Interleukin 10 Inhibits Hydrogen Peroxide-induced Autophagy Of Hepatic Stellate Cells Via The Akt/mTOR/STAT3 Signaling Pathway

Objective:Liver fibrosis is an abnormal hyperplasia of connective tissue in the liver caused by various pathogenic factors,which can eventually develop into liver cirrhosis,which is a serious threat to human health.Inhibits the activation and proliferation of hepatic stellate cells（HSCs）,and promoting the phenotypic conversion and apoptosis of activated HSCs are the main therapeutic strategies for anti-hepatic fibrosis.Autophagy is an important factor in maintaining the activation of HSCs.Interleukin 10（IL-10）has a significant anti-fibrotic effect.This study intends to explore the mechanism of Akt/mTOR/STAT3 signaling pathway in IL-10 regulating cell autophagy so as to provide a new basis for the prevention and treatment of liver fibrosis.Methods:（1）Induction of autophagy by hydrogen peroxide（H₂O₂）:Rat hepatic stellate cells（HSC-T6）and human hepatic stellate cells （HSC-LX2）were induced by different concentrations of H₂O₂ to establish cells autophagy model.Cell Counting Kit-8（CCK-8）and lactate dehydrogenase（LDH）test were used to detect the viability and mortality of cells after H₂O₂ treatment.The expression of autophagy-related proteins LC3I and LC3II was detected by Western Blot.（2）Regulation of IL-10 on H₂O₂-induced HSCs autophagy:HSC-T6 and HSC-LX2 cells were co-treated with H₂O₂and different concentrations of IL-10.Using Westernblot to detect the expression of autophagy related protein LC3I and LC3II,and immunofluorescence and transmission electron microscopy（TEM）were used to detect the number of autophagosomes.（3）The role of Akt/mTOR/STAT3 pathway in IL-10 regulating H₂O₂-induced autophagy in HSCs:Western Blot was used to detect the expression of autophagy-related proteins LC3I and LC3II and the total and phosphorylated protein expression of Akt,mTOR and STAT3 in HSCs after co-treatment with IL-10 and H₂O₂.The number of autophagosomes in cells was detected by immunofluorescence and TEM.HSCs were co-treated with IL-10 and H₂O₂ after pretreatment with mTOR inhibitor rapamycin or STAT3 inhibitor cryptotanshinone,then Western Blot assay was used to evaluate the regulatory effect of IL-10 on H₂O₂-induced HSCs autophagy.Results:（1）Autophagy in HSCs was successfully induced by H₂O₂:CCK-8 assay showed that the viability of HSC-T6 and HSC-LX2 were no significantly changed when the concentration of H₂O₂ is less than 100μmol/L and 500μmol/L,respectively.And LDH assay showed that the mortality of HSC-T6 was no significantly changed when the concentration of H₂O₂ is less than 100μmol/L.Western Blot assay showed that the ratio of autophagy-related protein LC3II/I in HSC-T6 treated with H₂O₂ for 3 h was significantly increased compared with control cells,while the LC3II/I ratio in HSC-LX2 was markedly increase after H₂O₂ treatment for 6 h.Compared with the H₂O₂-induced autophagy group,LC3II/I ratio was significantly decreased in autophagy inhibitor 3-MA treatment group.（2）The inhibitory effect of IL-10 on H₂O₂-induced HSCs autophagy:HSCs were co-treated with different concentrations of IL-10 and 100μmol/L H₂O₂.The results showed that the ratio of autophagy-related protein LC3II/I was significantly in IL-10（10 ng/m L）treatment group compared with H₂O₂-induced autophagy group.Immunofluorescence assay showed that numbers of autophagosomes in HSCs were significantly increased in H₂O₂ treatment group or markedly decreased in IL-10 co-treatment with H₂O₂ group compared with control group.TEM test confirmed that H₂O₂ treatment could increase the number of autophagosomes while IL-10 treatment could decrease these.（3）The role of Akt/mTOR/STAT3 pathway in IL-10 regulating H₂O₂-induced autophagy:Western Blot was used to detect the autophagy-related protein LC3II/I and signal pathway protein expressed in HSCs with or without IL-10 treatment.The results showed that H₂O₂ can decrease the phosphorylation of Akt,mTOR and STAT3（Y705）,and increase autophagy-related protein LC3II/I compared with the control group;while IL-10 can promote the phosphorylation of Akt,mTOR and STAT3（Y705）,and inhibit the expression of autophagy-related protein LC3II/I in HSCs.Rapamycin,which is a special inhibitor of mTOR,could abrogate the effect of IL-10 on H₂O₂-induced autophagy in HSCs.Cryptotanshinone,which is a special inhibitor of STAT3,can significantly inhibit the phosphorylation of STAT3 in HSC-T6 and HSC-LX2 cells treated with IL-10,however,it could only increase autophagy-related protein LC3II/I expression in HSC-T6.Immunofluorescence and TEM assay showed that the number of autophagosomes in HSCs treated with rapamycin or cryptotanshinone were more than those in IL-10 co-treated with H₂O₂ group.Conclusions:（1）Autophagy in HSCs was successfully induced by H₂O₂;（2）IL-10could inhibit H₂O₂-induced autophagy in HSCs;（3）IL-10 could inhibit H₂O₂-induced autophagy in HSCs by up-regulating phosphorylated protein in Akt/mTOR/STAT3 pathway.