The Study Of Curative Effect And Molecular Of NI XIAN FANG On Experimental Liver Fibrosis

1 ObjectiveHepatic fibrosis is the inevitable mediate stage and a common pathological process ofvaried chronic liver disease to cirrhosis. Now, there isn't any effective method to cure thediseases leading to hepatic fibrosis, so prevent and delay the development of hepaticfibrosis is a very important strategy. In our country, the main three factors leading tofibrosis are virus hepatitis, ethanol, and schistosomiasis. According to the serumepidemiology survey on virus hepatitis in the whole country in 1992, the Positive rate ofHBsAg is 9.75%, approximate 120 million people, in which there are nearly 30 millionhepatitis patients, most of them have developed to hepatic fibrosis. So the hepatic fibrosis isa kind of common disease which is severely impairing the health of Chinese people; aneffective therapy for hepatic fibrosis is very necessary and exigent.There is no effective special medicine for liver fibrosis treatment, but distinctive effecthas been achieved in prevention and treatment of it on Chinese herbs.The compound recipeof NI XIAN FANG(NXF), which has the effect of heat-clearing and detoxicating,strengthening body resistance, promoting blood flow, is made up by 11 kinds Of herbs.It hasbeen confirmed the exact effects of anti-HBV in previous empirical study. We set up theanimal model useing DMN to study the therapic molecular mechanism of NXF on rat liverfibrosis and provide theory evidence for the herbs.2 MethodsThe experiment is made up of three parts:2.1 Study of NXF in vitro and in vivo100 SD rats were brought into the experiment and 94 of them were administratedDMN every three days in a week. The bland groups were injected with sodium chloridesimultaneously. The injection lasted 4 weeks and the body weights of the rats were recordedon the first day of every week. In the end of the forth week, the rats were divided into 7 groups: control group, BIE JIA RUAN GAN PIAN(BJRGP) group, hydro-extracted of NXFhigh dose group, hydro-extracted of NXF low dose group, alcohol-extracted of NXF highdose group, alcohol-extracted of NXF low dose group. Each group had 12 rats except thecontrol group with 14.The intragastric administration dose is 2g/KG of BJRGPgroup,30g/KG of hydro-extracted, alcohol-extracted of NXF high dose group, 15g/KG ofhydro-extracted,alcohol-extracted of NXF low dose group respectively. The body weightsof all groups were recorded and intragastric administration for 21 days. The death recordwas taken notes in detail from the beginning to the end of the experiment.In the end of thestudy, the serum was collected by remove the eyeball after doping the rats with SU MIANXIN. The condition of ascites and the liver was observed and taken the left anterior lobe ofliver, part of the liver was placed into the Bouin fixation fluid to staining with HE, Massonand immunohistochemistry of a-SMA,collagen 1.HSC-T6 was used as the model in vitro. The stock solution of NXF was diluted into 10densities, DanShen, the positive control medicine was diluted into 7 densities respectivelly.The cell toxicity was detected using MTT to evaluate the inhibitory action of NXF on HSC.2.2 The effect of NXF on the expression of HSC Fas/FasL mRNA in vitro and in vivoIn this part, we plan to approach the curative mechanisms of NXF from HSC apoptosis.Annexin V/PI staining combining flow cytometry detect the apoptosis condition ofHSC.The method of Real-time PCR was applied to quantitate the expression of Fas/FasLmRNA of HSC in vivo and vitro.2.3 The effect of NXF on the expression of HSC uPA,PAI-1,TIMP-1,MMP-13 mRNAin vitroThe third experiment studied the expression of uPA and PAI-1 in the rat livers and theregulation of NXF by the method of inmmunohistochemistry. The method of Real-timePCR was applied to quantitate the expression of uPA,PAI-1,TIMP-1 and MMP-13 mRNAof HSC. On the base of the result, we approach the relationship between the uPAplasminogen system and liver fibrosis and the regulation of NXF.3 Results3.1 Study of NXF In vitro and in vivoThe hydro-extracted of NXF high and low dose group can both degrade the level ofserum HA,LN,Câ…£,PCâ…¢and HyP of liver. The high dose group surpass than the lowgroup. The alcohol-extracted of NXF high and low dose group failed to degrade the thelevel of serum HA,LN,Câ…£,PCâ…¢and HyP of liver. The BJRGP group is not as good asthe the hydro-extracted of NXF high and low dose group. The result of HE staining show that the liver of the normal group was integrated. Theliver of the control group lost the normal lobule and the collagen fibers extended into thehepatic lobule, which led to the form of fake lobule. To some degree, all the slices could beobserved zone of necrosis and hemorrhagic focus following inflammatory cell infiltrate inthe center or some part. The louble of the hydro-extracted of NXF high dose group weredegraded not obviously. There were two samples with the deposit of fibrosis around theconverge duct and one sample formed the false lobule. The rest can be observed fiber trabsand had the regularly arranged liver cells. The low dose group can also be observed thedeposition of fibrosis around the converge duct and inflammatory cell infiltrate. All thesample of the alcohol-extracted of NXF high dose group can be observed bulk fiberdeposition around the converge duct and inflammatory cell infiltrated. Three samples hadthe hemorrhagic focus. The low dose had the same pathological changes as the high dose.In vitro, the TC₅₀ of NXF on HSC was 62.15mg/ml and that of DanSHen solution, thepositive control medicine was the dilute strength 1:10. Both NXF and DanSHen solutionshow strong inhibition on HSC-T6 and the inhibition is strengthen with the drug densitywhich illustrating that there is the symbiosis between the dosage and effect. In conclusion,the anti-liver fibrosis effect of NXF is relevant with its inhibition on HSC.3.2 The effect of NXF on the expression of HSC Fas/FasL mRNA in vitro and in vivoThe ratio of cell apoptosis detected by the flow cytometry was as following: 08% of thecontrol group, 75.4% of the hydro-extracted of NXF high dose group, 10.9% of thehydro-extracted of NXF low dose group, 7.6% of DanShen high density, 2.8% of DanShenlow density.The result of Real-time PCR shows that both the hydro-extracted of NXF high dosegroup and low dose can up-regulate the expression of Fas/FasL mRNA, but the high dosegroup has the better ability. The expression of Fas/FasL mRNA of the alcohol-extracted ofNXF is approximate that of the control group.In vitro, both the hydro-extracted of NXF high and low dose group and DanShensolution can regulate the expression of Fas/FasL(P＜0.05或P＜0.01).The hydro-extractedof NXF high dose group regulate the expression of Fas/FasL better than other groups.3.3 The effect of NXF on the expression of HSC uPA,PAI-1,TIMP-1,MMP-13 mRNAin vitroThe result of inmmunohistochemistry show that the expression of uPA in thehydro-extracted of NXF high dose gruop is higher than other groups,PAI-1 can be observedexpressing in four samples. The expression of uPA in the hydro-extracted of NXF low dosegruop is obviously lower than the high group and PAI-1 expresses in all samples. The expression of uPA in BJRGP gruop is obviously lower than that of the low group and PAI-1expresses in all samples. The alcohol-extracted of NXF high and low dose group has thehigh expression of PAI-1, while the expression of uPA is higher in the alcohol-extracted ofNXF low dose group than that of the high dose group.The result of Real-time PCR shows that both NXF and DanShen solution can regulatethe expression of uPA,PAI-1,TIMP-1及MMP-13 mRNA (P＜0.05或P＜0.01). Thehydro-extracted of NXF high dose group regulates the expression of Fas/FasL better thanother groups and shows the symbiosis between the dosage and effect.4 Conclusions4.1 The NXF can degrade the serum indicatrix of liver fibrosis and the content of HyP inrats. The histopathology shows that the NXF can lighten the damage of liver and reverse theproceeding of liver fibrosis. In vitro, the NXF has obvious inhibition on HSC which showsthe the symbiosis between the dosage and effect.4.2 The NXF can induce the apoptosis of HSC by Fas/FasL system, the induce ability hasthe relation with the dose.4.3 The NXF can reverse liver fibrosis by regulating the uPA plasminogen system directlyand the activity of MMPs indirectly.