Zhao Mei, He Qingman, Shu Xinyao, Xu Ruitong, Zhang Zhongyi, Mou Yu, Liao Wenhao, Zhang Yong, Zhou Zubing, Shen Tao
Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, PR China.
Institute of Traditional Chinese Medicine, Sichuan Academy of Chinese Medicine Sciences, Chengdu, 610014, PR China.
Phytomedicine. 2025 Mar;138:156439. doi: 10.1016/j.phymed.2025.156439. Epub 2025 Jan 28.
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.
This study investigated the molecular mechanism of ZYP in treating MAFLD through both in vivo and vitro 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.
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.
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.
代谢相关脂肪性肝病（MAFLD）是全球主要的慢性肝病，影响着很大一部分人群。逐瘀丸（ZYP）是一种传统中药，已在临床上用于治疗MAFLD，其有效性在人类患者和动物模型中均得到证实。然而，ZYP治疗MAFLD的潜在机制仍需进一步研究。
本研究通过体内和体外方法探讨ZYP治疗MAFLD的分子机制。
采用高脂高糖饮食诱导小鼠MAFLD模型12周。给予ZYP治疗4周，非诺贝特作为阳性对照。分别用自动生化分析仪和ELISA检测血清和肝组织中的脂质代谢指标。采用HE和油红O染色对肝组织切片进行组织病理学评估。采用转录组学进一步研究ZYP治疗MAFLD的机制。此外，使用ELISA、流式细胞术、免疫组织化学和免疫荧光（IF）分析肝脏中的巨噬细胞及其极化情况。通过蛋白质印迹法和IF在体内和体外验证候选蛋白和信号通路。使用抑制剂和共培养策略在体外验证信号通路。
ZYP显著改善了MAFLD小鼠的肥胖和肝脂肪变性，降低了体/肝重量，并调节了血清和肝组织中的脂质代谢指标。转录组数据的生物信息学分析突出了脂质代谢调节和炎症控制是ZYP治疗MAFLD的关键作用。体内实验结果表明，ZYP抑制了MAFLD小鼠巨噬细胞的M1极化（促炎）并促进了巨噬细胞的M2极化（抗炎）。进一步的体内和体外实验表明，ZYP与脂多糖竞争结合Toll样受体4（TLR4），抑制肝巨噬细胞的M1极化，并改善MAFLD。体外共培养系统也证实，ZYP通过调节M1巨噬细胞极化减少肝脏脂质沉积。
ZYP通过抑制肝巨噬细胞的M1极化减轻MAFLD，表明ZYP可能是一种有前途的MAFLD治疗药物。其作用机制是抑制TLR4/MyD88/TRAF6信号通路，调节巨噬细胞极化，改善炎症反应。
