Effect of bioactive peptide of Carapax Trionycis on TGF-β1-induced intracellular events in hepatic stellate cells.

In traditional Chinese medicines for hepatic fibrosis therapy, Carapax Trionycis is used usually as an indispensable component and has a long history of medical use in China. Previous studies have demonstrated that extracts of Carapax Trionycis were able to protect liver against fibrosis in CCl4 animal models. The purpose of this study is to verify the inhibitory effect and the underlying mechanisms of Carapax Trionycis extract peptide (CTEP) on activated hepatic stellate cells which play a central role in liver fibrogenesis. Hepatic stellate cells induced by TGF-β1 were applied to evaluate the anti-fibrotic effect of CTEP in vitro. MTS assay, enzyme-linked immunosorbent assay and western blotting were then used to further investigate the molecular mechanisms. The results show that the contents of collagen I, collagen III and TIMP-1 were significantly inhibited and the level of collagen I, collagen III, p-Smad 3, TIMP-1 and α-SMA proteins decreased significantly in a concentration-dependence manner after treatment with CTEP. Interestingly, the level of Smad 3 protein was not different significantly. Our data indicate that CTEP efficiently inhibits cultured HSC-T6 cell activation and proliferation via the TGF-β1/Smad pathway as well as by the elimination of the extracellular matrix.

Tang Y ，Hu C ，Liu Y 《-》

Carapax Trionycis extracts inhibit fibrogenesis of activated hepatic stellate cells via TGF-β1/Smad and NFκB signaling.

Carapax Trionycis is used as a traditional Chinese medicine with a long history of clinical application in China, and it represents an essential medication used for liver fibrosis treatment. Previous studies demonstrated that Carapax Trionycis extracts protect liver against fibrosis in CCL4-induced animal models. This study investigated the anti-fibrotic molecular mechanisms exerted by Carapax Trionycis extracts with molecular weight less than 6 KD (CT6) in rat hepatic stellate cell line HSC-T6 activated by TGF-β1. HSC-T6 cells induced by TGF-β1 were used to evaluate CT6 anti-fibrotic effect in vitro. CCK8 was used to evaluate cell viability and CT6 effect on HSC-T6 proliferation. ELISA was performed to detect the presence of inflammatory cytokines. Western blot and q-PCR were performed to explore the molecular mechanisms. Our data demonstrated that CT6 did not clearly affect cell viability but suppressed TGF-β1-induced HSC-T6 proliferation. Collagen I and α-smooth muscle actin (α-SMA) protein levels were decreased by CT6 in TGF-β1-induced HSC-T6, followed by the inhibition of TIMP1, TIMP2 and TGF-β1/Smad pathway. Furthermore, CT6 decreased Jun D and p-p65 protein levels, down-regulated Tgf-β1, Tnf-α, Il-1β, Il-6 mRNA and TNF-α, IL-1β and IL-6 expression in TGF-β1-treated HSC-T6. These results suggested that CT6 inhibited HSC-T6 activation induced by TGF-β1, indicating the potential therapeutic effect of these extracts against liver fibrosis.

Hu Z ，You P ，Xiong S ，Gao J ，Tang Y ，Ye X ，Xia Y ，Zhang D ，Liu Y ... -  《-》

Synthesis of peptides of Carapax Trionycis and their inhibitory effects on TGF-β1-induced hepatic stellate cells.

Hu CL ，Peng XZ ，Tang YP ，Liu YW ... -  《-》

Oligo-peptide I-C-F-6 inhibits hepatic stellate cell activation and ameliorates CCl(4)-induced liver fibrosis by suppressing NF-κB signaling and Wnt/β-catenin signaling.

Oligo-peptide I-C-F-6 is a Carapax trionycis extract component that has an effect on hepatic fibrosis, however, its mechanism of action is still unclear. This study investigated whether oligo-peptide I-C-F-6 could inhibit liver fibrosis by suppressing NF-κB and Wnt/β-catenin signaling, which are important in liver fibrosis. HSC-T6 cells were treated with oligo-peptide I-C-F-6, and rats were divided randomly into five groups: control (saline), CCl4, CCl4 plus oligo-peptide I-C-F-6 (0.12 and 0.24 mg/kg), and CCl4 plus colchicine (0.11 mg/kg). Here, we demonstrated that oligo-peptide I-C-F-6 ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl4. Oligo-peptide I-C-F-6 also inhibited the activation of hepatic stellate cells (HSCs) in vivo and in vitro, as evaluated by the expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA), which is a specific marker of HSC activation. Moreover, oligo-peptide I-C-F-6 significantly reduced the expression and distribution of β-catenin, P-AKT, phospho (P)-GSK-3β, nuclear factor κB (NF-κB) P65, phospho-P65, and IκB kinase α/β (IKK-α/β) levels; additionally, IκB-α level was elevated both in vivo and in vitro. Together, these results indicate that oligo-peptide I-C-F-6 has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be related to modulating NF-κB and Wnt/β-catenin signaling.

Sun H ，Chen G ，Wen B ，Sun J ，An H ，Pang J ，Xu W ，Yang X ，He S ... -  《-》

Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-β1/Smad and NOX4/ROS pathways.

Liver fibrosis is a pathological wound-healing response caused by chronic liver damage due to a virus, autoimmune disorder, or drugs. Hepatic stellate cells (HSCs) play an essential role in the pathogenesis of liver fibrosis. Methyl ferulic acid (MFA), a biologically active monomer, has a protective effect on liver injury. However, the effects and roles of MFA in liver fibrosis remain unknown. The purpose of the current study was to investigate the effect of MFA on hepatic fibrosis and the underlying mechanisms. Human hepatic stellate LX-2 cells were exposed to 5 μg/L TGF-β1 for 48 h to stimulate liver fibrosis in vitro. Using MTT, RT-PCR and Western blot analysis, we revealed that MFA significantly inhibited the proliferation of LX-2 cells as well as decreased the expressions of α-SMA and type I collagen in LX-2 cells. SD rats were fed with ethanol, and this combined with the intraperitoneal injection of CCl4 induced liver fibrosis in vivo. We found that the administration of MFA markedly decreased the levels of hyaluronic acid (HA), procollagen type III (PC-III), type IV collagen (CIV) and laminin (LN) in the serum, inhibited the expression of α-smooth muscle actin (α-SMA) as well as type I and type III collagen, and up-regulated the ratio of MMP-2/TIMP-1 in rats. The antifibrotic effects of MFA were also evaluated by H&E staining and Masson's trichrome staining. In addition, further studies suggested that this protection by MFA from liver fibrosis was possibly related to the inhibition of TGF-β1/Smad and NOX4/ROS signalling. In conclusion, our results demonstrate that MFA attenuated liver fibrosis and hepatic stellate cell activation by inhibiting the TGF-β1/Smad and NOX4/ROS signalling pathways.

Cheng Q ，Li C ，Yang CF ，Zhong YJ ，Wu D ，Shi L ，Chen L ，Li YW ，Li L ... -  《-》