Unravelling the mechanisms of erchen decoction in the treatment of nonalcoholic fatty liver disease (NAFLD): an integrative study combining network pharmacology, molecular docking, molecular dynamics simulation, multi-omics analysis, and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Erchen decoction (ECD) is a traditional Chinese herbal formula widely used in clinical practice for the treatment of nonalcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying its therapeutic effects have not been fully clarified.

AIM OF THE STUDY: To explore the pharmacological efficacy and potential molecular mechanisms of ECD in the treatment of NAFLD.

MATERIALS AND

METHODS

UPLC-Q-E-Orbitrap-MS was used to identify the chemical composition of the ECD and ECD-containing serum (ECDS). Network pharmacology, molecular docking, molecular dynamics simulation, transcriptomics, and metabolomics were integrated to predict the underlying mechanisms. In vivo and in vitro models-including high-fat diet- and glucose-fructose water-fed mice, as well as oleic acid- and palmitic acid-induced HepG2 cells-were used to evaluate the therapeutic effects of ECD. An inhibition strategy was applied to investigate the functions of the validated pathways.

RESULTS

ECD attenuated NAFLD progression both in vivo and in vitro. Thirty-eight active ingredients were identified in the ECDS. Network pharmacological analysis predicted that PPARG and BCL-2 might be central targets, whereas AMPK signalling and apoptosis are likely the main pathways mediating the anti-NAFLD effects of ECD. Molecular docking and molecular dynamics simulation suggested that multiple ECDS ingredients exhibited favourable binding affinities with AMPK and BCL-2, potentially offering therapeutic effects in NAFLD treatment. RNA sequencing analysis revealed that the differentially expressed genes were enriched primarily in the AMPK pathway. Liver metabolomics analysis revealed that the differentially expressed metabolites were associated mainly with oxidative phosphorylation and AMPK signalling. In experimental validation, ECD treatment increased phosphorylated AMPK levels, thereby promoting PPARα activation and upregulating key enzymes involved in β-oxidation (CPT1A and ACOX1) while downregulating PPARγ and its downstream genes associated with fatty acid synthesis (SREBP-1 and FASN), thus improving hepatic lipid metabolism both in vivo and in vitro. Moreover, the combination of ECD with the AMPK inhibitor compound C counteracted the lipid synthesis-reducing and lipid degradation-promoting effects of ECD both in vivo and in vitro. Additionally, ECD reduced the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1β), suppressed ROS production, increased the mitochondrial membrane potential, and inhibited cell apoptosis by modulating the expression of BCL-2, Bax, cleaved PARP and cleaved caspase-3 both in vivo and in vitro.

CONCLUSION

ECD attenuates NAFLD progression by targeting AMPK-mediated hepatic lipid metabolism and reducing hepatic apoptosis, providing new insights into the anti-NAFLD mechanism of ECD.