NRF2 and FXR dual signaling pathways cooperatively regulate the effects of oleanolic acid on cholestatic liver injury.

Background: Previous studies have shown that the anti-cholestatic effect of oleanolic acid (OA) is associated with FXR and NRF2. However, how the two signaling pathways cooperate to regulate the anti-cholestatic effect of OA remains unclear.

Purpose: This study aimed to further demonstrate the effect of OA on alpha-naphthyl isothiocyanate (ANIT)-induced cholestatic liver injury and the interaction mechanism between NRF2 and FXR signaling pathways in maintaining bile acid homeostasis.

Methods: Gene knockout animals and cell models, metabolomics analysis, and co-immunoprecipitation were used to investigate the mechanism of OA against cholestatic liver injury.

Results: The effect of OA against ANIT-induced liver injury in rats was dramatically reduced after Nrf2 gene knockdown. With the silencing of Fxr, the hepatoprotective effect of OA was weakened, but it still effectively alleviated cholestatic liver injury in rats. In L02 cells, OA can up-regulate the levels of NRF2, FXR, BSEP and UGT1A1, and reduce the expression of CYP7A1. Silencing of NRF2 or FXR significantly attenuated the protective effect of OA on ANIT-induced L02 cell injury and its regulation on downstream target genes, and the influence of NRF2 gene silencing on OA appeared to be greater. The NRF2 activator sulforaphane, and the FXR activator GW4064 both remarkably promoted NRF2 binding to P300 and FXR to RXRα, but reduced β-catenin binding to P300 and β-catenin binding to FXR.

Conclusion: The effect of OA on cholestatic liver injury is closely related to the simultaneous activation of NRF2 and FXR dual signaling pathways, in which NRF2 signaling pathway plays a more important role. The dual signaling pathways of NRF2 and FXR cooperatively regulate bile acid metabolic homeostasis through the interaction mechanism with β-catenin/P300.