Exploration of the Combined Mechanism of Direct and Indirect Effects of Paeoniflorin in the Treatment of Cholestasis

Cholestasis is a multifactorial hepatobiliary disorder, characterized by obstruction of bile flow and accumulation of bile, which in turn causes damage to liver cells and other tissues. In severe cases, it can result in the development of life-threatening conditions, including cirrhosis and liver cancer. Paeoniflorin (PF) has been demonstrated to possess favourable therapeutic potential for the treatment of cholestasis. The objective of this research was to examine the molecular mechanism of PF in the treatment of ANIT-induced cholestasis and to propose novel avenues for further research on the pharmacological effects of PF. In vivo and in vitro models of cholestasis were developed. The histopathological changes in the bile ducts and liver were evaluated through the use of hematoxylin and eosin (HE) staining. The extent of apoptosis was evaluated through the use of immunofluorescence (IF), immunoblotting (WB), and electron microscopy. The JNK signalling pathway was identified as the direct mechanism of action of PF through the utilisation of HuProt™ 20 K chips and other technologies. The present study demonstrated that PF markedly alleviated liver injury in an ANIT-induced cholestasis model. Specifically, PF was observed to attenuate cholestasis-induced liver injury by reducing the abnormal elevation of liver function indices and suppressing the expression of inflammatory mediators. Furthermore, PF exhibited anti-apoptotic properties in both in vivo and in vitro experiments, thereby mitigating cholestasis-induced hepatocyte apoptosis. These protective effects are attributable to the fact that PF exerts its action through direct interaction with the JNK pathway. It has been demonstrated that PF is capable of binding directly to MAPK8 (JNK1) and MAPK9 (JNK2), thereby inhibiting JNK activation and reducing apoptosis. With regard to the protection of bile ducts, PF may indirectly inhibit hepatocyte apoptosis by maintaining the structural integrity and tight junctions of bile duct cells. PF improved cholestasis by inhibiting hepatocyte apoptosis directly by targeting the JNK signaling pathway and indirectly inhibited hepatocyte apoptosis by improving the tight junctions of bile duct cells to regulate the bile duct microenvironment.