Hepatoprotective effect of phillygenin on carbon tetrachloride-induced liver fibrosis and its effects on short chain fatty acid and bile acid metabolism

Forsythiae fructus, the dried fruit of Oleaceae plant Forsythia suspensa (Thunb.) Vahl, is a traditional Chinese medicine widely used in clinical practice and has a variety of pharmacological activities, such as anti-inflammation, antioxidation, and hepatoprotection. Phillygenin (PHI), an important fingerprint lignan component of Forsythiae fructus, has prominent hepatoprotective, anti-inflammatory and antioxidant effects. Previously, it was shown that PHI could exert anti-fibrotic effects by modulating inflammation and gut microbiota. Therefore, given the important roles of SCFAs and BAs in the development of liver fibrosis, as well as their close links with gut microbiota, we aimed to determine the protective effects of PHI on carbon tetrachloride (CCl4)-induced liver fibrosis and its effects on the metabolism of SCFAs and BAs based on metabolomics. In C57BL/6J mice, liver fibrosis model was established by intraperitoneal injection of olive oil containing 10% CCl4 for 4 weeks. Firstly, the mouse liver tissues were subjected to histological analysis and biochemical index assay to evaluate the protective effect of PHI on CCl4-induced liver fibrosis. Subsequently, the effects of PHI on the metabolism of SCFAs and BAs in CCl4-induced liver fibrosis mice were determined using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) for metabolomics analysis. Finally, the levels of the closely related proteins and genes were detected by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) to explore the underlying mechanisms of the protective effect of PHI on CCl4-induced liver fibrosis. The histological analysis and the determination of relevant biochemical indexes of liver tissues showed that PHI could attenuate CCl4-induced liver fibrosis. The metabolomic analysis on SCFAs showed that PHI could promote SCFA production in the gut of mice with CCl4-induced liver fibrosis, especially acetic acid, propionic acid and butyric acid. It has been reported that the increased production of SCFAs was possibly beneficial to health. The metabolomic analysis on BAs found that PHI could restore the disturbance of BA metabolism in mice with CCl4-induced liver fibrosis. The immunohistochemistry and RT-qPCR results confirmed that PHI could ameliorate intestinal epithelial barrier disruption, and reverse the expression of BA metabolism-related genes in mice with CCl4-induced liver fibrosis. Promoting the production of SCFAs in the gut and restoring the disturbance of BA metabolism may be the potential mechanisms by which PHI alleviated CCl4-induced liver fibrosis.