| OBJECTIVE:Establish rat liver heat ischemia in perfusion injury model, use the reagent of Copp and Zopp which can specific inducer and inhibit HO-1on the pretreatment of experimental animal, and analysis the protein、liver tissue inflammatory factor and pathological changes of liver tissue of TLR2-mediated MyD88signaling pathway. This paper discusses the protection mechanism of HO-1in the hepatic ischemia-reperfusion injury (HIRI) of TLR2-mediated MyD88signaling pathway, we want to reduce HIRI by inducting or inhibiting HO-1expression, and further to provide ideas and new therapeutic targets for reduce clinical hepatic ischemia reperfusion injury.Method:1. To establish the models of70%part of the HIRI by the method of operation which Nauta RJ had reported.2. Male Sprague-Dawley (S-D) Rats weighing220-250g were used and divided randomly into four groups:(1) sham operation group (n=9):no drugs were applied, only Laparotomy and anatomy of hepatic portal operation.(2) Ischemia reperfusion group (n=9):no drugs were applied, Hepatic ischemia reperfusion.(3) CoPP group (n=9):Rats received intraperitoneal injection CoPP, an HO-1inducer (i.p.)24h prior to Hepatic ischemia reperfusion.(4) ZnPPgroup (n=9):Rats received intraperitoneal injection ZnPP, an HO-1inhibitor (i.p.)24h prior to Hepatic ischemia reperfusion. Separate groups of rats were killed at24h after their Hepatic ischemia reperfusion and portal vein were opened, and the samples were collected for further analysis.Extraction of inferior vena cava blood5ml and the acquisition of fresh liver tissue, The hepatocellular function(ALT, AST) was analyzed; The expressions of HMGB1in the liver were detected by ELISA.HO-lmRNA, IL-1βmRNA, IFN-ymRNA, CXCL-1mRNAand CXCL-2mRNA expression levels of Liver tissue were examined by RT PCR.The levels of TLR2, MyD88, Caspase-3, p/t-p38, p/t-ERK, p/t-JNK, p-c-Jun and NF-κB protein were detected by Western Blotting. And further to detect the expression of related molecules of TLR2signaling pathway in Hepatic ischemia reperfusion, In order to explore the relationship between HO-1which protect of liver ischemia reperfusion injury and TLR2signaling pathway.The hepatocellular damage was evaluated by liver histology.Immunohistochemical (SP method) test CD3+T lymphocytes on hepatic periportal infiltration.Results:1. Thirty-six cases of ischemia-reperfusion injury models of liver were successfully established and partial liver ischemia time was sixty minutes.2. Serum ALT and AST levels:Compared with S group, the levels of ALT and AST in I group, C group and Z group were significantly higher(P<0.05); the levels of ALT and AST in C group were significantly lower than I group and Z group (P<0.05); However the levels of ALT and AST in Z group were more higher than I group (P<0.05); The difference was statistically significant.3. The content of HMGB1in serum:Compared with S group, the levels of HMGB1in I group, C group and Z group were significantly higher(P<0.05); the levels of HMGB1in C group were significantly lower than I group and Z group (P<0.05); while the levels of HMGB1in Z group were more higher than I group (P<0.05); The difference was statistically significant.4. The results of RT-PCR:(1) Compared with S group, the levels of IL-1β, IFN-γ, CXCL-1, CXCL-2mRNA in I group, C group and Z group were significantly higher(P<0.05); the levels of IL-1B, IFN-y, CXCL-1, CXCL-2mRNA in C group were significantly lower than I group and Z group (P<0.05); while the levels of IL-1B, IFN-y, CXCL-1, CXCL-2mRNA in Z group were more higher than I group (P<0.05); The difference was statistically significant.(2) Compared with S group, the levels of HO-1mRNA in I group and C group were significantly higher(P<0.05); the levels of HO-1mRNA in Z group were significantly lower than S group (P<0.05); while the levels of HO-1mRNA in C group were more higher than I group and Z group(P<0.05); the levels of HO-1mRNA in Z group were significantly lower than I group (P<0.05); The difference was statistically significant.5.The results of Western blot:(1) Compared with S group, the levels of TLR2, MyD88, p-ERK, p-c-Jun, Caspase-3, NF-κB in I group and Z group were significantly higher(P<0.05); the levels of TLR2, MyD88, p-ERK, p-c-Jun, Caspase-3, NF-κB in C group were significantly lower than S group, I group and Z group (P<0.05); while the levels of TLR2, MyD88, p-ERK, p-c-Jun, Caspase-3, NF-κB in Z group were more higher than I group (P<0.05); The difference was statistically significant.(2) Compared with S group, the levels of p-p38in I group and C group were significantly higher(P<0.05); the levels of p-p38in Z group were significantly lower than S group (P<0.05); while the levels of HO-1mRNA in C group were more higher than I group and Z group(P<0.05); the levels of HO-1mRNA in Z group were significantly lower than I group (P<0.05), The difference was statistically significant。(3) Compared with S group, the levels of t-p38, t-ERK, p/t-JNK in I group C group and Z group had no statistical difference (P>0.05).6. Histological results:S group the structure of hepatic lobule and Liver cell morphology were normal, there are a few inflammatory cells infiltration around the portal; I group the structure of hepatic lobule were disorder, Liver cell morphology were extensive edema while different degrees of vacuolar degeneration, others were partial liver cell spotty necrosis and eosinophil increase.Z group the structure of hepatic lobule were extensive edema, Liver cells were cytoplasm osteoporosis. There are part of a ballooning and eosinophilic necrosis, while some inflammatory cells infiltration around the portal. While C group the structure of hepatic lobule and Liver cell morphology were normal, there are some inflammatory cells infiltration around the portal.7. The results of CD3+T immunohistochemistry:HE staining in S group no significant inflammatory cell infiltration, I group showed many inflammatory cells infiltration, C group showed a small amount of inflammatory cell infiltration, while Z group showed a large number of inflammatory cell infiltration. The cytoplasm appeared brown staining for CD3+T lymphocyte positive cells, Compared with S group, the levels of CD3+T lymphocyte positive cells in I group, C group and Z group were significantly higher(P<0.05); the levels of CD3+T lymphocyte positive cells in C group were significantly lower than I group and Z group (P<0.05); while the levels of CD3+T lymphocyte positive cells in Z group were more higher than I group (P<0.05).Conclusion:1. Rat liver warm ischemia reperfusion injury model is a stable, reliable, simple, the model is workable.2. High expression of HO-1which induced by CoPP can inhibit cell death after HIRI, so then reduce the HIRI, may be related to the down-regulation of TLR2.3. In liver ischemia reperfusion after TLR2activation.use of ZnPP before reperfusion pretreatment can cause the intracellular adaptor protein MyD88, p-ERK, p-c-Jun, Caspase-3express increased, however use of CoPP before reperfusion pretreatment can cause the intracellular adaptor protein MyD88, p-ERK, p-c-Jun, Caspase-3express decreased, which means that TLR4participates in the pathogenesis of HIRI through upregulating the expression of MyD88, p-ERK, p-c-Jun, Caspase-3.4. In liver ischemia reperfusion after TLR2activation. use of ZnPP before reperfusion pretreatment can cause the intracellular adaptor protein p-p38express increased, however use of CoPP before reperfusion pretreatment can cause the intracellular adaptor protein p-p38express decreased, which means that TLR4participates in the pathogenesis of HIRI through upregulating the expression of p-p38.5. NF-κB was activated and inflammatory factors IL-1β, IFN-γ, CXCL-1and CXCL-2were overexpressed in HIR, NF-κB activity and the expression of IL-1β, IFN-γ, CXCL-1and CXCL-2was decreased significantly while TLR2was blocked by Copp; which indicates that HO-1can inhibit the activation of TLR2, Further by inhibiting activation of NF-κB to downregulate inflammatory factos of IL-1β, IFN-γ, CXCL-1and CXCL-2.6. TLR2-mediated MyD88signaling pathway was activated in HIRI,the inflammatory reaction was palliated while TLR2was blocked, which means that HO-1probably by down-regulating the expression of TLR2, Inhibiting of the TLR2-MyD88signal transduction pathway activation, reducing the release of inflammatory factors to protect the HIRI. |
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