Solamargine alleviates liver fibrosis by regulating YAP signaling in hepatic stellate cells.

OBJECTIVES: Solamargine is a natural compound derived from the traditional Chinese medicinal plant Solanum nigrum L., and exhibits a variety of biological activities, including anti-inflammatory, antimalarial, pro-apoptotic, analgesic, and antitumor effects. However, the role of solamargine in liver fibrosis remains unclear. This study aims to investigate the anti-fibrotic effects and potential mechanisms of solamargine using human hepatic stellate cells (LX-2 cells) and a carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis.

METHODS: LX-2 cells were treated with solamargine (1.25 and 2.50 μmol/L) for 24 h. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) assay. The expression levels of fibrosis-related genes and proteins were detected by real-time quantitative PCR (RT-qPCR) and Western blotting analysis. C57BL/6J mice were randomly divided into a control group, CCl4 group, CCl4+0.5 mg/kg solamargine group, and CCl4+1.0 mg/kg solamargine group. The control group received intraperitoneal injections of olive oil, whereas the CCl4 and solamargine-treated groups received intraperitoneal injections of CCl4 3 times per week for 4 weeks to induce liver fibrosis. Meanwhile, the solamargine-treated groups were intraperitoneally administered solamargine at low and high doses every other day. At the end of the experiment, liver tissues and plasma samples were collected for analysis. Hematoxylin and eosin (HE), Sirius Red, Masson trichrome, and α-smooth muscle actin (α-SMA) immunohistochemical staining were performed to evaluate pathological changes and the degree of fibrosis. RT-qPCR and Western blot analysis were used to detect the expression of fibrosis-related genes and proteins. Commercial assay kits were used to measure serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic hydroxyproline content. To explore whether solamargine regulates Yes-associated protein (YAP) signaling pathway during liver fibrosis, LX-2 cells were treated with solamargine (1.25 and 2.50 μmol/L) for 24 h. Western blotting analysis was performed to detect the expression of large tumor suppressor kinase 1 (LATS1), p-LATS1 (Thr1079), YAP and p-YAP (Ser397), while RT-qPCR was used to determine the mRNA expression levels of YAP, Ctgf, and Ankrd1.

RESULTS: CCK-8 assays showed that treatment with 1.25 and 2.50 μmol/L solamargine for 24 and 48 h significantly reduced the proliferative activity of LX-2 cells compared with the control group (all P<0.05). RT-qPCR and Western blotting results demonstrated that, compared with the control group, solamargine treatment significantly reduced the mRNA expression levels of fibrosis-related genes, including Acta, Col1a1, TGF-β1, Timp1, and PDGF-A, as well as inflammatory cytokines IL-6 and TNF-α (all P<0.05). Protein expression levels of smooth muscle actin α (α-SMA) and collagen type I alpha 1 chain (Col1a1) were also significantly downregulated (all P<0.05). Compared with the CCl4 group, mice treated with solamargine exhibited markedly alleviated liver pathological injury and reduced inflammatory cell infiltration. Quantitative analyses of Sirius Red and Masson staining showed significantly decreased collagen deposition areas and reduced hepatic hydroxyproline content (all P<0.05). Serum ALT and AST levels were also significantly decreased (all P<0.05). In addition, the mRNA expression levels of Acta and Col1a1, as well as α-SMA protein expression in liver tissues, were significantly reduced (all P<0.05). In LX-2 cells, solamargine treatment significantly decreased YAP protein expression (P<0.05) and increased p-LATS1 (Thr1079) protein levels (P<0.05) compared with the control group. No statistically significant differences were observed in total LATS1 or p-YAP (Ser397) protein expression (all P>0.05). Furthermore, the mRNA expression levels of YAP, Ctgf, and Ankrd1 were significantly decreased (all P<0.05).

CONCLUSIONS: Solamargine attenuated liver fibrosis by suppressing YAP signaling and downregulating downstream targets such as Ctgf and Ankrd1. These findings suggest that solamargine holds promise as a potential antifibrotic therapeutic agent.