The Mechanism Of Hic-5 Gene Regulates Hepatic Ischemia-reperfusion Injury Through NF-κb And TLR4 Signaling Pathways

Objective: To explore the effect of Hic-5 gene in HIRI and its potential mechanism,enrich the mechanism of HIRI,and provide potential possibility for the prophylaxis and cure of HIRI in practice.Methods: 1.C57BL/6 mice(Male,6 to 8 weeks),including WT and Hic-5knockout(Hic-5 KO),were shuffled grouping.70% hepatic ischemia mouse model was established by blocking the left and middle lobe of the liver,and mice specimen were gathered at different reperfusion time points.The level of Hic-5 in liver tissues of WT and Hic-5 KO mice at different reperfusion time points were tested by WB and PCR.The morphological changes of liver tissue were observed by HE staining.The extent of ALT and AST were measured by ELISA.The extent of inflammatory cytokines was further examined.Immunohistochemical staining of CD11 b positive cells was performed to observe the number of pro-inflammatory Kupffer cells.The levels of chemokines in liver tissues and serum were detected by PCR and ELISA.Cell apoptosis was evaluated by TUNEL staining.The extent and activity of apoptosis-related proteins were test by Western Blot and ELISA.TLR4-positive cells in liver tissue sections were observed by immunohistochemical staining.TLR4,FADD and p-p65 protein expressions were detected by Western Blot.2.Cell experiments: liver tissues were digested by in situ digestion and in vitro digestion.HSC of WT and Hic-5 KO mice were isolated by Nycodenz gradient centrifugation.HSC H/R model was established by mixed gas culture method.The level of Hic-5 in WT and Hic-5 KO HSC was detected by PCR.The inflammatory cytokines,TLR4 were detected by PCR.Results: 1.Animal experiments: In WT Sham group,Hic-5 gene expression was scruple.In the HIRI model,the degree of Hic-5 in the WT mice was increased.The peak of Hic-5 gene appeared about 6h after reperfusion.HE staining showed that HIRI caused local liver tissue necrosis in mice,and Hic-5KO mice had fewer necrotic areas.ALT and AST were infinitesimal in the sham group,but aggrandize in HIRI group,and the phenomenon was more apparent in WT mice than in Hic-5 KO mice.Inflammation-related cytokines were less expressed in the Sham group.In the HIRI model mice,IL-1 and IL-6 levels were apparent increased,especially in WT mice.IL-10 was increased more in Hic-5 KO than WT mice.CD11 b staining results indicated that more CD11 b positive cells(M1-phenotype Kupffer cells)appeared in the liver tissues of mice in the HIRI group,and the number of CD11 b positive cells in the liver tissues of Hic-5 KO mice was less.Further studies showed that the expression of chemokines increased in HIRI mice,and Hic-5 deficiency inhibited the upregulation of chemokines.The TUNEL staining results showed that hepatocyte apoptosis occurred after HIRI,and Hic-5 deficiency could alleviate the hepatocyte apoptosis caused by HIRI.Meanwhile,Cleaved caspase-3 and 8level was up-regulated,and the absence of Hic-5 suppressed this phenomenon.The level of TLR4,FADD and p-p65 proteins were up-regulated in HIRI mice,and the degree of up-regulation was low in Hic-5 KO mice.2.Cell experiment:HSC of WT and Hic-5 KO mice was successfully isolated.PCR results indicated that Hic-5 expression was low in normal WT-HSC,but significantly increased in H/R model.Both HSCs showed increased IL-1 and IL-6 level in H/R,which was consistent with animal level,and WT-HSC expression was significant.Finally,PCR detection of related signaling molecules indicated that H/R increased the expression of TLR4,and Hic-5 deletion could inhibit the up-regulation of TLR4.Conclusion: 1.HIRI can increase the level of Hic-5 in mouse liver tissue;2.H/R can increase the level of Hic-5 in HSC.2.Hic-5 deficiency alleviates inflammation,apoptosis and liver tissue injury induced by hepatic ischemia/reperfusion through NF-κB and TLR4 signaling pathways.