Forsythiaside-A improved bile-duct-ligation-induced liver fibrosis in mice: The involvement of alleviating mitochondrial damage and ferroptosis in hepatocytes via activating Nrf2

Liver fibrosis is a key and reversible stage in the progression of many chronic liver diseases to cirrhosis or hepatocellular carcinoma. Forsythiaside-A (FTA), a main compound isolated from Forsythiae Fructus, has an excellent liver protective activity. This study aims to investigate the efficacy of FTA in improving cholestatic liver fibrosis. Bile-duct-ligation (BDL) was conducted to induce liver fibrosis in mice. Hepatic collagen deposition was evaluated by Masson and Sirus red staining. The bile acid spectrum in the liver and serum was analyzed by mass spectrometry. Liver oxidative stress injury and mitochondria damage were observed by using Mito-Tracker Red fluorescence staining, transmission electron microscopy, etc. The level of ferrous iron (Fe2+) and the expression of ferroptosis-associated molecules were detected. The binding between FTA and its target protein was confirmed by Co-immunoprecipitation (Co-IP), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR). Our results demonstrated that FTA alleviated BDL-induced liver fibrosis in mice. FTA did not decrease the elevated amount of bile acids in BDL-treated mice, but reduced the bile acid-induced mitochondrial damage, oxidative stress and ferroptosis in hepatocytes, and also induced nuclear factor erythroid 2-related factor-2 (Nrf2) activation. In Nrf2 knock-out mice, the FTA-provided protection against BDL-induced liver fibrosis was disappeared, and FTA's inhibition on mitochondrial damage, oxidative stress and ferroptosis were lowered. Further results displayed that FTA could directly bind to Kelch-like ECH-associated protein-1 (Keap1), thereby activating Nrf2. Moreover, the BDL-induced liver fibrosis was markedly weakened in liver-specific Keap1 knockout mice. Hence, this study suggests that FTA alleviated the BDL-induced liver fibrosis through attenuating mitochondrial damage and ferroptosis in hepatocytes by activating Nrf2 via directly binding to Keap1.