Integrated serum metabolomics reveal molecular mechanism of Xietu Hemu prescription on metabolic dysfunction-associated steatotic liver disease-related obesity

Background: Xietu Hemu prescription (XHP), a Chinese patent formula, is optimized based on the theory of "phlegm-dampness" and has been clinically validated to effectively combat metabolic dysfunction-associated steatotic liver disease (MASLD). It notably reduces visceral fat and body mass index. However, the molecular mechanisms underlying its regulation of lipid metabolism homeostasis remain unexplored.

Aim: To elucidate the mechanisms by which XHP inhibits adipocyte differentiation and maintains lipid metabolism homeostasis.

Methods: The therapeutic efficacy of XHP in metabolic-related disorders was analyzed using HepG2 cells and 3T3-L1 cells, along with transcriptomics to assess gene expression alterations during white adipogenesis. The primary metabolites of XHP were identified through ultra-performance liquid chromatography, and metabolic pathways were examined via serum metabolomics. Network analysis was employed to predict therapeutic targets. The accumulation of lipid droplets and the expression of associated proteins were confirmed using oil red O staining and Western blotting, respectively. Molecular docking was utilized to identify core targets and signaling pathways, which were substantiated through immunofluorescence and siRNA interference.

Results: XHP-containing serum (XHPS) significantly inhibited the transformation of normal HepG2 cells into fatty liver cells. Concurrently, the treatment suppressed the differentiation of 3T3-L1 cells, reduced lipid droplet accumulation and total Cholesterol/triglyceride levels, and downregulated the expression of PPARγ, C/EBPα, and FABP4. Through transcriptomics and network pharmacological intersectionality analyses, 24 core targets were identified, predominantly enriched in the AMPK signaling pathway. Molecular docking validated the strong binding affinity of XHP metabolites to targets such as Leptin (-11.3 kcal/mol) and ADIPOQ (-9.4 kcal/mol). ELISA results indicated that XHPS augmented Leptin autocrine secretion, thereby activating the AMPK signaling pathway (P < 0.05). Conversely, LEPR knockdown negated this effect (P < 0.05).

Conclusion: XHP effectively inhibits adipogenesis and enhances lipid metabolism homeostasis through the LEP/AMPK/PPARγ pathway, presenting a promising multi-target therapeutic strategy for MASLD by mitigating lipotoxicity.

Leptin; Lipotoxicity; Metabolic dysfunction-associated steatotic liver disease; Obesity; Xietu Hemu prescription.