The Effect Of Gan-fu-kang On Apoptosis Related Proteins In Liver Tissues Of Hepatic Fibrosis Rats And Hepatic Stellate Cell

Research Objectives：proliferation and apoptosis of hepatic stellate cells plays akey role, inhibiting the activation of hepatic stellate cell proliferation and induceapoptosis of hepatic stellate cells, is one of the important measures for treatment of liverfibrosis. This study observed from two aspects of the overall and in vitro liver fibrosisin liver tissue and the change of hepatic stellate cell apoptosis related proteins, and theGan-fu-kang within the study of the impact, so as to provide experimental basis oftraditional Chinese medicine treatment of liver fibrosis.Materials and method:In vivo：SD rats were randomly divided into normal control group, model group,Chinese medicine treatment group (high, medium and low dose group). To the modelgroup and treatment group with olive oil dilution to10%of the Chinese traditionalmedicine preparation of subcutaneous injection of carbon tetrachloride to liver fibrosismodels,5ml/kg,2times/week,90days. In normal control group, the injection materialby carbon tetrachloride into saline solution, residual method with the former. Buildingin60days with different dose of Chinese medicine liver convalescent lavage on the ratmedication. Dose used for high, medium and low respectively, namely3125,312.5,31.25mg/kg/day, treatment time for60days. All rats were feed standard, free drinkingwater activities. All rats in last for24hours after death, quickly take the left hepatic lobeof rats, on the molecular biological and histological examination.In vitro：the HSC T6cell lines at37°C,5%CO2incubator of mixed gas, withpenicillin (100u/ml), fetal bovine serum (10%) and streptomycin (100u/ml) of culturedDEMN medium and batches. When cells after reaching good, all the cells cultured inserum-free DEMN medium for24h, and then were randomly divided into three groups,the first set of the normal control group, with10%of normal rat serum cultured DEMN medium; A second group of liver convalescent treatment group, with containing10%dose group in liver convalescent treatment rats serum and acetaldehyde200umol/LDEMN culture medium; The third group is acetaldehyde, with containing10%normalrat serum and acetaldehyde200umol/L DEMN medium culture. Will the above threegroups of cells in the incubator for training at the same time, the culture conditions for37°C and5%CO2gas mixture, incubation time for48h.This experiment by HE dyeing method to observe the morphology of liver tissuestructure change; Using immunohistochemical method to observe each group of alphaSMA expression in liver tissue; By reverse transcription-polymerase chain reactionmethod (reverse transcriptase polymerase chain reaction, RT-PCR) assay liver andhepatic stellate cells (caspase-3, caspase-9, Cyt-C, ɑ-SMA，type Ⅰ collagen and typeⅢ collagen gene expression levels.The result:(1) experimental animals in general observation: Normal control group rats growthin good shape, active and lively, different levels of weight gain; Model group rats afterbuilding spiritual malaise, curled up, fur often dull, irritability, and feeding waterreduction, slow growth or even different levels losing weight; Spirit of traditionalChinese medicine treatment group rats, activity, growth and development, etc, is worsethan the normal group, but are better than that of model group. Will kill rats after visible:normal control group rats liver texture is soft, the surface is ruddy; More than modelgroup rats liver surface rough, nodules, color dark hair hard, part of the abdominalcavity with ascites, liver itself; Liver convalescent treatment group rats liver comparedwith model group, the quality of a material is relatively soft, the surface is relativelysmooth.(2) the liver HE staining observation: Normal control group rats hepatic lobularstructure complete, texture clear, radiate out in hepatic cord and no inflammatory cellinfiltration; Structure of hepatic lobules in model group rats liver disorders, liver cellswelling, fatty degeneration, a large amount of fibrous tissue hyperplasia, fiber intervalhas a large number of monocyte, lymphocyte and acidophilic granular cells andfibroblasts, part of the fibrous tissue surrounding the segmentation of liver cells formfalse lobule; Liver convalescent liver compared with model group, the treatment grouprats hepatic lobule structure improvement, fatty degeneration of liver tissue loss, fiberintervals and with dose in the treatment group rats improved obviously, low dose groupand high dose group liver organizational structure also improved. Each score were significantly lower than the model group, three in the treatment group with lower dosegroup was most pronounceD.(3) immunohistochemical detection of ɑ-SMA expressed in liver tissue:Normal control group of ɑ-SMA expression only in arterioles and venules, noexpression in bile duct; Model group ɑ-SMA is long fusiform or elliptic, mainlyexpress in the portal area and fiber intervals. Results found: ɑ-SMAimmunohistochemical staining area is expressed in model group increased significantly,and expressed in Chinese medicine treatment group was significantly lower (comparedwith model group), of which the dose treatment group down more obvious.(4) the RT-PCR detection of apoptosis related proteins in rat liver tissue mRNAexpression:Compared with normal control group, model group of Cyt-c expression wassignificantly lowered (P <0.01); And compared with model group, the expression ofliver convalescent treatment of Cyt-c significantly raised, with the dose increaseobviously (P <0.01).Compared with normal control group, model group of apoptosis proteins caspase-3,and the expression of caspase-9significantly lowered (P <0.01); And compared withmodel group, the liver caspase-3in the convalescent treatment group, the expression ofcaspase-9rise significantly, the dose in the most significant (P <0.01).In order to further confirmed that Chinese medicine liver convalescent resistinghepatic fibrosis, this experiment also tested the ɑ-SMA, Collagen type Ⅰ (ColleganⅠ), Collagen type Ⅲ (Collagen Ⅲ) mRNA expression. Compared with normalcontrol group, model group of ɑ-SMA, Collegan Ⅰ, Collagen Ⅲ expressionsignificantly raised (P <0.01); And compared with model group, the liver convalescenttreatment group for each index expression were significantly lowered, the dose in themost significant (P <0.01).(5) within the liver of acetaldehyde to stimulate apoptosis related proteins in rathepatic stellate cell lines.To find a more ideal way to stimulate the HSC T6cells, respectively in thepreliminary experiments with CCl4and acetaldehyde of HSC stimulated cells. Foundthat acetaldehyde (200umol/L) group of hepatic stellate cells (HSC) in the ɑ-SMA,Collagen type Ⅰ (Collegan Ⅰ) and Collagen type Ⅲ (Collagen Ⅲ) indicators suchexpressions were significantly increased (P <0.01); Acetaldehyde stimulation group cytc, caspase-3, the expression of caspase-9compared with the normal group significantly lowered.And close to liver convalescent treatment group and normal group, ɑ-SMA, Ⅰexpression of type collagen and collagen Ⅲ index compared with the aldehyde groupdecreased significantly (P <0.01); Liver convalescent treatment group of cyt-c,caspase-3, the expression of caspase-9significantly raised compared with aldehydegroup.The results of the cell experiment basic consistent with the results of rat liver tissue(6) Cyt-c and caspase-3, caspase-9, Collagen I, Collagen Ⅲ and correlationanalysis between ɑ-SMACorrelation analysis results show that the cyt c and caspase-3, the expression ofcaspase-9were positively correlated, and Collagen I, Collagen Ⅲ, ɑ-SMA expressionshowed a negative correlation.Correlation analysis Correlation analysis results show that the cyt c and caspase-3,the expression of caspase-9were positively correlated, and Collagen I, Collagen Ⅲand alpha between SMA between negatively correlated (p <0.01).Conclusion:1. Liver fibrosis in rat hepatic stellate cells and liver tissue caspase3, caspase-9apoptosis related proteins expression changes may happen in liver fibrosis developmentplay an important role in the process.2. Hepatic stellate cells induced the expression of apoptosis related proteins may isone of the mechanisms of liver convalescent treatment of hepatic fibrosis, and its role indose in the treatment effect is best.