helicobacter pylori have infected nearly half of the world s population . in the developing countries , more than 90% of adults
are typically infected during their early childhood , and once acquired the infection would last for several decades ( 1 ) .
helicobacter pylori infection is the main cause of gastritis and persistent chronic inflammation may lead to peptic ulcers or gastric cancer ( 2 - 4 ) .
helicobacter pylori gastritis is associated with the expression of various cytokines and immune cells infiltration ( 1 ) .
it is recently demonstrated that il-17 , il-8 and il-18 are associated with h. pylori - related gastritis diseases ( 5 - 7 ) .
recently , it is found that il-17 , a pro - inflammatory cytokine that can recruit neutrophils by inducing the production of cxc chemokines , plays a vital role in the inflammatory response to the h. pylori - related diseases ( 8) .
furthermore , it is also reported that il-17 , il-8 , and il-18 are important to maintain the chronic gastric inflammation in the mongolian gerbils model induced by the h. pylori ( 8 - 10 ) .
considering that up - regulation of il-17 , il-8 and il-18 plays a crucial role in h. pylori infection , it was explored that the expression of il-17 , il-8 and il-18 in peripheral blood may be involved in the inflammatory response to h. pylori .
the current study aimed to obtain evidence regarding the association between serum levels of il-17 , il-8 and il-18 and the stomach pathological changes induced by h. pylori infection in mongolian gerbils .
three - week - old male specific pathogen - free mongolian gerbils were purchased from wenzhou medical university animal experiment center / china ( approximately 25 g ) .
they were kept in polypropylene cages on saw dust bedding in groups of two per cage , and caged under a ld 12:12 cycle .
the animals used in the current study were cared for according to the institutional guidelines .
the experimental protocol was approved by the animal care and use committee of wenzhou medical university / china .
the bacteria were cultivated in columbia agar ( oxoid england ) with 10% sheep blood ( wenzhou kont biology and technology , ltd , china ) for two to three days at 37c under microaerobic conditions ( 15% co2 ) and high humidity , on a shaker at 100 rpm . after fasting ,
animals were inoculated every other day for three counts with 1 ml medium of h. pylori ( 109 cfu / ml ) using a metal stomach catheter as one cycle .
mongolian gerbils were randomly divided into four groups ( n = 6 in each ) .
groups a , b and c were firstly inoculated by gavage with h. pylori nctc 11637 for one cycle .
group b was enhanced inoculation for two counts , and group c was enhanced inoculation for three counts after the first inoculation .
all the animals were sacrified under anesthesia at 35 weeks . immediately after scarifying , the stomach was quickly removed and used for macroscopic , histological and electron microscopy examinations .
the serum levels of il-17il-8 and il-18 were measured using an elisa kit ( invitrogen , carlsbad , ca , usa ) according to the manufacturer s instructions .
differences between the groups were tested by an anova with subsequent scheffe post - hoc test and the relationship between il and gastritis serious were tested using spearman test .
three - week - old male specific pathogen - free mongolian gerbils were purchased from wenzhou medical university animal experiment center / china ( approximately 25 g ) .
they were kept in polypropylene cages on saw dust bedding in groups of two per cage , and caged under a ld 12:12 cycle .
the animals used in the current study were cared for according to the institutional guidelines .
the experimental protocol was approved by the animal care and use committee of wenzhou medical university / china .
the bacteria were cultivated in columbia agar ( oxoid england ) with 10% sheep blood ( wenzhou kont biology and technology , ltd , china ) for two to three days at 37c under microaerobic conditions ( 15% co2 ) and high humidity , on a shaker at 100 rpm . after fasting ,
animals were inoculated every other day for three counts with 1 ml medium of h. pylori ( 109 cfu / ml ) using a metal stomach catheter as one cycle .
mongolian gerbils were randomly divided into four groups ( n = 6 in each ) .
groups a , b and c were firstly inoculated by gavage with h. pylori nctc 11637 for one cycle .
group b was enhanced inoculation for two counts , and group c was enhanced inoculation for three counts after the first inoculation .
all the animals were sacrified under anesthesia at 35 weeks . immediately after scarifying , the stomach was quickly removed and used for macroscopic , histological and electron microscopy examinations .
the serum levels of il-17il-8 and il-18 were measured using an elisa kit ( invitrogen , carlsbad , ca , usa ) according to the manufacturer s instructions .
differences between the groups were tested by an anova with subsequent scheffe post - hoc test and the relationship between il and gastritis serious were tested using spearman test .
the assessment was performed for inflammation , neutrophil infiltrate , mucosal atrophy and intestinal metaplasia using the semi quantitative scales (  , none ; + , mild ; + + , moderate ; + + + , severe ) of the updated sydney system ( 11 ) ( table 1 ) .   note .
n= number of animals ; = none ; + = mild ; + + = moderate ; + + + = severe .   for control and the other groups ( n = 6 ) .
the results of the analysis showed the correlation between enhance inoculation counts and gastritis serious .
the serum levels of il-17 , but not il-8 and il-18 showed a correlation with the severity of gastritis ( figure 1 )   there was a correlation between serum levels of il-17 and the degree of gastritis .
the current study showed that the serum levels of il-17 , il-8 and il-18 obviously increased compared to those of control group .
moreover , it indicated that the serum levels of il-17 continuously increased after repetitive intragastric administration of h. pylori and were obviously correlated with the severity of gastritis .
however , the serum levels of il-8 and il-18 were up - regulated after intragastric administration of h. pylori for one cycle and without increase after intragastric administration ( figure 2 ) . a : il-17 ; b : il-8 ; c : il-18 .
the gastric mucosa of the control group and group a showed no visible changes . in group
b , the stomach had several small ulcers and mucosa expanded and thickened slightly . in group c , erosive lesions with more ulcers were detected , covered by abundant mucus ( figure 3 ) a : control group ; b : group a ; c : group b ; antral mucosa slightly expanded and several ulcers were detected ( arrows ) ; d : group c ; mucosa was covered by abundant mucus ( arrows ) .
histology examination was taken in the gastric mucosa of mongolian gerbils . in the control group , inflammatory cell infiltration in the lamina propria was inconspicuous . in group
the normal gland cells disappeared , leaving elongated pseudopyloric glands with a little of mucins in the transitional zone mucosa . in the lamina propria of the pyloric mucosa , moderate amount of inflammatory infiltrating cells was observed . in group
the submucosa also located similar mucous glands surrounded by interstitium with a conspicuous infiltration of lymphocytes . in group c ,
several ulcers were found near the transitional zone and some of them reached the muscularis propria . in the vicinity of transitional mucosa between pylori and fundic mucosa large erosion was observed , which consisted of numerous inflammatory cells ( figure 4 ) . a : control group ; b : group a
; few neutrophils infiltrated the epithelium in the transitional zone ; c : group b ; erosion was observed ; d : group c ; the glandular structures were damaged ( h & e x400 ) .
electron microscopy observation was conducted on the mongolian gerbils gastric mucosa . in the control group ,
a , the nucleus showed mild pyknosis , mild expansion could be observed in rough endoplasmic reticulum , and the mitochondria edema could be observed in cellular . moreover , gastric mucosa microvilli showed edema and falling off .
in group b , the nucleus showed pyknosis and deformation , the mitochondria vacuoles appeared , and the mucus particles of glands epithelial cells became fusion and aggregation . in group c ,
gap of nuclear membrane and rough endoplasmic reticulum expanded , the edema , degeneration and necrosis could be observed in mitochondria ( figure 5 ) .
b : group a ; the gastric mucosa microvilli edema and falling off ( arrows ) ; c : group b. the mucus particles of glands epithelial cells became fusion and aggregation ( arrows ) ; d : group c ; autophagosome and autophagic vacuoles formatiom ( arrows ) .
in the present study , it was known that the regulation of cytokines in the gastric mucosa plays a significant role in the h. pylori infections such as il-17 , il-6 , il-8 , il-18 , and tnf- ( 1 , 5 , 9 , 10 , 12 ) .
mongolian gerbils are perfect animals to study gastric disease pathogenesis details ( 13 , 14 ) .
the current research enriches the cytokine profiles of the chronic phases of h. pylori infections . to the authors ` best knowledge , there are no reports on the serum levels of il-17 , il-8 , il-18 and the gastric mucosa pathological changes to date .
helicobacter pylori infections in the mongolian gerbils induced an antral - predominant gastritis and gradually progressed to the whole corpus ( 14 , 15 ) .
some mucosal cytokines expression was paralleled with the acute inflammation such as il-1 ( 16 ) .
in addition , gastric corporal mucosal chronic inflammation was paralleled with ifn- expression ( 17 ) .  
pro - inflammatory gene activity was highly sublimated and the risk of gastric disease was promoted for the increased inflammatory cytokines . il-17 as an emerging family member expressed in the t memory cells plays a key role in the pathogenesis of some chronic inflammatory diseases ( 18 - 20 ) .
il-17 can also stimulate the expression level of il-8 and facilitate the chemotaxis of neutrophils ( 20 , 21 ) .
some clinical studies found that the levels of il-17 increased in gastric mucosa of the patients infected with h. pylori ( 20 ) .
it was also known that the gastric mucosal levels of il-17 increased in mongolian gerbils with h. pylori infection , especially in the chronic inflammatory phase ( 8 , 18 - 20 ) .
up to now , almost all investigators only focused on studying the gastric mucosal levels of il and the mrna expression .
the current study focused on studying the serum levels of il-17 and it was confirmed to be bound up with the amount of neutrophils as well as the development of gastric disease .
numerous small ulcers appeared in the animals stomach and mucosa expanded and thickened slightly in the macroscopic observation .  
an obvious neutrophils infiltration on the background of chronic inflammation could be perceived by the ordinary microscope and the edema , degeneration and necrosis in subcellular organelles could be observed through electron microscopy .
the contemporaneous appearance of pathology changes and il-17 level suggest that the inflammation of cells infiltration may be linked to the serum cytokines levels .
these results suggest that il-17 plays a significant role in the inflammatory response to h. pylori infection and may serve as a potential biomarker to diagnose and predict the chronic active gastritis .
il-8 acts as a chemo attractant which could enhance the migration of neutrophils and leukocytes , and regulate the processes of chronic inflammation ( 22 ) .
it is also engaged in human gastric cancer cells adhesion , migration , and invasion in vitro ( 23 ) and highly expressed in gastric cancer cells ( 24 ) .
yamaoka y , et al . reported that il-8 and il-18 are associated with h. pylori - related gastritis diseases ( 5 , 7 ) .
the current study also demonstrated that il-8 was a marked up regulation compared with the control group , and the increased serum levels of il-8 may not promote the inflammatory response .
il-18 as a member of il-1 superfamily induced by ifn- was correlated with the severity of gastric inflammation ( 25 , 26 ) .  
some researchers reported that il-18 is important to maintain the chronic gastric inflammation in the mongolian gerbils model induced by the h. pylori ( 8 - 10 ) .
however , other studies indicated that the mrna levels of il-18 are independent of h. pylori infections ( 25 , 27 ) . in the current study ,
however , the serum levels of il-18 were not correlated with counts of enhanced inoculation or the severity of pathology changes . therefore , in order to understand the role of il-18 in human h. pylori infections further studies are needed . in conclusion , the current study results demonstrated that the serum levels of il-17 , il-8 and il-18 play significant roles in the progression and regulation of mongolian gerbils with h. pylori infections .
moreover , the serum levels of il-17 differed among each group of animals , and depended on the severity of gastritis disease .
therefore , il-17 might be related to the gastritis clinical prognosis and be used as a marker to predict the prognosis . however , in order to understand more correlation of the cytokines in h. pylori infections additional studies are required .
 background : persistent helicobacter pylori infection confers an increased risk for serious illnesses such as peptic ulcers and gastric cancer . 
 various cytokines are involved in the regulation of inflammatory immune response in h. pylori - infected gastric mucosa.objectives:the current study aimed to obtain evidence regarding the association between il-17 , il-8 and il-18 expression in peripheral blood and h. pylori infection in mongolian gerbils.materials and methods : mongolian gerbils were inoculated with h. pylori by a metal stomach catheter . 
 after sacrifice , their gastric mucosae were examined in macroscopic , histological and electron microscopy levels . in addition , enzyme linked immunosorbent assay ( elisa ) assay was performed on the il-17 , il-8 and il-18 cytokines in the blood samples.results:serum levels of il-17 , il-8 and il-18 were remarkably up - regulated compared to those of the control group . 
 there was an obvious correlation between the increase of il-17 and the serious extent of gastritis in the current study . 
 however , the serum levels of il-8 and il-18 without getting increasingly more for repetitive intragastric administration . 
 there were plenty of neutrophils infiltrating in the infected group mucosal . 
 intestinal metaplasia and gastric ulcers were also founded in h. pylori infected animals after enhanced inoculation . 
 the edema , degeneration and necrosis changes could be found in organelles by transmission electron microscopy . 
 more serious pathological changes were detected in the enhanced inoculation groups compared to the cycle group.conclusions:the serum levels of il-17 , but not il-8 and il-18 may serve as a potential biomarker for diagnosis and predicting the prognosis of gastritis caused by h. pylori . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Animals
3.2. Bacterial Inoculation and Helicobacter pylori Colonization 
3.3. Experimental Design
4. Results
5. Discussion