reactive oxygen species ( ros ) play a vital role in lps - triggered macrophage activation by regulating intracellular reduction - oxidation ( redox ) sensitive signaling pathways and nuclear transcription factors , such as nuclear factor-b ( nf-b ) and nuclear factor - erythroid 2 p45-related factor 2 ( nrf2 ) [ 13 ] .
overproduction of ros in macrophages leads to excessive expression of cytokines and inflammatory factors , resulting in atheromatous plaques , acute inflammation , tissue injury , and sepsis .
however , most cell types have developed defensive mechanisms to counteract ros generation [ 5 , 6 ] .
hemeoxygenase-1 ( ho-1 ) , a member of the intracellular phase ii enzyme family , is thought to play an essential role in maintaining cellular redox homeostasis against ros generation and oxidative stress [ 7 , 8 ] .
ho-1 , which is expressed in cells at a low level without stimulation , can be rapidly induced by various oxidative - inducing agents , including lps , heme , hypoxia , and subsequent lethality . in oxidative stress and inflammation conditions ,
enhancement of ho-1 expression plays an important role in cell protection [ 9 , 11 ] .
this important cytoprotective action in response to various cellular stimuli makes it conceivable to target ho-1 induction as a promising therapeutic intervention in treating a variety of disorders related to oxidative stress and inflammation .
nrf2 , an upstream transcription factor modulating phase ii enzyme activity , interacts with the antioxidant response element ( are ) in the nucleus to induce are - dependent gene expression . under physiological conditions ,
nrf2 is sequestered by binding to kelch - like ech - associated protein 1 ( keap1 ) . upon oxidative stress , nrf2 parts from keap1 and translocates into the nucleus to induce the expression of ho-1 .
suppression of nrf2 by the specific nrf2 inhibitor , brusatol , has been reported to attenuate the nrf2-mediated defense mechanism and weaken its antioxidant ability , indicating the importance of nrf2 in antioxidant activity .
previous studies have suggested that phosphatidylinositol 3-kinase ( pi3k)/akt is a key survival signaling pathway that enhances cellular defense , making it a potential treatment target not only by promoting cell survival , but also by modulating nrf2 as an upstream signaling molecule [ 1417 ] . cross - talk between the pi3k / akt and nrf2 signaling pathways has been reported to govern the cellular defense system against inflammatory and oxidative damages .
ly294002 , a specific pi3k / akt inhibitor , could significantly attenuate the pi3k / akt - mediated cell defense mechanism by suppressing phosphorylation of akt and thus inhibiting activation of nrf2/ho-1 expression
. therefore , the pi3k / akt pathway plays a vital role in the nrf2-mediated antioxidant response , making it a potential target for medical intervention .
many polyphenols that scavenge free radicals have been identified and proposed as therapeutic agents to counteract oxidative stress - induced diseases .
recently , pomegranate extract , composed of abundant tannins , has been reported to exhibit antioxidant , anti - inflammation , and lipase inhibitory activities [ 2023 ] .
punicalagin ( 2,3-hexahydroxydiphenoyl - gallagyl - d - glucose ; pun ) is the major polyphenol isolated from pomegranate ( punica granatum l. ) , but few studies have been carried out focusing on its bioactivities . our previous research has shown that pun inhibits lps - induced mitogen - activated protein kinases ( mapks ) and nf-b activation and thus suppresses overproduction of cytokines and inflammatory factors , including nitric oxide ( no ) , prostaglandin e2 ( pge2 ) , interleukin- ( il- ) 1 , il-6 , and tumor necrosis factor- ( tnf- )  .
however , the antioxidant activity and mechanisms of pun in macrophages remain unknown . in this study
furthermore , we tried to uncover the molecular mechanism by which the pi3k / akt pathway regulates the pun - induced nrf2/ho-1 activation and antioxidant activity . moreover , we tried to reveal the underlying defense mechanism of pun in lps - stimulated macrophage oxidative stress .
pun ( > 98% hplc purity ) and brusatol were purchased from tauto biotech ( shanghai , china ) .
lps ( escherichia coli 055:b5 ) and insulin were purchased from sigma - aldrich chemical ( st . louis , mo , usa ) . fetal bovine serum ( fbs ) , antibiotic - antimycotic , and trizol reagent
bicinchoninic acid ( bca ) protein assay kit was purchased from pierce ( rockford , il , usa ) .
antibodies against gapdh , akt , p - akt , keap1 , nrf2 and ho-1 , and ly294002 were purchased from cell signaling technology ( danvers , ma , usa ) .
the goat anti - mouse antibody was purchased from li - cdr odyssey ( lincoln , ne , usa ) .
the probe , 2,7-dichlorodihydrofluorescein diacetate ( dcfh2-da ) , was purchased from invitrogen ( carlsbad , ca , usa ) .
raw264.7 cells were purchased from the american type culture collection ( rockville , md , usa ) .
cells were cultured in dmem medium supplemented with 10% fbs and antibiotics ( 100  u / ml penicillin and 100  u / ml streptomycin ) at 37c in a humidified incubator with 5% co2 .
the nitrite accumulated in the culture medium was measured as an indicator of no production based on the griess reaction .
raw264.7 cells , cultured in 96-well plates for 24  h , were treated with or without lps ( 1  g / ml ) in the presence of various doses of pun ( 25 , 50 , or 100  m , 1  h prior to lps treatment ) .
after 24  h , culture supernatants were mixed with griess reagent ( equal volumes of 1% ( w / v ) sulfanilamide in 5% ( v / v ) phosphoric acid and 0.1% ( w / v ) naphthylethylenediamine - hcl ) and incubated at room temperature for 10  min .
absorbance values were measured at 550  nm and no concentration was calculated with reference to a standard curve of sodium nitrite .
briefly , raw264.7 cells , cultured in 24-well plates for 24  h before the experiment , were pretreated with pun ( 100  m ) for 1  h and then stimulated with lps ( 1  g / ml ) for 12  h to induce ros production .
cells were washed twice with pbs and then incubated with dcfh2-da probe ( 20  nm ) for 15  min .
fluorescence staining was visualized using a fluorescence microscope ( olympus , ix71 ) , and fluorescence assays were measured with a fluorescence microplate reader ( tecan , sunrise ) at excitation / emission 525/610  nm .
raw264.7 cells ( 1  10 ) cultured in tissue culture flasks for 24  h were treated with the desired agents .
cells were then harvested on ice , washed twice with ice - cold pbs , and suspended in 500  l of lysis buffer supplemented with protease inhibitors ( jiancheng , nanjing , china ) .
after 30  min incubation on ice , cell extracts were subjected to centrifugation ( 12,000  g ) at 4c for 15  min to get cell protein .
proteins were separated by sds - page , electrotransferred to nitrocellulose membranes ( pierce ) , and then hybridized with the specific antibodies .
densitometric values of immunoblot signals were obtained from three separate experiments using image j ( national institutes of health , bethesda , md , usa ) .
raw264.7 cells were preincubated in 6-well plates ( 1  10 ) and pretreated with pun ( 25 , 50 , or 100  m ) 1  h prior to 1  g / ml lps treatment for 12  h in a 37c and 5% co2 incubator .
the concentration and integrity of the rna were measured at a 260/280  nm ratio .
quantitative pcr analysis was carried out using the dna engine mx3000p fluorescence detection system ( agilent , santa clara , ca , usa ) against a double - stranded dna - specific fluorescent dye ( stratagene , la jolla , ca , usa ) according to optimized pcr protocols .
-actin was amplified in parallel with the target genes and used as a normalization control .
95c for 3  min , followed by 40 cycles of 95c for 10  s , 60c for 20  s , and 72c for 60  s. expression levels were determined using the relative threshold cycle ( ct ) method as described by the manufacturer ( stratagene ) .
the pcr reaction system ( 25  l in total ) contained 12.5  l of sybr green pcr mix ( stratagene ) , 0.375  l of reference dye , 1  l of each primer ( both 10  m ) , 1  l of cdna template , and 9.125  l of depc - treated water .
quantitative real - time rt - pcr was performed using the following primers : -actin , f : 5-cccatctatgagggttacgc-3 , r : 5-tttaatgtcacgcacgattt c-3 ; sod1 , f : 5-ccacgtccatcagtatgggg-3 , r : 5-cgtcctttccagcagtcaca-3 ; sod2 , f : 5-gtgtctgtgggagtccaagg-3 , r : 5-ccccagtcatagtgctgcaa-3 ; ho-1 , f : 5-cacgcatatacccgctacct-3 , r : 5-ccagagtgttcattcgagca-3 ; nrf2 , f : 5-aacagaacggccctaaagca-3 , r : 5-tgggattcacgcataggagc-3.
the results were expressed as mean  sem and differences between mean values of normally distributed data were assessed by the one - way analysis of variance ( anova ) followed by duncan 's test for multiple comparisons .
ho-1 expression was measured to determine whether pun exhibits potential antioxidant activity by upregulating the intracellular phase ii enzyme , ho-1 , in raw264.7 cells .
western blot analysis was performed to detect the expression of ho-1 induced by different concentrations of pun and as a function of time .
the results showed that pun started to significantly increase ho-1 protein levels from 6  h in a time - dependent manner ( figure 1(a ) ) .
furthermore , pun enhanced ho-1 protein levels at doses of 50 to 200  m in a dose - dependent manner ( figure 1(b ) ) . to further characterize the molecular mechanism of pun activity , nrf2 ,
the data demonstrated that 100  m pun treatment significantly increased nrf2 expression from 4  h to 8  h in a time - dependent manner ( figure 1(c ) ) . also , an 8  h treatment with 50 to 200  m pun increased nrf2 expression in a dose - dependent manner ( figure 1(d ) ) .
keap1 functions as a suppressor of nrf2 by retaining it in the cytoplasm and enhancing its proteasomal degradation via ubiquitination .
however , in our study pun treatment also increased keap1 accumulation from 4  h to 12  h ( 100  m ) and at doses of 50 to 200  m ( 8  h ) ( figures 1(c ) and 1(d ) ) .
rt - pcr results also showed that pun could significantly increase the nrf2 and ho-1 mrna expression since 4  h treatment .
however , we found that 24  h treatment still showed an enhancement in nrf2 mrna expression but not in nrf2 protein expression , indicating that the inhibition effect of pun on keap1-nrf2 could not last more than 24  h ( figure 1s available online at http://dx.doi.org/10.1155/2015/380218 ) .
thus , we speculated that pun increased nrf2 accumulation and activation by inhibiting keap1 ubiquitination of nrf2 and/or the physical interaction , thereby preventing nrf2 from being degraded . in the 12  h pun treatment group ,
the keap1 level significantly increased , while the nrf2 level notably decreased , suggesting that the inhibitory effect of pun ( 100  m ) on the keap1-nrf2 ubiquitination sustained no longer than 12  h , as keap1 rapidly accumulated and degraded nrf2 protein , keeping nrf2 at a low concentration .
brusatol is a specific nrf2 inhibitor , suppressing its translocation and preventing it from inducing are - mediated antioxidant genes , including ho-1 .
we used brusatol to block nrf2 activity and found that the pun - induced ho-1 accumulation was significantly attenuated ( figure 2 ) , suggesting that pun induced ho-1 upregulation via nrf2-mediated signaling .
recent studies have demonstrated that the pi3k / akt pathway acts as an important upstream regulator of ho-1 expression ; thus we investigated whether the pi3k / akt pathway also plays a central role in the pun - induced ho-1 expression .
western blot analysis showed that pun treatment notably enhanced akt phosphorylation after 2  h of treatment in a time - dependent manner , but not the total akt protein level , suggesting that the pun - induced ho-1 expression may be associated with the pi3k / akt signaling pathway ( figure 3(a ) ) . to further reveal the mechanism , we used ly294002 , a specific pi3k / akt inhibitor .
the results showed that pun treatment significantly increased ho-1 expression ; however , inhibiting akt phosphorylation by ly294002 significantly attenuated the pun - induced ho-1 activation ( figure 3(b ) ) .
therefore , we concluded that pi3k / akt is essential for the pun antioxidant activity by regulating pun - induced ho-1 expression .
lps treatment triggers ros generation in macrophages , so we used lps to determine whether pi3k / akt signaling regulates the ros scavenging property of pun .
as expected , both fluorescence staining and assay values demonstrated that the pi3k / akt pathway regulates the ability of pun to scavenge ros ( figure 4 ) , which is in accordance with its effect on ho-1 expression .
the results showed that ly294002 inhibited the pun - induced activation of nrf2 ( figure 3(b ) ) , indicating that nrf2 may be a vital molecule in the pi3k / akt - mediated ho-1 expression in pun - treated cells . to further understand this mechanism , brusatol was used to suppress the nrf2-mediated signaling pathway in the presence of pi3k / akt mediators .
we found that upregulation ( insulin ) or downregulation ( ly294002 ) of pi3k / akt in presence of pun did not show a significant modulation effect on ho-1 expression when nrf2 was blocked ( figure 5 ) , indicating that nrf2 plays a central role in the pi3k / akt - regulated ho-1 expression in pun - treated cells .
hence , we concluded that nrf2 is downstream pi3k / akt , but upstream ho-1 in the pun - regulated antioxidant signaling pathway .
when stimulated with lps , the intracellular ros level in macrophages increased rapidly , causing oxidative stress .
we then tried to evaluate the antioxidant activity of pun by examining ros generation and sod1/sod2 mrna expression .
lps stimulation significantly increased the ros level in macrophages , but pretreatment with pun notably prevented the lps - induced ros generation .
as a well - established ros scavenger , n - acetyl cysteine ( nac ) treatment also showed a significant inhibitory effect on the lps - induced ros overproduction .
the inhibitory effect of 200  m pun and 50  m nac treatment was comparable ( figures 6(a ) and 6(b ) ) .
rt - pcr analysis showed that lps significantly reduced sod1 mrna expression and enhanced sod2 mrna expression .
pun pretreatment significantly reversed the reduction in sod1 mrna caused by lps but showed no effect on sod2 mrna expression ( figure 6(c ) ) .
no is another important free radical molecule and proinflammatory factor in lps - stimulated macrophages . because we revealed that the pi3k / akt pathway is essential for pun to scavenge intracellular ros , we examined the role of pi3k / akt in the reduction of no production in pun - treated cells .
the results showed that pun pretreatment notably decreased the lps - induced no overexpression in macrophages ; however , upregulation or downregulation of pi3k / akt did not affect the pun - induced reduction in no , suggesting that pun inhibited the lps - induced no expression via another signaling pathway figure 6(d ) .
pomegranate extracts have been reported to have many beneficial health effects , exhibiting antioxidant , anti - inflammation , antiproliferative , and dna repair activities , which are generally attributed to the high polyphenol content [ 2628 ] .
the pomegranate husk is rich in polyphenols such as punicalagin ( pun ) , punicalin , gallagic acid , ellagic acid , and ea - glycosides .
pun is a hydrolysable polyphenol in which gallagic acids are linked to a sugar moiety with a molecular weight of 1084 .
previous studies have shown that pun composes 85% of the total pomegranate tannins and accounts for more than 50% of the antioxidant activity of pomegranate juice [ 29 , 31 ] .
an increasing number of studies indicated that the bioactivities of pomegranate extracts are associated with pun [ 32 , 33 ] .
our recent study has demonstrates that pun inhibits lps - induced inflammatory factors and cytokine overexpression , including no , pge2 , il-1 , il-6 , and tnf- , via suppression of toll - like receptor 4-mediated mapks and nf-b activation in macrophages , which may contribute to the inhibition effect of pomegranate on inflammation .
oxidative stress caused by lps or other stimuli may trigger the activation of macrophages , leading to an excessive inflammatory process .
however , as a polyphenol possessing most of the pomegranate 's antioxidant activity , only limited studies have been carried out on the antioxidant property of pun and its underlying mechanism in macrophages .
ho-1 can be stimulated by a variety of factors , including heme , hyperoxia , and ros in most cell types , and studies have revealed that upregulation of ho-1 contributes to the cellular defense mechanism in response to stimuli .
lps challenge rapidly increases the ros level in macrophages , not only directly damaging dna , but also inducing overproduction of inflammatory factors as well as cytokines , which may lead to severe tissue injury .
macrophages are essential for recognizing and eliminating microbial pathogens , and thus the survival of macrophages may directly contribute to the host defense system .
therefore , ho-1 , which is important for protecting macrophages from ros , has recently become a popular target for antioxidant medicine development [ 3638 ] .
the present study showed that pun treatment markedly increased ho-1 protein level in macrophages in a time- and dose - dependent manner , indicating that pun exhibited antioxidant activity by upregulating ho-1 expression .
nrf2 is known as an upstream mediator of are - dependent phase ii enzyme expression , including ho-1 [ 3941 ] . under quiescent conditions ,
nrf2 is anchored in the inactive form to the cytoplasm through binding to keap1 , which in turn facilitates the ubiquitination and subsequent proteolysis of nrf2 protein .
the sequestration and further degradation of nrf2 are mechanisms for the repressive effect of keap1 and nrf2 . to investigate whether pun upregulates ho-1 via enhancing nrf2 accumulation and activation , protein was extracted and the nrf2 protein level was examined by western blot analysis .
the results showed that pun treatment significantly increased nrf2 protein level in the nucleus from 4  h to 8  h and in a dose range of 50  m to 200  m , suggesting that pun may regulate ho-1 expression by mediating nrf2 signaling .
we used brusatol , a specific nrf2 inhibitor , to determine whether blocking nrf2-mediated signaling attenuates pun - induced ho-1 expression .
as expected , we found that inhibition of nrf2 markedly suppressed pun - induced ho-1 expression .
however , as shown in figures 1(c ) and 1(d ) , the protein level of keap1 also increased by pun treatment . as a cul3-rbx1-dependent e3 ubiquitin ligase
, keap1 degrades nrf2 by directly interacting with it and conjugating ubiquitin onto the n - terminal neh2 domain of nrf2 .
thus , we hypothesized that pun may also affect the interaction between keap1 and nrf2 .
therefore , we investigated the possibility that pi3k / akt mediates pun - induced ho-1 expression .
as shown in figure 3(a ) , pun treatment notably increased the phosphorylation level of akt protein , while no significant changes were found in the total akt protein level , suggesting that enhancement of akt protein phosphorylation may contribute to the pun - induced ho-1 expression . to further identify the role of the pi3k / akt pathway , ly294002 , a specific pi3k / akt inhibitor , was used to treat macrophages together with pun .
the results showed that inhibition of the pi3k / akt signaling pathway markedly blocked ho-1 expression in the presence of pun .
furthermore , we investigated whether pi3k / akt plays an important role in ros scavenging by pun , in accordance with its regulation of ho-1 expression .
fluorescent probes showed that blocking pi3k / akt signaling by ly294002 attenuated the ros scavenging capability of pun in lps - induced oxidative stress in macrophages , and this may be related to the downregulation of ho-1 expression . because the pi3k / akt and nrf2 pathways are both implicated in the transcriptional regulation of the antioxidant enzyme ho-1 , we tried to reveal their underlying mechanisms in the pun - induced ho-1 protein expression .
the data demonstrated that inhibition of pi3k / akt significantly suppressed nrf2 protein expression induced by pun .
then , we examined whether blocking nrf2 inhibits the pi3k / akt - mediated ho-1 expression in the presence of pun .
the results demonstrated that inhibition of nrf2 suppressed the upregulation of ho-1 by pun treatment in macrophages ; however , neither ly294002 nor insulin had a significant effect when the nrf2 signaling was blocked .
therefore , we concluded that the pi3k / akt signaling pathway played a vital role in pun - induced ho-1 expression by regulating nrf2 . ros overproduction plays a central role in lps - induced macrophage activation , leading to an excessive inflammatory process . in this study , we assessed the inhibitory effect of pun on ros production .
nac , a well - established ros scavenger , was used to treat macrophages as a positive control , and 50  m nac significantly decreased the lps - induced ros overproduction , to a similar extent as 200  m pun treatment .
sod , which catalyzes the dismutation of superoxide radical ( o2 ) to hydrogen peroxide ( h2o2 ) and oxygen ( o2 ) , is another kind of phase ii enzyme mediated by nrf2 that serves as a defense mechanism against oxidative damage .
sod1 ( cu / zn - sod ) locates primarily in the cytoplasm , and sod2 ( mn - sod ) , a structurally distinct protein , locates in the mitochondria .
our results demonstrated that a 12  h lps challenge significantly reduced the sod1 mrna expression and increased sod2 mrna expression in macrophages .
however , pun pretreatment only notably reversed the sod1 mrna expression , with no effect on sod2 mrna expression .
no is another important molecule regulated by the nrf2-mediated pathway , whose excessive generation has been shown to result in oxidative stress and inflammation during an lps challenge [ 37 , 47 ] .
previous studies have indicated that the pi3k / akt pathway is a potential target of medicines that exhibit an no inhibition effect in lps - induced macrophages [ 48 , 49 ] .
hence , we investigated whether pi3k / akt signaling also modulates the inhibition property of pun on no generation in macrophages .
however , comparing ly294002 treatment with insulin treatment , we concluded that upregulation or downregulation of pi3k / akt showed no significant changes in decreasing the lps - induced no expression in the presence of pun , indicating that pi3k / akt signaling was not involved in the inhibitory effect of pun on no expression in macrophages . our previous study has revealed that pun inhibits lps - induced no overproduction by suppressing mapks / nf-b activation . in that study , we inferred that pun inhibited no expression through mapks signaling , but not through pi3k / akt signaling .
thus , we concluded that pun exhibited antioxidant properties , including scavenging ros , potentiating sod1 expression , and inhibiting no production . taken together ,
these results show that pun enhances ho-1 expression by upregulating the nrf2-mediated pathway in macrophages and that the pi3k / akt pathway plays a central role in this mechanism .
our data also demonstrated that pun exhibits considerable antioxidant properties in lps - stimulated macrophages by inhibiting ros generation and no overproduction and by enhancing the sod1 mrna expression .
these findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage , sepsis , and endotoxemic shock .
 reactive oxygen species ( ros ) and oxidative stress are thought to play a central role in potentiating macrophage activation , causing excessive inflammation , tissue damage , and sepsis . 
 recently , we have shown that punicalagin ( pun ) exhibits anti - inflammatory activity in lps - stimulated macrophages . 
 however , the potential antioxidant effects of pun in macrophages remain unclear . 
 revealing these effects will help understand the mechanism underlying its ability to inhibit excessive macrophage activation . 
 hemeoxygenase-1 ( ho-1 ) exhibits antioxidant activity in macrophages . 
 therefore , we hypothesized that ho-1 is a potential target of pun and tried to reveal its antioxidant mechanism . here , pun treatment increased ho-1 expression together with its upstream mediator nuclear factor - erythroid 2 p45-related factor 2 ( nrf2 ) . however , specific inhibition of nrf2 by brusatol ( a specific nrf2 inhibitor ) dramatically blocked pun - induced ho-1 expression . 
 previous research has demonstrated that the pi3k / akt pathway plays a critical role in modulating nrf2/ho-1 protein expression as an upstream signaling molecule . here , ly294002 , a specific pi3k / akt inhibitor , suppressed pun - induced ho-1 expression and led to ros accumulation in macrophages . furthermore , pun inhibited lps - induced oxidative stress in macrophages by reducing ros and no generation and increasing superoxide dismutase ( sod ) 1 mrna expression . 
 these findings provide new perspectives for novel therapeutic approaches using antioxidant medicines and compounds against oxidative stress and excessive inflammatory diseases including tissue damage , sepsis , and endotoxemic shock . 
1. Introduction
2. Materials and Methods
3. Statistical Analysis
4. Results
5. Discussion