Mou Yu, Tang Yao, Zheng Xiuyan, Liu Xiang, Wu Xuemei, Wang Hongji, Zeng Jie, Rao Qing, Ben-David Yaacov, Li Yanmei, Huang Lei
State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Guizhou Medical University, Guiyang, China.
Natural Products Research Center of Guizhou Province, Guiyang, China.
Front Pharmacol. 2025 Mar 12;16:1542143. doi: 10.3389/fphar.2025.1542143. eCollection 2025.
Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease, characterized by hepatic lipid accumulation. The Fufangduzhong formula (FFDZ) is a traditional Chinese medicine (TCM) formulation composed of Oliv. (Lour.) S. Y. Hu, Linn (Miq.) Miq. ex Havil., Georgi. It has demonstrated hepatoprotective effects and the ability to reduce lipid accumulation. However, its mechanisms against NAFLD remain unclear.
UPLC-MS/MS was used to identify FFDZ metabolites. C57BL/6J mice were fed a high-fat diet (HFD) supplemented with or without FFDZ (HFD+L, 0.45 g/kg/d; HFD+H, 0.9 g/kg/d) for 12 weeks. Biochemical indicators and histopathological observations were utilized to assess the extent of metabolic homeostasis disorder and hepatic steatosis. An analysis of differentially expressed genes and regulated signaling pathways was conducted using hepatic transcriptomics. Metabolomics analysis was performed to investigate the significantly changed endogenous metabolites associated with NAFLD in mice serum using UPLC-Q-TOF/MS. Western blot was employed to detect proteins involved in the lipid metabolism-related signaling pathways. Oleic acid-induced hepatic steatosis was used to examine the lipid-lowering effect of FFDZ-containing serum .
A total of eight active metabolites were identified from the FFDZ formula and FFDZ-containing serum through UPLC-MS/MS analysis. FFDZ reduced body weight, liver weight, and levels of inflammatory cytokines, and it ameliorated hepatic steatosis, serum lipid profiles, insulin sensitivity, and glucose tolerance in mice with HFD-induced NAFLD. Transcriptomics revealed that FFDZ modulated the lipid metabolism-related pathways, including the PPAR signaling pathway, Fatty acid metabolism, and AMPK signaling pathway. Meanwhile, Western blot analysis indicated that FFDZ downregulated the expression of lipid synthesis-related proteins (Srebp-1c, Acly, Scd-1, Fasn, Acaca, and Cd36) and upregulated the fatty acid oxidation-related proteins (p-Ampk, Ppar-α, and Cpt-1). Furthermore, metabolomics identified FFDZ-mediated reversal of phospholipid dysregulation (PC, PE, LPC, LPE). Additionally, FFDZ-containing serum remarkedly reduced OA-induced lipid accumulation in HepG2 cells.
The present results demonstrate that FFDZ exerts anti-NAFLD effects by enhancing glucose tolerance and insulin sensitivity, as well as regulating the Ampk signaling pathway to ameliorate lipid metabolism disorder, lipotoxicity, hepatic steatosis, and inflammatory responses.
非酒精性脂肪性肝病（NAFLD）是一种常见的慢性肝病，其特征为肝脏脂质蓄积。复方杜仲方（FFDZ）是一种中药配方，由杜仲（Lour.）S.Y.Hu、淫羊藿（Miq.）Miq.ex Havil.、巴戟天Georgi组成。它已显示出肝脏保护作用以及减少脂质蓄积的能力。然而，其抗NAFLD的机制仍不清楚。
采用超高效液相色谱-串联质谱法（UPLC-MS/MS）鉴定FFDZ的代谢产物。将C57BL/6J小鼠喂以含或不含FFDZ的高脂饮食（HFD+L，0.45 g/kg/d；HFD+H，0.9 g/kg/d），持续12周。利用生化指标和组织病理学观察评估代谢稳态紊乱和肝脏脂肪变性的程度。使用肝脏转录组学对差异表达基因和调控信号通路进行分析。采用代谢组学分析，利用超高效液相色谱-四极杆飞行时间质谱（UPLC-Q-TOF/MS）研究小鼠血清中与NAFLD相关的显著变化的内源性代谢产物。采用蛋白质印迹法检测参与脂质代谢相关信号通路的蛋白质。用油酸诱导的肝脏脂肪变性来检测含FFDZ血清的降脂效果。
通过UPLC-MS/MS分析，从FFDZ配方和含FFDZ血清中总共鉴定出8种活性代谢产物。FFDZ降低了高脂饮食诱导的NAFLD小鼠的体重、肝脏重量和炎性细胞因子水平，并改善了肝脏脂肪变性、血脂谱、胰岛素敏感性和葡萄糖耐量。转录组学显示，FFDZ调节脂质代谢相关通路，包括过氧化物酶体增殖物激活受体（PPAR）信号通路、脂肪酸代谢和腺苷酸活化蛋白激酶（AMPK）信号通路。同时，蛋白质印迹分析表明，FFDZ下调脂质合成相关蛋白（固醇调节元件结合蛋白-1c、ATP柠檬酸裂解酶、硬脂酰辅酶A去饱和酶-1、脂肪酸合酶、乙酰辅酶A羧化酶α和脂肪酸转运蛋白CD36）的表达，并上调脂肪酸氧化相关蛋白（磷酸化的AMPK、PPAR-α和肉碱棕榈酰转移酶-1）的表达。此外，代谢组学鉴定出FFDZ介导的磷脂失调（磷脂酰胆碱、磷脂酰乙醇胺、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺）的逆转。此外，含FFDZ血清显著降低了油酸诱导的HepG2细胞中的脂质蓄积。
目前的结果表明，FFDZ通过增强葡萄糖耐量和胰岛素敏感性，以及调节AMPK信号通路来改善脂质代谢紊乱、脂毒性、肝脏脂肪变性和炎症反应，从而发挥抗NAFLD作用。
