Total Flavonoids of <i>Hovenia dulcis</i> Seeds Ameliorate Hepatic Fibrosis via Modulating PI3K/AKT Signaling Pathway In Vitro and In Vivo

Hovenia dulcis is commonly consumed as a herbal traditional Chinese medicine (TCM). Although existing studies have documented its hepatoprotective effects, the bioactive compounds and underlying mechanisms responsible for its anti-hepatic fibrosis (HF) activity remain poorly understood. In this study, total flavonoids from H. dulcis seeds (H. dulcis total flavonoid [HDTF]) were purified using macroporous resin D101 and analyzed by ultra-performance liquid chromatography-Orbitrap-mass spectrometry (UPLC-Orbitrap-MS). The model of HF was established by mice induced by CCl₄ and hepatic stellate cells (HSCs) stimulated with transforming growth factor-β1 (TGF-β1) to evaluate the anti-hepatic fibrotic effects of HDTF. A combination of Cell Counting Kit-8 (CCK-8) assay, Western blot, wound-healing assay, immunofluorescence staining, molecular docking, and confirmatory experiments was employed to elucidate the mechanisms involving the phosphoinositide 3-kinase (PI3K)/AKT pathway in the treatment of HF. As a result, 58 flavonoids were identified in the HDTF extracts. In both CCl₄-induced liver injury in mice and TGF-β1-activated HSCs, HDTF significantly attenuated HF by inhibiting HSC proliferation and migration. It also reduced the expression of α-smooth muscle actin (α-SMA) and Collagen I (COL-I) in hepatic tissues and cell cultures, highlighting its anti-fibrotic potential. Furthermore, HDTF decreased oxidative stress markers, including glutathione (GSH) and superoxide dismutase (SOD). Mechanistically, HDTF promoted apoptosis by modulating the expression of cleaved-caspase-3 and phosphorylated AKT (p-AKT). It also inhibited the PI3K/AKT signaling pathway by reducing the levels of p-AKT and phosphorylated PI3K (p-PI3K), both in vivo and in vitro. This study demonstrates that HDTF ameliorates HF by inhibiting proliferation and promoting apoptosis through the PI3K/AKT signaling pathway. These findings provide valuable insights into the therapeutic potential of H. dulcis seeds in the management of HF.