IL-10 Promotes The Senescence Of Activated Hepatic Stellate Cells Via STAT3/P53/P21 Signal Pathway

Background and Aim: Hepatic fibrosis is a common pathological process involved in various chronic liver injuries,which is characterized by excessive accumulation of extracellular matrix.Activated hepatic stellate cells(HSCs)are the main source of extracellular matrix.Studies have found that promoting the activated HSCs senescence can inhibit proliferation and secretion of extracellular matrix and become one of important strategies to reverse liver fibrosis.Our previous studies have shown that IL-10 can attenuate carbon tetrachloride-or porcine serum-induced liver fibrosis in rats,however,little is known about the anti-fibrotic effects of IL-10 on the senescence of activated HSCs.Therefore,the aims of this study are to investigate the effects and underlying pathway of IL-10 on activated HSCs senescence,and then to further elucidate the anti-fibrotic effects of IL-10.Methods: Primary rat HSCs were isolated by the collagenase perfusion and density-gradient centrifugation method and cultured in vitro.The activation of primary rat HSCs was identified by expression of collagen type I and ?-smooth muscle actin(?-SMA)in HSCs.Primary rat activated HSCs were divided into control group and IL-10 treatment group.SA-?-Gal staining,flow cytometry and Western blot were used to analyze the activity of ?-galactosidase,cell cycle and expression of senescenceassociated protein P53 and P21 in activated HSCs treated with or without IL-10.Pifithin-?(a specific inhibitor of P53)and cryptotanshinone(a specific inhibitor of signal transducers and activator of transcription 3,STAT3)were used to inhibit the expression of p53 and phosphorylation of STAT3 in activated HSCs,respectively.SA-?-Gal staining and Western Blot were used to detect the activity of ?-galactosidase and senescent protein marker in primary HSCs or HSC-T6.Immunofluorescence stain and Western Blot were used to detect the expression and distribution of p-STAT3 in activated HSCs after IL-10 treatment.Results: Primary rat HSCs were successfully isolated and activated after cultured on plastic plate for 7 days.Western Blot showed that the expression of collagen type I and ?-SMA in primary HSCs cultured for 7 days were significantly increased compared to 3 days(p<0.001);Compared to control group,IL-10 treatment can significantly increase the activity of ?-galactosidase(p<0.001),induce the cell cycle arrest in G1 phrase(p<0.001),elevate the expression of senescence-associated proteins P53 and P21(p<0.05).Pifithin-? inhibited SA-?-Gal activity(p<0.001)and decreased the expression of p53 and p21(p<0.05)in activated HSCs treated with IL-10.IL-10 also increased the expression of total STAT3 and p-STAT3 in activated HSCs and promoted translocation of p-STAT3 from cytoplasm to nucleus.Cryptotanshinone blocked the phosphorylation of STAT3,decreased expression of P53 and P21 and inhibited the activity of SA-?-Gal in activated HSCs treated with IL-10.Conclusions: IL-10 promotes the senescence of activated hepatic stellate cells via STAT3 / p53 / p21 signal pathway...