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 ... -  《-》

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 《-》

Liuweiwuling tablets attenuate BDL-induced hepatic fibrosis via modulation of TGF-β/Smad and NF-κB signaling pathways.

Liuweiwuling (LWWL) tablets contain a six-herb Chinese formula and are commonly prescribed to facilitate nourishment of the liver and kidneys, clear away toxic materials and activate blood circulation. Administration of LWWL is well known for its protective effects on the liver and its capacity to confer long-term stability in patients exhibiting reduced transaminase levels. Clinical studies have reported that LWWL can also be used for the treatment of liver fibrosis with associated treatment regimens resulting in a concomitant reduction in transforming growth factor β1 (TGF-β1) levels in the serum of patients with hepatic fibrosis. TGF-β1 plays a prominent role in stimulating liver fibrogenesis and this effect is mediated by myofibroblasts (MFB) derived from hepatic stellate cells (HSCs). It is likely that this phenomenon underpins the antifibrotic effects associated with LWWL. The purpose of this study was to investigate the antifibrotic effects and mechanisms pertaining to LWWL. Hepatic fibrosis was induced in rats following bile duct ligation (BDL). Rats that underwent BDL received daily gavage administration of colchicine (0.2mg/kg per day), LWWL (0.4, 1.6, 6.4g/kg per day) or PBS (for the control group). Pathological changes in hepatic tissue were examined using hematoxylin and eosin (HE) and sirius red staining. Immunohistochemical analysis was performed to monitor α-SMA and type I collagen (Collagen I) protein expression. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analyses were used to monitor the expression of genes and proteins in the TGF-β/Smad signaling pathway, including TGF-β1, bone morphogenic protein and activin membrane-bound inhibitor (Bambi), Smad3, phosphorylated Smad3 (p-Smad3) and Smad7. We also monitored the expression of genes and proteins in the nuclear factor-κB (NF-κB) signaling pathway, including NF-κB p65, IκBα and phosphorylation of IκBα (p-IκBα), tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β). LWWL dose-dependently inhibited BDL-induced liver injury and hepatic fibrosis in rats. Furthermore, LWWL reduced liver tissue collagen deposition, hydroxyproline content, liver dysfunction and α-SMA expression in BDL-induced hepatic fibrosis rats. Moreover, LWWL markedly prevented activation of the TGF-β/Smad signaling pathway by inhibiting expression of Smad2/3 and phosphorylation of Smad3, and upregulating the expression of Bambi and Smad7. In addition, LWWL regulated the expression of the inflammatory cytokines IL-1β, TNF-α and IL-6 by inhibiting the activation of NF-κB p65 and the phosphorylation of IκBα, and increasing the expression of IκBα. LWWL can attenuate BDL-induced hepatic fibrosis in rats, and this effect may be due to modulation of the NF-κB-dependent inflammatory response and activation of HSC and TGF-β/Smad-mediated synthesis and degradation of Collagen I.

Liu H ，Dong F ，Li G ，Niu M ，Zhang C ，Han Y ，He L ，Yin P ，Wang B ，Sang X ，Li R ，Wang J ，Bai Z ，Xiao X ... -  《-》

Mistletoe alkaloid fractions alleviates carbon tetrachloride-induced liver fibrosis through inhibition of hepatic stellate cell activation via TGF-β/Smad interference.

Mistletoe (Viscum coloratum (Kom.) Nakai) has long been categorized as a traditional herbal medicine in Asia. In addition to its application in cancer therapy, mistletoe has also been used in the treatment of chronic hepatic disorders in China. In the present study, we investigated the antifibrotic effect and mechanisms of action of mistletoe extracts in a rat model of carbon tetrachloride (CCl4)-induced hepatotoxicity. An experimental model of hepatic fibrosis was established by intraperitoneal injection of rats with CCl4 for 8 weeks. Rats were subsequently treated with a mistletoe alkaloid fraction preparation via oral administration (120mg/kg daily for 8 weeks) or with distilled water as a control. Histopathological changes were observed by hematoxylin and eosin staining and Masson׳s trichrome staining. The expression of markers relevant to hepatic stellate cell (HSC) activation in the liver was assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry and western blotting. The anti-fibrosis activity and mechanisms of action of mistletoe alkaloid fractions were further investigated in the HSC-T6 HSC line, following treatment with mistletoe alkaloid fractions (12mg/ml) for 48h. Hepatic fibrosis decreased markedly in CCl4-treated animals following treatment with mistletoe alkaloid fractions, compared to controls. The mRNA levels of transforming growth factor-β1 (TGF-β1), procollagen I and tissue inhibitors of metalloproteinases (TIMPs) were significantly downregulated, by about 40%, 40% and 45%, respectively, in liver tissues from rats treated with mistletoe alkaloid fractions. Furthermore, significant downregulation of TGF-β1, TGF-β1 receptor, phosphorylated Smad 2 and alpha smooth muscle actin (α-SMA) proteins, by about 45%, 30% and 40%, respectively, was also observed in liver tissues from mistletoe alkaloid fractions-treated rats. In contrast, Smad 7 levels were significantly increased by about 30% in mistletoe alkaloid fractions-treated rats. Treatment of HSC-T6 cells with mistletoe alkaloid fractions significantly induced Smad 7 expression and inhibited the expression of α-SMA, TGFβ1, TGF-β1 receptor, Smad 2 and TIMP-1, in vitro. We demonstrate that mistletoe alkaloid fractions decrease extracellular matrix accumulation by inhibiting HSC activation. Mechanistically, this may occur via inhibition of TGF-β1/Smad 2 and Smad 7 signal transduction, thereby blocking the synthesis of procollagen I and TIMP-1. These findings suggest that mistletoe alkaloid fractions may be a potential therapeutic agent for the treatment of hepatic fibrosis.

Jiang Y ，Wang C ，Li YY ，Wang XC ，An JD ，Wang YJ ，Wang XJ ... -  《-》

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 ... -  《-》