Experimental Study Of Hujin Granules And Emodin On The Anti-hepatic Fibrisis Mechanism

Hepatic fibrosis is regarded as a pathologically repair reaction followed various kinds of chronic liver disease. Fibrosis mainly presents the imbalance of the ECM compound production and degradation, which makes the ECM deposit pervadingly in liver. Hepatic fibrosis is the necessary pathologic phase of chronic liver injury from a variety of cause including virus (HBV, HCV), hemadsorption, alcohol abuse, autoimmune, drug induced, cholestatic and metabolic disease. With the advance of fibrosis, nodule formation and altered hepatic function result to cirrhosis at the end stage, and hepatic cirrhosis can seriously harms patient' s physical and mental health.Recently researches show that the advance of fibrosis is regulated by a compact connectly network consisted of some hepatic parenchyma cell, cytokine and extracellular matrix . Cores among these has been the identification of hepatic stellate cells (HSCs) , and a series of basic researchs exploring mechanism of hepatic fibrosis has made steadly progression about activation , proliferation, apoptosis of HSCs. The achievements have culminated in clear evidence that cirrhosis can be reversible, and in realistic expection that effective antifibrotic therapy will significantly alter the management and prognosis of patients with liver disease.Hepatic stellate cells play a key role in liver fibrogenesis regardless of what the pathogenesis is;therefore it is becoming cardinal therapentic target. Reducing the transformation of quiescent stellate cells to activated myofibroblasts, inhibiting stellate cells proliferation, stimulating stellate cells apoptosis and increasing the degradation of scar matrix are attractive strategy against liver fibrosis.There is a high incidence of virosis hepatitis in China. According to statistical data, about 10 percent people were chronic sufferer infected with HBV or HCV, and 1/4 among them will inevitable suffer from cirrohosis or hepatoma. At present, drugs against HBV and HCV still have no conspoicuous effect. To find the valid way and medicine for curing hepatic fibrosis not only is a urgent need but also have deeply significance and great value to our society.The research works about anti-hepatic fibrosis applying the Chinese medicine is getting extensive reconstruction .Because of its special feature of poly-target, Poly-lint, poly-channel, and its compound effects, the Chinese medicine can aim to the complicated mechanism of hepatic fibrosis accordingly which makes it have traditional and clearly prevalence against the western medicine. The Chinese medicine carry it effect on anti -hepatic fibrosis through the following mechanism: 1. inhibiting the hepatic stellate cell activation and proliferation;2. regulating the metabolism by blocking matrix synthesis and accelerating matrix degradation;3. interfering the effect of cytokines and their receptor on hepatic stellate cells;4. inhibiting the cell cycle progression;5. stimulating the stellate cell apoptosis;6. depressing the hepatic stellate cells contraction and decreasing portal resistance;7. inter -fering the intercellular signaling pathways;8.the compound intervention effect on the stellate cell.Hujin Granules is a experienced prespcription and consisted with six herbs of Rhizoma Polygonicuspidate, Radix Curcumae, Fructrs crataegi, Radix Notoginserg, Rhizoma Alismatis, and Ganoderma. All of them together have the effect of clearing heat and detoxicating, promoting blood to resolve stagnation , replenishing qi to reinforce deficiency. Experimental and clinic researchs have showed that the compound have better effect to prevent and treat hepatic fibrosis .The aim of study is to explore the possible effects and mechanism of HJG on experimental hepatic fibrosis .Method :1. Experiment in vivoThe hepatic fibrosis rat model was induced by dimethylnitrosamine (DMN) .The details of method are as follows :Animals were intraperitoneally injectedwith 0. 5%DMN(2ml/kg) at 2/3 of total dosage for successive three days at first week, and at total dosage at the latter 3 weeks .In addition , thecontrol group' s animals were simultaneously injected saline water at the same administrationg route and dosage .4 weeks later, the experimental rats were randomly divided into 8 groups: normal control group, model control group ,Dahuangzhechongwan(DHZCW), the low dose group of HJG (0. 6g/kg), the mean dose group of HJG (1.2g/kg), the high dose group of HJG (2. 4g/kg), the low dose group of Emodin (20mg/kg), the high dose group of Emodin (40mg/kg ). All the rats were administrated once per day with corresponding drugs and physiological saline for successive 4 weeks. At the end of the experimention, rats in each group were sacrificed. The blood and liver of rats were collected for further determinations. We valued the liver function by measuring the indexes of ALT(alanine transaninase), AST(aspartic acid transaminase ), TP(total protein), ALB(globulin) in serum by biochemical examination, chose HA(hyaluraic acid ), LN(lamin) , PCllKprocollagen III) in serum as the hepatic fibrosis indexes by radioimmunological examination, and measured the indexes of MDA(malondialdehyde), SOD (Superoxidedismutase), Hyp(hydroxyproline), Angll(Angiotensinll) in liver tissue according to the introduction of kit. The liver tissue was fixed by 10%formalin, and then we observed the liver tissue under the light microscope via HE (hermatoxylin-eosin) and VG (van gieson) dyeing method. Furthermore, the expression of ATiR (Angiotensinlltype 1 receptor) and ERKi (Extracellular signal-regulated kinase 1) in liver tissue were detected by immunhistochemical examination.2. Experiment in vitroThe immortalized rat hepatic stellate cell line HSOT6 was commonly cultured after revival, and was allocated into normal control group and treated groups with different concentrations Emodin. The cell growth and proliferation were assessed via MTT assay, and cell proliferative cycle and apoptosis rate were analyzed by flow cytometry, and intracellular Ca2+ concentration of HSC-T6 were detected by Furo-3/Am fluorescence assay and confocal laser microscopy, and the expression of ATiR mRNA and ERK, mRNA were measured by semi-quantitative RT-PCR, and the expression of A^R and ERKi by immunohistochemistry. All of measured data were analyzed by statistic software SPSS11.0 for windows, and numeration data by software epi6. 0.Results:1. After successfully induce the hepatic fibrosis model using DMN , the serum level of liver function , ALT and AST, rise obviously in the model,/group .compared with the normal group , the difference seems significant (P<0. 01). But the content of ALT and AST in serum in the HJG treated groups and the Emodin treated groups resumed in different degree decreased obviously (compared with the model group, P<0. 05, P<0. 01), and appear the dose-dependent effect.The level of TP and ALB in the model group is lower than the normal one (P<0.01), after exposure to HJG and Emodin, the serum level of these clearly increase (compared with the model group, P<0. 05, P<0.01).The level of hepatic fibrosis indexes, HA and LN and PCIII, were higher than the normal group (P<0. 01), after the intervention of HJG and Emodin, the level decrease significantly (compared with the model group, P<0. 05, P<0. 01) in a dose-dependent manner.2 The content of lipid peroxidation index MDA remarkable increase in the model group compared with the normal one (P<0. 01), but the other index SOD on the contrary . After treatment with HJG and Emodin , the level of SOD in liver tissue significantly rise , but MDA fall down(compared with the model group, P<0.05, P<0.01).The Hyp and Ang II level of liver tissue in the model group are higher than the normal one (P<0. 01) . After exposure to HJG and Emodin, these in the treatment groups obviously decrease, and it appear significant difference (compared with the model group P<0. 05, P<0. 01)3. The optical microscope observation results indicate that hepatic steatosis, necrosis, and fibre tissue proliferation were more severe than the normal one, partly extend to the hepatic lobules in model group. Each dose of HJG and Emodin can lessen cell steatosis and necrosis, reduce hyperplasia of fibrolast.4. In the hepatic fibrosis rats indudced by DMN the expression of ATrR become argmented or fortified, and distribute couglobately. After treated with HJG and Emodin , it is slight and distribute sporadicly. The expression of ERKi in model rats' liver tissue .after exposure to HJG and Emodin, it devepe to feeble or depauperate, and its distribution scaney.5. The best density of HSC-T6 is 1X 105/ml. After intervention of different concentration Emodin (the extreme concentration are lOumol/ml, 20umol/ml, 40umol/ml, 60umol/ml, and 80u mol/ml), the inhibition rate of proliferation are 18%, 18%, 25%, 46%, 67% respectively. And the increasing inhibition rateresponse to the increasing concentration.6. After intervention of Emodin , the proliferation index (PI) of HSC-T6 drop out (compared with the normal group , P<0. 05), and appear dose-dependent manner. In cell cycle progression , Emodin can inhibit cell cycle and the cell at Go/Gi phase increase obviously but descend at S phase , and G2/M phase decrease when Emodin concentration at 40~60nmol/ml(compared with the normal group , P<0. 05). The apoptosis rate of HSC-T6 induced by Emodin at different concentration are 4.3 + 0.6%, 6.9±1. 1%, 15.2 + 1.9% respectively, and appear significantly difference compared with the control group(P<0.05, P<0. 01).7. Intracellular Ca2+ concentration of HSC-T6 were significantly increased by incubation with Emodin at different concentration (10u mol/ml, 20umol/ml, 40u mol/ml), the vary ratio of Ca2+ are 102.50%, 125. 10%, 215. 00% respectively, and there is significantly difference compared with the control group(P<0. 01).8. All of the HSC-T6, either in the normal group or in the treatment with Emodin ones, can express strongly ATiR mRNA and ERKi mRNA. After exposure to different concentration Emodin (20u mol/ml, 40u mol/ml, 60 V- mol/ml), the expression level of the ATjR mRNA and ERK! mRNA are obviously downregulate, and there are statistical significance compared with the normal group (P<0. 05, P<0.01).9. Immunohistiochemical results showed that the entire HSC-T6 cell expresses remarkablely ATiR and ERKt in normal group and Emodin treatment one. After Intervention of different concentration Emodin (20 u mol/ml, 40 u mol/ml, 60u mol/ml) the position cell expressed ATiR and ERKi reduce obviously and the strength of its decrease.Cone I us i on:1. HJG and Emodin have better effect of lowering enzyme action, alleviating the damage of hepatic cell, protecting liver function, and at some extent anti-hepatic fibrosis.2. HJG and Emodin have the effect of curing the hepatic fibrosis , which may relate to depress the lipid peroxidation and decrease the level of Ang II in liver tissue . .,___......3. HJG and Emodin can protect the hepatic cells and inhibit th$ proliferati/ -on of collagen fibro.4. HJG and Emodin can depress the expression of ATIR and ERKI in rat J/Lvertissue5. Emodin can inhibit the proliferation onf HSC-T6, reduce the proliferati -on index of cells, depress cell cycle at G0/Gi phase and induce the apoptosis.6. Emodin has clearly increased the intracellular Ca2+ concentration of HSC-T6.7. Emodin can restrain the expression of ATiR and ATiR mRNA, ERKi and ERKi mRNA in HSC-T6.To sum up, HJG and Emodin have good effect on rat hepatic fibrosis induced by DMN, and can protect the hepatic cell, inhibit the proliferation of collagen fibro. Which concerned with their ability of anti-oxidotion .reducing the level of Ang II in liver tissue .restraining the expression of AT\R and ERKi in liver tissue .The results indicated that emodin is one of the effective component of HJG. The mechanism of Emodin is possiblly to inhibit the HSOT6 proliferation , to induce the cell apoptosis , to depress the expression of AT,R and ERK, .