Effect And Mechanism Of Erythropoietin Derivative HBSP On Hepatic Ischemia-reperfusion Injury And Fibrosis In Mice

Background: Ischemia/Reperfusion(I/R)injury is one of the main causes of complications after transplantation and even grafts failure,and liver fibrosis affects long-term graft survival after liver transplantation.Both are closely related to the complications of liver transplantation,which seriously affects the success rate of surgery and the prognosis of patients.There is no effective treatment.HBSP(Helix B surface peptide)is a short peptide consisting of 11 amino acids derived from EPO(Erythropoietin)with the function of strong tissue protection.In this study,HBSP was used as an intervention to explore its role and related mechanisms in liver I/R injury and liver fibrosis in mice.Methods: The study was divided into two parts to illustrate the role of HBSP in hepatic ischemia/reperfusion injury and bile duct ligation induced liver fibrosis.1.HBSP in hepatic I/R injury In the first part of this paper,we described the regulation of autophagy in hepatocytes by HBSP in a mouse liver I/R model and a model of cell apoptosis induced by Cobalt chloride(Co Cl2).C57BL/6J mice were randomly divided into 6 groups:(1)sham operation group,(2)I/R group,(3)I/R + HBSP group,(4)I/R + HBSP + 3-MA(inhibitor of autophagy)group,(5)I/R + HBSP + Rapamycin(inhibitor of m TOR)group,(6)I/R + HBSP + 3BDO(agonist of m TOR)group.Among them,from(2)to(6)group performed partial hepatic ischemia/reperfusion surgery,from(3)to(6)group underwent HBSP intervention,(4)group used 3-MA to inhibit autophagy level,and(5)group used Rapamycin to inhibit m TOR then enhanced levels of autophagy,and group(6)uses 3BDO to activate m TOR then inhibit autophagy.Serum was used to detect the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH).H&E(hematoxylin-eosin)staining was used to detect the degree of injury from the liver tissue.In addition,for the purpose of evaluating the effect of HBSP on autophagy and apoptosis of hepatocytes,we detected liver microtubule-associated protein 1 light chain 3α(Map1lc3 or LC3)Beclin 1,p-m TOR,m TOR by the method of immunohistochemistry,immunofluorescence and western blot.These assays and indicators were used to evaluatethe level of autophagy and the degree of apoptosis.In addition,we used Co Cl2 to mimic hypoxic environment to induce hypoxia injury in hepatic cell line AML12.We designed(1)control group,(2)Co Cl2 group(Co Cl2 stimulated AML12 cells)and(3)Co Cl2+HBSP group(Co Cl2 stimulated after pre-treated by HBSP in AML12 cells).For evaluating the protective effect of HBSP on hepatocyte,the levels of ALT,AST and LDH in supernatants were detected,and the degree of apoptosis was detected by flow cytometry.Moreover,double fluorescent virus was used for detecting autophagy flux of hepatocytes to verify the regulation role of autophagy induced by HBSP.2.HBSP in liver fibrosis injury In the second part of this paper,we explored the role of HBSP in a mouse model of liver fibrosis induced by bile duct ligation(BDL)and macrophage-promoting mouse hepatic stellate cells(m HSCs)activation cell model.We demonstrated that,mechanically,HBSP inhibited the profibrotic effects of macrophage.In detail,C57BL/6J mice were randomly divided into(1)sham operation group,(2)bile duct ligation group(BDL),(3)BDL+HBSP group.The levels of ALT,AST and alkaline phosphatase(ALP)in mouse serum were used to evaluate liver function.H&E staining,masson staining,and sirius red staining were used to evaluate the fibrosis degree.Fibrosis indicators,such as α-SMA,Collagen I,FN1,were detected by q PCR.Western blot was used to detect fibrosisassociated proteins to assess the extent of liver fibrosis in mice.Furthermore,F4/80,the biomarker of macrophage,was detected by immunohistochemistry.q PCR was conducted to detect the m RNA expression levels of cytokines such as Ly6 c,IL-1β,TNF-α and TGF-β.The aim of this part was to explore the role of HBSP on regulating macrophages and their secreted cytokines.For in vitro cell experiments,the experiment was divided into(1)LPS group(LPSstimulated macrophages),(2)LPS+HBSP group(macrophage co-stimulated by HBSP and LPS),and(3)m HSCs-LPS group(using the supernatant in the first group to cultur m HSCs),(4)m HSCs+LPS+HBSP group(using the supernatant in the second group to cultur m HSCs).The m RNA expression levels of IL-1β,TNF-α,and IL-6 in(1)and(2)group were tested.The m RNA and protein expression levels of α-SMA,Collagen I and FN1 in(3)and(4)groups were detected.All of detections were used to validate whetherHBSP has an effect on the secretion function of macrophages and further explore whether HBSP could regulate the activation of m HSC via macrophages.In addition,in order to study whether HBSP directly affects the activation of m HSCs,m HSCs treated with HBSP and without HBSP.The expression level of α-SMA,Collagen I and FN1 genes was also detected.Results: The results in first part showed that HBSP significantly reduced liver I/R injury,and the expression of LC3Ⅱ,LC3 I and Beclin 1 and quantification of the formed autophagosome in I/R + HBSP group were higher than those in I/R group.The protective effect of HBSP was inhibited to some extent by 3-MA and 3BDO,but was further strengthened by rapamycin.Furthermore,for the in vitro cell experiments,HBSP attenuated Co Cl2-induced hepatocyte injury and apoptosis,and induced an increase in autophagy levels.The second part of the results found that: in the mouse liver fibrosis model induced by BDL,we found that HBSP can reduce the degree of liver fibrosis in mice.The expression level of fibrosis indicators,like α-SMA,Collagen I,and FN1 in the group of HBSPtreated mice is down-regulated.In addition,we also found that the protein expression of F4/80 in the BDL+HBSP group was decreased,and the m RNA levels of cytokines,like IL-1β,TNF-α and TGF-β,were all decreased.For the cell biology experiment,it was found that the supernatant,from macrophages cells pre-treated by HBSP,significantly reduced the activation of m HSCs.However,while HBSP directly acted on TGF-β stimulated the m HSCs,could not reduce their activation.Conclusion: Firstly,we proved that HBSP alleviated hepatic ischemia/reperfusion injury by inhibiting the phosphorylation of m TOR and enhancing autophagy.Moreover,we found that HBSP could alleviate BDL induced liver fibrosis in mice.Mechanically,HBSP might regulate the function of macrophages via limiting the secretion of cytokines and reducing the activation of m HSCs.