Interleukin-10 regulates starvation-induced autophagy through the STAT3-mTOR-p70s6k axis in hepatic stellate cells.

Abstract

The degree of activation of hepatic stellate cells (HSCs) is closely related to the level of autophagy in HSCs. We previously showed that interleukin-10 (IL-10) strongly inhibits HSC activation in rat fibrotic liver. However, little is known about the effect of IL-10 on HSC autophagy. For investigation of the effect of IL-10 on starvation-induced autophagy in immortal rat hepatic stellate cells (HSC-T6) and the molecular mechanism, HSC-T6 cells were incubated with serum-free DMEM for different periods and treated with IL-10 at different concentrations. Transmission electron microscopy (TEM), analysis of autophagic flux and Western blotting (WB) assays were used to observe changes in autophagosome morphology and number and autophagy-related protein expression in HSC-T6 cells and to evaluate the regulatory effect of IL-10 on starvation-induced autophagy. Cryptotanshinone (CPT) and rapamycin (Rapa) were used to block activation of the signal transducer and activator of transcription 3 (STAT3) and mTOR signaling pathways, respectively. STAT3-mTOR-p70s6k signaling pathway proteins were analyzed by WB to assess the signaling pathway by which IL-10 regulates autophagy. WB showed an increased LC3II/I ratio, increased Beclin1 expression, and decreased p62 expression in HSC-T6 cells starved for 3 h (p < 0.05). IL-10 inhibited the increases in the LC3II/I ratio and Beclin1 expression and upregulated p62 expression (p < 0.05), and the optimal IL-10 concentration was 20 ng/mL. TEM and double-labeled immunofluorescence analysis showed that IL-10 inhibited autophagosome formation and autophagic flux, as indicated by the decreased numbers of double-membrane autophagosomes and yellow autophagic puncta. Further examination of signaling pathway molecules showed that phosphorylation of the mTOR, STAT3, and p70s6k proteins was significantly decreased during starvation-induced autophagy, but IL-10 could increase mTOR, STAT3, and p70s6k protein phosphorylation (p < 0.05). Blocking either the mTOR or STAT3 pathway reversed the inhibitory effect of IL-10 on starvation-induced autophagy in HSC-T6 cells (p < 0.05). IL-10 suppresses starvation-induced autophagosome formation through activation of the STAT3-mTOR-p70s6k axis in HSC-T6 cells.