Therapeutic Effect Of Dahuangzhechong On The Experimental Hepatic Fibrosis And It's Mechanism

Hepatic fibrosis,is the common pathological changes of chronic liver disease which caused by a variety of etiological factoers,and the main pathological basis of the development for each chronic liver disease to cirrhosis even liver cancer.Its main eyiological factor is hepatitis B virus and alcohol-based,but in different districts it have a certain difference. China is a region of the high incidence for chronic liver disease region,and the chronic hepatitis B always occupies the first place of high incidence. According to the statistics by WHO,there have 350 million chronic HBV infected persons currently,and about 75%patient is dirtributed to the Asia-Pacific region.China,as the region which has the higher infection rate of HBV,have about 120 milliom infected persons by not mis-classification satistics,and the detection rate of HBsAg in crowd is high as 9.75%HBV infection has reached 120 million people around the crowd of hepatitis B surface antigen(HBsAg) detection rate as high as 9.75 percent.On the opposite,alcohol is the main reason for liver fibrosis in Western European countries,in China as people's living standard.Increased alcohol-induced liver fibrosis also showed an upward trend.According to recent statistics,China has more than 20 million liver disease patients now.Hepatic fibrosis,because of its higher incidence rate,becomes a serious health problem which must to be treat seriously in our country.China's higher incidence rate is the people of our country must deal with.Therefore,finding the effective ways and drugs which used for the prevention and treatment of liver fibrosis,is the urgent requirement of our country,and it has the far-reaching significance and major social value.Hepatic fibrosis is not only a healing repair process after the chronic liver injury,but also the common pathologic basis of the process that is from all the chronic liver disease to cirrhosis.Effective treatment for liver fibrosis can prevent the delopment from all kinds of chronic liver disease to liver cirrhosis.In recent years,the research for the priming and progress mechanism of hepatic fibrosis have been got some advancements,but in the field of clinical treatment of hepatic fibrosis.Although there have a number of Chinese and Western medicines for the clinical treatment of hepatic fibrosis, but the effect of them is also not ideal,some drug side effects are larger and the price of them are more expensive.So the researchers are trying to find a drug,which have the exact effect,low prices and adverse effects,in order to prevent and reverse the process of hepatic fibrosis,even cure most chronic liver disease eventually.In China,most of the hepatic fibrosis and cirrhosis have relation with the development of the chronic viral hepatitis.To aim directly at the pathological features of chronic hepatitis liver fibrosis,this text,based on the previous work,prepare to use the DMN-induced hepatic fibrosis model to clarified the curative effect of JIAWEI DAHUANGZHECHONG PILL by pathological observations and serum biochemical indicators,and adopt immunohistochemistry and cell culture technology,flow cytometry,molecular biology Methods(RealTime-PCR technology)in order to research the mechanism of action for the drug from apoptosis and fibrinolysis.It would provid the experimental basis for the further generalization,exploitation and application.1.There is the observation for the change of the HA,LN,IVC,PCIINP and HyP in the DMN hepatic fibrosis rat model,which caused by JIAWEI DAHUANGZHECHONG PILL.The results showed that:the level of the serum HA,LN,IVC,PCIII HyP,for the model group and the administered group, are increase.There are significant differences when compared with the normal group(P＜0.01).Compared with the model group,the level of the serum HA,PCIII,LN,CIV and HyP for compound water large and small dose group,are decrease obviously(P＜0.05 or 0.01),especially the large dose group.But the descending level of the index for large and small dose group,which drug are extracted by alcohol,is not significant(P＞0.05).The level of the index for the BIEJIARUANGAN TABLET have no significant difference with model group(P＞0.05).2.There is the observation for the effect of the hepatic histomorphology in the DMN hepatic fibrosis rat model,which caused by JIAWEI DAHUANGZHECHONG PILL:(1)HE staining;(2)α-SMA immunohistochemistry;(3)typeⅠimmunohistochemistry of collagen. This study shows that the JIAWEI DAHUANGZHECHONG PILL can cure the experimental hepatic fibrosis by reducing the extent of hepatic fibrosis,improving the liver structure and promoting ECM degradation, which based on objective change of liver structure,for example,the damage of acini hepatis,the fake lobulation and the accrementition of collagen fibers.3.There is the observation for the inhibitory action to the growth of HSC-T6 in vitro by JIAWEI DAHUANGZHECHONG PILL.The compound TC50 is 42.15 mg,the TC50 of the salvia is the dilution of 1:10.(1)TIMP-1, MMP-2,MMP-13mRNA expressed;(2)cells RNA extracted.After the drug action for 24 h,the culture cell is extracted the cell RNA by Trizol. The expressed information,which is acquire from detecting the TIMP-1, MMP-2,MMP-13mRNA of HSC-T6 cells by fluorescence quantitative PCR, indicated that JIAWEI DAHUANGZHECHONG PILL can relive the inflammation in liver and the extent of hepatic fibrosis.According to the experimental data,it is confirmed that JIAWEI DAHUANGZHECHONG PILL can be used to cure the hepatic fibrosis by regulating uPA fibrinolysis, indirect adjusting the degradation of ECM and promoting the deposition of ECM in the liver.4.There is the observation for the expression of Fas / FasL,TNF-αapoptosis gene mRNA and the intervention of JIAWEI DAHUANGZHECHONG PILL. The result shows that:JIAWEI DAHUANGZHECHONG PILL can increase the expression of HSC Fas / FasL gene mRNA significantly in the liver tissue of rat and vitro,and the effect is better than the positive control drug.So,we consider that JIAWEI DAHUANGZHECHONG PILL can reverse the hepatic fibrosis by regulating HSC apoptosis which is induced by the Fas / FasL system.5.There is the Observation for the apoptosis gene mRNA expression of HSC Fas / FasL and TNF-α,and the effect to apoptosis of the HSC-T6 cell by intervention of JIAWEI DAHUANGZHECHONG PILL..Experimental method:analysis by Annexin V / PI double combination with flow cytometry.The results:in the scatterplot Of two-variable flow cytometry,the lower left quadrant shows that living cells, (FITC-/PI-),right upper quadrant of right are un-Living cells,which arenecrosis cells,(FITC+/PI+);and the lower right quadrant are apoptosis cells,showing(FITC + / PI-).The results show that:both the JIAWEI DAHUANGZHECHONG PILL and salvia injection can induce the apoptosis of HSC,especially,the 30 mg / ml dose group of JIAWEI DAHUANGZHECHONG PILL.It indicated that inducing HSC apoptosis may be the mechanism of the anti-fibrosis action by using the JIAWEI DAHUANGZHECHONG PILL.6.There is the observation of the relation between the fibrolysis system and the effect of anti-fibrosis by the JIAWEI DAHUANGZHECHONG PILL. The results:(1)immunohistochemical picture for the liver uPA, PAI-Irats in each group.The positive expressions of the uPA and PAI-1 are not be detected in the normal control group.The uPA of the liver tissue in model group are distributed inside of the fake folials and the hyperplastic fabric diaphragmaliver.PAI-1 are distributed in the district of the cross hepatic ducts,where the liver fibrosis is active,and there have significant positive staining in the place of the necrotic liver cell.JIAWEI DAHUANGZHECHONG PILL can reduce the inflammation of the liver effectively,improve and reduce the degree of liver fibrosis.According to the experimental data,it is confirmed that the JIAWEI DAHUANGZHECHONG PILL can cure the hepatic fibrosis by regulating uPA fibrinolysis,adjusting ECM degradation indirectly and promoting ECM deposition in the liver.(2)the amount of relative expression for HSC uPA,PAI-1,TIMP-1 and MMP-13 mRNA:comparing with the control group,each salvia injection groups,the large and small gose group of JIAWEI DAHUANGZHECHONG PILL can regulate the expression of uPA,PAI-1,TIMP-1 and MMP-13 mRNA,especially the 30 mg / ml dose group of JIAWEI DAHUANGZHECHONG PILL.So,we consider that JIAWEI DAHUANGZHECHONG PILL can reversed the liver fibrosis by regulating uPA fibrinolysis way,adjusting the activity of MMPs indirectly,and promoting ECM degradation,.Using the DMN-induced rat model of liver fibrosis,this research evaluates the therapeutic action of JIAWEI DAHUANGZHECHONG PILL for experimental hepatic fibrosis,by liver defibrillators indicators from the serum,liver HyP and the change of the liver shape.It is confirmed that the JIAWEI DAHUANGZHECHONG PILL can be reversed the by the hepatic fibrosis of rat model which caused by DMN.The exoteric experiment use the HSC-T6 cells as a model to detect the effect of the JIAWEI DAHUANGZHECHONG PILL for the generation of HSC-T6 by MTT,and comfire the effect on restrainng the generation and activation of HSC by JIAWEI DAHUANGZHECHONG PILL.Though the experiment in vivo and vitro,the research for the action of JIAWEI DAHUANGZHECHONG PILL to the induced action and the apoptosis pathway of HSC, indicate that JIAWEI DAHUANGZHECHONG PILL can cure the hepatic fibrosis by HSC which is induced by Fas / FasL system.The experiment in vitro is based on HSC-T6 cells,studied the regulated action of JIAWEI DAHUANGZHECHONG PILL to the uPA,and comfired that JIAWEI DAHUANGZHECHONG PILL can reverse the hepatic fibrosis by increasing the activity of uPA and MMPs,and promoting ECM degradation.