OBJECTIVE To investigate the mechanism by which Linggui Zhugan Granules regulate lipid metabolism, modulate oxidative stress and inflammatory responses, and improve metabolic dysfunction-associated steatohepatitis (MASH).

METHODS A mouse model of MASH was established by using a western diet (high-fat, high-cholesterol, and high-fructose diet) to detect its oxidative stress, lipid metabolism, and inflammation-related indicators. Human hepatocellular carcinoma cells HepG2 were induced with 0.75 mol·L^-1 free fatty acids (sodium oleate∶sodium palmitate=2∶1) to establish a hepatocyte steatosis model, and oxidative stress, lipid metabolism, and inflammation-related indicators were detected in lipid-stained cells.

RESULTS Compared to the normal group, the model group mice exhibited significantly increased body weight and liver weight (P < 0.001); serum levels of GLU, LDL-C, HDL-C, TC, ALT, and AST were significantly elevated (P < 0.05, P < 0.001); liver tissue pathology was severe, with significantly increased levels of TC, TG, and MDA in the liver (P < 0.001), while GSH content and SOD activity were significantly decreased (P < 0.001). At the molecular level, mRNA expression of PPARα was significantly downregulated in the liver tissue of the model group mice (P < 0.001), while mRNA expression of SREBP-1c, FAS, Toll-like receptors (TLR-2/4/9), IL-1β, TNF-α, TGF-β1, COL1A1, and α-SMA was significantly upregulated (P < 0.001), and the expression of NLRP3 and Cleaved caspase-1 protein was significantly increased (P < 0.05, P < 0.001). In the HepG2 cell model, the relative area of lipid droplets, TC, TG, and MDA content were significantly increased (P < 0.001), while GSH content and SOD activity were significantly decreased (P < 0.001). The protein expression levels of CPT1A, ACOX1, PPARα, ACADM, SIRT1, Nrf2, and HO-1 were significantly decreased (P < 0.05, P < 0.01, P < 0.001), while the protein expression levels of NLRP3, Caspase-1, and Cleaved caspase-1 were significantly increased (P < 0.05, P < 0.01). Compared with the model control group, the serum levels of ALT and AST in each administration group were significantly reduced (P < 0.001), the serum levels of LDL-C, HDL-C and GLU in the high-dose group were significantly reduced (P < 0.05, P < 0.01), the pathological damage of the liver was significantly improved, the levels of TC, TG, and MDA in the liver were significantly decreased (P < 0.01, P < 0.001), while GSH content and SOD activity in the liver of the high-dose group were significantly increased (P < 0.05, P < 0.01). Additionally, mRNA expression of PPARα in the liver tissue in high dose group was significantly upregulated (P < 0.001), while mRNA expression of SREBP-1c, FAS, Toll-like receptors (TLR-2/4/9), TNF-α, TGF-β1, COL1A1, and α-SMA in all dose groups was significantly downregulated (P < 0.001), and the expression of NLRP3, Caspase-1, and Cleaved caspase-1 proteins in the high-dose group was significantly decreased (P < 0.05, P < 0.01, P < 0.001). In the cell experiments, 200, 400 μg⋅mL^-1 drug treatment significantly reduced the relative area of lipid droplets, TG, and MDA content in HepG2 lipid-degenerate cells (P < 0.001), and increased GSH content and SOD activity (P < 0.01, P < 0.001). The expression of CPT1A, ACOX1, PPARα, ACADM, SIRT1 and Nrf2 proteins was significantly upregulated (P < 0.05), while the expression of NLRP3 and Caspase-1 proteins was significantly downregulated (P < 0.05, P < 0.001).

CONCLUSION The compound Linggui Zhugan Granules can effectively improve liver tissue pathology and lipid degeneration in HepG2 cells of MASH mice. The mechanism may involve the regulation of SREBP and PPARα signaling pathways, promoting lipid metabolism regulation. Additionally, it may improve oxidative stress by regulating the SIRT1/Nrf2/HO-1 pathway and inhibit the NLRP3 inflammasome pathway to reduce inflammatory responses.