Chinese Medicine The Xinshu Injection Fluid On The Abdominal Aorta After Balloon Injury Of Endothelial Damage, Intimal Hyperplasia, Smooth Muscle Cell Proliferation Effects Of Experimental Research

Purpose:To observe the endothelial injury,endothelial hyperplasia,and the smooth muscle cell proliferation of rabbits' abdominal aorta after injured by sacculus proprius,under the influence of Xinshu injection,a kind of Chinese herbal medicine,which has effect of supplementing Qi and nourishing Yin;promoting blood circulation to remove meridian obstruction.Also,to investigate the mechanism of Xinshu injection preventing and curing the restenosis after PCI operation.Material and method:The first part:theories studies.In this part the main point is to explain how traditional Chinese medicine recognize the restenosis after PCI operation. the tutor's academic thought;the way of design in this experiment;the choice of medicine that are used to comparation in this experiment;the research of the composition,effect,modern pharmacology of Xinshu injection;and also the relationship between these detective index and the restenosis.The second part:experiment study.It is divided into internal and external experiments.The first experiment,internal test.The effect of Chinese herbal medicine - Xinshu injection on the endothelial injury and endomembrane hyperplasia after injured by the sacculus proprius.The new-zealand big ear white rabbits were divided into 4 groups by random, A the normal group(n=12);B the model group(n=12);C the Chinese herbal medicine -Xinshu injection group(chinese herbal medicine for short,n=12);D the Atorvastatin group(western medicine group for short,n=12).The A group hasn't yet done any surgical operation or uses any medicine,where as B,C,D group were done with some surgical operations to build a mode.Used a sacculus proprius pipe to strip the endothelium of abdominal aorta of the rabbits in the B,C, and D group.The model group were not given medicine treatment before or after the operation.The animals in Chinese medicine group were given Xinshu injection(4ml/kg/d) through the vein of ear edge ever since the day before operations for 15days;the animals in western medicine group were given Atorvastatin(1mg/kg/d) by oral application also for 15days.After 2 weeks of building the mode,these animals were these animals were anesthetized and taken blood from the abdominal aorta.After that detected the serum NO by using the method of nitro-reduction,detected the serum ET by using the method of radio-immunity.And then collected the specimen of abdominal aorta,used the immunity组化method and Western Blot blotting analysis to detect the expression of the protein VEGF,bFGF and TGFβ1,and used the RT-PCR to detect the expression of VEGF,bFGF,TGFβ1mRNA of the abdominal aorta.Besides observe the area of endomembrane,tunica media and the index of endomembrane hyperplasia under the light microscope.Then observe the microstructure of the abdominal aorta under the TEM.The second experiment.External test.The influence of Xinshu injection on the proliferation of primary culture smooth muscle cell.Preparation of drug serum:After being molded for 2 weeks,all the animals were anesthetized.Then blood samples were taken from the abdominal aorta of the animals from control group,the TCM group and the western medicine group. We separated serum from the blood samples and kept it in a -70℃freezer.It should be melted under 37℃and be filtered through a 0.22μm membrane for sterilization before use.Cultivation of SMC:We acquired the first generation of SMC from the abdominal aorta of New Zealand Rabbit by means of tissue-piece inoculation.Identification of SMC:immunohistochemical identification ofα-SM actin.Observation of 24hr drug influence on SMC:First,determine the SMC survival rate by MMT chromatometry,then find the most suitable plasma concentration, finally detect the growing circle of SMC by flow cytometry.Statistical treatment:All statistics from this research are analyzed with SPSS 10.0.We did homogeneity test for variance first on basis of which analysis of variance was done between each group.Results:1 the results of internal test.1.1 The results of serum NO and plasma ET:After the rabbits' abdominal aorta hurted by ball bag.The serum NO descended obviously,while the plasma ET ascended obviously(compared with the normal group p＜0.01);The serum NO ascended in chinese medicine group(compared with normal group p＜0.01),and the serum NO in western medicine group also ascended(comepared with normal group p＜0.05),compared with the western medicine group,Chinese herbal medicine group was advanced.(p＜0.05),and the plasma ET descended in both of the western medicine group and the chinese hebal medicine group(compared with the normal group p＜0.01).1.2 The immunohistochemistry results of VEGF,bFGF,TGFβ1:After injured by sacculus proprius,the expression of VEGF protein in abdominal aorta were higher,but there is no significant variance compared with the normal group(p＞0.05),the expression of abdominal aorta protein bFGF,TGFβ1 ascended obviously(compared with normal group p＜0.01).Both TCM and western medicine can lead to the ascending of the expression of protein VEGF(compared with the normal group and the model group,p＜0.01),and both TCM and western medicine can lead to the descending of the expression of protein bFGF,TGFβ1(compared with model group,p＜0.05).1.3 The Western blot results of VEGF,bFGF,TGFβ1:After the rabbits ' abdominal aorta injured by sacculus proprius,although the expression of protein VEGF was higher,compared with normal group there were no significant variance.(p＞0.05);and the exression of protein bFGF,TGFβ1 ascended obviously(compared with normal group p＜0.01).Both TCM and western medicine can lead to the ascending of the expression of protein VEGF(compared with the normal group and the model group,p＜0.01),and both TCM and western medicine can lead to the descending of the expression of protein bFGF,TGFβ1(compared with model group,p＜0.01). 1.4 The RT-PCR results of VEGF,bFGF,TGFβ1:After the rabbits ' abdominal aorta injured by sacculus proprius,although the expression of VEGFmRNA was higher,compared with normal group there were no significant variance.(p＞0.05);and the expression of bFGFmRNA,TGFβ1mRNA ascended obviously(compared with normal group p＜0.01).Both TCM and western medicine can lead to the ascending of the expression of VEGFmRNA(compared with the normal group and the model group.p＜0.01),and both TCM and western medicine can lead to the descending of the expression of bFGFmRNA,TGFβ1mRNA(compared with model group,p＜0.01).1.5 Results of light microscope:In the control group,the area of vascular endothelial cell(VEC) is unable to measure because its tight attachment to the elastic layer of tunica media.The VEC area of the TCM group and the western medicine group contracts remarkably because of the hyperplasy of VEC caused by sacculus proprius damage.(P＜0.01 compared with the control group) There is no variance of tunica media area between each group.(P＞0.05) Traditional Chinese Medicine is able to reduce the hyperplasy of VEC,(P＜0.01 compared with the control group) so is Western medicine.(P＜0.05 compared with the control group)1.6 Results of electron microscope:TCM can improve the ultramicrostructure of the impaired sacculus proprius to restore the consistency of VEC and the normal form of SMC.2.Out-of-body experiment:2.1 Identification of SMC:Under microscope,the endochylema of SMC reveals to be reddish-brown and in which lies the actin filament parallel to the longitudinal axis of the SMC.2.2 MTT chromatometry:to do flow cytometry with 25%plasma concentration serum.2.3 The detecting results of cell cycle:The percentage of cell number in G₀/G₁ phase is higher in both TCM and western medicine group,compared with normal group p＜0.05;and in TCM group,the percentage of cell number in S and G₂/M phase is lower,compared with normal group p＜0.05;In western medicine group,the percentage of cell number in G₂/M phase is lower,compared with normal group p＜0.05.The percentage of cell number in G₂/M phase is higher in TCM group than in western medicine group,compared with western medicine group p＜0.05.Conclusion:1 After despaired by sacculus proprius,the serum NO of abdominal aorta descends while the plasma ET ascends,and the expression of bFGF,TGF-β1 also ascends,which take part in the restenosis;VEGF does not take part in the restenosis,and it can promote the hemal endothelial reparation to prevent the development of restenosis;Xinshu injection can elevate the level of serum NO,while can degrade the level of plasma ET;Xinshu injection group is better than western medicine group in elevating the level of the serum NO,so that, it is possible that the TCM is better than western medicine in the hemal endothelial reparation;Xinshu injection can elevate the protein VEGF expression,while can degrade the expression of bFGF,TGF-β1.2 Xinshu injection can reduce the area and the index of endomembrane hyperplasia.3 Xinshu injection can improve the super microstructure of vessel wall.4 Xinshu injection can inhibit the SMC proliferation,which probably related to its effect of inhibiting endothelial hyperplasia;Xinshu injection can inhibit VSMC hyperplasia from controlling the reversal point of the cell G₁-S,G₂-M phases.5 Combination of TCM and western medicine that prevent restenosis after PCI operation can take endothelial reparation as a respect.6 Xinshu injection can prevent and cure the restenosis after PCI operation,its effect is possibly related to the these mechanisms that promote hemal endothelial reparation,inhibit endothelial hyperplasia,improve the super microstructure of vessel wall and inhibit the SMC proliferation.