Saikosaponin-d (SSd) is one of the major triterpenoid saponins derived from Bupleurum falcatum L., which has been reported to possess antifibrotic activity. At present, there is little information regarding the potential target of SSd in hepatic stellate cells (HSCs), which serve an important role in excessive extracellular matrix (ECM) deposition during the pathogenesis of hepatic fibrosis. Our recent study indicated that SSd may be considered a novel type of phytoestrogen with estrogen-like actions. Therefore, the present study aimed to investigate the effects of SSd on the proliferation and activation of HSCs, and the underlying mechanisms associated with estrogen receptors. In the present study, a rat HSC line (HSC-T6) was used and cultured with dimethyl sulfoxide, SSd, or estradiol (E2; positive control), in the presence or absence of three estrogen receptor (ER) antagonists [ICI-182780, methylpiperidinopyrazole (MPP) or (R,R)-tetrahydrochrysene (THC)], for 24 h as pretreatment. Oxidative stress was induced by exposure to hydrogen peroxide for 4 h. Cell proliferation was assessed by MTT growth assay. Malondialdehyde (MDA), CuZn-superoxide dismutase (CuZn-SOD), tissue inhibitor of metalloproteinases-1 (TIMP- 1), matrix metalloproteinase-1 (MMP-1), transforming growth factor-β1 (TGF-β1), hydroxyproline (Hyp) and collagen-1 (COL1) levels in cell culture supernatants were determined by ELISA. Reactive oxygen species (ROS) was detected by flow cytometry. Total and phosphorylated mitogen-activated protein kinases (MAPKs) and α-smooth muscle actin (α-SMA) were examined by western blot analysis. TGF-β1 mRNA expression was determined by RT-quantitative (q)PCR. SSd and E2 were able to significantly suppress oxidative stress-induced proliferation and activation of HSC-T6 cells. Furthermore, SSd and E2 were able to reduce ECM deposition, as demonstrated by the decrease in transforming growth factor-β1, hydroxyproline, collagen-1 and tissue inhibitor of metalloproteinases-1, and by the increase in matrix metalloproteinase-1. These results suggested that the possible molecular mechanism could involve downregulation of the reactive oxygen species/mitogen-activated protein kinases signaling pathway. Finally, the effects of SSd and E2 could be blocked by co-incubation with ICI-182780 or THC, but not MPP, thus indicating that ERβ may be the potential target of SSd in HSC-T6 cells. In conclusion, these findings suggested that SSd may suppress oxidative stress-induced activation of HSCs, which relied on modulation of ERβ.

NLRP3 inflammasome activation results in severe liver inflammation and injury. Saikosaponin-d (SSd) possesses anti-inflammatory and hepatoprotective effects. This study aimed to determine the protective effects of SSd on carbon tetrachloride (CCl4)-induced acute liver injury in mice, and whether oxidative stress and NLRP3 inflammasome activation participate in the process. The CCl4 mice model and controls were induced. The mice were treated with SSd at 1, 1.5, or 2.0 mg/kg in a total volume of 100 µl/25 g of body weight. Liver injury was assessed by histopathology. Oxidative stress was determined using mitochondrial superoxide production (MSP), malondialdehyde (MDA) content, and superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities. NLRP3, ASC, and Caspase 1 were determined by real-time PCR and western blot. IL-1β and IL-18 levels were determined by ELISA. Significantly elevated oxidative stress was induced in the liver by CCl4, as demonstrated by histopathology and increases of MDA and MSP levels and decreases of SOD, GPx, and CAT activities (all P < 0.01). SSd significantly decreased the MDA and MSP levels and increased the activities of SOD, GPx, and CAT (all P < 0.05). The mRNA expression of NLRP3, ASC, and Caspase 1, and the protein expression of Caspase 1-p10, NLRP3, ASC, IL-1β, and IL-18 were significantly increased after CCl4 induction (all P < 0.01). These changes were reversed by SSd (all P < 0.05). Suppression of the oxidative stress and NLRP3 inflammasome activation were involved in SSd-alleviated acute liver injury in CCl4-induced hepatitis.

Decursin possesses the potential to alleviate transforming growth factor (TGF)-β-induced hepatic stellate cells (HSCs) activation. However, the mechanisms by which decursin alleviates hepatic fibrosis remain not fully understood. Our aim is to explore the function of decursin on regulating HSCs activation and hepatic fibrosis. The anti-fibrotic effect of decursin was evaluated by Masson and Sirius red staining, and immunohistochemical (IHC) and quantitative real-time PCR (qRT-PCR) analysis for alpha-smooth muscle actin (α-SMA) and collagen types I (Col1a1) expression. Ferroptosis was assessed by measuring iron concentration, glutathione peroxidase 4 (Gpx4) and Prostaglandin endoperoxide synthase 2 (Ptgs2) expression, glutathione (GSH) level, lipid peroxidation, and reactive oxygen species (ROS) level. We found that decursin treatment decreased CCl4-induced liver fibrosis. The primary HSCs isolated from decursin-treated group showed an increased Fe2+, lipid ROS level, and decreased Gpx4 and GSH levels compared with HSCs from model group. Moreover, decursin promoted ferroptosis in activated HSCs in vitro, as evidenced by declined Gpx4 and GSH levels, increased Fe2+, ROS, and Ptgs2 levels compared with control. More important, ferroptosis inhibitor destroyed the anti-fibrosis effect of decursin on HSCs. In summary, these data suggest that decursin has potential to treat hepatic fibrosis.

Saikosaponin‑d (SSd) the primary active component of triterpene saponin derived from Bupleurum falcatum L., possesses anti‑inflammatory and antioxidant properties. The present study aimed to examine the potential therapeutic effects of SSd on carbon tetrachloride (CCl4)‑induced acute hepatocellular injury in the HL‑7702 cell line and its underlying mechanisms. HL‑7702 cells were treated with SSd at different doses (0.5, 1 or 2 µmol/l). Cell viability was determined using an MTT assay. Injury was assessed by the levels of serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Oxidative stress was assessed using malondialdehyde (MDA) content and total‑superoxide dismutase (T‑SOD) activity. The expression of nucleotide‑binding domain, leucine‑rich‑containing family, pyrin domain‑containing‑3 (NLRP3), apoptosis‑associated speck‑like protein (ASC), caspase‑1 and high mobility group protein B1 (HMGB1) was assessed by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Interleukin (IL)‑1β and IL‑18 were determined by RT‑qPCR and ELISA. SSd attenuated the inhibition of cell viability and the high AST and ALT levels induced by CCl4 in HL‑7702 cells. Oxidative stress was induced in HL‑7702 cells by CCl4, as demonstrated by the increase of MDA and the decrease of T‑SOD activity. These changes were reversed by SSd. SSd significantly downregulated the mRNA and protein expression of NLRP3, ASC, caspase‑1, IL‑1β, IL‑18 and HMGB1 induced by CCl4. In conclusion SSd alleviated CCl4‑induced acute hepatocellular injury, possibly by inhibiting oxidative stress and NLRP3 inflammasome activation in the HL‑7702 cell line.

Liver fibrosis is the ultimate common pathway in most types of chronic liver damage characterized by imbalance of extracellular matrix degradation and synthesis. Saikosaponin-d (SSd) possesses anti-inflammatory and anti-liver fibrosis effects. However, the underlying mechanism of SSd in repressing hepatic stellate cells (HSCs) activation remains unclear. Here we found that SSd alleviated remarkably carbon tetrachloride (CCl4)-induced liver fibrosis, as evidenced by decreased collagen level and profibrotic markers (COl1a1 and α-smooth muscle actin (SMA)) expression. SSd repressed CCl4-induced NOD-like receptor family pyrin-domain-containng-3 (NLRP3) activation in fibrotic livers, as suggested by decreased level of NLRP3, IL-18, and IL-β. The primary HSCs of CCl4 mice exhibited a significant increase in profibrotic markers expression and NLRP3 activation, but SSd treatment reversed the effect. SSd also repressed TGF-β-induced profibrotic markers expression and NLRP3 activation in vitro. Mechanistically, TGF-β decreased the expression of Estrogen receptor-β (ERβ) in HSCs, whereas SSd treatment reversed the effect. ERβ inhibition enhanced NLRP3 activation in HSCs. More important, ERβ or NLRP3 inhibition destroyed partially the function of SSd on anti-liver fibrosis. In summary, the current data suggest that SSd prevents hepatic fibrosis through regulating ERβ/NLRP3 inflammasome pathway, and suggest SSd as a potential agent for treating liver fibrosis.