The Role Of Toll Like Receptor-4 Signal Pathways Activation In Gastric Ischemia-Reperfusion Injury

The Role of Toll Like Receptor-4 Signal Pathways activation in Gastric Ischemia-Reperfusion InjuryBackgroundRecently, along with the progress in clinical medicine, the importance of ischemia-reperfusion injury was generally realized. Gastric mucosal was hypersensitive to redox, and was the most likely be involved in and express some clinical symptoms. Stomach and intestine endured ischemia-reperfusion injury at certain degree, when body suffered from trauma illness and operation stress.Our recent research found that Mitogen-activated protein kinases (MAPKs) and NF-kB signaling pathway were involved in gastric mucosa ischemia-reperfusion injury. Depressing the p38 MAPK, JNK and NF-kB pathways could attenuate stress induced stomach mucosa bleeding, lesion, inflammatory cytokines expression and apoptosis in rats. Some evidences showed the mechanism of ischemia-reperfusion and inflammatory injury were sameToll like receptor 4 (TLR4) played an important role in inflammation, and it had been found in gastric mucosa cells and many other tissues in mammalian. TLR4 was the upstream of MAPKs and NF-kB pathways, and the deficiency of it would depress the downstream pathways activation. TLR4 knockdown or mutant mouse strain showed some protective affection on ischemia-reperfusion stress, in brain, heart, liver and kidney. And TLR4 selected inhibitor Eritoran had been proved effective in protecting heart from ischemia-reperfusion induced injury. MAPKs and NF-kB pathways were involved in gastric ischemia-reperfusion injury (GIRI), but the role of TLR4 in GIRI is still unclear. Thus, the first part of this study was to compare the TLR4 mutant and wild type mice on GIRI, further investigated the activation of critical pathways downstream of TLR4, apoptosis and inflammation at the same time.In clinical cases, antiacid was widely used to prevent and cure gastric ischemia-reperfusion injury, which just get rid of the external part or aggravate factor of gastric ischemia-reperfusion injury. The intrinsic factor of gastric ischemia-reperfusion injury was redox stress, caused pathways activation, then inflammatory cytokines expressing and apoptosis, at last the barrier shield function of gastric mucosa was breakdown. Thus the second parts of the present study were to compare the effect of p38 MAPK inhibitor and proton pump inhibitor on gastric ischemia-reperfusion injury, and to investigate the mechanism of such effect. We expect that awareness of the role that TLR4 downstream signal transduction pathways in gastric ischemia-reperfusion induced gastric injury will add something useful to the pathogenesis of GIRI.Part I:The mechanism of TLR4 deficient reduced gastric ischemia-reperfusion injury in miceObjectives:To observe TLR4 deficient mice on gastric ischemia-reperfusion injury versus wild type mice, and investigate the mechanism of TLR4 deficient attenuated gastric ischemia-reperfusion injury.Methods:Wild type mice C3H/HouJ and TLR4 mutant mice C3H/HeJ,8-12 weeks old, were randomly divided into wild type sham group (WT sham), mutant sham group (MU sham), wild type (WT) and TLR4 mutant (MU) reperfusion Oh, 0.5h, 1h,2h,4h and 12h groups. Mice were starved but allowed free access to water for 12 h before the experiments. I/R injury of the stomach were produced by the occlusion and releasing of the celiac artery as Wada et al. described. Briefly, under pentobarbital sodium (40 mg/kg i.p.) anesthesia, the celiac artery was occluded with a small clamp for 0.5 h, and reperfusion was achieved by removing the clamp. Sham-operated animals underwent the same surgical procedure but without clamping the celiac artery. At different reperfusion time mice were killed, and tissue samples were collected. Four mice stomachs of each semi-group were kept in 10%neutral formalin solution for gastric mucosa bleeding area analysis by Image J 1.4, pathological examination, and the other six stomachs were kept in liquid nitrogen for further analysis. Thereafter, the gastric injury was evaluated by Masuda E's double-blind pathological score. The total RNA was extracted from the stomach tissues using Trizol and the TNF-a, IL-10 IL-1βand IFN-γmRNA expression was tested by the meaning of real time PCR. The location and counts of apoptosis cell in stomach was detected by TUNEL staining. Western blot was accessed to exam the protein content of caspase-8, Cleaved Caspase-3 and phosphorylation of p38 MAPK, JNK, ERK, IKB-a in stomach tissue. Activation of NF-kB was dectected by EMSA.Results:(1)The stomach showed significant bleeding area after 1 hour of reperfusion injury versus sham group both in wild type and mutant mice, and mutant mice showed obvious less bleeding than wild type mice after 1 hour of reperfusion injury. Results of Masuda E's double-blind pathological scores evaluation showed that, the sores of ischemia-reperfusion group were significant higher than the sham group's, at the same time mutant mice showed obvious lower scores than wild type mice after 1 hour of reperfusion injury. (2)According to the TUNEL examination results, TUNEL stained positive cells had been found expressed in the nucleus of stomach mucosa epithelial cells, and the positive cells count result was corresponding with caspase-8 and Cleaved caspase-3 expression. The apoptosis of ischemia-reperfusion group were significant higher than the sham group's, at the same time mutant mice showed obvious lower apoptosis than wild type mice after 1 hour of reperfusion injury. (3)TNF-a mRNA from the wild type 0.5 hours and 1 hour reperfusion groups expressed significantly higher than the wild type sham group, TNF-a mRNA from the mutant mice 0.5 hours and 1 hour reperfusion groups expressed significantly lower than the wild type mice to each correspond time point did by real time PCR test. IL-10 mRNA from the wild type 0.5 hours reperfusion groups expressed significantly higher than the wild type sham group, IL-10 mRNA from the mutant mice 0.5 hours reperfusion groups expressed significantly lower than the wild type mice, and 1 hour reperfusion groups expressed significantly higher than the wild type mice did by real time PCR test. IL-1(3 and IFN-γmRNA expression in TLR4 mutant mice were lower than wild type mice after 1 hour of rperfusion. (4)The expression of phosphorylated p38 MAPK (0,0.5,1,2,4 hours) and JNK (0.5 and 1 hours) were significant higher in wild type mice than in mutant mice after reperfusion. The expression of phosphorylated ERK was significant lower in wild type mice than in mutant mice after 0.5,2 and 4 hours of reperfusion, and there is no difference in other reperfusion group. EMSA analysis showed the activation of NF-kB was significant higher in wild type mice than in mutant mice after 1 hour of reperfusion. The expression of phosphorylated IKB-a were significant higher in wild type mice than in mutant mice after 1 hour of reperfusion, which were corresponding with the activation of NF-kB.Conclusions:TLR4 deficient reduced gastric ischemia-reperfusion injury versus wild type mice. The possible mechanisms are that the activation of p38 MAPK and NF-kB pathway lower in TLR4 mutant mice than in wild type mice, and TLR4 mutant mice has less apoptosis than wild type mice after gastric ischemia-reperfusion injury. TLR4 might be a potential strategy for prevention the pathological changes in gastric ischemia-reperfusion injury.Part II:The mechanism of p38 MAPK inhibitor reduced gastric ischemia-reperfusion injury in miceObjectives:To observe the effect of p38 MAPK inhibitor and proton pump inhibitor on gastric ischemia-reperfusion injury, and investigate the mechanism of attenuate effect on gastric ischemia-reperfusion injury of two reagents in mice. Then give some guideline on prevention and cure of gastric ischemia-reperfusion injury in clinic casesMethods:C57BL/6J mice,8-12 weeks old, were randomly divided into sham group (sham), ischemia-reperfusion+vehicle group (I/R+vehicle), ischemia-reperfusion+proton pump inhibitor group (I/R+proton pump inhibitor) and ischemia-reperfusion+p38 MAPK inhibitor group (I/R+p38 MAPK inhibitor). Mice were starved but allowed free access to water for 12 h before the experiments. Physiological saline (as vehicle) (0.9%NaCl lOml/kg), Pantoloc solution (20ml/kg) or SB239063 solution (20ml/kg,) were injected intraperitoneally 1 hour before the operation. I/R injury of the stomach were produced by the occlusion of the celiac artery as Wada et al. described. Briefly, under pentobarbital sodium (40 mg/kg i.p.) anesthesia, the celiac artery was occluded with a small clamp for 0.5 h, and reperfusion was achieved by removing the clamp. Sham-operated animals underwent the same surgical procedure but without clamping. After 1 hour of reperfusion mice were killed, and tissue samples were collected, four mice stomachs of each semi-group were kept in 10%neutral formalin solution for gastric mucosa bleeding area analysis by Image J 1.4, pathological examination, and the other six stomachs were kept in liquid nitrogen for further analysis. Thereafter, the gastric injury was evaluated by Masuda E's double-blind pathological score. The total RNA was extracted from the stomach tissues using Trizol and the TNF-a, IL-1β, INF-y and ICAM-1 expression was tested by the meaning of real time PCR. Western blot was accessed to exam the protein content of Cleaved Caspase-3 and phosphorylation of p38 MAPK, JNK and ERK in stomach tissue. For investigating the effect of proton pump inhibitor or p38 MAPK inhibitor on gastric fluid pH, mice were randomly divided into physiological saline group; Proton pump inhibitor group and p38 MAPK inhibitor group. Physiological saline (as vehicle), Pantoloc solution or SB239063 solution was administered immediately after pylorus was ligated. Mice were sacrificed 1 hour after solution injection and the stomachs were washed with 3 ml deionized water for measurement of gastric fluid pH.Results:(1)The stomach showed significant bleeding area after 1 hour of reperfusion injury versus sham group, both proton pump inhibitor and p38 MAPK inhibitor pretreatment group showed obvious less bleeding area than I/R+vehicle group. Results of Masuda E's double-blind pathological scores evaluation showed that, the sores of I/R+vehicle group were significant higher than that of sham group, at the same time pretreatment with proton pump inhibitor and p38 MAPK inhibitor showed obvious lower scores than I/R+vehicle. (2)MDA content from I/R+vehicle group expressed significantly higher than the sham group. At the same time pretreatment with proton pump inhibitor and p38 MAPK inhibitor showed obvious lower MDA content than I/R+vehicle. TNF-a, IL-1β, INF-y and ICAM-1 expression mRNA from I/R+vehicle group expressed significantly higher than the sham group, pretreatment with proton pump inhibitor significantly reduced TNF-α, IL-1βand INF-y mRNA expression versus I/R+vehicle group, pretreatment with p38 MAPK inhibitor significantly reduced TNF-a, mRNA expression versus I/R+vehicle group did by real time PCR test. (3)The expression of phosphorylated p38 MAPK, JNK and ERK from I/R+vehicle group expressed significantly higher than those of sham group, pretreatment with proton pump inhibitor significantly reduced activation of p38 MAPK, JNK and ERK versus I/R+vehicle group, pretreatment with p38 MAPK inhibitor significantly reduced activation of p38 MAPK and JNK versus I/R+vehicle group. Cleaved caspase-3 from I/R+vehicle group expressed significantly higher than the sham group, pretreatment with proton pump inhibitor had no obvious effect on Cleaved caspase-3 expression versus I/R+vehicle group, pretreatment with p38 MAPK inhibitor significantly reduced Cleaved caspase-3 expression versus I/R+ vehicle group.Conclusions:The p38 MAPK inhibitor attenuated gastric ischemia-reperfusion injury and had no obvious affect on gastric fluid pH in mice, the possible reasons were that p38 MAPK inhibitor depressing the p38 MAPK and JNK pathways, but had on obvious affect on activation of ERK pathway, finally breakdown of mucosa barrier was attenuated. Inhibition of p38 MAPK activation might be a potential strategy for prevention the pathological changes in gastric ischemia-reperfusion injury.