JAK1-STAT3-SOCS3信号通路在白介素10调节肝星状细胞衰老中的作用与机制

Senescence is associated with the development of liver fibrosis. Senescence of activated Hepatic stellate cells (HSCs) limits liver fibrosis. Interleukin-10 (IL-10) is a pleiotropic cytokine that downregulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. Our previous study had reported that IL-10 attenuates liver fibrosis in rats by inhibiting the activation and proliferation of HSCs, which associated with the increase of cells senescence number in the area of fibrogenesis. However, little is known regarding the effect of IL-10 on senescence of activated HSCs. In this study, the primary HSCs in different state of activation and the fibrotic rats were treated with IL-10，the change of senescence and cell cycle of HSCs were assessed by immunocytochemistry and flow cytometry analysis. Real-Time PCR and Western Blot analysis were used to detect the gene and protein expression of JAK1-STAT3-SOCS3 signal pathway and senescence of HSCs. This study was to investigate the effluence of IL-10 on the senescence of HSCs and its signal pathway, and then to assess the effect of shRNA interference targeting SOCS3 gene and a specific inhibitor for STAT3 on senescence of primary HSCs treated with IL-10.This study is designed to give a interpretation for the anti-fibrotic mechanism of IL-10 and will provide a new insight and experience data for anti-fibrotic therapy of IL-10.

细胞衰老与肝纤维化的形成密切相关，活化的肝星状细胞（HSCs）衰老能限制肝纤维化发展。白介素-10（IL-10）是一多效性炎症抑制因子及对肝纤维化产生具有调节作用。我们前期研究证实IL-10可抑制HSCs活化、增殖而拮抗肝纤维形成，伴随纤维沉积区细胞衰老增多，但IL-10是否能直接促进活化HSCs衰老，未见报道。课题拟通过IL-10干预对不同活化状态的原代HSCs及纤维化大鼠，细胞化学与流式细胞观察HSCs衰老与细胞周期改变，定量PCR与免疫印迹检测JAK1-STAT3-SOCS3信号与衰老指标的变化，评估IL-10对HSCs衰老及信号通路的影响；采用RNA干扰沉默SOCS3基因与特异性抑制剂阻断STAT3信号，再次评估IL-10对HSCs衰老的影响。课题有助于阐明IL-10抗纤维化机制，为IL-10临床抗纤维化治疗提供一种新视角和实验数据。

细胞衰老与肝纤维化的形成密切相关，活化的肝星状细胞（HSCs）衰老限制肝纤维化发展。白介素-10（IL-10）是一多效性炎症抑制因子及对肝纤维化产生具有调节作用。前期研究证实IL-10可抑制HSCs活化、增殖而拮抗肝纤维形成。本研究目的是探讨IL-10对活化肝星状细胞衰老的影响及抗纤维化的潜在机制。方法：胶原酶灌注和密度梯度离心法分离原代大鼠HSCs，体外培养原代大鼠HSCs并行不同浓度IL-10干预，通过SA-β-Gal染色，流式细胞术及CCK8试验检测各组细胞β-半乳糖苷酶的活性，细胞周期及细胞增殖能力的变化；Western Blot法检测各组Collagen typeⅠ和α-SMA表达、信号蛋白STAT3和p-STAT3与衰老相关蛋白p53、p21的表达情况；免疫细胞荧光检测p-STAT3蛋白细胞内分布并经细胞核质蛋白验证；通过p53与STAT3特异性药物抑制剂阻断p53与STAT3的磷酸化表达，再次检测活化HSCs细胞衰老与衰老蛋白的表达，评价STAT3-p53信号通过在IL-10诱导HSCs衰老中的作用；体内IL-10干预CCL4诱导肝纤维化大鼠，组织学评估纤维化程度与活化HSCs衰老情况。结果:成功分离大鼠原代HSCs；Western Blot结果显示体外培养7d的原代HSCs完全活化；与对照组比较，SA-β-Gal染色显示IL-10显著增强HSCs的β-半乳糖苷酶的活性；流式细胞术结果显示IL-10干预后G1期细胞比例明显增加，S期细胞比例明显减少；Western Blot结果显示IL-10干预后活化的HSCs表达信号蛋白 STAT3、p-STAT3与衰老相关蛋白p53、p21显著上调；免疫细胞荧光和核质抽提实验结果显示IL-10干预后细胞内p-STAT3主要定位于核内并经核质蛋白验证；p53抑制剂显著抑制IL-10对的活化HSCs衰老及p53和 p21表达的激活作用；STAT3抑制剂显著抑制IL-10诱导的HSCs衰老并显著抑制STAT3、p-STAT3、p53及p21的表达；体内实验证实IL-10抗纤维化作用与活化HSCs衰老相关。结论：IL-10通过STAT3-p53通路诱导化的HSCs衰老并抑制肝纤维化形成,为IL-10抗纤维化提供新的理论依据。