Production of gastric ulcers**

Gastric ulcers were produced in rats using our modification (19) of acetic acid method originally proposed by Okabe et al. (1971) with our group modification [Konturek et al., 1987]. Animals were anesthetized with ether, the stomach was exposed and 75 l of acetic acid was poured through the plastic mold (6 mm diameter) onto serosal surface of anterior wall of the stomach just proximal to the antral gland area for 25 s. This produced an immediate necrosis of the entire mucosa and submucosa (but not serosa) within the area where the acetic acid was applied, i.e., about 28 mm2. The excess of acetic acid was then removed and the serosa was gently washed out with saline. Our previous studies documented that these ulcers became chronic within 2-3 days and healed completely within 2-3 weeks without perforation or penetration to the surrounding organs as described in original technique [Konturek et al., 1987; Okabe et al., 1971]. After the application of acetic acid the animals were allowed to recover from anesthesia and received only water at the day of operation (day 0). Then, they were divided into various groups and received normal chow and water *ad libitum* for the next 9 days and then were sacrificed.

### **2.2 Effect of orexin-A, ghrelin without and with the antagonists of ghrelin and orexin-A receptors on ulcer healing and the alterations in GBF at ulcer margin**

Several groups of rats with gastric ulcers, each consisting of 6-8 animals, were treated daily either with 1) vehicle (saline), 2) ghrelin or orexin-A (2.5-30 g/kg-d i.p.), 3) D-Lys3-GHRP-6 (200 g/kg i.p.), the ghrelin receptor antagonist (Peeters, 2005), or SB 334867 which is a selective non-peptide OX-R1 receptor antagonist [Smart, *et al.*, 2001; Holland, *et al.*, 2006] administered alone or in the combination with ghrelin and orexin-A (standard dose of 30 g/kg-d i.p.), respectively. SB 334867 was dissolved in DMSO and kept in a 20 mM solution as recommended by manufacturer [Tocris Cookson Ltd., Bristol, UK]. The dose of OX-R1 antagonist used in this study was selected on the basis of our earlier studies (unpublished

Role of New Appetite Hormones Ghrelin, Orexin-A

A and obestatin administration.

weight.

**radioimmunoassay**

researcher blind to the experimental grouping.

and Obestatin in the Mechanism of Healing of Chronic Gastric Ulcers 191

concentration with isotonic saline. Control rats received the corresponding vehicle. Our preliminary studies (data not included) showed that none of the COX inhibitors used in this study produced by itself any gastric lesions at the doses tested. At the dose used in the present study, indomethacin has been shown previously to inhibit gastric PGE2 generation

In another group of animals with gastric ulcers and treated with COX-1 and COX-2 inhibitors with or without ghrelin, orexin-A and obestatin administration, the effect of prostaglandin replacement therapy using 16, 16 dimethyl PGE2 (Upjohn, Kalamazoo, MI, USA) applied i.g. in a dose of 5 g/kg-d was examined. This dose of dimethyl PGE2 was found in our preliminary study to be without any influence on ulcer healing and accompanying fall in GBF at ulcer margin. For this reason, synthetic PGE2 analog was administered together with each COX-1 or COX-2 inhibitor with or without ghrelin, orexin-

Upon the termination of experiment, the animals were anesthetized with pentobarbital, their abdomen was opened by midline incision and the stomach exposed for measurement of GBF by means of the H-gas clearance technique as described previously [Brzozowski et al., 2000 a]. For this purpose two electrodes of an electrolytic regional blood flow meter (Biotechnical Science, Model RBF-2, Osaka, Japan) were inserted into the gastric mucosa. The measurements were made in three areas of the oxyntic mucosa at ulcer margin and the mean values were calculated and expressed as percent changes of those recorded in the control animals. After GBF measurement, the stomach was removed, rinsed with saline and pinned open for macroscopic examination. The area of gastric lesions was determined by computerized planimetry (Morphomat, Carl Zeiss, FRG) [Brzozowski et al., 2000b] by a

Samples of the oxyntic gland area from the ulcer margin were taken by biopsy (about 100 mg) immediately after the animals were sacrificed to determine the mucosal generation of PGE2 by specific radioimmunoassay (RIA) as described previously [Konturek et al., 1995]. PGE2 was measured in duplicate using RIA kits (New England Nuclear, Munich, Germany). The mucosal generation of PGE2 was expressed in nanograms per gram of wet tissue

At the termination of some experiments with obestatin applied i.p. alone or coadministered with ghrelin, the rats were anesthetized with pentobarbital and blood samples (about 3 ml) taken from the *vena cava* for the measurement of plasma ghrelin, orexin-A and obestatin levels by RIA. Intact rats fasted overnight and given only vehicle (saline) i.p. were measured similarly in order to determine control values for plasma ghrelin, orexin-A and obestatin concentration. Blood samples were collected in heparin coated polypropylene tubes and centrifuged at 3000 rpm for 20 minutes at 4oC. The supernatant was then stored at -80C until measurement of plasma ghrelin, orexin-A and obestatin levels using an RIA-kit for rat ghrelin, orexin-A and obestatin (Phoenix Peptide, Belmont, CA, USA) [Brzozowski et al., 2004; Brzozowski et. al., 2006 b]. Briefly, the ghrelin, orexin-A and obestatin RIA involved the competition of each rat peptide sample with 125I-rat ghrelin, orexin-A and obestatin tracers for binding to a specific rabbit anti-ghrelin-, anti-orexin-A- and antiobestatin polyclonal antibody. The limit of assay sensitivity for ghrelin, orexin-A and

**2.4 Determination of plasma ghrelin, orexin-A and obestatin levels by**

by ~ 90 % without itself causing any mucosal damage [Konturek et al., 1987].

observation) with dose-dependent inhibition of orexin-A protection against WRS damage obtained with this orexin-A antagonist.

In another group of animals with gastric ulcers and treated with COX-1 and COX-2 inhibitors with or without ghrelin, orexin-A and obestatin administration, the effect of prostaglandin replacement therapy using 16,16 dimethyl PGE2 [Upjohn, Kalamazoo, MI, USA] applied i.g. in a dose of 5 g/kg-d was examined. This dose of dimethyl PGE2 was found in our preliminary study to be without any influence on ulcer healing and accompanying fall in GBF at ulcer margin. For this reason, synthetic PGE2 analog was administered together with each COX-1 or COX-2 inhibitor with or without ghrelin, orexin-A and obestatin administration.

Upon the termination of experiment, the animals were anesthetized with pentobarbital, their abdomen was opened by midline incision and the stomach exposed for measurement of GBF by means of the H-gas clearance technique as described previously [Brzozowski et al., 2000 a]. For this purpose two electrodes of an electrolytic regional blood flow meter (Biotechnical Science, Model RBF-2, Osaka, Japan) were inserted into the gastric mucosa. The measurements were made in three areas of the oxyntic mucosa at ulcer margin and the mean values were calculated and expressed as percent changes of those recorded in the control animals. After GBF measurement, the stomach was removed, rinsed with saline and pinned open for macroscopic examination. The area of gastric lesions was determined by computerized planimetry (Morphomat, Carl Zeiss, FRG) [Brzozowski et al., 2000 b] by a researcher blind to the experimental grouping.

Samples of the oxyntic gland area from the ulcer margin were taken by biopsy (about 100 mg) immediately after the animals were sacrificed to determine the mucosal generation of PGE2 by specific radioimmunoassay (RIA) as described previously [Konturek et al., 1995; Brzozowski et al., 2000]. PGE2 was measured in duplicate using RIA kits (New England Nuclear, Munich, Germany). The mucosal generation of PGE2 was expressed in nanograms per gram of wet tissue weight.

### **2.3 Effect of ghrelin and orexin-A on the healing of acetic acid ulcers and accompanying changes in the GBF at ulcer margin. Involvement of the COX-PG and NO-NOS systems in ghrelin-induced acceleration of ulcer healing**

The involvement of the COX-PG system in the ulcer healing effects of ghrelin was studied in rats treated with (or without) indomethacin, a non-selective COX-1 and COX-2 inhibitor, and with SC-560, a selective COX-1 inhibitor or rofecoxib, a specific COX-2 inhibitor [Brzozowski et.al., 1999]. In the experimental protocol, the following groups of rats, each consisting of 6-8 animals, were used: 1) vehicle (saline 1 ml i.p.); 2) ghrelin, orexin-A or obestatin, each applied in a standard dose of 30 g/kg-d i.p.; 2) indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg-d i.g.) and rofecoxib (10 mg/kg-d i.g.), each applied alone or coadministered with ghrelin, orexin-A or obestatin (30 g/kg-d i.p.). The doses of SC-560 and rofecoxib were selected on the basis of previous studies showing that these agents almost completely suppress PGE2 generation in exudates of air-pouch inflammation and inhibit gastric PGE2 production in mucosa with preexisting gastric ulcer [Futaki et al., 1993; Lesch et al., 1998]. SC-560 (Cayman Chemical Co., Ann Arbor, Michigan, USA) was first dissolved in absolute ethanol to obtain a stock solution of 50 mg/ml and then diluted to the desired concentration with isotonic saline. Rofecoxib [Sharp & Dhome, Warsaw, Poland] was first dissolved in methanol to obtain a stock solution 75 mg/ml and then diluted to the desired

observation) with dose-dependent inhibition of orexin-A protection against WRS damage

In another group of animals with gastric ulcers and treated with COX-1 and COX-2 inhibitors with or without ghrelin, orexin-A and obestatin administration, the effect of prostaglandin replacement therapy using 16,16 dimethyl PGE2 [Upjohn, Kalamazoo, MI, USA] applied i.g. in a dose of 5 g/kg-d was examined. This dose of dimethyl PGE2 was found in our preliminary study to be without any influence on ulcer healing and accompanying fall in GBF at ulcer margin. For this reason, synthetic PGE2 analog was administered together with each COX-1 or COX-2 inhibitor with or without ghrelin, orexin-

Upon the termination of experiment, the animals were anesthetized with pentobarbital, their abdomen was opened by midline incision and the stomach exposed for measurement of GBF by means of the H-gas clearance technique as described previously [Brzozowski et al., 2000 a]. For this purpose two electrodes of an electrolytic regional blood flow meter (Biotechnical Science, Model RBF-2, Osaka, Japan) were inserted into the gastric mucosa. The measurements were made in three areas of the oxyntic mucosa at ulcer margin and the mean values were calculated and expressed as percent changes of those recorded in the control animals. After GBF measurement, the stomach was removed, rinsed with saline and pinned open for macroscopic examination. The area of gastric lesions was determined by computerized planimetry (Morphomat, Carl Zeiss, FRG) [Brzozowski et al., 2000 b] by a

Samples of the oxyntic gland area from the ulcer margin were taken by biopsy (about 100 mg) immediately after the animals were sacrificed to determine the mucosal generation of PGE2 by specific radioimmunoassay (RIA) as described previously [Konturek et al., 1995; Brzozowski et al., 2000]. PGE2 was measured in duplicate using RIA kits (New England Nuclear, Munich, Germany). The mucosal generation of PGE2 was expressed in nanograms

**accompanying changes in the GBF at ulcer margin. Involvement of the COX-PG and**

The involvement of the COX-PG system in the ulcer healing effects of ghrelin was studied in rats treated with (or without) indomethacin, a non-selective COX-1 and COX-2 inhibitor, and with SC-560, a selective COX-1 inhibitor or rofecoxib, a specific COX-2 inhibitor [Brzozowski et.al., 1999]. In the experimental protocol, the following groups of rats, each consisting of 6-8 animals, were used: 1) vehicle (saline 1 ml i.p.); 2) ghrelin, orexin-A or obestatin, each applied in a standard dose of 30 g/kg-d i.p.; 2) indomethacin (5 mg/kg i.p.), SC-560 (5 mg/kg-d i.g.) and rofecoxib (10 mg/kg-d i.g.), each applied alone or coadministered with ghrelin, orexin-A or obestatin (30 g/kg-d i.p.). The doses of SC-560 and rofecoxib were selected on the basis of previous studies showing that these agents almost completely suppress PGE2 generation in exudates of air-pouch inflammation and inhibit gastric PGE2 production in mucosa with preexisting gastric ulcer [Futaki et al., 1993; Lesch et al., 1998]. SC-560 (Cayman Chemical Co., Ann Arbor, Michigan, USA) was first dissolved in absolute ethanol to obtain a stock solution of 50 mg/ml and then diluted to the desired concentration with isotonic saline. Rofecoxib [Sharp & Dhome, Warsaw, Poland] was first dissolved in methanol to obtain a stock solution 75 mg/ml and then diluted to the desired

**2.3 Effect of ghrelin and orexin-A on the healing of acetic acid ulcers and**

**NO-NOS systems in ghrelin-induced acceleration of ulcer healing**

obtained with this orexin-A antagonist.

A and obestatin administration.

per gram of wet tissue weight.

researcher blind to the experimental grouping.

concentration with isotonic saline. Control rats received the corresponding vehicle. Our preliminary studies (data not included) showed that none of the COX inhibitors used in this study produced by itself any gastric lesions at the doses tested. At the dose used in the present study, indomethacin has been shown previously to inhibit gastric PGE2 generation by ~ 90 % without itself causing any mucosal damage [Konturek et al., 1987].

In another group of animals with gastric ulcers and treated with COX-1 and COX-2 inhibitors with or without ghrelin, orexin-A and obestatin administration, the effect of prostaglandin replacement therapy using 16, 16 dimethyl PGE2 (Upjohn, Kalamazoo, MI, USA) applied i.g. in a dose of 5 g/kg-d was examined. This dose of dimethyl PGE2 was found in our preliminary study to be without any influence on ulcer healing and accompanying fall in GBF at ulcer margin. For this reason, synthetic PGE2 analog was administered together with each COX-1 or COX-2 inhibitor with or without ghrelin, orexin-A and obestatin administration.

Upon the termination of experiment, the animals were anesthetized with pentobarbital, their abdomen was opened by midline incision and the stomach exposed for measurement of GBF by means of the H-gas clearance technique as described previously [Brzozowski et al., 2000 a]. For this purpose two electrodes of an electrolytic regional blood flow meter (Biotechnical Science, Model RBF-2, Osaka, Japan) were inserted into the gastric mucosa. The measurements were made in three areas of the oxyntic mucosa at ulcer margin and the mean values were calculated and expressed as percent changes of those recorded in the control animals. After GBF measurement, the stomach was removed, rinsed with saline and pinned open for macroscopic examination. The area of gastric lesions was determined by computerized planimetry (Morphomat, Carl Zeiss, FRG) [Brzozowski et al., 2000b] by a researcher blind to the experimental grouping.

Samples of the oxyntic gland area from the ulcer margin were taken by biopsy (about 100 mg) immediately after the animals were sacrificed to determine the mucosal generation of PGE2 by specific radioimmunoassay (RIA) as described previously [Konturek et al., 1995]. PGE2 was measured in duplicate using RIA kits (New England Nuclear, Munich, Germany). The mucosal generation of PGE2 was expressed in nanograms per gram of wet tissue weight.

### **2.4 Determination of plasma ghrelin, orexin-A and obestatin levels by radioimmunoassay**

At the termination of some experiments with obestatin applied i.p. alone or coadministered with ghrelin, the rats were anesthetized with pentobarbital and blood samples (about 3 ml) taken from the *vena cava* for the measurement of plasma ghrelin, orexin-A and obestatin levels by RIA. Intact rats fasted overnight and given only vehicle (saline) i.p. were measured similarly in order to determine control values for plasma ghrelin, orexin-A and obestatin concentration. Blood samples were collected in heparin coated polypropylene tubes and centrifuged at 3000 rpm for 20 minutes at 4oC. The supernatant was then stored at -80C until measurement of plasma ghrelin, orexin-A and obestatin levels using an RIA-kit for rat ghrelin, orexin-A and obestatin (Phoenix Peptide, Belmont, CA, USA) [Brzozowski et al., 2004; Brzozowski et. al., 2006 b]. Briefly, the ghrelin, orexin-A and obestatin RIA involved the competition of each rat peptide sample with 125I-rat ghrelin, orexin-A and obestatin tracers for binding to a specific rabbit anti-ghrelin-, anti-orexin-A- and antiobestatin polyclonal antibody. The limit of assay sensitivity for ghrelin, orexin-A and

Role of New Appetite Hormones Ghrelin, Orexin-A

and Obestatin in the Mechanism of Healing of Chronic Gastric Ulcers 193

Fig. 1. The area of gastric ulcer induced by acetic acid and gastric blood flow (GBF) at ulcer margin at 8 day upon ulcer induction in rats treated with vehicle (saline) or with various doses of orexin-A (1 - 30 g/kg-d i.p.) or ghrelin (30 g/kg-d i.p.). Means SEM of 6-8 rats. Asterisk indicates a significant change (p<0.05) as compared to the vehicle-control values. area of gastric ulcer and significantly increased the GBF at ulcer margin starting from the dose of 5 g/kg (Fig. 2). The maximal reduction of ulcer area accompanied by a significant rise in the GBF at ulcer margin was recorded at the dose of 30 g/kg of obestatin where 35% reduction of ulcer area and an increase by about 19% of GBF at ulcer margin were observed

**3.2 Role of cyclooxygenase (COX)-1 and COX-2 inhibition and the blockade of nitric oxide (NO) synthase on ulcer healing activity and alterations in the GBF at ulcer**

As shown in Fig. 3, ghrelin and orexin-A given i.p. in dose 30 g/kg-d significantly decreased ulcer area and enhanced GBF as compared to those observed in vehicle. Treatment with D-Lys3-GHRS-6 (200 g/kg i.p.), the ghrelin GHS-R1a receptor antagonist, by itself failed to affect the area of gastric ulcers and GBF at ulcer margin at day 8 upon ulcer induction. The decrease in the area of these ulcers and accompanying rise in the GBF at ulcer margin induced by ghrelin and orexin-A were significantly attenuated by concurrent treatment with ghrelin and orexin-A receptor antagonists, D-Lys3-GHRS-6 and SB334867. The administration of D-lys3GHRP, which is a specific ghrelin receptor antagonist or SB334867, an antagonist of orexin-A OXR-1 receptors when applied alone, failed to affect the area of gastric ulcer and the GBF at ulcer margin as compared to those measured in vehicletreated rats. Treatment with D-lys3GHRP significantly attenuated ghrelin-induced acceleration of ulcer healing and the accompanying increase in GBF at ulcer margin.

as compared to the respective values in vehicle-treated animals.

**margin in rats treated with ghrelin, orexin-A and obestatin**

obestatin were 3 pg, 6pg and 5 pg per tube, the intra-assay variation was less than 8%,7% and 9%, and the interassay variation less than 5%, 4% and 7%, respectively.

### **2.5 Reverse-transcriptase-polymerase chain reaction (RT-PCR) for detection of messenger RNA (mRNA) for ghrelin and proinflammatory cytokines IL-1 and TNF in rats without and with gastric ulcers**