Multi-omics analysis provides new insights into mechanism of didymin on non-alcoholic fatty liver disease in rats

Background: Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases accompanied by lipid and glucose metabolism disorder. Didymin has been reported to have various hepatoprotective effects, however, its potential effects and mechanisms on NAFLD remain unclear from the perspective of the whole. Purpose: To investigate the underlying mechanism of didymin against NAFLD using multi-omics technologies. Methods: Rats were fed with a high-fat diet (HFD) for 8 weeks to induce NAFLD, followed by didymin treatment for 8 weeks. Next, biochemical analysis and histopathological examinations were performed to evaluate the effects of didymin. The key regulating pathways were predicted using transcriptomics, metabolomics and proteomics, and the target pathways were then verified by detecting the key genes/proteins using various experiments. Results: Didymin markedly mitigated liver injury and excessive lipid droplet accretion. An integrative multiomics analysis suggested that the PPAR signaling cascade and insulin signaling pathway might serve as pivotal mechanisms underlying the modulation of lipid and glucose homeostasis by didymin. Further dissection identified five pivotal genes (PPAR alpha, PPAR beta, FABP4, ANGPTL4, and PLIN2) and four genes (HK1, HK3, GCK, and PTPN1) as potential hubs within these pathways. Subsequent validation experiments, including qPCR and Western blot, demonstrated upregulated expression of PPAR alpha and PPAR beta, indicating the activation of the PPAR pathway by didymin. Concurrently, didymin appeared to modulate the insulin signaling pathway, as evidenced by the upregulated expression of HK1 and downregulated expression of PTPN1. Notably, the manipulation of PPAR alpha, PPAR beta, and PTPN1 expression in LO2 cells through silence or overexpression confirmed that didymin significantly reduced lipid accumulation, with its molecular targets likely being the PPAR and insulin pathways. Conclusions: Our findings demonstrate that didymin has a protective effect on NAFLD, and its underlying mechanism may be associated with the regulation of the PPAR and insulin signaling pathways.