The Effect Of Artemisia Argyi Extract Solution On ECM Of Hepatic Fibrosis Rats And HSC

Background:The formation of the central link of liver fibrosis is caused by activation and proliferation of hepatic stellate cells, inhibitting the activation and proliferation of hepatic stellate cells is an important way to treat liver f ibrosis. TGFβ1-Smad sig-nal transduction pathway plays a very important role in the forma-tion and development of liver fibrosis.Objective:To observe the effect of traditional Chinese medicine the Artemisia argyi extract solution treating hepatic fibrosis in experimental rats and pharmacodynamics; To clarify the level of Artemisia argyi extract solution mechanism of anti-fibrosis from molecular biology by studying the Artemisia argyi extract solution drug serum on hepatic stellatecells proliferation and on the TGFβ1-Smad signaling pathway influence.Methods(一) The Artemisia argyi extract solution on Extracelluar matrix of hepatic fibrosis in experimental rats:Rat liver fibrotic models were established by intraperitoneal injection with hetero-serum.60 experimental rats were randomly by divided into normal control group, model control group, Artemisia argyi extracting sol-ution high, moderate and low-dose group. After the liver tissue was stained with hematoxylin and eosin(HE), the collage's proliferation of the liver tissue were observed under the light microscope; the contain of collagenⅠandⅢ,TIMP-1 were observed by immunal hist-ochemistry, the results of which were analysed by image system.(二) The Artemisia argyi extract solution drug serum on hepatic stellate cells:1.Artemisia argyi extract solution drug serum on hepatic stellate cell proliferation and its mechanisms:Subculture of rat hepatic stellate cells (HSC-T6)and Artemisia argyi extract solut-ion drug serum (5%,10%,20%)co-cultured for 24 hours, Cell prolife-ration using MTT assay, cell cycle by flow cytometry, Western-blot, real-time quantitative RT-PCR detection of Cyclin D1 protein and mRNA expression.2. Artemisia argyi extract solution drug serum on hepatic stell-ate cells apoptosis and its mechanism:Using Annexin V/PI double staining with flow cytometry apoptosis detected rate of HSC-T6 and Western-blot, real-time quantitative RT-PCR detected apoptotic protein Bcl-2 protein and mRNA.3. Artemisia argyi extract solution drug serum on hepatic stellate cells TGFβ1-Smad signal transduction pathway effects: Elisa detection of TGFβ1 protein expression,Western-blot and real-time fluorescence quantitative RT-PCR detection of TGFβ1, Smad3, Smad4, Smad7 protein and mRNA.Results:1. In extracelluar matrix aspects:compared to the model group, the degree of hepatic fibrosis in rats in Artemisia argyi extract solution therapeutic groups reduced significantly, the contains of collagenⅠandⅢand TIMP-1 mRNA in the model group of hepatic f ibrosis increased more significantly than that of the normal group (P<0.01), and the contains of collagenⅠandⅢand TIMP-1 mRNA in Artemisia argyi extract solution therapeutic groups decreased significantly than that of the model group (P<0.05, P<0.01).2. In cell proliferation aspects:different concentrations of Artemisia Argyi extract solution drug serum (5%,10%,20%) compared with blank control group and the saline serum group, could significantly inhibit the proliferation of HSC-T6 (P<0.01), its inhibition in a concentration-dependent manner. Flow cytometry analysis showed that Artemisia argyi extract solution drug serum group had a significant increase in G0/G1 phase cells (P<0.05, P<0.01), the S-phase cells decreased signif icantly(P<0,05,P<0.01), G2 phase but had no effect. And the results showed that the cell apoptosis decreased signif icantly(P<0,05, P<0.01). At the same time Artemisia argyi extract solution drug serum could inhibit the cyclin D1 mRNA and protein expression (P<0.01).Moreover, with the increase of serum concentration, the stronger the inhibition,a dose-de pendent manner.3. In apoptosis aspects:the percentage of early apoptotic cells of Artemisia argyi extract solution drug serum group was signifi-cantly higher than blank control group and saline serum group (P<0.01).With the increase of serum concentration, HSC-T6 cell early apoptosis rate was gradually increased in a dose dependent manner. At the same time Artemisia argyi extract solution drug serum group could down regulate antiapoptotic gene Bcl-2 mRNA and protein expression (P<0.05, P<0.01). Moreover, with the increase of serum concentration,the lower of Bcl-2 mRNA and protein expression, in dose dependent manner. 4. In the cell TGFβ1-Smad signal transduction pathway aspects: with the blank control group and saline serum group compared to Artemisia argyi extract solution drug serum could significantly inhibit HSC TGFβ1, Smad3, Smad4 protein and mRNA expression(P<0.05, P<0.01) and increase Smad7 protein and mRNA expression (P<0.05, P<0.01),but with the increased concentration of drug serum on the HSC-T6 TGFβ1, Smad3, Smad4 protein and mRNA expression of inhibition gradually increased,while the expression of Smad7 protein and mRNA gradually increased, showing dose dependent manner.Conclusions:1. The Artemisia argyi extract solution can decrease the expres-sion of collagenⅠandⅢand TIMP-1, and reduce depoistance of the hepatic collagen.2. Artemisia argyi extract solution drug serum can inhibit HSC proliferation and promote apoptosis of HSC.3. Artemisia argyi extract solution drug serum can inhibit HSC TGFβ1, Smad3, Smad4 protein and mRNA expression and increase Smad7 protein and mRNA expression.4. Artemisia argyi extract solution drug serum takes the role of anti-f ibrosis mechanisms may be through inhibiting the prolifer-ation of HSC and promoting HSC apoptosis, regulating Smad-related protein and its mRNA expression, reducing depoistance of the heap-tic collagen.