| Purpose:This topic is based on the treatment of "Quyushengxin",and the treatment principle is to “Xingqihuayufuzheng”,and begin an experimental study on ischemic coronary angiogenesis was conducted with Xuanbitongyu formula.The myocardial ischemia model was selected for the method of anterior descending branch of the coronary artery was ligated.To explore the regulation mechanism of the Notch/Dell4 signaling pathway and angiogenesis in myocardial ischemia rats which was fed Xuanbitongyu formula.To clarify the mechanism of Xuanbitongyu formula for the treatment of myocardial ischemia in patients with coronary heart disease,to enrich the research idea of myocardial ischemia in coronary heart disease,meanwhile to provide experimental basis for the clinical application of traditional Chinese medicine in the treatment of angiogenesis.Method:1.Experimental Study on the mechanism of Xuanbi tongyu Decoction on experimental myocardial ischemia in ratshalf male and half female SD rats(weight 220g-250g)were selected,all of the rats were fed for 1 weeks,after that the rat model of myocardial ischemia was prepared.The myocardial ischemia model was prepared by the method of ligation of the anterior descending artery of the coronary artery,and the sham operation group was not ligated(n=16).The standard II lead electrocardiogram was recorded before and after the molding,and ST segment deviation was observed.The 60 rats that were successful in the model were randomly divided into myocardial ischemia model control group(n=15),the positive control group of Shexiang baoxin pills(n=15),and The low-medium and high dose group of xuanbi tongyu formula(n=45).The dosage of the drug intervention group was as follows.the positive control group give Shexiang baoxin pills 24.3mg/(kg·d),the low-medium and high dose group of xuanbi tongyu formula give the dose 0.8g?1.6g?3.2g/kg·d,myocardial ischemia model group and sham operation group were given equal volume of distilled water.the duration of drug intervention was 4 weeks.At the fourth weekend the rats were observed and recorded we should recorded rat mortality statistical,AST,CK,LDH,cTn?,SOD,MAD equatorial plane for paraffin pathological sections were cut by hematoxylin and eosin(HE),and transmissionelectron microscopy and immunohistochemistry to detect the expression of VEGF,Notch,CD34,fluorescence quantitative PCR detection of VEGF,Notch1,Dell4 gene,expression of myocardial tissue protein extraction for Western blot detection of VEGF protein,Notch1 protein,Dell4 protein.2.Effect of Xuan Bi Tong Yu recipe on Notch-Dell4 Pathway of HUVEC Vascular Endothelial cells stimulated by ischemia and hypoxia.Apply MEMa to do culture,cultivate HUVEC in the situation being short of oxygen(95%N2,5%C02),and then respectively cultivate them for 2 hours,4 hours and 8hours.Based on the different culture time use CCK8 to test the impact on HUVEC activity in the situation lack of oxygen.According to the experiment results,select HUVEC which goes through 4h culture and make ischemia and hypoxia model.Cells in vitro experiment divides into five groups:ischemia and hypoxia model control group:ischemia and hypoxia culture cells 4h shexiang baoxin pills positive control group:ischemia and hypoxia culture cells4h+10%shexiang baoxin pills including medicine serum culture 4h xuanbitongyu low dosage group:ischemia and hypoxia culture cells 4h+2%Xuanbi tongyu Decoction including medicine serum culture 4h Xuanbi tongyu Decoction middle dosage group:ischemia and hypoxia culture cells 4h+10% Xuanbi tongyu Decoction including medicine serum culture 4h Xuanbitongyu high dosage group:ischemia and hypoxia culture cells 4h+15% Xuanbi tongyu Decoction including medicine serum culture 4h.Use RT-PCR Assay to test the expressions of VEGF,Notch1,Dell4 and Hes1 mRNA in HUVEC.Western blot was used to detect the expressions of VEGF,Notch1,Dell4 and Hes1 protein in HUVEC.The Transwell migration assay was carried out on the cells of each experimental group,and the lumen formation test was carried out on the cells in each experimental group.Result:Therapeutic effect and mechanism of Xuanbitongyu recipe on myocardial ischemia rats prepared by coronary ligation in vitro empirical study.1.1 At 5 min after coronary artery ligation,St segment of electrocardiogram of ischemic model group was significantly elevated(P<0.001,P<0.01 or P<0.05)compared with sham operation group.In the medium dose group of Xuanbitongyu decoction,the amplitude of St segment up-shift decreased(P<0.05);from the 3rd to the 5th day of administration,thechanges of St segment were compared with those of the control group(P< 0.05).Compared with the model group,the St segment up-shift amplitude in the low dose group of Xuanbi Tongyu prescription decreased significantly(P<0.05).From the second to the fifth day after administration,compared with the modell group,the St segment up-shift amplitude of ECG in the positive control group(Shexiang Baoxin pills)decreased significantly(P<0.05).1.2 At the beginning of 2 weeks,4 weeks and 6 weeks,the rats in each experimental group died to different degrees.Compared with the ischemic modell group,the mortality of rats in the high dose group of Xuanbi Tongyu decoction decreased significantly(P<0.05).The release of CK and LDH in the modell group was significantly higher than that in the sham operation group(P<0.001).Compared with the modell control group,the high dose of Xuanbi Tongyu prescription significantly decreased the contents of CK and LDH in ASTU(P<0.01 or P<0.05)and the activity of AST and LDH in the medium dose of Xuanbi Tongyu recipe significantly decreased(P<0.01 or P<0.05).The low dosage of Xuanbi Tongyu prescription could obviously reduce the activity of LDH(P<0.05).Compared with the ischemic modell control group,Xuanbi Tongyu prescription group could significantly reduce the content of cTn in the animal tissues(P<0.001,P<0.01 or P<0.05),and showed a good dose-effect relationship according to the dosage of the drug.Compared with the ischemic modell control group,the myocardial infarction area in the high and middle dose group decreased significantly(P<0.01 and P<0.05).HE staining shows that Sham operation group had clear arrangement of myocardial cells and clear structure.Imodel group,cardiac myocytes can be seen coagulant necrosis and nuclear fragmentation.Myocardial injury is improved and myocardial cell arrangement is regular in Xuanbi Tongyu high dose group and shexiang baoxin pills group.1.3 Immunohistochemical results: compared with the model group,the content of VEGF in the myocardium of the Shexiang Baoxin pills group,the Xuanbi Tongyu high dose group,the middle dose group,the low dose group and the low dose group were significantly increased(P<0.01,P<0.05),and compared with the model group,the content of VEGF was significantly increased in the Shexiang Baoxin Pill group.The content of Notch1 in myocardial tissue of medium dose and high dose of Xuanbi Tongyu decreased significantly(P<0.01,P<0.05).1.4 The results of immunofluorescence: compared with the model group,the positive control group of Shexiang Baoxin pills had a low level of Xuan Bi and Tong Yu,and the content of CD34 in myocardial tissue of rats treated with medium and high dose group was significantly higher than that of the control group(P<0.01).Compared with the model group,the positive control group of Shexiang Baoxin pills,Xuan Bi,Tong Yu low,medium and high dose group after treatment of myocardial tissue Dell4 content was significantly higher than that of the control group(P<0.05).1.5 RT-PCR results: compared with sham operation group,VEGFmRNA-Notch1 mRNA expression in myocardial ischemia model group was significantly up-regulated and down-regulated(P<0.01),compared with myocardial ischemia model group,the positive control group of Shexiang Baoxin Pill,Xuan Bi Tong Yu recipe,The expression of VEGFmRNA in myocardium was up-regulated in high dose group(P<0.01),and the expression of Notch1 mRNA in high dose group was significantly down-regulated(P<0.05,P<0.001)in the positive control group of Shexiang Baoxin pills and Xuanbi Tongyu group.The positive control group of Shexiang Baoxin Pill,Xuanbi Tongyu prescription,low,medium and high dose group and Dell4 mRNA expression upregulation(P<0.01,P<0.05)1.6 The results of Western blot showed that the expression of VEGFN Notch1 protein was significantly up-regulated in model rats compared with sham-operation group(P<0.05),and the expression of Dell4 protein was significantly down-regulated(P<0.001),and the expression of Xuanbi Tongyu prescription was lower and lower than that of model group,while the expression of VEGFN Notch1 protein was significantly lower than that of sham-operated group.In the high dose group and the positive control group of Shexiang Baoxin pills,the expression of VEGF protein increased significantly(P<0.05,P<0.01),and in the positive group of Shexiang Baoxin Pill,the expression of Notch1 protein decreased significantly in the Xuanbi Tongyu recipe,and the expression of Notch1 protein in the high dose group was significantly down-regulated,and the expression of Notch1 protein in the Shexiang Baoxin Pill positive group was significantly down-regulated.The expression of Dell4 protein in myocardium of positive group,Xuanbi Tongyu prescription and high dose group was upregulated significantly(P<0.01,P<0.001).2.Effect of Xuan Bi Tong Yu recipe on Notch-Dell4 Pathway of HUVEC VascularEndothelial cells stimulated by Ischemia and hypoxia2.1 Effect of ischemia and hypoxia on cell survival rate measured by CCK8 at different time pointsCompared with the model group,the cell survival rate of the low,middle,high dose group,the positive drug group,were not significantly changed.Compared with the model group,the cell survival rate of the positive drug treatment group increased significantly(P<0.01 and P<0.001)in a dose-dependent manner.2.2 RT-PCR results:compared with the control group,the expression of cell hypoxia ischemia model group VEGFmRNA decreased significantly(P<0.001),significantly increased the expression of Notch1mRNA(P<0.01),significantly increased the expression of Hes1mRNA(P< 0.001);compared with the ischemia group,Shexiang Baoxin Pills positive control group,Xuan Tong Bi blood stasis,low and high dose group significantly increased the expression of VEGF in cells of mRNA(P<0.01,P<0.001),SXBXW Xuanbi positive control group,high dose group Notch blood cells.The expression of 1mRNA decreased significantly(P<0.01).The expression of Hes1 mRNA in the positive control group of Shexiang Baoxin pill,Xuanbi Tongyu recipe and high dose group decreased significantly(P<0.05,P<0.01).2.3 Western blot results:The expression of VEGF protein increased significantly in the high dose group of Xuan Bi Tong Yu recipe(P<0.01).The expression of Notch1 protein decreased significantly in the positive control group of Shexiang Baoxin Pill,Xuan Bi Tong Yu recipe and the high dose group.(P<0.01,P<0.05),the positive control group of musk Baoxin Pill,the expression of Hes1 protein in the high dose group was significantly decreased in the high dose group(P<0.05,P<0.001).2.4 Cell tube-forming detection:Compared with the blank control group,the ability of HUVEC cells to form the lumen decreased under hypoxia,which was mainly due to the increase of the broken lumen and the serious damage to the integrity of the lumen.Compared with the ischemia and hypoxia group,with the increase of the serum dosage of Xuan Bi Tong Yu,the damage of the lumen was gradually weakened,but the ability of forming the lumen was weaker than that of the blank control group.2.5 Cell migration detection: compared with the blank control group,the number of cells passing through the micropore membrane of the positive drug treatment group wassignificantly decreased compared with the control group and the high dose group of traditional Chinese medicine(P<0.001,P<0.01 and P<0.05),and compared with the ischemia and hypoxia group,the number of cells in the positive drug treatment group was significantly lower than that in the ischemia and hypoxia group.With the increase of serum dosage of Xuanbi Tongyu,cell migration ability was gradually enhanced,but the migration ability was weaker than that of normal group.Conclusion:1.Xuanbi Tongyu prescription can reduce the mortality of experimental myocardial ischemia in rats,and improve theabnormal electrocardiogram changes of myocardial ischemia rats.2.Xuanbi Tongyu recipe can reduce myocardial trienzyme and troponin in rats with experimental myocardial ischemia,reduce the infarct size of heart,promote angiogenesis in myocardial infarction area,and protect myocardial tissuestructure and function.3.Xuanbi Tongyu prescription can significantly increase the expression of VEGF Dell4 in myocardial tissue of myocardial ischemia rats,and inhibit the expression of Notch1 in myocardial tissue of myocardial ischemia rats.4.Xuan Bi Tong Yu recipe can obviously up-regulate the expression of VEGF gene and protein in HUVEC vascular endothelial cells and down-regulate the expression of Notch1Hes1 gene and protein in HUVEC vascular endothelial cells.5.Xuan Bi Tongyu recipe can promote the migration and formation of ischemic anoxia HUVEC.6.Xuanbi Tongyu prescription can promote angiogenesis in myocardial ischemia tissue of coronary heart disease,and its mechanism may be related to the increase of VEGF expression and regulation of Notch-Dell4 signal pathway. |
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