Long-chain Fatty Acid Activates Hepatocyte Through CD36 Mediated Oxidative Stress

Objective:Liver fibrosis is a result of a variety of chronic liver damages characterized by abnormal liver connective tissue hyperplasia and deposition of the excess extracellular matrix?ECM?,its continuing development can lead to liver cirrhosis.Accumulating evidence suggests that activated hepatocytes are involved in the deposition of the excess extracellular matrix during liver fibrosis via epithelial to mesenchymal transition?EMT?.Lipid accumulation in hepatocytes and abnormal lipid metabolism are implicated in the pathogenesis of chronic liver injury.Fatty acid translocase,belongs to the scavenger of pattern recognition receptors,also known as Cluster of Differentiation 36,whose abnormal expression was associated with chronic liver injury.CD36 is known to mediate long-chain fatty acid?LCFA?uptake and participates in the progression of myofibroblast-like cell activation.However,it is unclear whether LCFA directly promotes hepatocyte activation or not,and the involved mechanisms have not been fully clarified.This study aim to investigate whether CD36 directly involved in the hepatocyte activation and liver fibrosis,and try to provide a new theoretical basis for prevention and treatment of liver fibrosis.Method:the first part,Animal models of 6-8-week-old male WT mice were fed with normal chow diet?NCD?or high fat diet?HFD?for 14weeks.Serum FFA and TG were detected to examine serum lipid content.RT-PCR was performed to detect the expression of profibrogenic gene Acta-2?col1 and col4;The hepatic protein and mRNA expression of CD36was examined by western blot and RT-PCR.The second part,hepatocytes were treated with PA at different concentrations.The cell proliferation assay was performed to detect hepatocyte proliferation in response to PA.We knocked down CD36 by transfecting CD36 siRNA into hepatocytes.Lipid accumulation was observed by BODIPY staining.The protein expression of CD36,a-SMA,and vimentin was examined by western blot.The mRNA expression of Acta2,Vimentin,Desmin and TGF-?signaling pathway related genes TGF-?,Snail,Twist,and Zeb1 was determined by real-time PCR.The third part,H₂O₂ levels and MDA levels in the livers of mice fed an NCD or HFD were detected.Hepatocytes and CD36-knockdown hepatocytes were treated with 0.2 mmol/L PA for 48h,and H₂O₂ levels and ROS levels were then determined.Hepatocytes were treated with 0.2mmol/L PA for 48h alone or with pretreatment of NAC at different concentrations or CD36-knockdown hepatocytes were treated with 0.2mmol/L PA for 48h in the absence or presence of H₂O₂.The mRNA expression of Acta2,Vimentin,Desmin and TGF-?signaling pathway related genes TGF-?,Snail,Twist,and Zeb1 were determined by real-time PCR.The protein expression of a-SMA and vimentin were examined by western blot.Result:the first part,Serum FFA and TG levels were increased in HFD-fed mice compared with those in NCD-fed mice.Hepatic mRNA expression of markers of fibrogenesis,including Acta2,Col1,and Col4,was significantly up-regulated in HFD-fed mice.Additionally,we also found that HFD increased hepatic CD36 protein and mRNA expression.The second part,Hepatocytes were treated with PA at different concentrations and times to determine the effect of PA on hepatocyte lipotoxicity and proliferation.We noticed that PA was comparatively non-toxic at the 0.1 mmol/L and 0.2 mmol/L concentrations for 48h.Furthermore,there was no stimulatory effect of PA on hepatocyte proliferation.Following BODIPY staining,we explored the concentrations of PA that enhanced lipid accumulation in hepatocytes but was clearly decreased in the CD36 knockdown hepatocytes.We found that CD36 protein expression was increased in PA-treated hepatocytes at both 0.1 mmol/L and 0.2 mmol/L concentrations,and CD36siRNA transfection resulted in markedly decreased CD36 protein and mRNA expression in PA-induced hepatocytes by western blot.Homoplastically,PA up-regulated the protein expression of a-SMA and vimentin and the mRNA expression of Acta2,Vimentin and Desmin,but down-regulated those in the CD36 knockdown hepatocytes.Moreover,this trend was consistent with the mRNA expression of TGF-?and key downstream transcription factors Snail,Twist and Zeb1.The third part,HFD-fed mice had greater H₂O₂ and MDA production in the liver than control mice.Similarly,the production of H₂O₂ and ROS was considerably higher in hepatocytes treated with PA than in the control group.Knockdown of CD36 considerably decreased the production of H₂O₂ and ROS in hepatocytes treated with PA.These results suggested that the level of oxidative stress depends on hepatocyte CD36 expression when treated with PA.We further found that NAC significantly decreased PA-induced hepatocyte activation and TGF-?signaling pathway related genes.In addition,the H₂O₂ supplement largely abrogated the improved effect of CD36 knockdown on hepatocyte activation.Conclusion:The purpose of our current study was to clarify the effects of lipids on hepatocyte activation.Here,we show that:1.LCFA treatment induced hepatocyte activation,evidenced by up-regulated expression of Acta2,Vimentin,Desmin,and TGF-?signaling pathway;2.CD36 inhibition attenuated LCFA-induced hepatocyte activation;3.Oxidative stress was critical in LCFA-induced hepatocyte activation,which is mediated by CD36.