The Role And Its Mechanism Of CD36 Gene In Acute Liver Injury Of Mice

The liver is the largest solid organ in the body,which has multiple biological functions that play a key role in maintaining steady state and maintain a metabolic balance.The liver is an important human immune organs,when the immune balance once destroyed,it will cause a variety of immune-related liver diseases,including HBV,HCV-induced persistent chronic hepatitis,primary liver cancer and autoimmune hepatitis(AIH).AIH is an autoimmune response mediated chronic progressive liver injury process,the body produces an immune response to the liver autoantigen,thereby destroying liver cells leading to inflammation and necrosis of the liver,the incidence rate in the world increased year by year,and severe cases can be rapid progress for the liver sclerosis and liver failure.The main treatment for AIH are immunosuppressive agents and liver transplantation,but the effect is poor,the potential pathophysiological mechanism is still unclear,so there is no clear and effective treatment.Additionaly,the liver has biotransformation function to a number of non-nutritive substances in vivo and in vitro,such as various drugs,poisons and certain metabolites in the body.In the use of various drugs in the process,due to the drug itself and/or its metabolites or due to special physical changes in the drug hypersensitivity or tolerance will lead to drug-induced liver injury(DILI).The current DILI has risen to the fifth cause of global death,become the world can not ignore the public health problems.Its pathogenesis involves metabolic protein adduct formation,mitochondrial dysfunction,oxidative stress,peroxynitrite formation and nuclear DNA cleavage and other key processes,the current clinical treatment of N-acetylcysteine detoxification have many limitations and lead to persistent disease progression,the development of liver fibrosis cirrhosis and so on.Cluster antigen 36(CD36)molecule is a single chain glycoprotein that is widely present on the cell surface and belongs to the B family scavenger receptor and can be used in a variety of cells such as macrophages,microvascular endothelial cells,platelets,adipocytes and epithelial cells.As a pattern recognition receptor,CD36 involved in a series of physiological and pathological processes,including immune,fatty acid metabolism,angiogenesis,atherosclerosis and phagocytosis.CD36 also binds to Toll-like receptors 4 and 6 to promote sterile inflammation.Notably,CD36 has been shown to co-express α13 integrin and CD133 on tumor stem cells to promote glioblastoma progression and inhibition of CD36 receptors will reduce tumor metastasis in human melanoma,oral cancer and breast cancer.In this study,the role and mechanism of CD36 in acute liver injury were discussed.Part I:CD36 Deficiency Attenuates Concanavalin A-induced Hepatitis in MiceConcanavalin A(Con A)-induced liver injury is a well-established murine model of T-cell mediated hepatitis.Intravenous injection of Con A induces acute liver injury and systemic immune activation in mice,which resembles the pathology of immune-mediated hepatitis in humans.The model showed clinical and pathological histological manifestations of acute hepatitis,including leukocyte and lymphocyte infiltration in the liver,hepatocellular necrosis,serum transaminase elevation and inflammation cytokine secretion and so on.Now this model has been widely used in the study of T cell-mediated hepatitis pathogenesis,which formed our current understanding of the process of liver injury.Objective:Although there is a large number of studies on the process of autoimmune liver injury,its exact cellular and molecular mechanisms are unclear.More and more data indicate that T cell activation plays a very important role in liver injury.The aim of this study was to investigate the regulation of CD36 gene on T cell-mediated autoimmune hepatitis and to provide a new direction for the prevention and treatment of current clinical hepatitis.Methods:To understand the role of CD36 in hepatitis,we tested the susceptibility of CD36-deficient(CD36-/-)mice to this model,injected at 12 mg/kg via the tail vein to induce acute hepatitis.(1)Effects of endogenous CD36 on Con A-induced liver injury in miceWT mice and CD36-/-transgenic mice were injected with Con A at 12 mg/kg via tail vein to induce liver injury.The serum was collected at 8 h and the activity of alanine aminotransferase(ALT)in serum was measured.The liver tissues were isolated and observed by hematoxylin and eosin(H&E)staining.The experiment was performed by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)analysis to evaluated apoptotic cells.(2)Immunoregulatory effects of endogenous CD36 on T cells activation induced by Con A in mice.Mononuclear cells(MNCs)were isolated from WT and CD36-/-mice at 8 h after Con A injection.Flow cytometry was used to detect T cells,NK cells,infiltrating macrophages and neutrophils.At the same time,the expression of inflammatory mediators(TNF-α,CXCL10,IL-1α,MCP-1,and IL-6)in liver tissues was analyzed by real-time reverse transcription polymerase chain reaction(such as IL-1α,MCP-1 and IL-6),and the expression of cytokines in serum was detected by enzyme-linked immunosorbent assay(ELISA).The levels of JNK,IKKα/β,ERK and STAT3 phosphorylation and the expression of Caspase-3 in liver tissue were detected by Western Blot.(3)Antagonists experiments were used to test the effect of endogenous CD36 on Con A-induced liver injury in miceWT mice were injected with tyrosine kinase inhibitor genistein that blocks the CD36-TLR4-TLR6 signaling.We verified the role of endogenous CD36 in Con A-induced liver injury through detecting ALT level,H&E staining and analyzing T cells,NK cells,macrophages and neurtrophils infiltration in WT mice.(4)Regulative effect of CD36 on CXCL10-induced hepatocyte apoptosis in vitroThe primary hepatocytes were isolated from WT mice and CD36-1-mice,incubated with 100 ng/ml Con A for 5min,20min,60min,4 h and 8h,stimulated with or without 5 μM genistein.The serum aspartate transaminase(AST)levels were tested at 8h and the expression of Akt,JNK,IKKα/β and Caspase-3 were measured by western blot at different time points.Results:CD36-/-mice were less sensitive to Con A-induced hepatitis,which showed a lower ALT levels and had a significantly lower number of liver MNCs,including CD4+,CD8+T cells,NK cells,infiltrating macrophages and neutrophils,as well as reduced expression of inflammatory mediators(TNF-a,CXCL10,IL-1α,MCP-1,and IL-6)compared with controls.Further,our data revealed that the CD36 receptor is essential for CXCL10-induced hepatocyte apoptosis and activation of IKK,Akt,and JNK.Moreover,treatment of WT mice with genistein,a tyrosine kinase inhibitor that blocks CD36-TLR4-TLR6 signaling,attenuated Con A-induced liver injury and reduced the number of MNCs.Conclusions:Our findings suggest that CD36 plays an important proinflammatory role in Con A-induced liver injury by promoting hepatic inflammation and mediating the proapoptotic effect of chemokine CXCL10,and therefore,may be a potential therapeutic target for immune-mediated hepatitis.Part Ⅱ:CD36 Deficiency Attenuates Acetaminophen-induced Hepatitis in MiceAcetaminophen(APAP)is the main component of antipyretic analgesics,and its common side effects are caused by drug-induced liver injury and even acute liver failure.APAP-induced drug-induced liver injury in Europe and the United States is particularly serious,in recent years,the incidence of our country has gradually increased,more and more attention.APAP-induced liver injury includes APAP metabolic protein adduct formation,mitochondrial dysfunction,oxidative stress,peroxynitrite formation and nuclear DNA cleavage and other key processes.Recent studies have shown that aseptic inflammation and innate immune cells may play an important role in APAP-induced liver injury and repair.Methods:To investigate whether CD36 gene plays a role in drug-induced liver injury and its mechanism,we established an APAP-induced acute hepatitis model in wild-type(WT)mice and CD3 6-deficient(CD36-/)mice.(1)Confirmation of the involvement of CD36 in APAP-induced liver injury WT mice were divided into two groups randomly,intraperitoneal injected with APAP(250mg/kg)or the same amount of PBS,the liver tissues were isolated at 1h,3h,6h,12h and 24h after treatment of mice,and the expression of CD36 mRNA in the liver was detected by real-time quantitative polymerase chain reaction(qPCR);the expression of CD36 protein in the liver was detected by Western blotting.(2)Effects of endogenous CD36 on liver injury induced by APAP in mice WT and CD36-/-mice were given intraperitoneal injection of APAP(250 mg/kg)after starving for 16 hours.Serum was collected at 8 h and 24h and serum alanine aminotransferase(ALT)activity was measured.Liver tissue was isolated and observed by hematoxylin and eosin(H&E)staining.The experiment was performed by terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)analysis to evaluated apoptotic cells.(3)The effect of endogenous CD36 on intracellular inflammatory response induced by APAP in miceWT mice and CD36-/-mice were injected intraperitoneally with APAP.The mononuclear cells(MNCs)were isolated from mice.Flow cytometry was used to detect the number of infiltrating macrophages and neutrophils.(RT-PCR)was used to analyze the expression of proinflammatory cytokines(such as TNF-α,IL-1β,IL-6,KC,and MCP-1)in real-time reverse transcription polymerase chain reaction(RT-PCR);and the expression of cytokines in serum was detected by enzyme-linked immunosorbent assay(ELISA).The expression of JNK,Caspase-3,the metabolite of APAP,N-acetyl-p-benzoquinone imine(NAPQI)protein adducts,and Cytochrome P450 2E1(CYP2E1)level were measured by Western blot.(4)Antagonist experiments were used to verify the role of endogenous CD36 in APAP-induced liver injury in miceWT mice were intraperitoneally injected with APAP,intraperitoneal injection of tyrosinase inhibitor PPIL We verified the role of endogenous CD36 in APAP-induced liver injury through detecting ALT level;and the detection of supernatant AST level in primary hepatocytes in WT mice.Results:CD36-/-mice were less sensitive to APAP-induced hepatitis,which showed a lower ALT levels and had a significantly lower number of liver MNCs,including infiltrating macrophages and neutrophils,as well as reduced expression of inflammatory mediators(KC,IL-β,MCP-1,and IL-6)compared with controls.Moreover,treatment of WT mice with PP1,a tyrosine kinase inhibitor,attenuated APAP-induced liver injury.Conclusion:Our findings suggest that CD36 plays an important proinflammatory role in APAP-induced liver injury by promoting hepatic inflammation and mediating the proapoptotic effect,and therefore,may be a potential therapeutic target for immune-mediated hepatitis.