Total Flavonoids from Carthamus tinctorius L. Reduce Liver Fibrosis by Influencing Autophagy via Hedgehog Signaling

Liver fibrosis is a critical determinant of the progression of chronic liver disease (CLD). Total flavonoids from Carthamus tinctorius L. (TFCTLs) exhibit diverse pharmacological activities while their effect on liver fibrosis remains incompletely understood. This study aimed to elucidate the effects and mechanisms of TFCTLs on liver fibrosis. To this end, we first established a carbon tetrachloride (CCl₄)-induced liver fibrosis model in mice. Histological analysis demonstrated that TFCTL treatment significantly alleviated CCl₄-induced liver collagen deposition (p < 0.001). Meanwhile, TFCTLs could also downregulate the expression levels of fibrosis markers α-SMA and collagen I in a dose-dependent manner (p < 0.05). In vitro, a cellular model of fibrosis was generated by treating HSC-T6 cells with TGF-β1. EdU incorporation assays revealed that TFCTLs significantly suppressed HSC proliferation (p < 0.05). Furthermore, immunofluorescence staining for α-SMA demonstrated a marked reduction in HSC activation upon TFCTL treatment. The inhibitory effect of TFCTLs on cell migration was confirmed by wound healing and transwell assays, which revealed a substantial decrease in the number of migrated cells (p < 0.001). Additionally, flow cytometric analysis indicated that TFCTL treatment promoted HSC apoptosis (p < 0.05). Further mechanistic investigations revealed that TFCTLs exert their antifibrotic effects by inhibiting Hedgehog pathway and activating autophagy process. The antifibrotic effect of TFCTLs was partially reversed by the autophagy inhibitor 3-MA. Furthermore, the Hedgehog agonist PUR not only counteracted the anti-fibrotic actions of TFCTLs but also suppressed TFCTL-induced autophagy activation. In conclusion, our study demonstrated that TFCTLs attenuate liver fibrosis by inhibiting Hedgehog signaling and subsequently promoting autophagy, highlighting their potential as a therapeutic agent for liver fibrosis.