Baicalin ameliorates high-fat diet induced MAFLD by inhibiting ERK/PPARγ/CD36 pathway

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a global health concern with limited therapeutic options. Baicalin (BA), a natural flavonoid, has shown promise in ameliorating lipid metabolism and liver function. This study employed an integrated strategy combining network pharmacology, molecular docking, and in vivo/in vitro validation to elucidate the novel protective mechanism of BA against MAFLD through modulation of the ERK/PPARγ/CD36 signaling axis in high-fat diet (HFD)-induced mice.

Potential targets of BA and MAFLD were identified from multiple databases, with common targets screened via Venn analysis. A protein-protein interaction (PPI) network was constructed and visualized based on degree values. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using R software. Molecular docking and surface plasmon resonance (SPR) experiments were performed to evaluate the binding affinity of BA to key targets, and a positive control was also added. For the in vivo study, MAFLD was induced in mice via 16-week HFD. Body weight, average food intake, glucose metabolism (GTT, ITT), serum lipid metabolism indexes (TG, TC, HDL-C, LDL-C), and serum liver function markers (ALT, AST, ALP, GGT) were assessed. Hepatic lipid deposition was analyzed using H&E, oil red O staining and NAS score. The protein and mRNA expression of p-ERK, PPARγ, CD36, SREBP-1c, FAS, ACC, and SCD1 were examined by Western blot or RT-PCR, with CD36 expression was further confirmed by immunofluorescence. In vitro, cell viability was measured using CCK8 at 12, 24, and 48 h after BA treatment. LO2 cells were treated with BA, free fatty acids (FFA), SCH772984 (a p-ERK inhibitor), or resomelagon (a p-ERK agonist), and lipid accumulation and protein expression were evaluated using Western blot and oil red O staining.

Network pharmacology analysis identified 37 common targets between BA and MAFLD, with PPARγ confirmed as a high-affinity BA target by molecular docking (pKi = -11.6096 µM) and SPR (K_D = 6.84 × 10^-7 M). In vivo, BA significantly reduced body weight (p < 0.05) and improved glucose metabolism (p < 0.05), lowered serum TG, TC, LDL-C levels (p < 0.05) and pro-inflammatory cytokines IL-1β, IL-6, TNF-α levels (p < 0.05). Additionally, BA attenuated hepatic steatosis and downregulated the expression levels of p-ERK, PPARγ and CD36 in liver tissues (p < 0.05). In vitro, BA or SCH772984 inhibited p-ERK, PPARγ, and CD36 expression (p < 0.05), as well as lipid accumulation in FFA-treated LO2 cells (p < 0.05). The resomelagon-treated cells were consistent with the effect of FFA, while BA could reverse the effects (p < 0.05).

This study by using integrates computational prediction demonstrated that PPARγ was the main target of BA, then with multi-level experimental validation, elucidated that BA could ameliorate MAFLD by modulating core ERK/PPARγ/CD36 signaling pathway, highlighting a novel therapeutic target for MAFLD.