Therapeutic Effect And Mechanism Of LR12 In TAA-Induced Acute Liver Failure

Acute liver failure(ALF)is a rare and serious disease,which threatens human health.Liver transplantation is the most effective treatment for ALF.However,due to the serious shortage of donor organs and high cost,the application of liver transplantation is limited.Therefore,novel effective therapies are urgently required.Hepatocyte necrosis leads to the recruitment of many inflammatory cells in the liver,which further triggers systemic inflammatory response syndrome(SIRS),and may be one of the pathological mechanisms of ALF.Therefore,inhibition of inflammatory response is essential for the treatment of ALF.LR12 can inhibit the activation of triggering receptor expressed on myeloid cells-1(TREM-1),which can be used to treat a variety of inflammatory diseases.However,the role of LR12 in ALF remains unclear.Liver regeneration is not enough to compensate for hepatocyte necrosis,leading to the loss of hepatic function,which is one of the pathological mechanisms of ALF.Liver is an organ with strong regeneration ability and significant ability to regulate final quality.Previous studies have demonstrated that liver regeneration is a critical determinant of survival in animal models of ALF.However,whether LR12 can promote liver regeneration remains unclear.In this study,we established thioacetamide-(TAA-)induced ALF model in mice and observed the effects of LR12 on liver injury and hepatocyte regeneration.We explored the mechanism of LR12 promoting hepatocyte regeneration by establishing TAA-induced hepatocyte injury model in vitro.In this study,the cytokine of LR12 promoting liver regeneration were detected by cytokine antibody arrays.We further verified the role of the cytokine in vitro and in vivo.This project mainly includes the following four parts:Part one LR12 alleviated the liver injury and promoted hepatocyte regeneration in mice with TAA-induced ALFObjective: To establish TAA-induced ALF model in mice and observe the therapeutic effect of LR12 on ALF.Methods: 1.The ALF model was established by intraperitoneal injection of TAA in mice.The mice were divided into control group,TAA group,LR12 group and TAA+LR12 group.The pathological changes of liver were detected by hematoxylin and eosin(H&E)staining.2.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum were detected by Lai's method.3.The survival rates of mice in TAA group and TAA+LR12 group were observed.4.The PCNA expression in liver was detected by Western blot.5.The co-localization and expression of cytokeratin18(CK18)and proliferating cell nuclear antigen(PCNA)were detected by confocal laser scanning microscopy(CLSM).Results: 1.Hemorrhagic necrosis was increased in the liver in TAA group than that in control group;while compared with TAA group,it was decreased in TAA+LR12 group(P<0.01).2.Compared with the control group,the levels of ALT and AST in TAA model group were significantly increased,while compared with TAA group,they were significantly decreased in TAA+LR12 group(P<0.01).3.Compared with TAA model group,survival rate of mice in TAA+LR12 group was increased(P<0.05).4.The PCNA expression in the liver in TAA group was down-regulated than that in control group,while compared with TAA group,it was up-regulated in TAA+LR12group(P<0.01).5.The number of CK18 and PCNA co-located cells in the liver in TAA + LR12 group was increased significantly than that in TAA group(P<0.01).Conclusions: LR12 alleviated the liver injury and promoted hepatocyte regeneration in mice with TAA-induced ALF.Part two LR12 promoted hepatocyte proliferation in TAA-induced hepatocyte injury by acting on macrophagesObjective: To establish TAA-induced hepatocyte injury model and study the mechanism of LR12 promoting hepatocyte regeneration.Methods: 1.To establish the hepatocyte injury model in LO2 cells with different concentrations of TAA.The PCNA expression in LO2 cells was detected by Western blot.2.LO2 cells were stimulated with TAA or combined with LR12.The PCNA expression in LO2 cells was detected by CLSM and Western blot.3.LO2 cells were stimulated with supernatant from macrophages treated with TAA or combined with LR12.The PCNA expression in LO2 cells and primary hepatocytes were detected by CLSM and Western blot.4.Flow cytometry(FCM)was used to detect the cell cycle of LO2 cells.Results: 1.The PCNA expression was significantly decreased both in 1mM TAA group and 2 mM TAA group compared with control group(P<0.05).2.The PCNA expression in LO2 cells in TAA group was significantly decreased than that in control group,and it was not significantly increased in TAA+LR12 group compared with TAA group(P>0.05).3.The PCNA expression in LO2 cells in Supernatant-TAA group was significantly decreased than that in control group,while it was significantly increased in Supernatant-TAA+LR12 group compared with Supernatant-TAA group.The results of primary hepatocytes were consistent with those of LO2 cells(P<0.01).4.The proportion of S phase LO2 cells in Supernatant-TAA group was significantly lower than that in control group,while it was significantly higher in Supernatant-TAA+LR12 group than that in Supernatant-TAA group(P<0.01).Conclusion: LR12 promoted hepatocyte proliferation in TAA-induced hepatocyte injury by acting on macrophages.Part there LR12 promoted hepatocyte regeneration by promoting macrophage secretion of CCL20 in mice with TAA-induced ALFObjective: To study the mechanism of LR12 promoting hepatocyte regeneration by acting on macrophages.Methods: 1.Cytokine microarray was used to analyze the proliferation related cytokines in the supernatant from macrophages stimulated by TAA or combined with LR12.2.The C-C chemokine ligand 20(CCL20)expression in supernatant from macrophages and macrophages was detected by enzyme-linked immunosorbent assay(ELISA)and quantitative real time PCR(q-RT-PCR)respectively.3.The co-localization and expression of macrophage marker F4/80 and CCL20 were detected by CLSM.4.The LO2 cells were assigned into control group,TAA group,CCL20 group and TAA+CCL20 group.The PCNA expression in LO2 cells was detected by Western blot.5.The LO2 cells were divided into two groups:Supernatant-TAA+LR12 group and Supernatant-TAA+LR12+anti-CCL20 group.The PCNA expression was detected by Western blot.6.The mice were divided into two groups: TAA+LR12+Ig G group and TAA+LR12+anti-CCL20 group.The pathological changes of liver were detected by H&E staining.7.The levels of ALT and AST in serum were detected by Lai's method.8.The co-localization and expression of CK18 and PCNA in the liver were detected by CLSM.Results: 1.The CCL20 expression in Supernatant-TAA was significantly down-regulated than that in control group,while it was significantly up-regulated in Supernatant-TAA+LR12 group compared with Supernatant-TAA group(P<0.05).2.This result was further verified by ELISA and PCR.3.The number of F4/80 and CCL20 co-located cells was significantly increased in TAA+LR12 group in the liver than that in TAA group(P<0.05).4.The PCNA expression in LO2 cells in TAA group was significantly decreased than that in control group,while it was significantly increased in TAA+CCL20 group compared with TAA group(P<0.01).5.The PCNA expression in Supernatant-TAA+LR12+anti-CCLl20 group in LO2 cells was significantly lower than that in Supernatant-TAA+LR12 group(P<0.01).6.Hemorrhagic necrosis was increased in the liver in TAA+LR12+anti-CCL20 group than that in TAA+LR12+Ig G group(P<0.01).7.The serum levels of ALT and AST in TAA+LR12+anti-CCL20 group were significantly higher than that in TAA+LR12+Ig G group(P<0.05).8.The number of CK18 and PCNA co-located cells in TAA+LR12+anti-CCL20 group was significantly decreased than that in TAA+LR12+Ig G group(P<0.05).Conclusion: LR12 promoted hepatocyte regeneration in mice with TAA-induced ALF by promoting macrophage secretion of CCL20.Part four LR12 promoted liver regeneration in mice with TAA-induced ALF by promoting secretion of CCL20 from macrophages and activating p38 MAPK pathwayObjective: To clarify the mechanism of LR12 regulating hepatocyte regeneration by promoting CCL20 secretion from macrophages.Methods: 1.The LO2 cells were stimulated with supernatant from macrophages treated with TAA or combined with LR12.The phosphorylation p38(p-p38)expression was detected by Western blot.2.The model of TAA-induced ALF in mice was established.The p-p38 expression in primary hepatocytes was detected by Western blot.3.The LO2 cells were divided into two groups: Supernatant-TAA+LR12 group and Supernatant-TAA+LR12+anti-CCL20 group,and p-p38 expression was detected by Western blot.4.The mice were assigned into two groups:TAA+LR12+Ig G group and TAA+LR12+anti-CCL20 group.The nuclear translocation of p38 in hepatocyte was detected by CLSM.Results: 1.The p-p38 expression in LO2 cells in Supernatant-TAA group was significantly decreased than that in control group,while it was significantly increased in Supernatant-TAA+LR12 group compared with Supernatant-TAA group(P<0.01).2.The p-p38 expression in primary hepatocytes in TAA+LR12 group was significantly up-regulated than that in TAA group(P<0.05).3.The p-p38 expression in LO2 cells in Supernatant-TAA+LR12+anti-CCL20 group was significantly down-regulated than that in Supernatant-TAA+LR12 group.4.The nuclear translocation of p38 in TAA+LR12+Ig G group was significant up-regulated compared with TAA+LR12+anti-CCL20 group(P<0.01).Conclusion: LR12 promoted hepatocytes regeneration in mice with TAA-induced ALF by promoting secretion of CCL20 from macrophages and activating p38 MAPK pathway.