| A common cardiovascular disease, coronary heart disease(CHD) belongs to the TCM category of “chest impediment” or “heart pain”, and is believed as a deficiency syndrome in origin(mostly because of Qi deficiency) and excess in symptom(mostly because of blood stasis). Currently, many of the studies on the effects of TCM treatment of CHD are evaluations of single clinical effect or studies of pharmacological mechanism. Systematic, comprehensive and step-by-step studies on the core ingredient, principles, clinical effects and pharmacological mechanism of TCM treatment of CHD are very few. Drawing from the author’s adviser’s rich clinical experiences and extensive TCM literature and data, this study explored the rules of TCM medication in treating CHD, found the primary principle in treating CHD being the Yiqihuoxue(enriching Qi and activating blood) method after studying representative formulas from Traditional Chinese Medicine. Further, adopting the systematic evaluation method from evidence-based medicine and examining the clinical experiments in treating CHD with Yiqihuoxue formulas, the author evaluated the clinical effects and safety of combining Yiqihuoxue formulas and identified two key representative ingredients of such formulas: Huangqi and Danshen. Adopting experimental methods from Cell and Molecular Biology, the author extracted active ingredients Astragaloside IV and Tanshinone IIA, observed their effects on the expression of Mesenchymal stromal cells Connexin protein, function regulation of Gap junction channel and promoting new angiogenesis ability after differentiation, in hope of providing research directions for the R&D of TCM treatment of CHD and effective intervention measures.Part one Rules of medication in Traditional Chinese Medicine · Famous Formulas for treating CHD1. PurposeStudy the rules and principles of medication in Traditional Chinese Medicine · Famous Formulas for treating CHD.2. Research methodSearch Traditional Chinese Medicine(CD ROM, 5th edition) with “chest impediment”, “heart pain”, “palpitation”, “coronary heart disease”, “angina pectoris” and “myocardial ischemia” as key words, content search; formulate a set of inclusion and exclusion standards; select and compile formula data for treating chest stuffiness and pains; set up an Excel data base; analyze data with SPSS19.0.3. Results264 documents were found from Traditional Chinese Medicine · Famous Formulas, which amount to 80,000 words. Sifting through inclusion and exclusion standards, 100 formulas treating coronary heart disease that meet our criteria were selected. Results: ①rules of treating chest stuffiness and pains: mainly to enrich Qi and tonify deficiency, activating blood and removing stasis; ② frequency of herbs used in CHD treatment:(from high to low) Danshen, Huangqi, Ginseng, Chuanxiong, Bingpian, etc.;③ effects of treatment: 16 categories of TCM herbs were identified according to functions: herbs that tonify deficiency appear the most frequent(33%), of which a high percentage(43%) tonifies Qi; herbs that remove blood stasis take up 14%; 34%herbs taste sweet, 30% acrid; mainly warming herbs. Channel tropism: mainly for the heart(23%)、spleen(18%)、liver(18%)and channels.4. ConclusionMost frequently used three herbs in Traditional Chinese Medicine · Famous Formulas for treating chest stuffiness and pains are Danshen, Huangqi and Ginseng. Therefor, key treating methods are enriching Qi, activating blood, at the same time regulating Qi and soothing nerves.Part two Systematic evaluation of the clinical effects of combining Yiqihuoxue formulas in treating CHD1. PurposeSystematic review of the literature of combining Yiqihuoxue formulas in treating CHD and evaluation of their clinical effects and safety.2. Research method2.1 Search in databases such as China Biology Medicine disc(CBM), CNKI, Pubmed etc.(search date: 2010-2014). Using evidence-based research method, obtain the randomized and semi-randomized controlled clinical trials in treating CHD with Yiqihuoxue formula combinations; select accurately the targeted literature through rigorous inclusion and exclusion standards.2.2 Compile data extraction form, extract basic research information, baseline information, complicating disease information, intervention measures and outcome indicators by two person independently; refer to the third party when results are inconsistent; evaluation of data with improved Jadad Scale.2.3 Combined analysis of the effect size of clinical effects with Rev Man 5.3 and systematic review of the clinical effectiveness of combining Yiqihuoxue formulas in treating CHD.2.4 Evaluation of clinical safety of combining Yiqihuoxue formulas in treating CHD with descriptive research methods.3. Results3.1In indicators such as overall clinical effects, TCM symptom effects, angina effects and electrocardiogram effects, the combination of western treatment and TCM Yiqihuoxue formulas is better than western methods alone, and statistically significant(P<0.05)in overall clinical curative effects, TCM syndrome, angina pectoris and ECG curative effects.3.2 Compared with pure western treatment, in the combination of western methods and TCM Yiqihuoxue formulas, the combined OR [95%CI]of the overall clinical curative effects and ECG curative effects are 3.74 [2.08,6.74]、2.23 [1.92,3.85]respectively and statistically significant(P<0.05). This suggests that the combination of western methods featuring Isosorbide Dinitrate and TCM Yiqihuoxue formulas in treating coronary heart disease is better than pure regular western methods featuring Isosorbide Dinitrate.3.3 Compared with regular western treatment featuring Isosorbide Dinitrate and Aspirin, in the combination of western methods and TCM Yiqihuoxue formulas, the combined OR[95%CI] of overall clinical curative effects, TCM syndrome, angina pectoris and ECG curative effects is 4.48 [3.25,6.19]、5.21 [2.13,12.75]、2.77 [1.57,4.88]、2.57 [1.92,3.45] respectively, which is significant statistically(P<0.05), which suggests that the combination of regular western methods featuring Isosorbide Dinitrate and Aspirin and TCM Yiqihuoxue formulas in treating coronary heart disease is better than pure regular western methods featuring Isosorbide Dinitrate and Aspirin.Observation of the adverse reaction of the 2900 cases of CHD patients included in our research showed that 5 cases in the group treated with the combination of western methods and TCM Yiqihuoxue formulas had adverse reactions which are gastrointestinal tract reactions such as nausea and vomiting; 14 cases in the regular western treatment group had adverse reactions such as dizziness, nausea, lack of strength, etc.4. Conclusion4.1The indicators of overall clinical effects, TCM syndrome, angina pectoris and ECG effects of the group with the combination of regular western treatment and TCM Yiqihuoxue formulas are better than the group with purely regular western methods featuring Isosorbide Dinitrate and Aspirin.4.2 The treatment of CHD with TCM Yiqihuoxue formula combinations has fewer adverse reactions, therefor, comparatively safer.Part Three Research on the effects of Tanshinone IIA, Astragaloside on MSC connexin expression and promoting angiogenesis1. PurposeBased on the regulating function of Tanshinone IIA, Astragaloside and their Combination on MSC Connexin Expression and GJC, we attempt to investigate whether this combination can promote angiogenesis, to provide empirical evidence for treating CHD with the combination of Tanshinone IIA and Astragaloside.2. Research method2.1Using the whole bone marrow adherent method, rats’ MSCs were cultured separately and observed under microscope; surface molecular identification of MSCs with flow cytometer; induced differentiation of endothelial cells; identification of FⅧ factor expression.2.2Separate the cells randomly into 4 groups and intervene for 72 hours: ① Tanshinone IIA; ② Astragaloside;③combination group;④blank group.2.3 Detection of induced MSCs for the mRNA and protein expression of Cx37、Cx40、Cx43 with real-time quantitative PCR and Western blot respectively.2.4 Detection of endothelial cells gap junctional intercellular communication through Wound Healing.2.5 Detection of induced endothelial cells multiplication through MTT.2.6Detection of cell cycle of induced endothelial cells with flow cytometer.2.7 Detection of endothelial cells’ capillary tube structure forming ability through stereoscopic cultivation of three dimensional matrix.3. Results3.1 The cells obtained through whole bone marrow adherent method looked like short spindle or fusiform. Flow cytometry detection suggests positive CD44、CD90expression and negative CD45、CD34 expression, showing they are mesenchymal stem cells. Induced by VEGF, Immunohistochemistry of them suggests positive Factor VIII-related antigen expression, which confirms that induced cells are endothelial cells.3.2 Real-time quantitative PCR suggests that the expression of Cx37、Cx40、Cx43m RNA can be seen from Yiqihuoxue combination group, Tanshinone IIA group, Astragaloside group and the blank group. The expression is strongest in the combination group, then the Tanshinone IIA group, then Astragaloside group, then the blank group. The difference is statistically significant(P<0.05). Western blot detection suggests that the protein expression of Cx37、Cx40、Cx43 of induced MSCs is higher in the combination group and exibits statistical difference; Astragaloside group a little lower than that of Tanshinone IIA group with no statistical difference.3.3 Dye wound healing experiment suggests that after 24 hours the migration distance of the combination group is 99±16 mm; Tanshinone IIA group, 72±6.5mm; Astragaloside group, 68±8.9 mm; the blank group, 50±10 mm. Difference between the latter two groups is statistically significant(P<0.05), which suggests that Chinese monomer herbs can improve intercellular gap junction communication function, of which the combination group is stronger than that of the Tanshinone IIA group and the Astragaloside group, and the lattwe two shows no significant difference.3.4 Cell multiplication detection through MTT suggests that the proportion of cell multiplication in the blank group is obviously lower than that of other groups, with that of thecombination group highest, and that of Tanshinone IIA group higher than the Astragaloside group. The difference is statistically significant(P<0.05).3.5Flow cytometer detection indicates that the proportion of s-phase cells in the blank group is obviously lower than that of other groups, with that of thecombination group highest, and that of Tanshinone IIA group higher than the Astragaloside group. The difference is statistically significant(P<0.05).3.6Detection of endothelial cells’ capillary tube structure forming ability through stereoscopic cultivation of three dimensional matrix suggests that there is no obvious tube structure formation in the blank group, whereas the other three groups all show visible tube structure formation, which that of the combination group the most complete.4. ConclusionTanshinone IIA, the Astragaloside and their combination can all improve Cx37, Cx40 and Cx43 protein and m RNA expression, gap junction communication function, endothelial cells’ multiplication ability and tube structure forming ability after differentiation induction, with those of the combination group the highest, those of Tanshinone IIA group higher than the Astragaloside group and statistically significant(P<0.05). |
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