Study On The Role And Mechanism Of HSP70-FTO Axis In Non-alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease(NAFLD)is a genetic-environmental-metabolic stress related disease,the pathogenesis is not yet fully clear.Heat shock protein 70(HSP70)is most abundant in most organisms and more sensitive to stress.Fat mass and obesity associated gene(FTO)is upregulated in NAFLD and promotes adipogenesis in liver cells.The study found that FTO knockdown can promote the synthesis of HSP70 in the case of heat shock.The purpose of this experiment is to study the mechanism of HSP70 regulating the pathogenesis of FTO-induced NAFLD,and to provide new ideas for the prevention and treatment of NAFLD.This issue explores the relationship between HSP70 and FTO and its role in NAFLD.1.The relationship between HSP70 and NAFLD Objective: To investigate the expression of HSP70 in human serum,the liver of NAFLD mice and the palmitic acid(PA)-induced hepatic steatosis model in HepG2 cells.Methods: the expression of HSP70 in serum of NAFLD population was detected by ELISA kit.The animal model of NAFLD was established by high-fat diet.The expression and histological location of HSP70 were detected by qPCR,Western blotting and immunohistochemistry.The steatosis model was induced by 0.5mM PA on HepG2 cells for 24 hours.The expression of HSPA1 A mRNA and HSP70 protein were detected by qPCR and Western blotting.Results: The expression of HSP70 in serum of NAFLD population was significantly increased,and positively correlated with BMI,waist circumference and serum ALT levels.Immunohistochemistry and Western analysis showed that the expression of HSP70 was up-regulated and nuclear entry was incresed in the liver of NAFLD-induced mice.In the HepG2 cell model of steatosis,HSP70 mRNA and protein levels were significantly increased.Conclusion: Upregulation of HSP70 expression in NAFLD may be closely related to the pathogenesis of NAFLD.2.The role of HSP70 in NAFLD Objective: To investigate the function and role of HSP70 in NAFLD Metdods:HSP70 overexpressed by Lenti-HSPA1 A and lenti-Vector transfection with lentivirus.lipid droplets in cells were determined by oil red O staining,and contents of triglyceride(TG)and cholesterol(TC)in the cell supernatants were detected by ELISA.The mRNA levels of Fatty synthetic-related enzymes(FAS,SCD,ACC,SREBP1C)and lipoxygenase-related enzymes(ACOX,CPT-1 and PPARα)were detected by RT-PCR.The above indicators were determined again by PA stimulation for 24 hours.After the expression of HSP70 was interfered by lentiviral transfection of Si-HSPA1 A and Si-Vector,the above indicators were determined on 0 hour and by PA stimulation for 24 hours.In vivo experiments,normal mice were divided randomly into two groups: positive control group(tail vein injection of AAV-CTL)and HSP70 over expression group(tail vein injection of AAV-HSPA1A).After 16 weeks of high fat diet,normal mice were divided into two groups: positive control group(tail vein injection of AAV-shCTL)and HSP70 interference group(tail vein injection of AAV-shHSPA1A).The liver steatosis was determined by HE and oil red O staining.Results: In vitro experiments,over expression of HSP70 slightly increased the number of lipid droplets in HepG2 cells compared with cells stably expressing empty lentiviral vector.After 0.5mM PA stimulation for 24 hours,the number of lipid drops in HSP70 over expressed cells was significantly increased compared with cells stably expressing empty lentiviral vector,and the lipid droplets in the control cells was also significantly increased.TG level of the cell supernatants over expressing HSPA1 A was slightly increased,whereas TC level was increased significantly.TG and TC levels in Cells stably expressing empty lentiviral vector did not change compared with the control cells.After PA stimulation,TC and TG levels in HSP70 over expressed cells is significantly increased.HSPA1A-overexpressing cells were treated with PA,.In addition,the mRNA levels of ACC and SREBP1 C were significantly increased in HSPA1 A over expressed cells,and PPARα expression was decreased regardless of whether cells were treated with PA.After HSP70 was knocked down,there was no significant difference in the number of lipid droplets in HepG2 cells compared with control cells and blank-transfected cells.After PA stimulation,the number of lipid droplets in HSP70 knockdown was decreased compared with control cells and blank-transfected cells.The levels of TG and TC in the supernatants of siHSPA1A-transfected cells were significantly decreased compared with the control cells,whether or not treated with PA.The expression of FAS,SCD and ACC in HSPA1 A knockdown cells were significantly decreased.We also found that mRNA levels of FAS,SCD,ACC and SREBP1 C were still decreased,but CPT-1 was increased in HSPA1 A knockdown cells after PA treatment.In addition,the expression of increased after PA treatment.In vivo experiments,there were all no significant pathological changes in the liver slices of normal mice and HSPA1 A over expressing mice.Oil red O staining results showed there were no steatosis of hepatic lobules in the normal mice and HSPA1 A over expressing mice.The AAV-mediated interfered HSPA1A/ B vector were injected by the tail vein of mice after high-fat diet for 16 weeks.After 3 weeks,Effects of the virus peaked and the liver tissues were taken.HE staining showed that a large number of hepatic steatosis were found in liver tissues of the high fat+AAV-shCTL mice,especially inflammatory cell infiltration in the portal areas with some punctate hepatocyte necrosis.The degrees of hepatic steatosis,inflammatory cell infiltration and hepatocyte necrosis in liver tissues of the high fat+ AAV-shHSPA1A/B mice were slightly reduced.In the meantime,Oil red O staining showed that a large number of lipid droplets dispersed in liver tissues of the high-fat + AAV-shCTL mice,and the shape of lipid droplets were also larger.The number of lipid droplets in liver tissues of the high-fat + AAV-shHSPA1A/B mice were decreased,the shape of lipid droplets was also decreased and the decrease in the portal area was more obvious,indicating that the degree of hepatic steatosis was reduced.Conclusion: Over expression of HSP70 promoted the pathogenesis of NAFLD and knockdown of HSP70 attenuated the degree of steatosis of NAFLD,indicating that HSP70 plays an important role in the pathogenesis of NAFLD.3.HSP70 promotes Hepatic Steatosis through up-regulating FTO in Non-alcoholic fatty liver disease Objective: To investigate the mechanism of HSP70 regulating FTO on human HepG2 cells steatosis Methods: The regulation of HSP70 on FTO and co-localization of HSP70 and FTO were determined by Western blotting and confocol methods,and the relationship between HSP70 and FTO was detected by GST-pull down method.Over expression of FTO by lentiviral transfection of Lenti-FTO and Lenti-Vector and knockdown of FTO by lentiviral transfection of si-FTO and si-Vector were done in human HepG2 cells.The intracellular lipid droplets were observed by oil red O staining.The levels of triglyceride(TG)and cholesterol(TC)in the cell supernatants were detected by ELISA.The mRNA levels of fatty synthetic-related enzymes(FAS,SCD,ACC)were detected by RT-PCR,and observe the impacts PA on lipid synthesis after PA stimulation for 24 hours.Results: In vivo experiments,Over expression of HSP70 in mouse liver promotes an increase in FTO expression.In vitro experiments,over expression of HSP70 increased the expression of FTO,and increased FTO levels after PA stimulation for 24 hours.Knockdown of FTO decreased the expression of FTO,and increased FTO levels after PA stimulation for 24 hours.Confocal analysis showed that HSP70 and FTO were distributed in the cytoplasm under normal conditions.HSP70 and FTO protein were enriched in the nucleus 24 hours after 0.5mM PA stimulation for 24 hours,indicating the activation of both proteins.FTO was also distributed in the cytoplasm after HSP70 knockdown,and FTO was enriched in the plasma and nuclear membranes except for the nucleus given after 0.5mM PA stimulation.Also after FTO knockdown,HSP70 remained in the cytoplasm,but HSP70 increased in the nucleus with 0.5mM PA stimulation.These results suggested that HSP70 promoted FTO nuclear transfer.GST-pull down showed that GST-FTO can bind HSP70,but GST protein cannot after GST-FTO and His-HSP70 protein purification,indicating that HSP70 can be directly bound FTO.Moreover,the lipid droplets in over expression FTO cells were slightly increased compared with the control cells,and the lipid droplets in the cells were significantly increased after 0.5mM PA stimulation for 24 hours.the contents of TC and TG in over expression FTO cell supernatants were increased,and the contents of TC and TG were significantly increased after 0.5mM PA stimulation on the basis of over expression of FTO.The mRNA levels of fatty synthetic-related enzymes(FAS,SCD and ACC)in over expression FTO cells were increased compared with the control cells,but on significant increases after 0.5mM PA stimulation.In contrast,knockdown of FTO reduced intracellular lipid synthesis.Conclusion: HSP70 can up-regulate the expression of FTO in NAFLD.HSP70 can bind directly to FTO and promoted FTO nuclear transfer under high fat,indicating that HSP70 is involved in the pathogenesis of NAFLD through regulating FTO.