Integration proteomics analysis to identify AMPK as key target pathways of TCM formula for high fat diet induced obesity in mice

Abstract

Background

Livsooth Authentic Herbal Formula (LAH) is a novel Chinese herbal medicine that has been previously shown to prevent non-alcoholic fatty liver disease (NAFLD). However, its efficacy in treating obesity and its underlying mechanisms remain unclear. This study uniquely investigates the therapeutic effects of LAH on high-fat diet (HFD)-induced obese mice, focusing on its multi-targeted regulation of metabolic pathways. This research highlights the potential of a multi-component herbal formula in simultaneously activating the AMPK pathway, regulating lipid metabolism, and enhancing antioxidant defenses. By integrating network pharmacology predictions with proteomics analysis, in vivo, and in vitro experiments, this study provides a comprehensive understanding of LAH's mode of action.

Materials and methods

Mice were fed a high-fat diet (HFD) for 8 weeks, followed by oral treatment with LAH at doses of 615 mg/kg and 2460 mg/kg for 10 weeks. Each treatment group consisted of 6 mice. Body weight, blood biochemistry, and antioxidant enzyme activities were measured. Network pharmacology and proteomics analyses were conducted to identify mechanisms, and in vitro studies validated molecular pathways.

Results

This study utilized network pharmacology to investigate the therapeutic effects and mechanisms of LAH on obesity. Through relevant databases, 19 major chemical components and 605 potential targets were identified. KEGG pathway analysis identified the AMPK signaling pathway as a key target of LAH. Animal experiments showed that LAH reduced body weight by 16.55 % compared to HFD-induced mice. In addition to weight reduction, LAH significantly improved serum metabolic parameters. Glucose, triglyceride, and cholesterol levels were significantly reduced, and liver function improved, with ALT decreasing from 142.00 ± 32.63 U/L (HFD) to 63.57 ± 33.16 U/L (H-LAH), and AST from 147.20 ± 12.92 U/L (HFD) to 81.71 ± 31.31 U/L (H-LAH). It also enhanced liver antioxidant enzyme activity and reversed oxidative stress. Proteomics analysis revealed that LAH treatment downregulated the expression of FASN, HMGCR, and SREBP1 while upregulating PRKAA1, PRKAA2, ACACA, SOD1, and GSTP1, which are linked to the AMPK pathway and antioxidant mechanisms. In vitro experiments confirmed that LAH ameliorates hepatic steatosis by activating the AMPK pathway, as evidenced by its regulation of p-ACC, p-AMPK, CPT1A, FAS, and SREBP1 protein expression, identifying it as a critical regulator of obesity and NAFLD.

Conclusion

LAH reduces the expression of FAS and SREBP1 proteins via the AMPK pathway, while enhancing the activity of antioxidant enzymes. These effects promote weight loss and improve hepatic lipid metabolism, supporting its potential as a therapeutic agent for obesity and related metabolic disorders. This provides a theoretical foundation for using LAH in weight management.