Induction of biliary epithelial cell apoptosis by toxic bile acids is involved in the development of cholestatic disease, but the underlying molecular mechanism is not clear. The purpose of this study was to investigate the molecular mechanisms involved in Sirt6 protection against the apoptosis of human intrahepatic biliary epithelial cells (HiBEC) induced by the bile acid glycochenodeoxycholate (GCDC). Sirt6 was either overexpressed or knocked down in HiBEC, with or without GCDC pretreatment. The CCK-8 assay was used to assess cell viability and, Hoechst 33258 staining was used to determine apoptotic rate. Mitochondrial DNA (mtDNA) copy number, malondialdehyde (MDA) and reactive oxygen species (ROS) production were detected to evaluate the severity of the mitochondrial dysfunction and oxidative stress. The mRNA and protein levels of PGC-1α, Nrf1, and Nrf2 were analyzed using RT-qPCR and western blot assay. The results showed that Sirt6 opposed GCDC-induced apoptosis in HiBEC via up-regulating PGC-1α expression and stabilizing mtDNA. We used agonists and inhibitors of AMPK to demonstrate that Sirt6 increased PGC-1α expression through the AMPK pathway whereas GCDC had the opposite effect. Finally, western blot, luciferase assay, and co-immunoprecipitation were used to describe a direct interaction and acetylation modification of PGC-1α by Sirt6. Our data illuminated that Sirt6 ameliorated GCDC-induced HiBEC apoptosis by upregulating PGC-1α expression through the AMPK pathway and its deacetylation effect.

中文翻译：

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Sirt6 通过 AMPK/PGC-1α 通路对抗糖鹅去氧胆酸盐诱导的胆管上皮细胞凋亡。

有毒胆汁酸诱导胆管上皮细胞凋亡参与胆汁淤积性疾病的发展，但其潜在的分子机制尚不清楚。本研究的目的是研究 Sirt6 保护胆汁酸鹅去氧胆酸盐 (GCDC) 诱导的人肝内胆管上皮细胞 (HiBEC) 凋亡的分子机制。在有或没有 GCDC 预处理的情况下，Sirt6 在 HiBEC 中过表达或被击倒。CCK-8 测定用于评估细胞活力，Hoechst 33258 染色用于确定细胞凋亡率。检测线粒体 DNA (mtDNA) 拷贝数、丙二醛 (MDA) 和活性氧 (ROS) 产生，以评估线粒体功能障碍和氧化应激的严重程度。PGC-1α、Nrf1、使用 RT-qPCR 和蛋白质印迹法分析 Nrf2。结果表明，Sirt6 通过上调 PGC-1α 表达和稳定 mtDNA 来对抗 GCDC 诱导的 HiBEC 细胞凋亡。我们使用AMPK的激动剂和抑制剂来证明Sirt6通过AMPK途径增加PGC-1α的表达，而GCDC具有相反的作用。最后，使用蛋白质印迹、荧光素酶测定和免疫共沉淀来描述 Sirt6 对 PGC-1α 的直接相互作用和乙酰化修饰。我们的数据表明，Sirt6 通过 AMPK 通路上调 PGC-1α 的表达及其去乙酰化作用来改善 GCDC 诱导的 HiBEC 细胞凋亡。我们使用AMPK的激动剂和抑制剂来证明Sirt6通过AMPK途径增加PGC-1α的表达，而GCDC具有相反的作用。最后，使用蛋白质印迹、荧光素酶测定和免疫共沉淀来描述 Sirt6 对 PGC-1α 的直接相互作用和乙酰化修饰。我们的数据表明，Sirt6 通过 AMPK 通路上调 PGC-1α 的表达及其去乙酰化作用来改善 GCDC 诱导的 HiBEC 细胞凋亡。我们使用AMPK的激动剂和抑制剂来证明Sirt6通过AMPK途径增加PGC-1α的表达，而GCDC具有相反的作用。最后，使用蛋白质印迹、荧光素酶测定和免疫共沉淀来描述 Sirt6 对 PGC-1α 的直接相互作用和乙酰化修饰。我们的数据表明，Sirt6 通过 AMPK 通路上调 PGC-1α 的表达及其去乙酰化作用来改善 GCDC 诱导的 HiBEC 细胞凋亡。