和血生络法对实验性心肌梗死大鼠的促血管新生作用及机制研究
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摘要
目的:
冠心病是中老年人的常见病和多发病,随着饮食结构等因素的变化,发病年龄日益年轻化,发病人数迅速上升,已成为引起死亡人数最多的十种疾病之一。现代医学虽然对此病有药物、手术、介入等多种疗法,但仍有很多患者不适用于上述疗法或效果不佳,治疗性血管新生疗法的产生为这些患者带来了希望。已有数千年历史的中医药虽然是这一新疗法的践行者,取得了很好的临床疗效,但由于理论认识的差异性,导致中医药疗法不能被广泛接受。我们依据传统中医理论认识,并结合文献及临床研究,提出血脉不和是冠心病的基本病机,以调和血脉为内涵的和血生络法是本病的基本治法。通过建立实验性心肌梗死模型,观察和血生络法对心梗模型血管新生的影响,利用逆转录-聚合酶链反应、蛋白质印迹等现代先进的检测技术观察了bFGF、MMP-2/TIMP-2、G-CSF、AKT、ERK等指标的变化,从生长因子、细胞外基质、内皮祖细胞、信号通路等多方面对促血管新生的机制进行了探讨,为和血生络法广泛应用于临床奠定了坚实的理论基础。
第一部分
中医对冠心病和血管新生的认识及和血生络法的提出
简述了冠心病的概念、表现、分类、治疗原则及常规治疗方法。鉴于冠心病治疗中存在的不足,介绍了本病治疗中的新思路,即治疗性血管新生。对其主要研究概况及应用前景进行了简要归纳。由于治疗性血管新生目前仅限于理论研究和临床前期阶段,并不能实际应用于临床,而中医的“脉络”与现代医学的“血管”对应,血管新生与中医学“生脉”等理论具有高度一致性,这就为重新审视中医药的治疗机制提供了新思路,也为中医药在冠心病的治疗中发挥其良好的作用提供了广阔前景。
整理查阅了大量的古今中医药与冠心病相关的文献,对与冠心病相关的中医病名、临床表现及鉴别进行了整理归纳,同时分析了古今对冠心病病因病机及治疗方法的认识,结合我们的临床体会提出了冠心病的基本病机是血脉失和,而“和血生络法”是冠心病的基本治法。
第二部分
心肌梗死大鼠模型制备及“和血生络法”促血管新生的作用
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏以TTC染色法测定大鼠左室心肌梗死范围;每组取另6只大鼠心脏免疫组化方法检测心肌梗死边缘区CD34表达,检测微血管数量和微血管密度。同时应用HE染色观察梗死边缘区的病理变化。
结果:
1大鼠一般状况的观察比较
大鼠呈蜷缩状态,精神萎靡,活动较少,毛发黄白色枯槁无光泽且多倒竖,进食量明显减少。抓握大鼠时无反抗或反抗较轻。假手术组大鼠手术后1天,食欲和运动量即恢复至术前状态,毛发平滑光泽无枯槁现象。实验进程中,模型组大鼠仍然表现出饮食、活动少的状态,因而出现增重缓慢,甚至无增重的情况。皮毛状态未见明显改观。各用药物组大鼠精神状态逐渐好转,表现出不同程度增重,尤以和血生络大剂量组最为明显。
2心肌梗死范围
假手术组大鼠未出现心肌梗死。模型组大鼠心肌梗死范围为(29.6±1.4)%,和血生络高剂量组、低剂量组及消心痛组大鼠心肌梗死范围分别为(19.5±2.2)%、(22.0±2.3)%、(26.1±2.5)%。各治疗组梗死范围均明显小于模型组(P<0.05),和血生络高剂量组、和血生络低剂量组明显优于消心痛组(P<0.05),差别具有统计学意义。
3心肌梗死边缘区微血管数量(MVC)和微血管密度(MVD)
假手术组大鼠MVC/MVD为75.50±4.29,模型组大鼠为88.50±2.44,较假手术组大鼠显著增高(P<0.05)。和血生络高剂量组、低剂量组及消心痛组大鼠MVC/MVD分别为133.90±6.50、122.03±2.68、117.50±3.68。各治疗组明显高于模型组(P<0.05)。和血生络高剂量组明显优于和血生络低剂量组及消心痛组(P<0.05)。
4病理组织变化
模型组高倍镜下部分心肌细胞肿胀,间质水肿,梗死灶可见心肌纤维溶解,纤维组织增生、炎细胞浸润。和血生络低剂量组、消心痛组表现为心肌细胞坏死较模型组少见,少量炎细胞浸润,纤维组织增生少见。和血生络高剂量组大多数心肌细胞无异常,个别心肌坏死、炎细胞浸润,无明显纤维增生。假手术组心肌细胞未见明显异常,无纤维组织增生及炎细胞浸润。
第三部分
“和血生络法”对碱性成纤维细胞生长因子的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏免疫组化法检测bFGF在大鼠心梗边缘区中的蛋白表达,用HMIAS-2000高清晰度彩色医学图文分析系统分析其平均灰度。每组取6只大鼠心脏组织RT-PCR法检测bFGF mRNA在大鼠左室心肌组织的表达,用法国VL公司BIO-PROFIF凝胶图象分析系统对目的电泳条带进行分析,以相应的内参电泳条带作为参照,结果以两者之积分吸光度的比值表示。每组取8只大鼠的血清ELISA法检测bFGF在血清中的含量。
结果:
1大鼠心梗边缘区bFGF的蛋白表达及其平均灰度
模型组、假手术组平均灰度分别为197.83±5.55、218.20±3.55,模型组bFGF蛋白表达明显高于假手术组(P<0.05)。和血生络高剂量组、和血生络低剂量组、消心痛组平均灰度分别为163.02±6.56、174.93±2.66、176.80±3.59,bFGF蛋白表达较模型组明显增高(P<0.05)。和血生络高剂量组较和血生络低剂量组与消心痛组表达明显增强(P<0.05)。
2 bFGF mRNA在大鼠左室心肌组织的表达及积分吸光度的比值
模型组(0.31±0.01)较假手术组(0.18±0.05)明显增强(P<0.05)。和血生络高剂量组(0.69±0.17)、和血生络低剂量组(0.49±0.04)、消心痛组(0.70±0.10)bFGF mRNA表达较模型组明显增强(P<0.05)。和血生络高剂量组与消心痛组较和血生络低剂量组表达明显增强(P<0.05)。
3 bFGF在血清中的含量
模型组(3.78±0.25)血清中bFGF含量较假手术组(2.99±0.42)增高,但无统计学差异(P>0.05)。和血生络低剂量组(4.32±1.29)亦较模型组增高但无统计学差异(P>0.05)。和血生络高剂量组(5.29±1.13)、消心痛组(5.04±2.10)较模型组bFGF含量明显增高(P<0.05)。
第四部分
“和血生络法”对基质金属蛋白酶-2及基质金属蛋白酶组织抑制剂-2的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏免疫组化法检测MMP-2及TIMP-2在大鼠心梗边缘区中的蛋白表达,用HMIAS-2000高清晰度彩色医学图文分析系统分析其平均光密度值。并与bFGF作相关性分析。
结果:
1 MMP-2在大鼠心梗边缘区中的蛋白表达
模型组(0.1490±0.0119)MMP-2蛋白表达较假手术组(0.0688±0.0072)明显增高(P<0.05)。和血生络高剂量组(0.2545±0.0161)、和血生络低剂量组(0.2174±0.0116)、消心痛组(0.2204±0.0109)MMP-2蛋白表达较模型组明显增高(P<0.05)。和血生络高剂量组强于消心痛组及和血生络低剂量组(P<0.05)。
2 TIMP-2在大鼠心梗边缘区中的蛋白表达
模型组(0.1328±0.0104)TIMP-2蛋白表达较假手术组(0.0835±0.0085)明显增高(P<0.05)。和血生络高剂量组(0.1878±0.0106)、消心痛组(0.1812±0.0105)较模型组TIMP-2蛋白表明显增高(P<0.05)。和血生络低剂量组(0.1690±0.0093)虽从数值看高于模型组,但并无统计学差异(P>0.05)。
3 MMP-2与TIMP-2比值变化
模型组(1.12±0.05)MMP-2/TIMP-2较假手术组明(0.82±0.03)显增高(P<0.05)。和血生络高剂量组(1.36±0.04)、和血生络低剂量组(1.29±0.04)、消心痛组(1.22±0.04)MMP-2/TIMP-2较模型组明显增高(P<0.05)。中药组强于消心痛组且和血生络高剂量组强于和血生络低剂量组(P<0.05)。
4 bFGF与MMP-2、MMP-2/TIMP-2的相关性分析
将实验二心肌bFGF蛋白表达与MMP-2、MMP/TIMP的表达进行相关性分析,结果bFGF与MMP-2、MMP/TIMP的表达均呈正相关,r分别为0.967、0.926,P<0.01。
第五部分
“和血生络法”对粒细胞集落刺激因子的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取8只大鼠的血清ELISA法检测G-CSF在血清中的含量。
结果:
1血清中G-CSF的含量
模型组血清G-CSF含量为31.15±5.12较假手术组15.34±3.02明显增高(P<0.05)。和血生络高剂量组、和血生络低剂量组、消心痛组血清G-CSF含量分别为66.61±8.46、38.23±9.76、52.59±7.18较模型组明显增高(P<0.05)。和血生络高剂量组、消心痛组较和血生络低剂量组明显增高(P<0.05),且和血生络高剂量组高于消心痛组(P<0.05)。
第六部分
“和血生络法”对PI3K/Akt和MEK/ERK信号转导通路的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏组织RT-PCR法检测AKT mRNA在大鼠左室心肌组织的表达,用BIO-PROFIF凝胶图象分析系统对目的电泳条带进行分析,以相应的内参电泳条带作为参照,结果以两者之积分吸光度的比值表示。每组取6只大鼠心脏组织Western blot检测ERK1/2在大鼠左室心肌组织的表达,用凝胶定量软件Quantity-One-version 4.6.2测定各条带的积分吸光度,以积分吸光度代表蛋白质的表达量。
结果:
1 AKT mRNA在大鼠左室心肌组织的表达
假手术组大鼠为0.17±0.01,模型组大鼠为0.26±0.02,较假手术组大鼠显著增高(P<0.05)。和血生络高剂量组、和血生络低剂量组及消心痛组大鼠分别为0.60±0.02、0.42±0.01、0.58±0.01。各治疗组明显高于模型组(P<0.05)。和血生络高剂量组与消心痛组明显优于和血生络高低剂量组。和血生络高剂量组明显优于消心痛组(P<0.05)。
2 ERK1/2在大鼠左室心肌组织的表达
假手术组大鼠为25470±1456,模型组大鼠为33703±488,较假手术组大鼠显著增高(P<0.05)。和血生络高剂量组、低剂量组及消心痛组大鼠分别为54335±1021、40146±289、38361±1200,各治疗组明显高于模型组(P<0.05)。和血生络高剂量组明显优于和血生络低剂量组及消心痛组(P<0.05)。和血生络低剂量组明显优于消心痛组(P<0.05)。
结论:
1“和血生络法”是冠心病的基本治法。
2和血生络法能够缩小心肌梗死范围,对大鼠心肌梗死具有治疗作用。
3和血生络法能够增加心肌梗死边缘区MVC/MVD,对缺血心肌具有促血管新生作用,从而有助于局部缺血状态的改善。
4和血生络法能增强心肌组织中bFGF蛋白和基因的表达,提高血清中bFGF的含量。由于bFGF是血管新生的重要生长因子,所以和血生络法促进bFGF产生是其促进血管新生的机制之一。
5和血生络法能够促进MMP-2的分泌,改变MMP-2、TIMP-2的比例,从而促进基底膜的降解,有利于内皮细胞的增殖和游走,为血管新生提供保障。
6和血生络法能提高血清中G-CSF含量,而G-CSF是骨髓中内皮祖细胞强有力的动员剂,可间接推论和血生络法通过动员内皮祖细胞进入外周血而促进血管新生。
7和血生络方能明显提高心肌中Akt及ERK1/2的表达量。说明和血生络法可通过激活Raf/MEK/ERK和PI3K/Akt两条途径促进内皮细胞的增殖、迁移而实现了血管新生。
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 I
Coronary Heart Disease and Angiogenesis in TCM and the Presentation of Hexue Shengluo Method
The 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 II
Establishment of Rat Model with Myocardial Infarction and Effects of Hexue Shengluo Method on Angiogenesis
Methods:
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 models
Rats 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 infarction
Myocardial 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 infarction
The 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 changes
High 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.
PartⅢ
Effects of Hexue Shengluo Method on bFGF
Methods:
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 infarction
The 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 absorbance
The 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 serum
The 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).
PartⅣ
Effects of Hexue Shengluo method on MMP-2 and TIMP-2
Methods:
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 infarction
The 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-2
The 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-2
There 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).
PartⅤ
Effects of Hexue Shengluo method on G-CSF
Methods:
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 serum
The 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).
PartⅥ
Effects of Hexue Shengluo method on PI3K/Akt and MEK/ERK signal transduction pathway
Methods:
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 rats
The 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 rats
Compared 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.
冠心病是中老年人的常见病和多发病,随着饮食结构等因素的变化,发病年龄日益年轻化,发病人数迅速上升,已成为引起死亡人数最多的十种疾病之一。现代医学虽然对此病有药物、手术、介入等多种疗法,但仍有很多患者不适用于上述疗法或效果不佳,治疗性血管新生疗法的产生为这些患者带来了希望。已有数千年历史的中医药虽然是这一新疗法的践行者,取得了很好的临床疗效,但由于理论认识的差异性,导致中医药疗法不能被广泛接受。我们依据传统中医理论认识,并结合文献及临床研究,提出血脉不和是冠心病的基本病机,以调和血脉为内涵的和血生络法是本病的基本治法。通过建立实验性心肌梗死模型,观察和血生络法对心梗模型血管新生的影响,利用逆转录-聚合酶链反应、蛋白质印迹等现代先进的检测技术观察了bFGF、MMP-2/TIMP-2、G-CSF、AKT、ERK等指标的变化,从生长因子、细胞外基质、内皮祖细胞、信号通路等多方面对促血管新生的机制进行了探讨,为和血生络法广泛应用于临床奠定了坚实的理论基础。
第一部分
中医对冠心病和血管新生的认识及和血生络法的提出
简述了冠心病的概念、表现、分类、治疗原则及常规治疗方法。鉴于冠心病治疗中存在的不足,介绍了本病治疗中的新思路,即治疗性血管新生。对其主要研究概况及应用前景进行了简要归纳。由于治疗性血管新生目前仅限于理论研究和临床前期阶段,并不能实际应用于临床,而中医的“脉络”与现代医学的“血管”对应,血管新生与中医学“生脉”等理论具有高度一致性,这就为重新审视中医药的治疗机制提供了新思路,也为中医药在冠心病的治疗中发挥其良好的作用提供了广阔前景。
整理查阅了大量的古今中医药与冠心病相关的文献,对与冠心病相关的中医病名、临床表现及鉴别进行了整理归纳,同时分析了古今对冠心病病因病机及治疗方法的认识,结合我们的临床体会提出了冠心病的基本病机是血脉失和,而“和血生络法”是冠心病的基本治法。
第二部分
心肌梗死大鼠模型制备及“和血生络法”促血管新生的作用
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏以TTC染色法测定大鼠左室心肌梗死范围;每组取另6只大鼠心脏免疫组化方法检测心肌梗死边缘区CD34表达,检测微血管数量和微血管密度。同时应用HE染色观察梗死边缘区的病理变化。
结果:
1大鼠一般状况的观察比较
大鼠呈蜷缩状态,精神萎靡,活动较少,毛发黄白色枯槁无光泽且多倒竖,进食量明显减少。抓握大鼠时无反抗或反抗较轻。假手术组大鼠手术后1天,食欲和运动量即恢复至术前状态,毛发平滑光泽无枯槁现象。实验进程中,模型组大鼠仍然表现出饮食、活动少的状态,因而出现增重缓慢,甚至无增重的情况。皮毛状态未见明显改观。各用药物组大鼠精神状态逐渐好转,表现出不同程度增重,尤以和血生络大剂量组最为明显。
2心肌梗死范围
假手术组大鼠未出现心肌梗死。模型组大鼠心肌梗死范围为(29.6±1.4)%,和血生络高剂量组、低剂量组及消心痛组大鼠心肌梗死范围分别为(19.5±2.2)%、(22.0±2.3)%、(26.1±2.5)%。各治疗组梗死范围均明显小于模型组(P<0.05),和血生络高剂量组、和血生络低剂量组明显优于消心痛组(P<0.05),差别具有统计学意义。
3心肌梗死边缘区微血管数量(MVC)和微血管密度(MVD)
假手术组大鼠MVC/MVD为75.50±4.29,模型组大鼠为88.50±2.44,较假手术组大鼠显著增高(P<0.05)。和血生络高剂量组、低剂量组及消心痛组大鼠MVC/MVD分别为133.90±6.50、122.03±2.68、117.50±3.68。各治疗组明显高于模型组(P<0.05)。和血生络高剂量组明显优于和血生络低剂量组及消心痛组(P<0.05)。
4病理组织变化
模型组高倍镜下部分心肌细胞肿胀,间质水肿,梗死灶可见心肌纤维溶解,纤维组织增生、炎细胞浸润。和血生络低剂量组、消心痛组表现为心肌细胞坏死较模型组少见,少量炎细胞浸润,纤维组织增生少见。和血生络高剂量组大多数心肌细胞无异常,个别心肌坏死、炎细胞浸润,无明显纤维增生。假手术组心肌细胞未见明显异常,无纤维组织增生及炎细胞浸润。
第三部分
“和血生络法”对碱性成纤维细胞生长因子的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏免疫组化法检测bFGF在大鼠心梗边缘区中的蛋白表达,用HMIAS-2000高清晰度彩色医学图文分析系统分析其平均灰度。每组取6只大鼠心脏组织RT-PCR法检测bFGF mRNA在大鼠左室心肌组织的表达,用法国VL公司BIO-PROFIF凝胶图象分析系统对目的电泳条带进行分析,以相应的内参电泳条带作为参照,结果以两者之积分吸光度的比值表示。每组取8只大鼠的血清ELISA法检测bFGF在血清中的含量。
结果:
1大鼠心梗边缘区bFGF的蛋白表达及其平均灰度
模型组、假手术组平均灰度分别为197.83±5.55、218.20±3.55,模型组bFGF蛋白表达明显高于假手术组(P<0.05)。和血生络高剂量组、和血生络低剂量组、消心痛组平均灰度分别为163.02±6.56、174.93±2.66、176.80±3.59,bFGF蛋白表达较模型组明显增高(P<0.05)。和血生络高剂量组较和血生络低剂量组与消心痛组表达明显增强(P<0.05)。
2 bFGF mRNA在大鼠左室心肌组织的表达及积分吸光度的比值
模型组(0.31±0.01)较假手术组(0.18±0.05)明显增强(P<0.05)。和血生络高剂量组(0.69±0.17)、和血生络低剂量组(0.49±0.04)、消心痛组(0.70±0.10)bFGF mRNA表达较模型组明显增强(P<0.05)。和血生络高剂量组与消心痛组较和血生络低剂量组表达明显增强(P<0.05)。
3 bFGF在血清中的含量
模型组(3.78±0.25)血清中bFGF含量较假手术组(2.99±0.42)增高,但无统计学差异(P>0.05)。和血生络低剂量组(4.32±1.29)亦较模型组增高但无统计学差异(P>0.05)。和血生络高剂量组(5.29±1.13)、消心痛组(5.04±2.10)较模型组bFGF含量明显增高(P<0.05)。
第四部分
“和血生络法”对基质金属蛋白酶-2及基质金属蛋白酶组织抑制剂-2的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏免疫组化法检测MMP-2及TIMP-2在大鼠心梗边缘区中的蛋白表达,用HMIAS-2000高清晰度彩色医学图文分析系统分析其平均光密度值。并与bFGF作相关性分析。
结果:
1 MMP-2在大鼠心梗边缘区中的蛋白表达
模型组(0.1490±0.0119)MMP-2蛋白表达较假手术组(0.0688±0.0072)明显增高(P<0.05)。和血生络高剂量组(0.2545±0.0161)、和血生络低剂量组(0.2174±0.0116)、消心痛组(0.2204±0.0109)MMP-2蛋白表达较模型组明显增高(P<0.05)。和血生络高剂量组强于消心痛组及和血生络低剂量组(P<0.05)。
2 TIMP-2在大鼠心梗边缘区中的蛋白表达
模型组(0.1328±0.0104)TIMP-2蛋白表达较假手术组(0.0835±0.0085)明显增高(P<0.05)。和血生络高剂量组(0.1878±0.0106)、消心痛组(0.1812±0.0105)较模型组TIMP-2蛋白表明显增高(P<0.05)。和血生络低剂量组(0.1690±0.0093)虽从数值看高于模型组,但并无统计学差异(P>0.05)。
3 MMP-2与TIMP-2比值变化
模型组(1.12±0.05)MMP-2/TIMP-2较假手术组明(0.82±0.03)显增高(P<0.05)。和血生络高剂量组(1.36±0.04)、和血生络低剂量组(1.29±0.04)、消心痛组(1.22±0.04)MMP-2/TIMP-2较模型组明显增高(P<0.05)。中药组强于消心痛组且和血生络高剂量组强于和血生络低剂量组(P<0.05)。
4 bFGF与MMP-2、MMP-2/TIMP-2的相关性分析
将实验二心肌bFGF蛋白表达与MMP-2、MMP/TIMP的表达进行相关性分析,结果bFGF与MMP-2、MMP/TIMP的表达均呈正相关,r分别为0.967、0.926,P<0.01。
第五部分
“和血生络法”对粒细胞集落刺激因子的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取8只大鼠的血清ELISA法检测G-CSF在血清中的含量。
结果:
1血清中G-CSF的含量
模型组血清G-CSF含量为31.15±5.12较假手术组15.34±3.02明显增高(P<0.05)。和血生络高剂量组、和血生络低剂量组、消心痛组血清G-CSF含量分别为66.61±8.46、38.23±9.76、52.59±7.18较模型组明显增高(P<0.05)。和血生络高剂量组、消心痛组较和血生络低剂量组明显增高(P<0.05),且和血生络高剂量组高于消心痛组(P<0.05)。
第六部分
“和血生络法”对PI3K/Akt和MEK/ERK信号转导通路的影响
方法:
健康雄性Wistar大鼠通过结扎冠状动脉左前降支建立实验性心肌梗死动物模型。选取造模成功大鼠48只,按照随机数字表法随机分为模型组(M)、消心痛组(Y)、和血生络低剂量组(D)、和血生络高剂量组(G),每组12只;另设假手术组(J)大鼠12只。术后24h灌胃给药,药物干预3周后,每组取6只大鼠心脏组织RT-PCR法检测AKT mRNA在大鼠左室心肌组织的表达,用BIO-PROFIF凝胶图象分析系统对目的电泳条带进行分析,以相应的内参电泳条带作为参照,结果以两者之积分吸光度的比值表示。每组取6只大鼠心脏组织Western blot检测ERK1/2在大鼠左室心肌组织的表达,用凝胶定量软件Quantity-One-version 4.6.2测定各条带的积分吸光度,以积分吸光度代表蛋白质的表达量。
结果:
1 AKT mRNA在大鼠左室心肌组织的表达
假手术组大鼠为0.17±0.01,模型组大鼠为0.26±0.02,较假手术组大鼠显著增高(P<0.05)。和血生络高剂量组、和血生络低剂量组及消心痛组大鼠分别为0.60±0.02、0.42±0.01、0.58±0.01。各治疗组明显高于模型组(P<0.05)。和血生络高剂量组与消心痛组明显优于和血生络高低剂量组。和血生络高剂量组明显优于消心痛组(P<0.05)。
2 ERK1/2在大鼠左室心肌组织的表达
假手术组大鼠为25470±1456,模型组大鼠为33703±488,较假手术组大鼠显著增高(P<0.05)。和血生络高剂量组、低剂量组及消心痛组大鼠分别为54335±1021、40146±289、38361±1200,各治疗组明显高于模型组(P<0.05)。和血生络高剂量组明显优于和血生络低剂量组及消心痛组(P<0.05)。和血生络低剂量组明显优于消心痛组(P<0.05)。
结论:
1“和血生络法”是冠心病的基本治法。
2和血生络法能够缩小心肌梗死范围,对大鼠心肌梗死具有治疗作用。
3和血生络法能够增加心肌梗死边缘区MVC/MVD,对缺血心肌具有促血管新生作用,从而有助于局部缺血状态的改善。
4和血生络法能增强心肌组织中bFGF蛋白和基因的表达,提高血清中bFGF的含量。由于bFGF是血管新生的重要生长因子,所以和血生络法促进bFGF产生是其促进血管新生的机制之一。
5和血生络法能够促进MMP-2的分泌,改变MMP-2、TIMP-2的比例,从而促进基底膜的降解,有利于内皮细胞的增殖和游走,为血管新生提供保障。
6和血生络法能提高血清中G-CSF含量,而G-CSF是骨髓中内皮祖细胞强有力的动员剂,可间接推论和血生络法通过动员内皮祖细胞进入外周血而促进血管新生。
7和血生络方能明显提高心肌中Akt及ERK1/2的表达量。说明和血生络法可通过激活Raf/MEK/ERK和PI3K/Akt两条途径促进内皮细胞的增殖、迁移而实现了血管新生。
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 I
Coronary Heart Disease and Angiogenesis in TCM and the Presentation of Hexue Shengluo Method
The 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 II
Establishment of Rat Model with Myocardial Infarction and Effects of Hexue Shengluo Method on Angiogenesis
Methods:
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 models
Rats 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 infarction
Myocardial 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 infarction
The 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 changes
High 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.
PartⅢ
Effects of Hexue Shengluo Method on bFGF
Methods:
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 infarction
The 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 absorbance
The 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 serum
The 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).
PartⅣ
Effects of Hexue Shengluo method on MMP-2 and TIMP-2
Methods:
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 infarction
The 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-2
The 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-2
There 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).
PartⅤ
Effects of Hexue Shengluo method on G-CSF
Methods:
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 serum
The 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).
PartⅥ
Effects of Hexue Shengluo method on PI3K/Akt and MEK/ERK signal transduction pathway
Methods:
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 rats
The 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 rats
Compared 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.
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