Abstract
BackgroundInduction 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).ResultsSirt6 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.ConclusionOur data illuminated that Sirt6 ameliorated GCDC-induced HiBEC apoptosis by upregulating PGC-1α expression through the AMPK pathway and its deacetylation effect.
Highlights
In cholestatic liver disease, both biliary epithelial cells and hepatocytes are exposed in their microenvironment to toxic bile acids arising from the disruption of bile acid homeostasis [1]
GCDC induced the down‐regulation of Sirt6 expression in human intrahepatic biliary epithelial cells (HiBEC) HiBEC were treated with GCDC at the concentrations and times indicated in Fig. 1a and CCK-8 assay was used to assess cell viability
Our results demonstrated that Sirt6 upregulated the mitochondrial biosynthesis regulator peroxidase-activated receptor gamma activator-1α (PGC-1α), which in turn enhanced the expression of downstream NRF1, NRF2 and Mitochondrial transcription factor A (TFAM)
Summary
Both biliary epithelial cells and hepatocytes are exposed in their microenvironment to toxic bile acids arising from the disruption of bile acid homeostasis [1]. Mitochondrial dysfunction and oxidative stress interact with each other and are important factors that induce cell death [11]. Preventing oxidative stress and mitochondrial dysfunction in hepatocyte and biliary epithelial cells can improve hepatic function and reduce cholestasis [13]. Bile acids inhibit mitochondrial respiratory chain complex activity and increase hepatocyte production of reactive oxygen species. 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 Sirt protection against the apoptosis of human intrahepatic biliary epithelial cells (HiBEC) induced by the bile acid glycochenodeoxycholate (GCDC)
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