Casticin Alleviates Liver Fibrosis By Targeting The TGF-β/Smad Pathway In Vitro And In Vivo

Background and aimsLiver fibrosis is characterized by the excessive deposition of extracellular matrix(ECM) components, mainly fibrillar collagen, that occurs in most types of chronic liver diseases. This pathological status, is a dynamic process usually preceded by liver injuries such as chronic inflammation, viral hepatitis, alcohol abuse, metabolic liver diseases and others. If the injuries persist, the fibrillar collagen accumulated, resulting in fibrosis and ultimately impairment of the liver function. Advanced liver fibrosis results in cirrhosis, hepatocellular carcinoma (HCC), liver failure, portal hypertension. Although a great progress has been made on the mechanisms underlying liver fibrosis, few opinions available for treatment for it.The activated Hepatic stellate cells (HSCs) are the major contributor to the production of fibrillar collagen in the injured liver In the normal liver, HSCs are quiescent, residing in the space of Disse, and function as the main storage of vitamin A. Following chronic injury, HSCs undergo a dramatic phenotypical transformation into α-SMA positive myofibroblast-like cells, and increased the expression of fibrillar collagen and MMPs such as MMP2 and MMP9, as well as TIMPs. Consequently the fibrillar collagen, especially collagen Ⅰ and collagenⅢ, are accumulated. Increasing evidence indicates that TGF-β1 has a key mediator in this pathogenesis by activating its downstream Smad signaling pathway. According to differences in structure and function, Smads were classified into three groups. Smad2/3 is named receptor-activated Smads(R-Smads), and Smad4 is categorized as a common Smad(Co-Smad). Smads 6,7,and 8 are an inhibitory Smad(I-Smad). When TGF-β1 binds to its receptor, Smad2/3 is phosphorylated and binds with Smad4, followed by translocation into the nucleus where these complexes activate transcription of profibrotic genes. Therefore, inhibition of the accumulation of activated HSCs by modulating either their activation and/or proliferation or promoting their apoptosis through the TGF-β/Smad signaling pathway is a potential target for therapy.Large amounts of natural compounds have been shown to have hepatoprotective effects. Vitex rotundifolia L. is a traditional herbal medicine in oriental countries. Its ripe fruits are called Fructus Viticis and has been used as folk medicine for headaches, colds, migraine and eye pain. Casticin (5,3’-dihydroxy-3,6,7,4’-tetramethoxyflavone) is one of the major flavonoids in Fructus Viticis extracts. It has been shown to have several biological activities, but most studies focus on its anti-tumor effects in different types of cancer. More recently, anti-inflammatory effect in vitro and in vivo by casticin has been reported. Casticin has been shown to ameliorate the cigarette smoke-induced acute lung inflammatory and reduce croton oil-induced ear dermatitis and edema in mice. Noteworthy, the latest research shows that casticin can induce HSC apoptosis in a dose-and-time-dependent manner in vitro. However, its biologic activity related to fibrosis in vivo has not yet been examined.This study aimed to identify the roles and potential mechanisms of casticin in liver fibrosis in mice and tried to provide a theoretical basis for its clinical application as a new therapeutic drug to treat hepatic fibrosis.Methods1. The C57 male mice were divided into 4 groups:Control group, Casticin group, CCl4 group and CCl4+Casticin group. To induce liver fibrosis, mice was given intraperitoneal (i.p) injections of CCl4 biweekly for 6 weeks. Then casticin was intragastric administrated daily to mice at the dose of 20mg/kg for 2 weeks.2. Blood biological assessment, histological and morphological analysis (HE, Sirius Red), and hydroxyproline detection to assess the effect of casticin in liver fibrosis in mice.3.Western blot, IHC and RT-PCR to examine the effects of casticin on activated HSC and TGF-β/Smad signaling pathway.4. CCK8 assay and Western Blot analysis to determine the effect of casticin on the proliferation and apoptosis of LX2 cells.5. To stimulate LX2 cells with TGF-β1, then test the inhibitory effects of casticin on the fibrotic effects on ECM expression by RT-PCR. Meanwhile, to assess the protein levels of p-Smad 2/3 by western blot analysis to investigate the potential mechanisms.Results1、 Casticin prevents CCl4-induced liver functional and histological damage.To investigate the effect of casticin in vivo, mice were given repeated injections of CCl4 for 6 weeks, and subsequently intragastric administrated casticin (20 mg/kg) everyday for 2 weeks after the final CCl4 treatment. Liver specimens were obtained 24 h after the last administration of casticin. Then morphological changes of liver injury and fibrosis were visualized in sections stained by H&E. As expected, serum aminotransferases of ALT and AST were also extremely evidently elevated in disease control when compared to normal control. Moreover, the thick fibrotic septa and pseudolobular formation was more significant in the mice exposed to CCl4 compared to normal control ones. The grades of fibrosis changes were identified between the groups. In contrast, treatment with casticin led to extenuation of both histological and functional injury. Normal mice treated with casticin displayed normal histological and serological changes similar to the normal control mice. These observations clearly demonstrated that casticin possessed an additional hepatoprotective effect.2、Casticin attenuates CCl4-induced liver fibrosis in vivo.Subsequently, the effect of casticin on regression of CCl4-induced hepatic fibrosis was observed in liver sections stained by Sirius red. Morphometrical analysis of Sinus red staining demonstrated that compared to normal control mice, CC14-treatment caused a remarkable collagen accumulation in the liver. In contrast, treatment with casticin markedly decreased hepatic collagen matrix accumulation. Further, the administration of casticin facilitated the decreases in hepatic hydroxyproline contents. Collectively, these findings indicated that casticin attenuates CCl4-induced hepatic fibrosis in vivo.3、Casticin inhibits HSC activation and collagen matrix expression by blocking TGF-β/Smad signaling in vivo.Next, we verificated antifibrotic potential mechanisms of casticin in vivo. Western blot analyses for a-SMA, and vimentin were performed. a-SMA, and vimentin protein levels were highly decreased in mice with administration of casticin compared to disease controls. In addition, immunohistochemical staining for a-SMA was performed. As expected, scarcely a-SMA-positive cells were detected in the normal control group. In these sections, however, a-SMA-positive cells were obviously detectable in disease controls. In sharp contrast, a-SMA-positive cells were markedly decreased in liver sections of mice treated with casticin. Thus clearly indicating that casticin inhibits HSCs proliferation and activation in vivo. Then we detected inhibitory function of casticin on the fibrotic effects of TGF-β1 on ECM expression in mice. Real-time PCR showed that casticin drastically suppressed the expressions of TGF-β1, and other fibrotic genes including MMP-2, MMP-9, TIMP-1 and TIMP-2. Furthermore, western blot analysis for p-Smad2 and p-Smad3 was performed to investigate the effects of casticin on TGF-β/Smad Signaling pathway. Casticin significantly decreased the phosphorylation of Smad 2/3, confirming that casticin ameliorates experimental hepatic fibrosis by inhibiting HSC activation and collagen matrix expression through TGF-β/Smad Signaling.4、Casticin inhibits proliferation and induces apoptosis in LX2 cells.To further determine the inhibitory role and potential antifibrogenic mechanisms of casticin on HSCs in vitro, we examined its effect on the LX2 cells. We first tested whether casticin affect the viability of LX2 cells. Casticin decreased the number of cells in 48 h in a concentration dependent manner. Casticin have been shown to promote apoptosis in a variety of cancer cells. We next examined whether casticin potentiate LX2 cells apoptosis in vitro. Western blot analyses for cleaved PARP were performed. Cleaved PARP was barely detectable in untreated LX2 Cells, while specific bands for full length PARP were clearly detected. In contrast, cleaved PARP was obviously detected in LX2 cells treated with casticin. These findings strongly indicated that casticin inhibits proliferation and induces apoptosis in a time and concentration dependent manner.5、Casticin inhibits the activation and collagen matrix expression by blocking TGF-β/Smad Signaling in LX2 Cells.We next examined whether casticin could suppress HSC activation. LX-2 cells were stimulated by TGF-β1 and meanwhile treated with casticin for 12 h after serum starvation. As expected, TGF-β1 increased LX-2 activation, as indicated by enhanced a-SMA levels compare to control, but this activation was suppressed in the presence of casticin. The expression of the a-SMA protein was assessed by immunofluorescence and the mRNA expression levels of a-SMA, MMP-2, MMP-9, TIMP-1, TIMP-2 and TGF-β1 was examined by real time-PCR. Indeed, casticin suppressed TGF-β1-mediated HSC activation in a concentration-dependent manner in LX2 cells.Previous studies have shown that TGF-β is a key mediator in human fibrogenesis. In HSCs, TGF-P favors the transition to myofibroblast-like cells, stimulates the synthesis of ECM proteins, and inhibits their degradation. The TGF-β1-mediated signaling pathway depends on the phosphorylation of Smad 2/3, which is reported as a potential target for antifibrotic therapy. To understand the molecular mechanisms responsible for the inhibition of HSCs activation, the protein levels of Smad 2/3 (intracellular mediators of TGF-β1 signal transduction) were assessed. Western blot analysis showed the significant increases in the phosphorylation of Smad 2/3 stimulated by TGF-β1, and the inhibition of these increases by casticin in LX-2 cells. These results indicated that casticin inhibited TGF-β1-induced HSC activation and collagen matrix expression.Conclusion1、Casticin prevents CCl4-induced liver functional and histological damage, and attenuates CCl4-induced liver fibrosis in vivo. It is likely that casticin inhibits TGF-β1 signaling pathways, leading to reduction of downstream phosphorylation of Smad 2/3, thereby causing inhibition of activation of HSCs in vivo.2、Casticin inhibits proliferation and induces apoptosis in LX2 cells. It inhibits the activation and collagen matrix expression by blocking TGF-β/Smad Signaling in LX2 Cells.3、Casticin is promising for a new treatment strategy of liver fibrosis, since this chemical most likely serves combinatory effects of hepatocyte-protection, anti-fibrogenesis, and chemo-prevention.