Department of Clinical Nutrition, Fujian Medical University Union Hospital, Fuzhou 350001, China.
Department of Gastroenterology, Fujian Medical University Union Hospital, Fuzhou 350001, China.
Exp Biol Med (Maywood). 2022 May;247(10):832-841. doi: 10.1177/15353702221080435. Epub 2022 Feb 24.
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 ( < 0.05). IL-10 inhibited the increases in the LC3II/I ratio and Beclin1 expression and upregulated p62 expression ( < 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 ( < 0.05). Blocking either the mTOR or STAT3 pathway reversed the inhibitory effect of IL-10 on starvation-induced autophagy in HSC-T6 cells ( < 0.05). IL-10 suppresses starvation-induced autophagosome formation through activation of the STAT3-mTOR-p70s6k axis in HSC-T6 cells.
肝星状细胞（HSCs）的激活程度与 HSCs 中的自噬水平密切相关。我们之前的研究表明，白细胞介素-10（IL-10）可强烈抑制大鼠纤维化肝脏中的 HSC 激活。然而，关于 IL-10 对 HSC 自噬的影响知之甚少。为了研究 IL-10 对永生化大鼠肝星状细胞（HSC-T6）饥饿诱导自噬的影响及其分子机制，用无血清 DMEM 孵育 HSC-T6 细胞不同时间，并以不同浓度的 IL-10 处理。透射电子显微镜（TEM）、自噬流分析和 Western blot（WB）检测观察 HSC-T6 细胞自噬体形态和数量的变化及自噬相关蛋白表达，评价 IL-10 对饥饿诱导自噬的调节作用。分别用 cryptotanshinone（CPT）和 rapamycin（Rapa）阻断信号转导和转录激活因子 3（STAT3）和 mTOR 信号通路的激活。用 WB 分析 STAT3-mTOR-p70s6k 信号通路蛋白，以评估 IL-10 调节自噬的信号通路。WB 显示，HSC-T6 细胞饥饿 3 小时（<0.05）后 LC3II/I 比值增加，Beclin1 表达增加，p62 表达减少。IL-10 抑制 LC3II/I 比值和 Beclin1 表达的增加，并上调 p62 表达（<0.05），最佳 IL-10 浓度为 20 ng/mL。TEM 和双标免疫荧光分析显示，IL-10 抑制自噬体形成和自噬流，双膜自噬体和黄色自噬斑点数量减少。进一步研究信号通路分子发现，饥饿诱导自噬时 mTOR、STAT3 和 p70s6k 蛋白磷酸化明显降低，但 IL-10 可增加 mTOR、STAT3 和 p70s6k 蛋白磷酸化（<0.05）。阻断 mTOR 或 STAT3 通路均可逆转 IL-10 对 HSC-T6 细胞饥饿诱导自噬的抑制作用（<0.05）。IL-10 通过激活 HSC-T6 细胞中的 STAT3-mTOR-p70s6k 轴抑制饥饿诱导的自噬体形成。
