Zhuyu pill attenuates metabolic-associated fatty liver disease by regulating macrophage polarization through TLR4 signaling pathway

BACKGROUND: Metabolic-associated fatty liver disease (MAFLD) is the leading chronic liver disease globally, impacting a large segment of the population. The Zhuyu Pill (ZYP), a traditional Chinese remedy, has been clinically used for treating MAFLD, with its effectiveness demonstrated in both human patients and animal models. However, the underlying mechanisms of how ZYP addresses MAFLD still require further investigation. OBJECTIVE: This study investigated the molecular mechanism of ZYP in treating MAFLD through both in vivo and vitro methods. METHODS: A murine MAFLD model was induced by a high-fat, high-fructose diet for 12 weeks. ZYP was administered for 4 weeks, with fenofibrate serving as a positive control. Indicators of lipid metabolism in serum and liver tissue were detected by automatic biochemical analyzer and ELISA, respectively. Histopathological evaluation of liver sections was performed using HE and oil red O staining. Transcriptomics was employed to further investigate the therapeutic mechanism of ZYP in MAFLD. Additionally, macrophages and their polarization in the liver were analyzed using ELISA, flow cytometry, immunohistochemistry, and immunofluorescence (IF). Candidate proteins and pathways were validated in vivo and in vitro by western blotting and IF. Validation of the pathway was performed in vitro using inhibitors and co-culture strategies. RESULTS: ZYP significantly improved obesity and hepatic steatosis in MAFLD mice, reducing body/liver weight and regulating lipid metabolism indicators in serum and liver tissue. Bioinformatics analysis of transcriptomic data highlighted lipid metabolism regulation and inflammation control as key effects of ZYP in treating MAFLD. The in vivo experimental results showed that ZYP inhibited M1 polarization of macrophages (pro-inflammatory) and promoted M2 polarization of macrophages (anti-inflammatory) in MAFLD mice. Further in vivo and vitro experiments indicated that ZYP competes with LPS to bind to Toll-like receptor 4 (TLR4), suppressing M1 polarization in liver macrophages, and improving MAFLD. The in vitro co-culture system also confirmed that ZYP reduces liver lipid deposition by modulating M1 macrophage polarization. CONCLUSIONS: ZYP alleviates MAFLD by inhibiting M1 polarization of liver macrophages, indicating that ZYP may be a promising treatment for MAFLD. Its mechanism of action is to inhibit the TLR4/MyD88/TRAF6 signaling pathway, modulate macrophage polarization, and improve inflammatory response.