Vitamin D receptor activation targets ROS-mediated crosstalk between autophagy and apoptosis in hepatocytes in cholestasis mice

Background and aims: Observational epidemiological studies have associated vitamin D deficiency with cholestasis. It has been reported by our lab that vitamin D/vitamin D receptor (VDR) aixs activation in cholangiocytes mitigates cholestatic liver injury by remodeling the damaged bile duct. However, the function of VDR in hepatocytes during cholestasis remains unclear.

Methods: Paricalcitol (PAL, VDR agonist, 200 ng/kg) was intraperitoneally injected into bile duct ligated (BDL) mice every other day for 5 days. Primary hepatocyte and HepG2 cell were transfected with Vdr shRNA, Control shRNA, Vdr plasmid, Control vector, Atg5 siRNA and Control siRNA. Liver histology, cell proliferation and Autophagy were evaluated.

Results: Treatment with the VDR agonist paricalcitol (PAL) improved BDL mouse liver injury by upregulating VDR expression in hepatocytes, which in turn reduced hepatocyte Apoptosis by inhibiting Reactive Oxygen Species (ROS) generation via suppressing Rac1/NOX1 pathway. Mechanistically, upon exposure to the ROS-inducing compound, Vdr siRNA contributed to Apoptosis events, whereas the Vdr plasmid led to Apoptosis resistance. Interestingly, upregulated VDR expression also appeared to increase autophagosome generation and macroautophagic/autophagic flux, which was the underlying mechanism for VDR activation-inhibited Apoptosis. Autophagy depletion completely impaired the upregulated VDR-inhibited Apoptosis events, whereas Autophagy induction showed a synergistic effect with VDR overexpression. Importantly, upregulation of VDR promoted Autophagy activation by suppressing the activation of the ERK/p38MAPK pathway. The p38MAPK inhibitor abrogated the Vdr siRNA-induced decrease in Autophagy and the Vdr siRNA-induced increase in Apoptosis. In contrast, the MEK/ERK Activator prevented the Vdr plasmid-enhanced Autophagy and decreased Apoptosis. Moreover, the ROS inhibitor NAC blocked Vdr siRNA-enhanced activation of the ERK/p38MAPK pathway.

Conclusions: VDR activation mitigated liver cholestatic injury by reducing autophagy-dependent hepatocyte Apoptosis and suppressing the activation of the ROS-dependent ERK/p38MAPK pathway. Thus, VDR activation may be a potential target for the treatment of cholestatic liver disease.

ERK/p38MAPK pathway; ROS generation; Vitamin D receptor activation; apoptosis; autophagy.