Lactucin reverses liver fibrosis by inhibiting TGF-β1/STAT3 signalling pathway and regulating short-chain fatty acids metabolism

TGF-β1 activation of hepatic stellate cells (HSCs), transcriptional activator 3 (Stat3) activation and short chain fatty acids (SCFAs), metabolite of intestinal bacteria, is closely associated with hepatic fibrosis (HF). This study was intended to investigate whether effect of Lactucin on liver fibrosis was mediated by TGF-β1/Stat3 and SCFAs. HSC-T6 cells were activated using TGF-β1 stimulation, followed by intervention of activated cells using Lactucin. The cells were collected for apoptosis assay, immunofluorescence and Western blot. The results showed Lactucin inhibited HSC-T6 cell activation induced by TGF-β1, promoted apoptosis in HSC-T6 cells, inhibited nuclear translocation of Stat3 and p-Stat3. And Smad3 and TGF-β1 protein expression was significantly inhibited, while TLR4 and Smad7 protein expression was significantly enhanced. For in vivo experiments, C57BL/6 mice were injected with CCl4 and treated with or without Lactucin. The serum was collected for biochemical indexes, liver tissues were collected for pathological analysis and protein expression analysis, and intestinal contents were collected and analyzed for metabolism of SCFAs. Lactucin alleviated liver fibrosis in mice, as evidenced by a reduction in inflammatory factors, collagen deposition, liver injury and fibrosis-related factors expression, especially the expression of Smad3 and TGF-β1 proteins was significantly suppressed and Smad7 protein expression was significantly increased in the liver. In addition, the levels of acetic acid, butyric acid and valeric acid in the intestine of Lactucin-treated mice were significantly higher than those in the intestine of liver fibrosis mice. In conclusion, based on the results of in vivo and in vitro experiments, preventive mechanism of Lactucin against liver fibrosis in mice may be to improve the enterohepatic circulation by regulating the metabolites of intestinal microorganisms, acetic acid and butyric acid, and to further regulate the Stat3 and TGF-β1 signalling pathway through the "gut-liver axis" to combat liver fibrosis.