Researches On The Effects Of Hexue Shengluo Method On Angiogenesis Of Rats With Myocardial Infarction And Mechanisms Involved

Objective:Coronary heart disease, a common and frequent disease in older adults, has become one of the top 10 causes of death. The mortality in younger population is increasing with the change of diet and other factors. Medicine, operation, and interventional therapy are used in the treatment, but the efficacy is not satisfied. However, therapeutic angiogenesis therapy brings hope to the patients. Traditional Chinese Medicine (TCM) with a history of thousands of years can not be accepted widely due to the theoretical diversity even if good efficacy has been witnessed in practice. Based on the theories of TCM and clinical researches, we find that disharmony of blood vessel is the basic pathogenesis in CHD. Hexue Shengluo Method which is to harmonize the blood vessel is the basic therapy.We observed the effects of Hexue Shengluo Method on angiogenesis by setting up myocardial infarction models, detected bFGF, MMP-2/ TIMP-2, G-CSF, AKT, ERK by RT-PCR, Western Blotting, discussed the mechanisms involved in angiogenesis from growth factor, ECM, EPCs, and signal path parts, which laid foundation for the clinical application of Hexue Shengluo Method.Part ICoronary Heart Disease and Angiogenesis in TCM and the Presentation of Hexue Shengluo MethodThe conception, symptoms, classification, therapeutic principles and conventional treatment of the coronary heart diseases are all stated in this paper. Considering the limitation of the conventional treatment, we summarized the general situation, perspective of therapeutic angiogenesis, a new way of treatment. Therapeutic angiogenesis can not be applied in clinic because it is still in the stage of theoretical research and pre-clinic. However, the similarities between"Luomai"and"vessel","angiogenesis"and"Shengmai"provide new thinking to the treatment of CHD.The name, clinical manifestation, and differentiation of the diseases related to CHD were summarized in this paper. Based on the etiology, pathogenesis, treatment and clinical researches, we find that disharmony of blood vessel is the basic pathogenesis in CHD. Hexue Shengluo Method which is to harmonize the blood vessel is the basic therapy.Part IIEstablishment of Rat Model with Myocardial Infarction and Effects of Hexue Shengluo Method on AngiogenesisMethods:The model was copied with the ligation of left anterior descending of coronary artery (LAD) in healthy Wistar male rats. 48 successful model rats were randomly divided into 4 groups (12 in each group): the model group (M), the isosorbide dinitrate group (Y), and low doses of Hexue Shengluo group (D), and high doses of Hexue Shengluo group(G). At the same time, sham operated group (J) was also prepared. They were treated with different therapies for 4 weeks, then we checked the area of the myocardial infarction by TTC staining method in 6 models every group, detected the expression of CD34 in the marginal zone of the infarction by immune histochemical method, studied the amount and density of capillaries in another 6 models every group. The pathological changes around the infarction area were observed by HE staining. Results:1 General condition of the modelsRats preferred to curled themselves up, and don't like to move. They were seemed to be lack of vitality, poor appetite, and the hair is lustreless and fell down. The resistance to be caught is not strong. The appetite and the hair condition recovered one day after the operation in J group. During the experiment, the rats in M group show poor appetite, lack of movement, slowly putting on weight, or even not putting on. And the condition of the hair made no difference. The mental state improved, the weight were increased in all groups intervened by drug, especially in G group.2 Size of myocardial infarctionMyocardial infarction size in all therapeutic groups[G-(19.5±2.2)%, D-(22.0±2.3)%,Y-(26.1±2.5)%] are significantly smaller than in model group ([29.6±1.4)%](P<0.05), and H and L group are better than Y groups(P<0.05).3 MVC and MVD in the marginal zone of myocardial infarctionThe amount of MVC/MVD in M group (88.50±2.44) is higher than in J group (75.50±4.29) (P<0.05); the amount of MVC/MVD in each therapeutic group (G-133.90±6.50, D-122.03±2.68, Y-117.50±3.68) is higher than in M group(P<0.05). And G group is significantly better than D group and Y group(P<0.05).4 Pathological changesHigh magnification showed that some myocardial cells bulged, interstitial cells swelled. And myocardial fiber dissolved or proliferated and inflammatory cell infiltration occurred in M group. Myocardial cell necrosis, inflammatory cell infiltration and fibrous proliferation were mild in D group and Y group. While most of the myocardial cells kept normal and only a few of the myocardial cells necrosis, inflammatory cells infiltration occurred, and the fibrous proliferation was scanty in G group. The myocardial cells and fibers kept normal in J group. PartⅢEffects of Hexue Shengluo Method on bFGFMethods:The models were established in the same way as in experiment 1. Successfully-established model rats were randomly divided into 4 groups (12 in each group): the model group (M),the isosorbide dinitrate group (Y), low doses of Hexue shengluo group (D), and high doses of Hexue Shengluo group (G). At the same time, sham operated group (J) was also prepared. They were treated with different therapies for 3 weeks, then we detected the expression of bFGF in the marginal zone of the infarction by immune histochemical method, studied the average gray by high-resolution color medical graphic analysis system. We observed the expression of bFGF mRNA in 6 rats every group by RT-PCR. The electrophoretic band was analyzed in BIO-PROFIF Gel image analysis system. The ratio of these two absorbances was showed according to relative electrophoretic band. And the content of bFGF was detected by ELISA in 8 rats serum every group.Results:1 Protein expression and average gray of bFGF in the marginal zone of the myocardial infarctionThe protein expression of bFGF in M group(197.83±5.55) is much higher than in J group(218.20±3.55)(P<0.05). The expression in G(163.02±6.56), D(174.93±2.66), Y(176.80±3.59) group is significantly higher than in M group(P<0.05). Among them, G group is much higher than D and Y group(P<0.05).2 Expression of bFGF mRNA in rats left ventricular tissue and the ratio of absorbanceThe expression in M group (0.31±0.01) is higher than in J group (0.18±0.05) (P<0.05). The therapeutic group are higher than M group(P<0.05). Among them, G(0.69±0.17) and Y(0.70±0.10) groups are higher than D(0.49±0.04) group(P<0.05). 3 Content of bFGF in serumThe content of bFGF in M group (3.78±0.25) is higher than in J group (2.99±0.42) but non-statistics significance (P>0.05). D group (4.32±1.29) is higher than M group but non-statistics significance (P>0.05). The content in G group (5.29±1.13) and Y group (5.04±2.10) are much higher than in M group (P<0.05).PartⅣEffects of Hexue Shengluo method on MMP-2 and TIMP-2Methods:The models were established in the same way as in experiment 1. Successfully-established model rats were randomly divided into 4 groups (12 in each group): the model group (M),the isosorbide dinitrate group (Y), low doses of Hexue shengluo group (D), and high doses of Hexue Shengluo group (G). At the same time, sham operated group (J) was also prepared. They were treated with different therapies for 3 weeks, then MMP-2 and TIMP-2 in the marginal zone of the infarction were detected by immune histochemical method in 6 rats every group. The average light density value was analyzed by high-resolution color medical graphic analysis system HMIAS-2000. And Correlation Analysis with bFGF was made.Results:1 Expression of MMP-2 in the marginal zone of the myocardial infarction The protein expression of MMP-2 in M group (0.1490±0.0119) is much higher than in J group (0.0688±0.0072) (P<0.05). The expression of MMP-2 in G (0.2545±0.0161), D (0.2174±0.0116), Y group (0.2204±0.0109) are much higher than in M group (P<0.05). The expression of MMP-2 in G group is higher than in Y group and D group (P<0.05).2 Expression of TIMP-2 in the marginal zone of the myocardial infarctionThe expression of TIMP-2 in M group (0.1328±0.0104) is much h- igher than in J group (0.0835±0.0085) (P<0.05). G (0.1878±0.0106), Y group (0.1812±0.0105) are higher than M group (P<0.05).D group (0.1690±0.0093)is higher than M group but non-statistics significance (P>0.05).3 Ratio of MMP-2 and TIMP-2The ratio in M group (1.12±0.05) is much larger than in J group (0.82±0.03) (P<0.05). The ratio in G (1.36±0.04), D (1.29±0.04), Y group (1.22±0.04)are much larger than in M group (P<0.05). The Chinese medicine groups are larger than Y group, and G group is larger than D group (P<0.05).4 Correlation Analysis between bFGF and MMP-2, bFGF and MMP-2/TIMP-2There is a positive correlativity between bFGF and MMP-2 (r=0.967), bFGF and MMP-2/TIMP-2 (r=0.926) at protein expression (P<0.01).PartⅤEffects of Hexue Shengluo method on G-CSFMethods:The models were established in the same way as in experiment 1. Successfully-established model rats were randomly divided into 4 groups (12 in each group): the model group (M),the isosorbide dinitrate group (Y), low doses of Hexue shengluo group (D), and high doses of Hexue Shengluo group (G). At the same time, sham operated group (J) was also prepared. They were treated with different therapies for 3 weeks, then we detected the content of G-CSF in 8 rats serum every group by ELISA method.Results:1 Content of G-CSF in serumThe content of G-CSF in M group(31.15±5.12) is higher than in J group(15.34±3.02) (P<0.05).The content of G-CSF in therapeutic groups is higher than in M group (P<0.05). The content of G-CSF in G (66.61±8.46) and Y (52.59±7.18) group is higher than in D (38.23±9.76) group (P<0.05).G group is higher than Y group (P<0.05). PartⅥEffects of Hexue Shengluo method on PI3K/Akt and MEK/ERK signal transduction pathwayMethods:The models were established in the same way as in experiment 1. Successfully-established model rats were randomly divided into 4 groups (12 in each group): the model group (M),the isosorbide dinitrate group (Y), low doses of Hexue shengluo group (D), and high doses of Hexue Shengluo group (G). At the same time, sham operated group (J) was also prepared. They were treated with different therapies for 3 weeks, then we detected the expression of AKT mRNA in left ventricular myocardial tissue in 6 rats every group by RT-PCR. The electrophoretic band was analyzed in BIO-PROFIF Gel image analysis system. The ratio of these two absorbances was showed according to relative electrophoretic band. The expression of ERK1/2 in left ventricular myocardial tissue in 6 rats every group was detected by Western blot. Absorbance was identified by Gel quantitative software Quantity-One-version 4.6.2, and the protein expression was represented by absorbance.Results:1 The expression of AKT mRNA in left ventricular myocardial tissue of the ratsThe expression of AKT mRNA in M group(0.26±0.02) is higher than in J group(0.17±0.01) (P<0.05). The expression of AKT mRNA in therapeutic groups are higher than in M group(P<0.05). G (0.60±0.02) and Y (0.58±0.01) group are higher than D (0.42±0.01) group(P<0.05). G group is much higher than Y group(P<0.05).2 The expression of ERK1/2 in left ventricular myocardial tissue of the ratsCompared to the expression of J group (25470±1456), that of M group (33703±488) is increased significantly (P<0.05). The expression of ERK1/2 in therapeutic groups is higher than in M group. G (54335±1021) group is higher than D (40146±289)and Y (38361±1200) groups (P<0.05). D group is much higher than Y group(P<0.05).Conclusions:1 Hexue Shengluo Method can be considered as the basic therapy for CHD.2 Hexue Shengluo Method can shrink the area of myocardial infarction.3 Hexue Shengluo Method can increase MVC/MVD in the marginal zone of the myocardial infarction, and can promote the angiogenesis, which can improve the condition of the local ischemia.4 Hexue Shengluo Method can increase the protein and genetic expression of bFGF in myocardial tissue, and can increase the content of bFGF in serum. bFGF is an important growth factor for angiogenesis, so Hexue Shengluo Method can promote angiogenesis.5 Hexue Shengluo Method decomposes the basilar membrane to allow the proliferation and migration of endotheliocytes, which is the precondition of angiogenesis, by means of promoting the secretion of MMP-2, and changing the proportion of MMP-2 and TIMP-2.6 Hexue Shengluo Method can increase the content of G-CSF in serum, which can promote endothelial progenitor cells into peripheral blood, therefore we get a conclusion indirectly that Hexue Shengluo Method can promote angiogenesis.7 Hexue Shengluo Method can increase the expression of Akt and ERK1/2 in myocardium, which indicates that this method can activate the two ways, Raf/MEK/ERK and PI3K/Akt to promote proliferation and migration of endotheliocytes. Angiogenesis come true by two ways.