Attenuation Effect Of Matrine Derivative-19 On Experimental Hepatic Fibrosis Of Rat And The Mechanism

【Background and Objective】Hepatic fibrosis is a compensation response of the liver to different chronic factors ,such as inflammation,insults,drug and heredity . It is characterized by the increasing fibroblast or myofibroblasts and the excess production and deposition of extracellular matrix (ECM) components, leading to tissue scarring and the destruction of normal hepatic parenchyma.Hepatic fibrosis is recognized as a dynamic and reversal process. Therefore, blocking, inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease.It has been widely accepted that hepatic stellate cells (HSCs) is the predominant source of myofibroblasts. Nevertheless, recent studies indicate that myofibroblasts can be generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells. In particular, myofibroblasts can be supplemented from cholangiocytes and hepatocytes by epithelial-mesenchymal transition (EMT) during hepatic fibrosis, indicating that the parenchymal epithelial cells of the liver have a significant role in the perpetuation of hepatic fibrosis.Matrine alkaloids ,isolated from Sophora flavescens Ait.(Kushen),have been found to possess a variety of pharmacological effects,including immunity-regulation activity,anti-inflammation ,protective effect on acute liver injury of mouse and experimental hepatic fibrosis of rat .However the activity of matrine is very low ,moreover the target protein of matrine is unknown .So it is very interesting for us to prepare of high potency and low toxicity matrine derivates ,and to investigate the target of matrine .Our preverious studies suggested that the pharmacological effect was largely increased ,when the oxy atom in the carbony group was substituted by sulfur atom .So we prepared 40 matrine derivatives under this guide . In this study , we firstly compared the toxicity and anti-inflammation effect of these derivatives , then the cure effect of matrine and matrine derivative-19 on experimental hepatic fibrosis of rat were investigated. In order to explore the mechanism of matrine's anti-fibrosis effect ,the relationship between EMT and the specific binding protein (Ribosom protein S5, RPS5) of matrine and matrine derivative-19 have been studied.【Methods】1. Comparison of inhibitory effects of matrine and matrine derivatives on TNF-αproduction and NF-кB transcriptional activity in LPS-stimulated macrophages RAW264.7The cytotoxicity of matrine and derivatives to RAW264.7 were detected by MTT based assay. After incubation with drugs for 6h ,the culture supernatants of LPS-stimulated RAW were collected,and the concentrations of TNF-αwere measured by enzyme-linked immunosorbent assay (ELISA) or assessed as the cytotoxicity against L929 cells. The NF-кB transcriptional activity in RAW264.7 were evalulated using Dual-Luciferase reporter gene system .2.The effect of Matrine derivative-19 on experimental hepatic fibrosisTwo distinct models of hepatic fibrosis were induced either by BDL or injection of DMN. For BDL model , Rats were randomly assigned to 7 groups , the number of each group was 10 ,except for the control with sham surgery was 6 . Common bile ducts of models were dissected bluntly and ligated. After ligated for 3 days , Matrine(50mg/kg),Oxymat(50mg/kg) or M-19(12.5 ,25 ,50 mg/kg)in saline was administered i.g. once daily for 3 weeks . Control and model group were administered i.g. same amount of saline . All animals were sacrificed at 25 d .For DMN model , rats were randomly assigned to 7 groups , the number of each group was 10 ,except for the control was 7 . Rats in normal control were received intraperitoneal saline injection. The remaining rats were injected intraperitoneally with 1% DMN (100μl/100g) for 3 consecutive days per week up to 5 weeks to reproduce hepatic fibrosis model . At the beginning of 5th week, Matrine(50mg/kg),Oxymat(50mg/kg) or M-19(12.5 ,25 ,50 mg/kg)in saline was administered i.g. once daily . Control and model group were administered i.g. same amount of saline . All animals were sacrificed 3 weeks after administration.The liver tissue were embedded by paraffin , then stained with H&E, Masson's trichrome, and Sirius red staining. For the semiquantitative analysis, connective tissues stained blue with Sirius red staining were measured on an image analyzer by a technician blinded to the samples. Five fields were selected randomly from each of two sections, and six rats from each group were examined. The percentage was calculated by an image analyzer. Immunohistochemistry was performed on paraffin-embedded liver sections according to manufacturer's recommendation. mRNA expression of EMT related genes was detected by real time RT-PCR.3.Mechanisms for anti-fibrosis of M-19 invitroPrimary hepatocytes or hepatic stellate cells (HSC)derived from male SD rats were cultured for 2 days, then the culture medium was replaced by serum-free medium containing 2 ng/ml TGFβ1. Simulately, M-19 (5,10 ,20μM) for hepatocytes and M-19 (2.5,5 ,10μM)for HSC were added. Total RNA in different groups were extracted and the expression of EMT related genes in the cells was detected by real time RT-PCR respectively after 48h . MTT based assay were used to detect the inhibition effect of M-19 on the proliferation of HSC-T6 cells. To investigate whether M-19 could induced HSC-T6 cells apoptosis , the flow cytometry(FCM) techniques were used.4. Identify specific binding protein of matrine and M-19The strategies of Serial affinity and competition affinity chromatography were used to isolate the specific binding protein of matrine from cell lysate of RAW264.7 and Jurkat by affinity resins bearing matrine . After identified by MALDI-TOF-TOF MS ,the specific binding protein was confirmed by Western blot technique . Biotin-Avidin System was used to isolate the specific binding protein of M-19 from cell lysate of HSC-T6 by M-19 covalented binding to biotin. M-19-biotin 10μM was added to HSC-T6 for 6h , then RPS5 and M-19-biotin were detected by using immunocytochemistry examination combined with laser scanning confocal microscope (LSCM) .5.The relationship between RPS5 and EMTAs previously described , Immunohistochemistry and Real-time PCR were performed to detect protein and mRNA levels of RPS5 in fibrotic liver . Primary hepatic stellate cell (HSC)derived from male SD rats were cultured for 7 days , cells were collected at 1d, 3d, 6d, 7d respectively, the mRNA expression level of RPS5 were measured by Real-time PCR at the indicted time point .The variation of RPS5 expression in TGF-β1 stimulated HSC-T6 cells was assessed by Western blotting . HSC-T6 was transfected with siRNA of RPS5(si 335) using lipofectamin TM2000 , 72h after transfection ,cells were collected ,and the expression of RPS5,vimentin and E-cadherin were detected by Western blotting .【Results】1. Comparison of the inhibitory effects of matrine and matrine derivatives on TNF-αproduction and NF-кB transcriptional activity in LPS-stimulated macrophages RAW264.7According to our result ,M-19 and M-24 are more potency and lower cytotoxity than matrine and other matrine derivatives .At the concentration of 30μM , there is no cytotoxity on RAW264.7 cells of M-19 and M-24 , and the inhibition ratio of them on the production of TNF-αin LPS-stimulated RAW264.7 cells were 70.0% and 83.6% of LPS alone respectively(P<0.001) .they could also decrease the NF-кB transcriptional activity to 48.2±7.3% (P=0.002)and 52±8.6%(P=0.002) respectively, Compared with LPS treatment along .2. Matrine derivative-19 attenuates experimental hepatic fibrosis(1) M-19 inhibits the development of hepatic fibrosis induced by BDLAs the Masson's trichrome stain showed that aniline blue-stained fibrils were associated with proliferating bile ducts that formed a continuous meshwork of connective tissue infiltrating the hepatic parenchyma with loss of the lobular architecture 3 weeks after BDL, suggesting that hepatic fibrosis was successfully established . The hydroxyproline content in M-19(50mg/kg) group (387.74±59.41μg/g liver tissue) decreased compared with that in model group (564.18±10~4.27μg/g, P =0.0003). Based on the analysis of image scan technique, the area of ECM in M-19(50mg/kg) group reduced by 37% (P<0.001) as compared with model group. The expression of TGF-β1,α-SAM and Desminand were decreased significantly, while the expression of HNF4αwas dramatically increased as compared with model group . The results of Real-time RT PCR demonstrated that the mRNA expression of TGF-β1,α-SAM and CollagenIII in liver of rat in M-19(50mg/kg) treatment group were decreased significantly as compared with model , but meanwhile mRNA expression of HNF4αwas increased .(2) M-19 attenuates hepatic fibrosis induced by DMN in ratsDMN injection induced prominent hepatic fibrosis in rats as shown by Masson's trichrome and Sirius red staining . M-19(50mg/kg) treatment reduced ECM (Masson's staining) by 64.5% (P =0.023) as compared with model group. The hydroxyproline content in M-19(50mg/kg) group (414.97±182.51μg/g liver tissue) also decreased compared with that in model group (720.30±289.33μg/g, P =0.021). The results of immunohistochemical staining showed that , M-19(50mg/kg) treated 3 weeks could block the increase of TGF-β1,α-SAM and Desmin expression , at the mean time up-regulated the expression of HNF-4αand E-cadherin. Real time-PCR indicated that the expression ofα-SMA,Vimentin,Snail,CollagenI mRNA was up-regulated in model(P<0.05), while the expression of E-cadherin, HNF4α, ALB were suppressed (P<0.05). These results demonstrated that DMN induce an EMT state in model , However M-19(50mg/kg) curing could block these changes of EMT .3. Mechanisms for anti-fibrosis of M-19 invitroThe results of Real time-PCR indicated that TGF-β1 2ng/ml could successfully induce an EMT state in primary hepatocytes , primary hepatic stellate cells and HSC-T6 cells ,while M-19 (10-20μM) could efficiently inhibit the expression of mesenchymal phenotype such asα-SMA,Vimentin, CollagenI , CollagenIII mRNA (P<0.05) ,and up regulate the expression of liver-specific genes and epithelial phenotype genes such as E-cadherin , HNF-4α(P<0.05) .Besides that ,M-19 (10-40μM) suppressed the proliferation of HSC-T6 in a concentration-dependent way . As the result of flow cytometry(FCM) techniques showed M-19 could also induce apoptosis of HSC-T6 cells at 40μM .4. Identify specific binding protein of matrine and M-19Serial affinity and competition affinity chromatography were used to isolate the specific binding protein of matrine from cell lysate of RAW264.7 or Jurkat by affinity resins bearing matrine .As the silver-stained polyacrylamide gel showed , there was a specific binding protein at about 23-KDa . The result of MALDI-TOF-TOF MS identification showed that there were at least 3 peptides(such as YLPHSAGR ,LTNSMMMHGR,QAVDVSPLR ) in this 23- KDa protein band ,which are identical to the peptides of ribosomal protein S5(RPS5) of human . RPS5 pulled down by matrine-resin was confirmed by western blot.The resultant proteins isolated by Biotin-Avidin System from HSC-T6 treated with biotin alone, biotin-M19, or biotin-M19 and M19, was detected by western blot using anti-RPS5 antibody.The result revealed that biotin-M19 pulled down much more RPS5 than that of biotin alone and that of biotin-M19 and M19 together. These results indicate that M19 can bind specifically with RPS5 in cells.Immunocytochemistry examination combined with laser scanning confocal microscope (LSCM) showed that M19 was colocalized with RPS5 around the cell nucleaus. Altogether, theses results revealed that RPS5 is a specific bingding protein of matrine and its derivative 19.5.The relationship between RPS5 and EMTTo investigate the expression variation of RPS5 in the fibrotic liver , Immunohistochemistry and Real-time PCR were performed on paraffin-embedded liver sections of both BDL and DMN induced fibrotic rat . Compared with normal group ,the mRNA and protein expression of RPS5 was greatly decreased in the liver of model group .Howevery ,the administration of M-19 50mg/kg for 3 weeks could significantly block the decrease in both model .To investigate the expression variation of RPS5 in the activation process of primary hepatic stellate cell (HSC) derived from male SD rats , the total RNA of primary hepatic stellate cell were collected at 1d, 3d, 6d, 7d respectively,and the mRNA expression level of RPS5 were measured by Real-time PCR at the indicted time point . the curve of RPS5 mRNA expression level showed that there was a rapid and persistent decrease of the level within the first 72h , followed with a constant low level until d7 .TGF-β1 also induced a decrease of RPS5 expression in HSC-T6 cells as the result of western blot indicated.but there seemed to be no time-dependent relationship exist. The expression of RPS5 decreased in the first 3 days(1-3d) ,then in the following three days(3-5) the expression level kept constantly with no decreasing , but decreased again in the last two days.To further investigate the relationship between RPS5 and EMT ,HSC-T6 cells were transfected with siRNA of RPS5 . when the expression of RPS5 have been down-regulated , the epithelial phenotype protein E-cadherin was also down-regulated , but, the mesenchymal phenotype protein Vimentin was up-regulated greatly.【Conclusion】1. M-19 and M-24 have a significant inhibitory effect on TNF-αproduction and NF-кB transcriptional activity in LPS-stimulated macrophages RAW264.7 than matrine and other derivatives of matrine2. M-19 could attenuate hepatic fibrosis induced by BDL or DMN in rats ,The mechanism maybe association with the anti-EMT effect of M-193. M-19 could inhibit the proliferation and induce apoptosis of HSC-T6 cells4. RPS5 is a specific bingding protein of matrine and its derivative 195. RPS5 is very important in hepatic fibrosis, the down-regulation of RPS5 will induce a EMT state in HSC-T6.