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INTRODUCTION Cirrhosis is a serious disease threat to human health, liver fibrosis is a variety of liver diseases (including viral, alcoholic, chemical, etc.) the only way to develop into cirrhosis. Development of hepatic fibrosis is the extracellular matrix (ECM) synthesis, decreased degradation results. Hepatic stellate cells (HSC) activation of hepatic fibrosis is the central link, HSC is a major cell secreting collagen synthesis. In the process of ECM degradation enzymes that play a major role in matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) and urokinase plasminogen activator system (uPA and PAI-1). HSC activation regulated by a variety of cytokines such as tumor necrosis factor (TNF-α), transforming growth factor β 1 (TGF-β 1 ), platelet-derived growth factor (PDGF), etc., which is the strongest PDGF mitogenic original, TGF-β 1 is the strongest factor promoting liver fibrosis. Now generally accepted that in alcoholic liver fibrosis and cirrhosis formation process, HSC play an important role, HSC success of liver fibrosis in vitro study provides an ideal cell model. Chinese herbal compound of multi-channel, multi-level, multi-target integrated pharmacological effects in the treatment of liver fibrosis shows unique advantages. This study was a Chinese anti-fibrosis Compound No. Ⅰ (KXI) acetaldehyde treated cultured rat HSC proliferation, collagen synthesis, MMPs, TIMPs and PAI-1mRNA expression and TGF-β 1 , PDGF secretion in order to elaborate KXI anti-alcohol-induced liver fibrosis mechanisms for clinical alcoholic liver disease (ALD) of traditional Chinese medicine to provide more theoretical basis. Materials and methods, the main materials and equipment (a) animals Wistar male rats. Type Ⅳ collagenase, density gradient centrifugation medium Nycodenz, NP-40 were purchased from Sigma, pronase for the German E. Merck tetramethylazodicarboxamide Mile Blue (M'IT) for the company's products Fluka, 40% acetic acid ( analytical grade) was purchased from China Medicine Group Shanghai Chemical Reagent Company, DMEM culture medium for the U.S. GeO Company. Rabbit anti-human desmin (Deslnin) antibody, SP kit was purchased from Fujian Mai new company, PClll, LN, HA radioimmunoassay kit for the Shanghai Institute of Naval Medicine products; RT-PCR kit hkaa products, primer by Beijing Bioko synthesis. Double-antibody sandwich ABC-EllSA kit was purchased from Hysen-Technology Industrial Co., Ltd.. (Two) Preparation of drug-drug serum: anti-fibrosis Compound Mutual No. (mainly by Salvia, Astragalus, safflower. Tetrandrine, arrowroot, peach, licorice and other drug composition) concentrated oral formulation and saline, rats who served . Under sterile conditions from the inferior vena cava blood, centrifuged serum. When used with serum-free DMEM medium was diluted into 10% of the drug and normal serum incubation medium. (Three) microplate reader E-Uza Mat-300: American-JC-12ho radioimmunoassay Y Shun instrument: made in China; w'n-100 PCR amplification: U.S. production; ID Kodak imaging analysis system: U.S. production; steady Pressure electrophoresis POWER/NPACJ00: BIO-RAD products. Second, the method (a) hepatic stellate cell isolation, culture and identification methods such reports with reference to Song Shaogang isolated hepatic stellate cells. Using pronase and type IV collagenase in situ perfused rat liver digest, after Billancourt he d by E density gradient centrifugation, lessons at the interface cells were suspended in 20% fetal calf serum in DMEM to IX10VInl were seeded in plastic culture M, the set CO. Incubator. This experiment uses pass 2-4 generations HSC. HSC Identification Methods: cells seeded Desndn with rabbit anti-human antibody, SP method for immunohistochemical staining, positive staining cytoplasm by HSC. (B) for determination of cell proliferation assay using the Mrt, passaged cells were trypsinized monolayer covered Rear 20% calf serum in DMEM culture medium to adjust the cell number is IX10 * L, added to each well 100gi one 96-well culture plates, and placing CO. After the pre-incubator for 72h, divided into normal control group, serum, normal serum ten aldehyde group and Drug serum ten acetaldehyde groups. Each hole 100J10% medicated serum incubation medium, plus people acetaldehyde make final concentrations of 100w on 00uM300uM. Control serum ten acetic acid group in the same way to add normal serum and acetaldehyde. Each concentration set four wells, the role of 24b, the addition of W solution 20 leaves, OD values ??measured with a microplate reader. (Three) were measured by radioimmunoassay nlll, LN, HA content with IX10 * bottles were seeded in culture flasks (25Cm2) covered with a monolayer of HSC, divided into positive · 2 · Chang serum control group, aldehyde group of ten normal serum acetaldehyde and Drug serum ten groups. Aldehyde groups added to a final concentration of acetaldehyde after Futami Gang, serum concentration of 10%, culture supernatants were collected after 24h, double-complex tube for each sample, in strict accordance with the instructions. JC-1200 RIA Y counting instrument measured directly PClll, LN, HA content, taking the mean. (Four) ELISA assay TGF *; and PDGF content by Method (three) processing and collection of HSC culture supernatant replicates of each sample were made in strict accordance with the instructions. Microplate detection OD value. (Five) RT PCR detection of an al (I), al (IV) collagen, MMP a 1,2,9, TIMP 1,2 and PAI expression by human methods (three) processing HSC 6h and 24h, other points extraction RNA Total RNA concentration and purity were determined. 2 pgRNA by BcaBEST Polylnerase reverse transcribed into cDNA. Primers were designed according to the literature, p-achn as an internal reference, PCR amplification conditions and different products (see the paper of people PCR products were 2% agarose gel electrophoresis, radiography, density scanning, and then the amount of the expression corresponding p-achn expression were compared. (six) statistical data of each group as mean standard deviation on k. S) said twenty-two were compared by t test. Experimental results (a) the identification of cultured rat HSCS immunocytochemistry showed passaged * SC purity> 98%. (See Figure 1-1) (two) acetaldehyde and serum on proliferation of HSC normal serum concentration of 10%, the concentration of acetaldehyde in 00pMJ00pM 100pM when there are right * SC proliferation Khan |