Activated hepatic stellate cells are reported to play a significant role in liver fibrogenesis. Beside the phenotype reversion and apoptosis of activated hepatic stellate cells, the senescence of activated hepatic stellate cells limits liver fibrosis. Our previous researches have demonstrated that interleukin-10 could promote hepatic stellate cells senescence via p53 signaling pathway in vitro. However, the relationship between expression of p53 and senescence of activated hepatic stellate cells induced by interleukin-10 in fibrotic liver is unclear. The purpose of present study was to explore whether p53 plays a crucial role in the senescence of activated hepatic stellate cells and degradation of collagen mediated by interleukin-10. Hepatic fibrosis animal model was induced by carbon tetrachloride through intraperitoneal injection and transfection of interleukin-10 gene to liver was performed by hydrodynamic-based transfer system. Depletions of p53 in vivo and in vitro were carried out by adenovirus-based short hairpin RNA against p53. Regression of fibrosis was assessed by liver biopsy and collagen staining. Cellular senescence in the liver was observed by senescence-associated beta-galactosidase (SA-β-Gal) staining. Immunohistochemistry, immunofluorescence double staining, and Western blot analysis were used to evaluate the senescent cell and senescence-related protein expression. Our data showed that interleukin-10 gene treatment could lighten hepatic fibrosis induced by carbon tetrachloride and induce the aging of activated hepatic stellate cells accompanied by up-regulating the expression of aging-related proteins. We further demonstrated that depletion of p53 could abrogate up-regulation of interleukin-10 on the expression of senescence-related protein in vivo and vitro. Moreover, p53 knockout in fibrotic mice could block not only the senescence of activated hepatic stellate cells, but also the degradation of fibrosis induced by interleukin-10 gene intervention. Taken together, our results suggested that interleukin-10 gene treatment could attenuate carbon tetrachloride-induced hepatic fibrosis by inducing senescence of activated hepatic stellate cells in vivo, and this induction was closely related to p53 signaling pathway.

Impact statement

This work further expanded the knowledge of the molecular mechanisms underlying IL-10 anti-fibrogenic effect by exploring the function of p53 in IL-10-induced activated HSCs senescence and fibrotic degradation in vivo. Our data showed that IL-10 gene intervention could lighten hepatic fibrosis induced by CCL₄ and induce the senescence of activated HSCs accompanied by up-regulating the expression of senescence-related proteins. In addition, depletion of p53 could abrogate up-regulation of IL-10 on the expression of aging-related proteins in vivo and vitro. Moreover, p53 knockout in fibrotic mice could block the senescence of activated HSCs and the degradation of fibrosis induced by IL-10 gene treatment. In summary, our results suggested that IL-10 gene intervention could attenuate CCL₄-induced hepatic fibrosis by inducing senescence of activated HSCs in vivo, and this induction was closely related to p53 signaling pathway. Our study sheds important light into the anti-fibrogenic therapy of IL-10.

中文翻译：

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沉默p53抑制白细胞介素10诱导的体内肝星状细胞活化衰老和纤维化降解

据报道，活化的肝星状细胞在肝纤维发生中发挥重要作用。除了活化的肝星状细胞的表型逆转和凋亡之外，活化的肝星状细胞的衰老也限制了肝纤维化。我们前期的研究表明，IL-10在体外可以通过p53信号通路促进肝星状细胞衰老。然而，纤维化肝脏中p53的表达与白细胞介素10诱导的活化肝星状细胞衰老之间的关系尚不清楚。本研究的目的是探讨p53是否在活化的肝星状细胞的衰老和白细胞介素10介导的胶原蛋白降解中发挥关键作用。采用腹腔注射四氯化碳诱导肝纤维化动物模型，并通过水动力转染系统将白细胞介素10基因转染至肝脏。通过基于腺病毒的针对 p53 的短发夹 RNA 进行了体内和体外p53 的去除。通过肝活检和胶原染色评估纤维化的消退。通过衰老相关β-半乳糖苷酶（SA-β-Gal）染色观察肝脏细胞衰老。采用免疫组织化学、免疫荧光双染和Western blot分析评估衰老细胞和衰老相关蛋白的表达。我们的数据表明，白细胞介素10基因治疗可以减轻四氯化碳诱导的肝纤维化，并诱导活化的肝星状细胞衰老，同时上调衰老相关蛋白的表达。 我们进一步证明，体内和体外p53的缺失可以消除白细胞介素10对衰老相关蛋白表达的上调。此外，在纤维化小鼠中敲除p53不仅可以阻止活化的肝星状细胞的衰老，还可以阻止白细胞介素10基因干预诱导的纤维化的降解。综上所述，我们的结果表明，白细胞介素10基因治疗可以通过诱导体内活化的肝星状细胞衰老来减轻四氯化碳诱导的肝纤维化，并且这种诱导与p53信号通路密切相关。

 影响报告

这项工作通过探索p53在IL-10诱导的活化HSC衰老和体内纤维化降解中的功能，进一步扩展了IL-10抗纤维化作用分子机制的知识。我们的数据表明，IL-10基因干预可以减轻CCL ₄诱导的肝纤维化，并诱导活化的HSC衰老，并上调衰老相关蛋白的表达。此外，p53的缺失可以消除IL-10对体内和体外衰老相关蛋白表达的上调。此外，纤维化小鼠中的p53敲除可以阻止活化的HSC的衰老以及IL-10基因治疗诱导的纤维化的降解。总之，我们的结果表明IL-10基因干预可以通过诱导体内活化的HSC衰老来减轻CCL ₄诱导的肝纤维化，并且这种诱导与p53信号通路密切相关。我们的研究为 IL-10 的抗纤维形成治疗提供了重要线索。