Experimental Study On The Prophylactic And Therapeutic Effect Of Xiongshao Decoction On HF And Its Mechanism

Hepatic fibrosis (HF) is mainly histopathological conception. It refers to the pathological changes of hyperplasia and abnormal deposition of the component of extracellular matrix in liver tissue, which resulting in liver structure or (and) the function abnormal. The majority of chronic hepatitis can cause HF, and Further development of HF can develop cirrhosis and liver cancer, which have serious impact on human health and life. At present there is no ideal drug on clinical which can cure HF.Therefore if or not the development of HF can be delayed, even be stoped or inversed to normal is of great significance.The formation of HF involves many factors and links, in versing the development of HF is possibly difficult by use medicine,which aim only directly at one link.Studies have shown that the pharmacologic actions of anti-HF chinese herbal compound recipe was comprehensive,and was multi-channels,Multi-targeting.Chinese herbal compound recipe was able to focus on Multiple links of the development of HF.Discovering anti-HF chinese herbal compound recipe whose therapeutic effect is better had been our important mission.Xiongshao decoction is a effective proved recipe of anti-HF,by which my advisor Prof. Men Jiuzhang treat HF.In this theses, on one hand we had an empirical study on the effects of Xiongshao decoction on Immune HF in rats and its mechanism,on the other hand,we had done an empirical study on the effects of Xiongshao decoction drugs blood serum on human hepatic stellate cell(HSC),which were cultivated in vitro.ObjectiveSynthetically evaluate the curative effect of Xiongshao decoction on HF by the symptoms and signs,assaying liver function and serumal marker of HF,observing the change of pathomorphology;Identify the anti-HF mechanism of Xiongshao decoction beginning with pathogenetic capital link of HF.Methods(1)In animal experiment,the HF model were induced by injecting porcine serum to the rats'abdominal cavity. We set up the normal control group,the model group,FZHY capsule group,the Xiongshao decoction preservation group, the Xiongshao decoction high-dose group and low-dose group.We had observed the general state of health of rats, including coat color, body weight and so on; observed the appearance of rats'fresh liver by naked eye; calculated the ratio of liver weight and body weight, the ratio of spleen weight and body weight;By routine methods detected the rats'serumal hepatic function parameter:ALT,AST,ALB, GLB and A/G; Using isotopes radio immunoassay detected the rats' serumal marker of HF:hyaluronic acid(HA),laminin(LN),ProcollagenⅢN-terminal peptide(PCⅢ) and collogen IV(CIV);Using xanthine oxidase detected the vitality of SOD in rats'serum,and using thiobarbituric acid(TBA) detected the content of MDA in rats'serum;Using enzyme linked immunosorbent assay(ELISA) detected the content of MMP-9, IgE, and C3 in rats'serum;By digestive method detected the content of Hyp in rats'hepatic tissue;Using HE and Masson staining observed the alteration of pathomorphology in rats'hepatic tissue;Using immunohistostaining measured the expression of collagenⅠ, collagenⅢ,α-SMA and TGF-β1 in rats'hepatic tissue;Using real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR) detected the expression of TIMP-1mRNA in rats'hepatic tissue;(2) In the cell experiment, we observed the effect of rats'drug serum in which there was Xiongshao decoction on the multiplication of human HSC LX-2 using MTT colorimetry; and measured the expression of collagenⅠ,collagenⅢ,α-SMA and TGF-β1 in LX-2 using immunohistostaining.Resultsl.The HF model in rats was successfully duplicated after injecting pig serum to rats'abdominal cavity for 15 weeks.2.Xiongshao decoction can improve the general status of immunitas HF rat,such as the coat color, the food-intake, the body weight, and so on.3.liver function test results:the content of AST,ALT and GLB in the model group rats'serum are obviously higher than in the normal group rats'serum (P<0.01), and the content of ALB and A/G in the model group rats'serum are obviously lower than in the normal group rats' serum (P<0.01); Besides the content difference of AST between in Xiongshao decoction low-dose group rats'serum and in model group rats'serum was not remarkable, the content of AST, ALT and GLB in every treatment group of Xiongshao decoction rats'serum were all obviously lower than in the model group (P<0.05 or P<0.01), and the content of ALB and A/G were all obviously higher than in the model group (P<0.01), the content of GLB in Xiongshao decoction preservation group rats'serum was obviously lower than in the FZHY group (P<0.05),the ratios of A/G in the Xiongshao decoction prevention group and high-dose group rats'serum was obviously higher than in the FZHY group (P<0.05)4.serum HF marker test results:the content of HA, LN, PCIII and CIV in model group rats'serum were obviously higher than in the normal group (P<0.01),the content of HA, PCIII and LN in every treatment group rats'serum were obviously lower than in the model group (P<0.05 or P<0.01),the content of HA and LN in Xiongshao decoction high-dose group rats'serum were obviously lower than in the prevention group (P<0.05),and the content of LN in Xiongshao decoction high-dose group rats'serum was obviously lower than in FZHY group (P<0.05), the content of CIV in prevention group rats'serum was obviously lower than in the model group (P<0.05), but the content difference of CIV between in other treatment group rats'serum and the model group were not remarkable.5.Liver heavy index and spleen heavy index result:the difference of all experimental groups liver heavy index were not remarkable;The spleen heavy index of the model group was obviously bigger than the normal group (P< 0.01),the Xiongshao decoction prevention group and low-dose group is smaller than the model group(P<0.01),the difference of spleen heavy index of the Xiongshao decoction high-dose group and the FZHY group were not remarkable.6.Hepatic tissue pathology results:observing through the microscope,every treatment group rats'hepatic inflammation and fibration was milder than the model group. Among every treatment group,Xiongshao decoction low-dose group rats'hepatic inflammation and fibration was the most severe.Analyzing through statistics,the model group rats'hepatic inflammation and fibration semiquantitative scoring were remarkably higher than the model group(P<0.01); Besides Xiongshao decoction low-dose group,each treatment group was obviously lower than the model group (P<0.05 or P<0.01), and was obviously higher than the normal group (P<0.05orP<0.01)7.Hyp in the liver organizes test results:the content of Hyp in the model group rats'hepatic tissue was obviously higher than the normal group (P<0.01), each treatment group was obviously lower than the model group (P<0.05 or P<0.01), both Xiongshao decoction prevention group and Xiongshao decoction high-dose group were lower than the FZHY group (P<0.05)8.SOD and MDA in rats'serum test results:the content of SOD in model group rats'serum was obviously lower than in the normal group (P<0.01),the content of MDA was obviously higher than in the normal group (P<0.01), besides the difference of the content of SOD between in FZHY group and in the model group was not remarkable,SOD in each treatment group was obviously higher than in the model group (P<0.05 or P<0.01),the content of MDA in every treatment group were obviously lower than in the model group (P<0.01),the content of SOD in Xiongshao decoction prevention group was obviously higher than in Xiongshao decoction low-dose group (P<0.05),and in Xiongshao decoction low-dose group was higher than in the Xiongshao decoction high-dose group,the content difference of MDA among treatment groups was not remarkable.9.IgE and C3 in rats'serum test results:the content difference of IgE and C3 in rats'serum between the model group and the normal group was not remarkable,the content of IgE in rats'serum of Xiongshao decoction prevention group was obviously higher than other all groups (P<0.05 or P<0.01),the content of C3 in rats'serum of Xiongshao decoction prevention group was obviously lower than the normal group and the model group.10.Immunohistostaining results ofⅠcollagen,Ⅲollagen,α-SMA,TGF-β1 in rats'hepatic tissue:the contents ofⅠcollagen,Ⅲcollagen,α-SMA,TGF-β1 in the model group rats'hepatic tissue were obviously higher than in the normal group (P<0.01),each treatment group was obviously lower than the model group (P<0.01). Among each treatment group,the contents ofⅠcollagen,Ⅲcollagen and a-SMA in the Xiongshao decoction prevention group rats'hepatic tissue were obviously lower than in the FZHY group (P<0.05 or P<0.01),the content ofⅢcollagen in Xiongshao decoction high-dose group was obviously lower than in the FZHY group (P<0.01),the contents ofⅢcollagen andα-SMA in the Xiongshao decoction high-dose group and Xiongshao decoction prevention group were obviously lower than in the Xiongshao decoction low-dose group (P<0.01).the content of TGF-β1 in the Xiongshao decoction low-dose group was obviously lower than in the Xiongshao decoction high-dose group (P<0.01)11.MMP-9 in rats' serum and TIMP-1mRNA in rats' hepatic tissue test results:the content of MMP-9 in the model group rats' serum was obviously lower than in the normal group (P<0.05),the expression of TIMP-1mRNA in the model group rats' hepatic tissue was obviously higher than in the normal group (P<0.01);the content of MMP-9 in the Xiongshao decoction prevention group rats'serum was obviously higher than in the model group(P<0.01),the content difference between other treatment groups and the model group was not remarkable;the expression of TIMP-1mRNA in Xiongshao decoction high-dose group rats' hepatic tissue was obviously lower than in the model group, the content difference between Xiongshao decoction high-dose group and the normal group was not remarkable.12.The inhibition ratio results of drug serum in which there was Xiongshao decoction to LX-2 reduplication:the inhibition ratio of 20% and 10% vacuity serum group was lower than homologue concentration FZHY group and Xiongshao decoction group (P<0.01), the inhibition ratio difference among 5% vacuity serum group,homologue concentration FZHY group and Xiongshao decoction group were not remarkable;the inhibition ratio of each concentration drug serum in which there was Xiongshao decoction was obviously higher than homologue concentration drug serum in which there was FZHY (P<0.05或P<0.01).the inhibition ratio difference between 20% and 10% drug serum in which there was Xiongshao decoction to LX-2 reduplication was not remarkable,but the inhibition ratio of 20% and 10% drug serum in which there was Xiongshao decoction were both higher than 5% drug serum in which there was Xiongshao decoction (P<0.01)13.Immunohistostaining results of the effect of drug serum in which there was Xiongshao decoction on the expression ofⅠcollagen,Ⅲcollagen,a-SMA,TGF-β1 in LX-2:the contents ofⅠcollagen,Ⅲcollagen,a-SMA and TGF-β1 in Xiongshao decoction serum group LX-2 and FZHY serum group LX-2 were obviously lower than in the vacuity serum group (P<0.01),the content difference of I collagen in the Xiongshao decoction serum group LX-2 and FZHY serum group LX-2 was not remarkable,the contents ofⅢcollagen and a-SMA in FZHY serum group LX-2 were obviously lower than in Xiongshao decoction serum group LX-2 (P<0.05),the content of TGF-β1 in Xiongshao decoction serum group LX-2 was obviously lower than in FZHY serum group (P<0.01)Conclusion1. Xiongshao decoction has exact curative effect on immune,the synthesis curative effect was best if Xiongshao decoction were used to prevent HF in prophase,its anti-HF mechanism was not identical with FZHY capsule, and under the controlled condition,its some aspects' anti-HF curative effect surpassed FZHY capsule.2.anti-HF mechanism of Xiongshao decoction was mainly the following several aspects.2.1 the effect of protecting the hepatic cells and improving hepatic function.2.2 both inhibiting the synthese of ECM,such as collagen and so on, and promoting its degradation.2.3 Sublimating the ability of HF rats eliminating oxygen radicals and suppressing lipid peroxidation. 2.4 adjusting immunologic function.2.5 promoting MMP-9 production and suppressing TIMP-1mRNA expression,thus promoting ECM degradation.2.6 suppressing the activating and multiplication of HSC and reducing the productions of cytokines,which could promote fibrosis.