Bioactive compounds and multitarget action mechanism of Erzhi pills in alleviating metabolic dysfunction-associated steatohepatitis

Ethnopharmacological relevance: Erzhi pills (EZP), as a traditional Chinese herbal prescription, have protective effects against various forms of liver injury. However, the therapeutic potential, bioactive ingredients and mechanism of action of EZP for metabolic dysfunction-associated steatohepatitis (MASH) have not been completely elucidated.

Aim of the study: This research aimed to evaluate the therapeutic effect of EZP and its bioactive compounds on MASH.

Methods: Two typical animal models of MASH, mice fed a methionine-choline-deficient (MCD) diet (representing non-obese MASH) and mice fed a high-fat and high-cholesterol diet (HFHC) (representing obese MASH), were used to investigate the effect of EZP on MASH in vivo. Transcriptomic and proteomic analysis were performed to elucidate the under lying mechanisms of EZP. The compositional analysis of EZP in MASH mice was conducted using UPLC-Q-Exactive-Orbitrap-MS. Free fatty acid (FFA)-stimulated HepG2 cells and transforming growth factor β1 (TGF-β1)-activated LX-2 cells were applied to further explore the effects and mechanisms of bioactive compounds from EZP.

Results: Our results indicate that the EZP mitigates hepatic lipid buildup, Insulin resistance, inflammation, Apoptosis, and fibrosis in different diet-induced MASH mice. Through multiomic analyses, UPLC-Q-Exactive-Orbitrap-MS analysis and molecular docking, we have identified that EZP exerts therapeutic effects on MASH involves activation of PPARα and PI3K/Akt/FOXO1 pathways, and inhibition of NLRP3, p53, and yes-associated protein (YAP) signaling pathways. It had been established that wedelolactone (wed), specnuezhenide (Spe), salidroside (Sal), and echinocystic acid-3-o-glucoside (Ech) served as the primary bioactive compounds in EZP for its therapeutic effect against MASH. In vitro experiments have confirmed that Spe, Sal, and Ech mitigate lipid accumulation by activating PPARα. Additionally, Spe and Sal improve Insulin resistance through the activation of the PI3K/Akt/FOXO1 signaling pathway. Furthermore, Wed, Sal, and Ech inhibit the NLRP3 inflammasome and p53 signaling pathways, thereby reducing inflammatory markers and providing anti-apoptotic effects. Moreover, Wed and Ech inhibit the activation of hepatic stellate cells (HSCs) by blocking the YAP signaling pathway. Notably, Sal and Ech were the primary bioactive components of EZP, and their combined anti-MASH efficacy was comparable to that of EZP.

Conclusion: This study is the comprehensive elucidation of the active ingredients and mechanism of action behind EZP in its anti-MASH properties. EZP, as well as the combination of Sal and Ech are potential treatment for MASH that targets multiple signaling pathways.

Bioactive compounds; MASH; Multi-pathway; Traditional Chinese medicine.