| The spectrum of nonalcoholic fatty liver disease(NAFLD)includes nonalcoholic simple fatty liver(NAFL),nonalcoholic steatohepatitis(NASH),associated fibrosis,and cirrhosis,which can eventually progress to end-stage liver disease and hepatocellular carcinoma.Currently,NAFLD has become one of the most common causes of chronic liver diseases worldwide.NASH is an intermediate stage in the progression of NAFLD to cirrhosis and hepatocellular carcinoma,and is considered to be a precipitating factor for cryptogenic cirrhosis and hepatocellular carcinoma.NASH cirrhosis rate and mortality were significantly higher than that of simple fatty degeneration,which posed a serious threat to human health.Therefore,it was of great significance to reverse and block the progression of NASH.At present,the pathogenesis of NASH,especially the mechanism of inflammation,is still unclear,and NASH lacks effective treatment.Therefore,it is of great scientific and clinical significance to explore the mechanism of NASH inflammation and new molecular targets.Autophagy is an intracellular cycle and degradation process,which is used to regulate cell survival and drug resistance.Autophagy is closely related to NASH inflammation.Autophagy dysfunction is consistent with chronic inflammation of M1 macrophage polarization,leading to progression from pure fat to NASH.In liver cells,autophagy helps to remove damaged mitochondria,quench various inflammatory cytokines in cells,and play an anti-inflammatory role.Therefore,activation of autophagy can inhibit hepatic inflammatory responses,and drug promoting autophagy may be a target for treatment of NASH inflammatory responses.Signal transduction and transcriptional activation factor 3(STAT3)is a classic transcription factor of mediating cell signal transduction.STAT3 is activated by phosphorylation of Tyr705,and translocated to the nucleus,binding to the target gene.STAT3 mainly regulates apoptosis,intermediate metabolism,participating in immune response,inflammation and tumorigenesis.STAT3,as a key mediator of inflammatory diseases,plays an important role in inflammatory response.STAT3 can interact with NF-B and promote the activation of NF-?B,which up-regulates a variety of pro-inflammatory mediators.Activated NF-B can produce IL-6 and further activate STAT3.The above positive feedback process can promote the occurrence and development of inflammation.In addition,STAT3 has an autophagy regulation effect,and it can inhibit autophagy by promoting STAT3 in a variety of cells.However,the relationship between STAT3 and autophagy in non-alcoholic steatohepatitis inflammation remains unclear.Metformin can improve insulin resistance in liver diseases,and reduce NAFLD blood glucose and lipid levels.It can reduce lipid peroxidation,and inhibit cell proliferation,inflammation and fibrosis.There is abundant evidence that metformin has autophagy activation.In addition,metformin inhibits the proliferation,metastasis and apoptosis of various tumor cells,which are related to its inhibition of STAT3 signaling pathway.At present,the evidence of metformin induced autophagy in the treatment of NAFLD is mainly limited to hepatic steatosis.Few studies have focused on the relationship between metformin,STAT3,NASH inflammation and autophagy.The exact role and possible mechanism of metformin in NASH inflammation are still unclear.We speculated that in NASH,metformin may activate autophagy by inhibiting p-stat3-tyr750,thereby inhibiting the inflammatory response and reducing the m RNA expression of inflammatory cytokines.To sum up,in this study,we used immunohistochemistry,RNA interference,q RT-PCR and Western Blot method in NASH patients,animal and cell experiments to elucidate the role and mechanism of STAT3 and autophagy in metformin's inhibition of NASH inflammation.Part one Expression characteristics of STAT3 and autophagy in liver tissues of patients with non-alcoholic steatohepatitis Objectives: To investigate the correlation between STAT3,p-STAT3 and autophagy inhibition in liver tissues of NASH patients.Methods: Serum GOT1/AST,GPT/ALT levels were determined by biochemical analyzer.Serum IL-1?,IL-6 and TNF-? levels were determined by ELISA.STAT3,p-STAT3 proteins,autophagy related protein LC3?and SQSTM1 expression of liver tissue were determined by immunohistochemical technique.For aminotransferase levels associated with inflammatory cytokines line analysis,LC3 ?,SQSTM1 express respectively and STAT3,p-STAT3 expression related analysis,STAT3,p-STAT3 is associated with whether autophagy inhibition.Aminotransferase levels and inflammatory cytokines were line analysised.LC3?,SQSTM1 expression were analysised respectively with STAT3 and p-STAT3 expression to improved whether autophagy inhibition was associated with STAT3 and p-STAT3.Results: 1.Serum AST,ALT,IL-1?,IL-6 and TNF-? in NASH group were significantly higher than those in healthy control group.(P < 0.0001)2.STAT3,p-STAT3,SQSTM1 protein expression were higher in NASH liver tissues than those in healthy controls.LC3 ? expression is lower than those in the healthy controls.(all P < 0.0001)3.The serum GOT1/AST and GOT/ALT levels in NASH group were positively correlated with the expression levels of IL-1?,IL-6 and TNF-?,respectively.(all P < 0.0001)4.SQSTM1 expression quantity was positively correlated with STAT3,p-STAT3 expression in NASH group of liver tissue.LC3 ? expression quantity is negatively related to the STAT3,p-STAT3 expression quantity.(all P < 0.0001)Conclusion: 1.The expressions of STAT3 and p-STAT3 proteins in liver tissues of NASH patients were increased and autophagy was inhibited.2.The autophagy status of liver tissue in NASH patients was negatively correlated with the expression of STAT3 and p-STAT3 protein.Part two The expression of STAT3 and autophagy in the liver of mice with nonalcoholic steatohepatitis Objectives: To investigate the expression characteristics of STAT3,p-STAT3 and autophagy in the liver tissues of NASH mice.Methods: NASH mouse model was established by feeding C57BL/6J mice with methionine and choline-deficient(MCD)diet for 4 weeks.Serum AST and ALT levels were determined by biochemical analyzer.The expression levels of STAT3,IL-1?,IL-6 and TNF-? were determined by fluorescence quantitative PCR.STAT3,p-STAT3,LC3? and,SQSTM1 protein expression were examined by Western blot(WB).Results: 1.The hepatocytes of the model group showed moderate to severe adiposis,a large number of inflammatory cells infiltration in the lobules,focal necrosis in some areas,and perisinus or portal area fibrosis to varying degrees.2.Serum GOT1/AST,GPT/ALT levels,and m RNA expression levels of IL-1?,IL-6 and TNF-? m RNA in liver tissues of the model group were significantly higher than those in the normal control group.(all P < 0.0001)3.The expressions of STAT3 m RNA,STAT3 and p-STAT3 protein in liver tissues of the model group were significantly higher than those of the normal control group.P-STAT3 activity(p-STAT3/STAT3)increased.(all P < 0.0001)4.Compared with normal controls,the liver tissue of the model group showed autophagy inhibition,increased SQSTM1 protein expression,and decreased LC3 ? protein expression.(all P < 0.0001)Conclusion: 1.NASH mouse model was successfully replicated in mice fed MCD for 4 weeks.2.In the NASH mouse model,STAT3 in liver tissue was activated and the activity increased,while autophagy was inhibited.Part three Study on the expression of STAT3 and autophagy in hepatocytes of non-alcoholic steatohepatitis and the mechanism of its effect on inflammation.Objectives: To investigate the expression characteristics of STAT3,p-STAT3 and autophagy in NASH cells.To investigate the effect of 3-MA(autophagy inhibition)on the expression of IL-1?,IL-6 and TNF-? m RNA in the model.To investigate the effect of STAT3 knockdown on the expression of IL-1?,IL-6 and TNF-? m RNA after transfected with si RNASTAT3 into NASH cell model.Results: 1.The intracellular lipid accumulation and the expression of IL-1?,IL-6 and TNF-? m RNA were increased in a time-dependent manner in AML12 cells treated with MCD medium for different time periods.2.The expression of STAT3 and p-STAT3 protein in AML12 cells were increased in a time-dependent manner in cells treated wih MCD medium for different time periods.3.The expression of LC3 ? protein was increased in AML12 cells treated with MCD medium±chloroquine for 24 h as compared to those in normal control group.LC3 net autophagy flux was decreased obviously.(all P < 0.0001)4.Knockdowned of STAT3 expression activated autophagy,characterized by decreased autophagy protein,SQSTM1 expression,and increased LC3? protein expression.(all P < 0.0001)5.After NASH cell model +3MA inhibited autophagy(MCD+3-MA),the expression levels of IL-1?,IL-6 and TNF-? m RNA in cells were significantly increased as compared to those of in the model group(MCD).(all P < 0.0001)6.Knockdowned of STAT3 expression+3-MA in NASH cell model(MCD+si STAT3),the expression levels of IL-1?,IL-6 and TNF-? m RNA in cells were significantly decreased as compared to those of in the model group(MCD).(all P < 0.0001)7.Knockdowned of STAT3 expression+3-MA in NASH cell model(MCD+si STAT3+3-MA),the expression levels of IL-1?,IL-6 and TNF-? m RNA in cells were not significantly decreased as compared to those of in the model group(MCD).(all P > 0.05)Conclusion: 1.NASH cell model was successfully replicated by treating AML12 cells with MCD medium for 24 h.2.In NASH cell model,the activity of STAT3 increased,and the expressions of STAT3 and p-STAT3 protein increased.Autophagy was inhibited.LC3 net autophagy flow in model group was significantly lower than that in normal control group.3.The expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH cell model were inhibited after autophagy was activated by knocking down the STAT3 expression.The expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH cell model were increased after autophagy was inhibited by 3-MA.Part four Study on the effect of metformin on non-alcoholic steatohepatitis inflammation Objectives: To investigate the effect of metformin on STAT3,p-STAT3 and autophagy in vivo and in vitro NASH models.To investigate the effect of 3-MA autophagy inhibition combined with metformin on the expression of IL-1??IL-6 and TNF-? m RNA in NASH cell model.To investigate the effect of STAT3 knockdown on autophagy activation combined with metformin on the expression of IL-1?,IL-6 and TNF-? m RNA in NASH cell models.Methods: 1.The expression levels of STAT3,IL-1?,IL-6 and TNF-? m RNA in liver tissues were determined by fluorescence quantitative PCR after the mice were fed with metformin +MCD for 4 weeks.STAT3,p-STAT3,LC3 ?,SQSTM1 protein expression level were measured by WB.2.The expression levels of STAT3,p-STAT3,LC3? and SQSTM1 protein in AML12 cells treated with different concentrations of metformin for 24 h were examined by WB.3.The expression levels of STAT3,IL-1?,IL-6 and TNF-? m RNA in AML12 cells treated with 20 m M metformin+MCD medium for 24 h were determined by fluorescence quantitative PCR.STAT3,p-STAT3,LC3 ? and SQSTM1 protein expression level were assessed by WB.4.After transfected AML12 cells with scramble and si STAT3 for 16 h,MET(20m M)were treated or not.Then,the expression levels of STAT3,p-STAT3,LC3 II,SQSTM1 protein and p-STAT3/STAT3 were measured by WB.5.The expression levels of IL-1?,IL-6 and TNF-? m RNA in AML12 cells treated with metformin +MCD culture + 3-MA for 24 h were determined by fluorescence quantitative PCR.6.The expression levels of IL-1?,IL-6 and TNF-? m RNA in AML12 cells treated with metformin +MCD culture + STAT3 si RNA for 24 h were determined by fluorescence quantitative PCR.Results: 1.The expression levels of STAT3,IL-1?,IL-6 and TNF-? m RNA in NASH mouse model treated with metformin were decreased.The expression levels of STAT3,p-STAT3 and SQSTM1 protein were decreased,while expression levels of LC3? protein was increased.(all P < 0.0001)2.The expression levels of STAT3,p-STAT3,LC3? and SQSTM1 protein in AML12 cells treated with different concentrations of metformin for 24 h were decreased in dose dependent manner.The expression levels of LC3? protein were increased in dose dependent manner.(all P < 0.0001)3.The expression levels of STAT3,IL-1?,IL-6 and TNF-? m RNA in AML12 cells treated with 20 m M metformin+MCD medium for 24 h,were decreased.The expression levels of STAT3,p-STAT3,LC3? and SQSTM1 protein were decreased,while those of LC3? protein was increased.(all P < 0.0001)4.Metformin significantly reduced STAT3,p-stat3 protein expression and p-stat3 /STAT3 levels in AML12 cells in the normal control group,and autophagy was activated.Compared with the MET group,STAT3,p-stat3 protein expression and p-stat3 /STAT3 levels were further decreased in the MET+si STAT3 group,and autophagy was further activated.Compared with si STAT3 group,STAT3,p-stat3 protein expression and p-stat3 /STAT3 levels were further decreased and autophagy was further activated in the MET+si STAT3 group.All of the above changes were statistically significant.5.The expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH cells treated with metformin +3MA(MET+3-MA+MCD)were significantly higher than those in the MCD+MET group(P < 0.0001),but not significantly lower than those in the NASH model group(MCD)(all P > 0.05).6.The expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH cells treated with metformin+ STAT3 si RNA(MET+si STAT3+MCD)were significantly lower than those in the NASH model group(MCD)(all P < 0.0001)Conclusion: 1.Metformin reduced the expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH mice and cell models,increased the expression of STAT3 and p-STAT3 proteins,and activated autophagy.2.Metformin reduced intracellular STAT3 and p-STATS protein expression and activated autophagy.3.When metformin was combined with autophagy inhibitor 3-MA,it counteracted its effect of reducing the expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH model.4.The effect of metformin in combination with STAT3 si RNA on reducing the expression levels of IL-1?,IL-6 and TNF-? m RNA in NASH model were enhanced.Conclusion: 1.STAT3 and autophagy played an important role in NASH inflammation.2.The activity of STAT3 was increased in NASH patients,mice and cell models.The expressions of STAT3 and p-STAT3 were increased,and autophagy was inhibited.3.Down-regulation of STAT3 expression could activate autophagy and reduce the expression levels of inflammatory cytokines IL-1?,IL-6 and TNF-? m RNA in NASH cell model.Autophagy inhibition increased the expression levels of inflammatory cytokines in NASH cell model.4.Metformin activated autophagy and reduced the expression levels of IL-1??IL-6 and TNF-? m RNA by inhibiting the expression of STAT3 and p-STAT3.5.STAT3 and autophagy might be a new target for metformin in the clinical in of NASH inflammation. |
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