肺心舒对野百合碱诱导肺心病肺动脉高压大鼠影响的药效机制研究
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摘要
目的:
慢性肺源性心脏病是严重危害人体健康的常见病、多发病,其发病率呈上升趋势,80%～90%由慢性阻塞性肺疾病(COPD)引起,已引起医学界的广泛重视。肺动脉高压是慢性肺源性心脏病发病过程中的重要环节,其产生及严重程度明显影响着COPD和肺心病的病程及预后。肺血管构型重塑是造成肺血管阻力持续升高和肺动脉高压发生、发展的主要原因。如何降低肺动脉高压,逆转肺血管重塑是当前肺心病或肺血管病研究的重要内容之一。
肺动脉高压的发生涉及细胞异常、分子介质和遗传因素等多个途径,内皮细胞、平滑肌细胞、成纤维细胞和血小板等多种细胞异常参与其形成,多种血管活性物质的失衡促进其发生,遗传因素在其发病中也起重要作用。
目前对肺动脉高压肺血管重塑机理的研究深入到细胞因子、信号转导通路的层面,但对这些细胞因子信号转导的研究多是在单一因子水平进行,对因子之间的级联通路的研究尚未完全明了。因此各种细胞生长因子在肺动脉高压及肺血管重构过程中的作用日益受到人们的关注。
在肺动脉高压的治疗上,目前尚无较成熟的治疗药物和措施,而中医药治疗肺心病肺动脉高压在临床工作中显示出了独特的优势,中医理论研究认为久病肺虚为肺心病发病的重要前提,感受外邪为其发病主要的诱发因素,疾病的发生发展与肺、脾、肾、心的关系密切,脏腑功能失调是加重病情、病程进展的主要原因,痰浊与瘀血交阻是肺心病肺动脉高压的主要病理基础,本虚与标实是其病机的主要特点。虚、痰、瘀贯穿疾病的自始至终。
本研究针对病机,总结张静生导师多年临床经验,结合现代药理研究,采用益气、活血、化痰法,拟定中药复方肺心舒,用于治疗慢性肺源性心脏病肺动脉高压症,并通过观察肺心舒对野百合碱致肺心病肺动脉高压大鼠肺血管内皮功能、肺血管重塑的影响以及肺心舒对野百合碱致肺心病肺动脉高压大鼠肺组织血管中VEGF、TGF-β1/Smad4传导通路分子表达的影响两个层面,从整体、器官、分子等不同水平,从形态学、病理学、功能、代谢等多方面探讨野百合碱诱导大鼠肺心病肺动脉高压的部分发病机制以及肺心舒干预肺心病肺动脉高压的可能的起效途径和作用靶点,为中药复方制剂治疗肺心病肺动脉高压的深入研究,也为抗肺动脉高压新药的开发提供一定的理论和实验依据。充分体现中医药的作用优势。
材料与方法:
1肺心舒对野百合碱致肺心病肺动脉高压大鼠肺血管内皮功能及肺血管重塑的影响本课题选用野百合碱(MCT)诱导的大鼠慢性炎性肺动脉高压动物模型,模拟肺心病急性发作期肺动脉高压。选用105只健康雄性Wister大鼠,按随机法均分为7组,即空白对照组、模型对照组、肺心舒高剂量组、肺心舒低剂量组、肺心舒预防组、西药对照组、中西药联合组。每组15只。第1天,空白对照组皮下注射生理盐水2ml/kg,其余6组均腹腔注射野百合碱60mg/kg制模。空白对照组、模型对照组于第2-30天每日一次蒸馏水10ml/kg灌胃;预防组第2-30天每日一次肺心舒煎剂(生药量1.4g/ml)10ml/Kg灌胃。中药高、低剂量组、西药对照组、中西药联合组于第2-15天每日一次蒸馏水10ml/Kg灌胃;第16-30天肺心舒高、低剂量组每日一次肺心舒煎剂高剂量(生药量5.6g/ml)、低剂量(生药量1.4g/ml),10ml/Kg灌胃。西药对照组每日一次硝苯地平20mg/kg,10ml/Kg灌胃。中西药联合组每日一次硝苯地平20mg/kg+肺心舒煎剂(生药量1.4g/ml)10ml/Kg灌胃。
通过对各组大鼠的一般情况与生存率、体重变化情况、肺动脉压(mPAP)及右室压(RSVP)、右心指数[RV/(LV+S)]和右室/体重(RV/BW)比、血ET-1和NO水平、肺血管病理形态学变化(采用HE染色及病理图象分析系统观察肺小动脉形态学改变及对肺小动脉管壁厚度与血管外径百分比(T/D)、管壁面积与血管面积(W/V)百分比进行统计学分析)的研究,观察肺心舒对肺心病肺动脉高压模型大鼠整体形态、生理、病理形态学、功能、代谢等方面的影响。
数据以“均数±标准差”( X±S)表示,采用SPSS 15.0统计学软件进行单因素方差分析,各组间两两比较采用LSD法,以P<0.05为具有统计学意义。
2肺心舒对肺心病肺动脉高压模型大鼠肺组织VEGF、TGF-β1/Smad4传导通路分子表达的影响
通过免疫组织化学观察研究,结合半定量分析技术,检测血管内皮生长因子(VEGF)、转化生长因子-β1(TGF-β1)、Smad4在各组大鼠肺血管中表达情况;采用逆转录聚合酶链法(RT-PCR)检测VEGFmRNA、TGF-β1mRNA、Smad4 mRNA在肺组织中的表达水平。进一步从免疫组化及分子生物学层面探讨肺心病肺动脉高压的部分发病机制及肺心舒的部分药效机制。
结果:
1肺心舒对野百合碱致肺心病肺动脉高压大鼠肺血管内皮功能及肺血管重塑的影响。大鼠一般情况观察结果显示:MCT造模组腹腔注射1周后与空白对照组比较不同程度地逐渐出现活动减少,倦卧,食量减少,第2周末开始出现毛发枯糙,活动明显减少,体重下降,甚至出现喘息,第3周末更趋严重,鼻部及唇周紫绀,甚至出现严重右心衰竭、死亡,各药物干预组均可不同程度的改善MCT模型大鼠精神状态、毛发色泽、呼吸、活动、食量、体重等状况,肺心舒预防组和高剂量组明显优于西药组,尤以中药预防组效果显著,表明肺心舒可明显改善模型大鼠的整体状态、提高生存率。
血流动力学指标测定、病理形态学观察及血中ET-1和NO指标检测结果显示:与空白对照组比较,模型组的mPAP、RVSP、RV/(LV+S)、RV/BW均明显增高;结合HE染色并病理图像分析,光镜下观察肺小动脉壁明显增厚,平滑肌增生肥厚明显,内膜增生和炎性细胞浸润,管腔明显狭窄,腺泡内肺微小动脉肌型化,血管周围炎性细胞浸润明显,肺血管损伤明显,内皮细胞变性肿胀,突向血管腔内,甚至坏死脱落;管壁厚度与血管外径百分比(T/D)、管壁面积与血管面积(W/V)百分比明显增高;血浆ET-1水平显著升高,血清NO水平显著降低,表明肺心病肺动脉高压模型复制成功。与模型组比较,各治疗组以上指标明显改善,说明各治疗组药物均能不同程度的降低大鼠肺动脉压、右室压、右心室重量,改善内皮功能、逆转肺血管重塑,中药各组及中西药联合组均优于西药组,组间比较以预防组与中西药联合组效果更好,且均优于中药高、低剂量组。中药高剂量组有优于低剂量组趋势,但无统计学意义。结果表明:肺心舒对MCT大鼠肺动脉高压有一定预防保护作用,中药预防和中西药联合治疗效果较显著。其可能的机制为:通过保护血管内皮功能、减少细胞外基质中生长因子(如转化生长因子β)等的生成,防止肺血管重构,改善肺血流动力学状态,以减低右心室的后负荷,改善右心功能,从而逆转肺动脉高压。
2肺心舒对肺心病肺动脉高压模型大鼠肺组织VEGF、TGF-β1/Smad4传导通路分子表达的影响
免疫组化检测结果显示:显微镜下可见阳性表达为棕黄色颗粒。在空白对照组肺组织中肺小动脉血管内皮细胞、部分的动脉壁的平滑肌细胞、毛细血管内皮细胞、肺泡内渗出的巨噬细胞和支气管的上皮细胞均呈弱阳性表达,模型组TGF-β1、VEGF、Smad4表达广泛,呈强阳性表达;与模型组比较,各治疗组TGF-β1、VEGF、Smad4阳性表达都有不同程度的抑制,结合蛋白半定量分析结果可见:模型组肺组织血管中TGF-β1、VEGF、Smad4表达显著高于空白对照组;各治疗组中TGF-β1、VEGF、Smad4表达均有不同程度的减少,肺心舒预防组、肺心舒高、低剂量组和中西药联合组TGF-β1、VEGF、Smad4表达与西药组比较显著降低;但肺心舒预防组与高、低剂量组和中西药联合组组间比较,以预防组与中西药联合组的表达降低明显,且优于高、低剂量组,预防组与中西药联合组组间比较无显著差异。高、低剂量组间无显著性差异。说明中药预防与中西药联合用药效果显著。
逆转录聚合酶链法(RT-PCR)检测结果显示:VEGFmRNA、TGF-β1mRNA、Smad4 mRNA蛋白半定量分析结果可见在空白对照组VEGFmRNA、TGF-β1mRNA、Smad4mRNA在肺组织中基因转录表达水平很低,模型组VEGFmRNA、TGF-β1mRNA、Smad4mRNA表达明显增强,两组比较有显著性差异;与模型组比较各治疗组VEGFmRNA、TGF-β1mRNA、Smad4mRNA表达水平都有不同程度的降低,说明各治疗组大鼠肺组织VEGFmRNA、TGF-β1mRNA、Smad4mRNA表达水平均受不同程度抑制,与西药组比较,预防组与肺心舒高、低剂量组和中西药联合组其表达水平降低明显,而以预防组与中西药联合组有更显著降低;肺心舒高剂量组略优于低剂量组,但中药高、低剂量组间比较无显著性差异。
结果表明,VEGF、TGF-β1及Smad4参与了MCT诱导大鼠肺心病肺动脉高压的发生,并且VEGF和TGF-β1、Smad4相互作用参与肺动脉高压及肺血管重塑,肺心舒能明显抑制肺心病肺动脉高压大鼠肺组织、血管中VEGF、TGF-β1、Smad4蛋白含量及mRNA的表达,从免疫组化和分子生物学层面显示了肺心舒降低肺心病肺动脉高压的发生和发展程度的部分机制。
结论:
1腹腔一次性注射野百合碱(60mg/kg)成功复制肺心病肺动脉高压模型,并模拟肺血管重塑的病理改变。模型大鼠内源性内皮素-1(ET-1)表达增加、一氧化氮(NO)表达减少并与肺动脉血压增高和右心肥厚并存。VEGF、TGF-β1及Smad4参与了MCT诱导大鼠肺心病肺动脉高压的发生与发展,并且VEGF和TGF-β1、Smad4相互作用参与了肺动脉高压及肺血管重塑,TGF-β1促进VEGF的表达,且受Smad4的调控。
2肺心舒对MCT诱导肺心病肺动脉高压大鼠有一定预防保护作用。其可能的作用机制与降低大鼠血浆ET-1,增加NO表达,保护血管内皮功能,减少细胞外基质中转化生长因子β的生成,从而防止肺血管重构,改善肺血流动力学状态,改善右心功能有关;可能通过对VEGF和TGF-β1的表达及其Smad4蛋白信号转导通路的抑制调节,阻止肺血管重塑,降低肺动脉高压,延缓病情进展。
3肺心舒预防治疗和中西药联合治疗肺心病肺动脉高压效果较显著。体现了中医药预防治疗及中西药联合治疗肺心病肺动脉高压的优势,充分展示了中医“治未病”思想在肺心病防治过程中应用的广阔前景。为中医多角度、多中心、多靶点防治肺心病肺动脉高压提供思路和方法。
Purpose:
Chronic pulmonary heart disease is a serious hazard to human health common disease, the Frequently occurring disease, and its incidence is upward trend, of which 80% to 90% is caused by chronic obstructive pulmonary disease (COPD), which has attracted extensive attention of the medical profession. Moreover pulmonary hypertension is an important part of the process of a chronic pulmonary heart disease, of which the generation and severity affect significantly the course and prognosis of COPD and pulmonary heart disease. And pulmonary vascular structural remodeling is the main reason of causing sustained increasing in pulmonary vascular resistance and the development of pulmonary hypertension. How to reduce pulmonary hypertension, and reverse pulmonary vascular remodeling are one of the important elements of the current research about pulmonary heart disease or lung disease.
Pulmonary hypertension involve abnormal cellular、molecular and genetic factors, and other media channels; And that endothelial cells, smooth muscle cells, fibroblasts and platelets, and other abnormal cells involve in the formation of it; And the imbalance of multiple vasoactive substances promote its occurrence; Further more genetic factors also play the important role in the pathogenesis of it.
The current mechanism of vascular remodeling in pulmonary hypertension research have been deep into the level of cell factor and signal transduction, but these studies of cytokines signal transduction mostly is the level of a single factor, moreover the studies of cascade pathway between factors is not yet fully understood. Therefore, the role of various cell growth factors in the process of pulmonary hypertension and pulmonary vascular remodeling are having got increasing number of researchers’attention.
In the treatment of pulmonary hypertension there is not more mature treatment drug and measures, while the TCM treatment of pulmonary hypertension demonstrated the unique advantages in clinical work. On the basis of Chinese medical theory ,it is argued that macronosia deficiency syndrome of the lung as an important prerequisite for the pathogenesis of pulmonary heart disease, invasion of exogenous pathogen as the main predisposing factor for the pathogenesis of pulmonary heart disease. The occurrence and development of pulmonary heart disease is closely linked to lung, spleen, kidney, heart. Phlegm and blood stasis obstructing cross is a major pathological basis of pulmonary hypertension, deficiency of Ben and repletion of Biao is actually the main features of its pathogenesis.
In this study, the therapies of tonifying qi, invigorating the circulation of blood and reducing phlegm were used to protocol a compound preparation named“Fei Xinshu”(FXS). It used to treat the chronic pulmonary heart disease caused by pulmonary hypertension. Discuss parts of the pathogenesis mechanism of pulmonary hypertension of the cor pulmonale rats induced by monocrotaline,and the possible means and targets in effect of FXS intervention in pulmonary hypertension, and fully reflect the predominance of Chinese medicine by observing the two levels of FXS on pulmonary vascular endothelial function, pulmonary vascular remodeling effect and on molecules expression of VEGF,TGF-β1/Smad4 conducting pathway in lung vascular effect of the rats of pulmonary hypertension induced by monocrotaline, from the morphology, function, metabolism and other aspects and from whole,organs,molecular and different levels. Provide the academic and experimental basics for that Traditional Chinese Medicine Compound cure pulmonary hypertension and exploitation of new drugs which against pulmonary hypertension.
Material and method:
1 The effect of Pulmonary diastolic on endothelial function and pulmonary vascular remodeling of the rats which with Pulmonary hypertension induced by monocrotaline.
The subject simulates pulmonary of acute chronic cor pulmonale with animal model, made with rats, in chronic and phylogenic pulmonary hypertension induced by monocrotaline. Select 105 health Wister rats of male, and then randomly detach in average 7 groups,15 rats in every group, which are control group and model group and the group with high-dose FXS and group with low-dose FXS and group with prevention of FXS and WM controlled group and TCM and WM combined group. On the first day injected saline, 2ml/kg, beneath the skin to the control group and the others received injection of monocrotaline, 60mg/kg, to their abdominal cavity to made model.
10ml/kg distilled water have been instilled into both control group and model group’s stomach once every day in the 2nd-30th days; 10ml/kg the FXS decoction (raw dose 1.4g/ml) have been instilled into prevention group’s stomach once a day for the 2nd-30th days. High and low dose group of Chinese medicine, WM controlled group, and TCM and WM combined group have been injected with 10ml/kg of distilled water into the stomach once a day for 2nd-15th days; High and low dose group of Chinese medicine have been separately injected with high-dose FXS (raw dose 5.6g/ml)and low-dose FXS(raw dose 1.4g/ml),both with 10ml/kg of decoction once-daily for 16th-30th days. WM controlled group have been injected with 20mg/kg Nifedipine of 10ml/kg into the stomach once-daily for 16th-30th days. TCM and WM combined group have been injected with Nifedipine 20mg/kg + the decoction of FXS (raw dose 1.4g/ml) into the stomach, 10ml/kg once-daily for 16th-30th days.
Through the study of the general case and survival rate of pulmonary hypertension rats induced by MCT,Weight changes, Pulmonary artery pressure (mPAP) and right ventricular pressure (RSVP),Right ventricular index [RV / (LV + S)] and right ventricle / body weight (RV / BW) ratio, Blood levels of ET-1 and NO, And morphological changes of Pulmonary blood vessel(Analysis of the HE staining pathology images: systematically observing morphological changes in pulmonary artery,and statistically analyzing the pulmonary artery wall thickness which is accompanying terminal bronchiole ), FXS’s effects on the rats of pulmonary hypertension were observed.
Data was symbolized by "mean±standard deviation" ( X±S). SPSS 15.0 statistical software was used for one-way ANOVA. LSD method was used for pairwise comparison of groups, with P <0.05 as statistically significant. Effect of FXS on the pulmonary hypertension rats lung tissue VEGF, TGF-β1/Smad4 pathway molecule expression
Detect VEGF,TGF-β1 and Smad4 in the lung vasculature which is belong to the pulmonary hypertension rats induced by MCT expression, through the observation of immunohistochemistry, and combining Semi-quantitative analysis. RT-PCR is used for detect the mRNA levels of VEGF, TGF-β1, Smad4 expression in lung tissue, and effects of Fei xinshu on VEGF, TGF-β1 and Smad4; Then further explore the mechanism of the Fei xinshu effect on pulmonary hypertension from immunohistochemical and molecular aspects.
Results:
1. Effect of FXS on rats induced by MCT pulmonary vascular endothelial function and vascular remodeling.
The observations on the rats general case showed that: the MCT model group after intraperitoneal injection of 1 week compared with the control group began to emerge in varying degrees reduced activities, tired of lying, food intake, dry coarse fur appeared after 2 weeks, decreased activity, weight loss, even wheezing, the 3rd weekend more serious, nose and lip cyanosis, severe right heart failure and even death. Each drug group could improve to the spirit of MCT rats, the fur color, breathing, activity, appetite, weight and other conditions in varying degrees; the prevention group and high-dose group were significantly better than WM controlled group. Particularly TCM prevention group acquired obvious effect, indicating that Fei xinshu can significantly improve overall state of rats and the survival rate.The results of Hemodynamic indexes , Pathological observation and ET-1 and NO blood test indicator show that: The statistics of the model group, compared with the control group’s, the mPAP, RVSP, RV/(LV + S) and RV/BW were significantly higher; Combined with HE staining image analysis, light microscopy of pulmonary artery wall thickening, smooth muscle hypertrophy significantly, endometrial hyperplasia and inflammatory cell infiltration, luminal stenosis, small intra-acinar pulmonary arterial muscularization, perivascular inflammatory cell infiltration, marked pulmonary vascular injury, endothelial cell degeneration and swelling, convex to endovascular,or even necrotic; wall thickness and vessel radius percentage (T/D), wall area and vascular area (W/V) percentage significantly higher ,blood levels of ET-1 significantly increased, NO level was significantly lower, all of the results indicated that pulmonary hypertension model reproduced successfully. Compared with the model group, as indicators all above of the treatment groups decreased significantly, indicate the drug of treatment group can significantly lower pulmonary artery pressure, right ventricular pressure, right ventricular weight, and improve endothelial function, reverse pulmonary vascular remodeling. TCM groups’and TCM and WM combined group’s were better than WM group’s. With the comparison between two groups, Prevention group and TCM and WM combined group had better effect than the others. There were no significant differences between TCM high and low-dose group. The results showed: FXS shows a certain protective effect on MCT rats; Its mechanism is relevant to function of Protecting vascular endothelial, reverse pulmonary vascular remodeling, improve pulmonary hemodynamic in order to reduce the right ventricular after-load, and improve right ventricular function; The protective effect was no significant dose-dependent, Combined treatment of traditional Chinese medicine and western medicine is more significant effect. The mechanism may be that: protection of pulmonary endothelial cell function, and to reduce the formation of growth factors in extracellular matrix (such as transforming growth factorβ)can prevent pulmonary vascular from remodeling and reversing pulmonary hypertension
2.Effect of FXS on the pulmonary hypertension rats lung tissue VEGF, TGF-β1/Smad4 pathway molecule expression
Immunohistochemistry results show that: the brown granules can be positively expressed under the Microscope. In control group’s lungs pulmonary artery endothelial cells, part of the arterial wall smooth muscle cells, capillary endothelial cells, macrophages exuding from alveolus and bronchial epithelium showed weak positive expression, TGF-model groupβ1, VEGF, Smad4 expression in a wide range, showing very strongly positive expression ; compare with model group, TGF-β1, VEGF, Smad4 expression have different-levels inhibition of the each treatment group, combine with semi-quantitative analysis of protein shows that: TGF-β1, VEGF, Smad4 in the pneumonic blood vessel of the model group whose expression was significantly higher than the control group; TGF-β1, VEGF, Smad4 expression of the treatment group have reduction with different levels, and TGF-β1,VEGF ,Smad4 of that which is prevention of FXS group, FXS high and low dose group and Chinese and Western medicine combined group whose expressions were significantly reduced compare with the single western medicine group’s; but comparison between the each two groups , prevention of FXS group and pulmonary diastolic high and low dose group and western medicine combined group have no significant difference. Practically, the expression of prevention group and Chinese and western medicine combined group were significantly lower, effectiveness of that is more effective than other treatment groups, no significant difference between the two groups. TGF-β1, VEGF, Smad4 of, high and low dose of FXS, the group’s expressional level lower than western medicine groups’, but no significant difference between the two groups. Explained that the effectiveness of prevention is remarkable, which with Chinese medicine. And Western medicine combines with Chinese medicine has significant effectiveness.
Results of analytical method, which with mRNA-PCR showed that: through the results of VEGF, TGF-β1, Smad4mRNA whose semi-quantitative analysis of protein could understand that VEGF, TGF-β1, Smad4 whose genetic- transcription expressive level in lung tissue of the control group was very low, the expressive level of VEGF, TGF-β1, Smad4 in model group was significantly increased. The two groups’results were significantly different. Compared with model group, VEGF, TGF-β1, Smad4 of treatment groups whose genetic-transcription level, which was expressed, in the lower has a different degree. The phenomenon indicate that VEGF, TGF-β1, Smad4m in the lung tissue of rats, which of the treatment group ,whose expressive level of RNA was restrained in varying degrees. Prevention group, Chinese and western medicine group whose expressive level decreased significantly, but there were no differences between the two groups. Compare with Western group, gene expressive level of high and low dose FXS group also have significant reduce, but high and low doses of Chinese medicine groups were no significant differences.
The results showed that, VEGF, TGF-β1 and Smad4 participate in the pulmonary hypertension which is induced by MCT occurring; and VEGF and TGF-β1, Smad4 interaction involves in pulmonary hypertension and pulmonary vascular remodeling, FXS can inhibit significantly VEGF,TGF-β1 and Smad4 protein content and mRNA expression in the pulmonary hypertension rats’pulmonary vessels. From the immunohistochemical and molecular aspects of pulmonary hypertension reduce the incidence and the degree of development.
Conclusion:
1. Intraabdominal injection of monocrotaline, 60mg/kg, successfully replicated model of pulmonary hypertension and simulated patho- logical changes of pulmonary vascular remodeling. Expression of model rats’endothelin-1 (ET-1) has increased and expression of nitric oxide (NO) has decreased and coexists with both pulmonary artery blood pressure increasing and right ventricular hypertrophy. VEGF, TGF-β1 and Smad4 participate in the pulmonary hypertension which is induced by MCT occurring and developing; and VEGF and TGF-β1, Smad4 interaction involves in pulmonary hypertension and pulmonary vascular remodeling, TGF-β1 could promote the expression of VEGF, and be controlled by Smad4.
2. FXS have some protective effect on the pulmonary hypertension rats induced by MCT. The possible mechanism has to do with the function of protecting vascular endothelial, reducing the generation of transforming growth factorβin the extracellular matrix, thereby preventing pulmonary vascular remodeling, improving the status of pulmonary hemodynamic and improving the function of right ventricular; Maybe through the expression of VEGF and TGF-β1, and regulating inhibition in Smad protein signaling pathway, prevent pulmonary vascular remodeling, improve pathological changes of pulmonary artery, reduce pulmonary hypertension and delay the progression of the disease.
3.Both FXS prophylactic curative effect and combination therapy effect with TCM and WM on pulmonary hypertension are significant. So reflects the advantages of the TCM prevention and treatment, and the advantages of combination therapy with TCM and WM about pulmonary hypertension; fully demonstrate broad prospects for the TCM“prevention of disease”thought in the applying of pulmonary heart disease prevention and control. The advantages offer ideas and methods for the TCM multi-angle, multi-center, multi-target controlling pulmonary hypert- ension.
慢性肺源性心脏病是严重危害人体健康的常见病、多发病,其发病率呈上升趋势,80%～90%由慢性阻塞性肺疾病(COPD)引起,已引起医学界的广泛重视。肺动脉高压是慢性肺源性心脏病发病过程中的重要环节,其产生及严重程度明显影响着COPD和肺心病的病程及预后。肺血管构型重塑是造成肺血管阻力持续升高和肺动脉高压发生、发展的主要原因。如何降低肺动脉高压,逆转肺血管重塑是当前肺心病或肺血管病研究的重要内容之一。
肺动脉高压的发生涉及细胞异常、分子介质和遗传因素等多个途径,内皮细胞、平滑肌细胞、成纤维细胞和血小板等多种细胞异常参与其形成,多种血管活性物质的失衡促进其发生,遗传因素在其发病中也起重要作用。
目前对肺动脉高压肺血管重塑机理的研究深入到细胞因子、信号转导通路的层面,但对这些细胞因子信号转导的研究多是在单一因子水平进行,对因子之间的级联通路的研究尚未完全明了。因此各种细胞生长因子在肺动脉高压及肺血管重构过程中的作用日益受到人们的关注。
在肺动脉高压的治疗上,目前尚无较成熟的治疗药物和措施,而中医药治疗肺心病肺动脉高压在临床工作中显示出了独特的优势,中医理论研究认为久病肺虚为肺心病发病的重要前提,感受外邪为其发病主要的诱发因素,疾病的发生发展与肺、脾、肾、心的关系密切,脏腑功能失调是加重病情、病程进展的主要原因,痰浊与瘀血交阻是肺心病肺动脉高压的主要病理基础,本虚与标实是其病机的主要特点。虚、痰、瘀贯穿疾病的自始至终。
本研究针对病机,总结张静生导师多年临床经验,结合现代药理研究,采用益气、活血、化痰法,拟定中药复方肺心舒,用于治疗慢性肺源性心脏病肺动脉高压症,并通过观察肺心舒对野百合碱致肺心病肺动脉高压大鼠肺血管内皮功能、肺血管重塑的影响以及肺心舒对野百合碱致肺心病肺动脉高压大鼠肺组织血管中VEGF、TGF-β1/Smad4传导通路分子表达的影响两个层面,从整体、器官、分子等不同水平,从形态学、病理学、功能、代谢等多方面探讨野百合碱诱导大鼠肺心病肺动脉高压的部分发病机制以及肺心舒干预肺心病肺动脉高压的可能的起效途径和作用靶点,为中药复方制剂治疗肺心病肺动脉高压的深入研究,也为抗肺动脉高压新药的开发提供一定的理论和实验依据。充分体现中医药的作用优势。
材料与方法:
1肺心舒对野百合碱致肺心病肺动脉高压大鼠肺血管内皮功能及肺血管重塑的影响本课题选用野百合碱(MCT)诱导的大鼠慢性炎性肺动脉高压动物模型,模拟肺心病急性发作期肺动脉高压。选用105只健康雄性Wister大鼠,按随机法均分为7组,即空白对照组、模型对照组、肺心舒高剂量组、肺心舒低剂量组、肺心舒预防组、西药对照组、中西药联合组。每组15只。第1天,空白对照组皮下注射生理盐水2ml/kg,其余6组均腹腔注射野百合碱60mg/kg制模。空白对照组、模型对照组于第2-30天每日一次蒸馏水10ml/kg灌胃;预防组第2-30天每日一次肺心舒煎剂(生药量1.4g/ml)10ml/Kg灌胃。中药高、低剂量组、西药对照组、中西药联合组于第2-15天每日一次蒸馏水10ml/Kg灌胃;第16-30天肺心舒高、低剂量组每日一次肺心舒煎剂高剂量(生药量5.6g/ml)、低剂量(生药量1.4g/ml),10ml/Kg灌胃。西药对照组每日一次硝苯地平20mg/kg,10ml/Kg灌胃。中西药联合组每日一次硝苯地平20mg/kg+肺心舒煎剂(生药量1.4g/ml)10ml/Kg灌胃。
通过对各组大鼠的一般情况与生存率、体重变化情况、肺动脉压(mPAP)及右室压(RSVP)、右心指数[RV/(LV+S)]和右室/体重(RV/BW)比、血ET-1和NO水平、肺血管病理形态学变化(采用HE染色及病理图象分析系统观察肺小动脉形态学改变及对肺小动脉管壁厚度与血管外径百分比(T/D)、管壁面积与血管面积(W/V)百分比进行统计学分析)的研究,观察肺心舒对肺心病肺动脉高压模型大鼠整体形态、生理、病理形态学、功能、代谢等方面的影响。
数据以“均数±标准差”( X±S)表示,采用SPSS 15.0统计学软件进行单因素方差分析,各组间两两比较采用LSD法,以P<0.05为具有统计学意义。
2肺心舒对肺心病肺动脉高压模型大鼠肺组织VEGF、TGF-β1/Smad4传导通路分子表达的影响
通过免疫组织化学观察研究,结合半定量分析技术,检测血管内皮生长因子(VEGF)、转化生长因子-β1(TGF-β1)、Smad4在各组大鼠肺血管中表达情况;采用逆转录聚合酶链法(RT-PCR)检测VEGFmRNA、TGF-β1mRNA、Smad4 mRNA在肺组织中的表达水平。进一步从免疫组化及分子生物学层面探讨肺心病肺动脉高压的部分发病机制及肺心舒的部分药效机制。
结果:
1肺心舒对野百合碱致肺心病肺动脉高压大鼠肺血管内皮功能及肺血管重塑的影响。大鼠一般情况观察结果显示:MCT造模组腹腔注射1周后与空白对照组比较不同程度地逐渐出现活动减少,倦卧,食量减少,第2周末开始出现毛发枯糙,活动明显减少,体重下降,甚至出现喘息,第3周末更趋严重,鼻部及唇周紫绀,甚至出现严重右心衰竭、死亡,各药物干预组均可不同程度的改善MCT模型大鼠精神状态、毛发色泽、呼吸、活动、食量、体重等状况,肺心舒预防组和高剂量组明显优于西药组,尤以中药预防组效果显著,表明肺心舒可明显改善模型大鼠的整体状态、提高生存率。
血流动力学指标测定、病理形态学观察及血中ET-1和NO指标检测结果显示:与空白对照组比较,模型组的mPAP、RVSP、RV/(LV+S)、RV/BW均明显增高;结合HE染色并病理图像分析,光镜下观察肺小动脉壁明显增厚,平滑肌增生肥厚明显,内膜增生和炎性细胞浸润,管腔明显狭窄,腺泡内肺微小动脉肌型化,血管周围炎性细胞浸润明显,肺血管损伤明显,内皮细胞变性肿胀,突向血管腔内,甚至坏死脱落;管壁厚度与血管外径百分比(T/D)、管壁面积与血管面积(W/V)百分比明显增高;血浆ET-1水平显著升高,血清NO水平显著降低,表明肺心病肺动脉高压模型复制成功。与模型组比较,各治疗组以上指标明显改善,说明各治疗组药物均能不同程度的降低大鼠肺动脉压、右室压、右心室重量,改善内皮功能、逆转肺血管重塑,中药各组及中西药联合组均优于西药组,组间比较以预防组与中西药联合组效果更好,且均优于中药高、低剂量组。中药高剂量组有优于低剂量组趋势,但无统计学意义。结果表明:肺心舒对MCT大鼠肺动脉高压有一定预防保护作用,中药预防和中西药联合治疗效果较显著。其可能的机制为:通过保护血管内皮功能、减少细胞外基质中生长因子(如转化生长因子β)等的生成,防止肺血管重构,改善肺血流动力学状态,以减低右心室的后负荷,改善右心功能,从而逆转肺动脉高压。
2肺心舒对肺心病肺动脉高压模型大鼠肺组织VEGF、TGF-β1/Smad4传导通路分子表达的影响
免疫组化检测结果显示:显微镜下可见阳性表达为棕黄色颗粒。在空白对照组肺组织中肺小动脉血管内皮细胞、部分的动脉壁的平滑肌细胞、毛细血管内皮细胞、肺泡内渗出的巨噬细胞和支气管的上皮细胞均呈弱阳性表达,模型组TGF-β1、VEGF、Smad4表达广泛,呈强阳性表达;与模型组比较,各治疗组TGF-β1、VEGF、Smad4阳性表达都有不同程度的抑制,结合蛋白半定量分析结果可见:模型组肺组织血管中TGF-β1、VEGF、Smad4表达显著高于空白对照组;各治疗组中TGF-β1、VEGF、Smad4表达均有不同程度的减少,肺心舒预防组、肺心舒高、低剂量组和中西药联合组TGF-β1、VEGF、Smad4表达与西药组比较显著降低;但肺心舒预防组与高、低剂量组和中西药联合组组间比较,以预防组与中西药联合组的表达降低明显,且优于高、低剂量组,预防组与中西药联合组组间比较无显著差异。高、低剂量组间无显著性差异。说明中药预防与中西药联合用药效果显著。
逆转录聚合酶链法(RT-PCR)检测结果显示:VEGFmRNA、TGF-β1mRNA、Smad4 mRNA蛋白半定量分析结果可见在空白对照组VEGFmRNA、TGF-β1mRNA、Smad4mRNA在肺组织中基因转录表达水平很低,模型组VEGFmRNA、TGF-β1mRNA、Smad4mRNA表达明显增强,两组比较有显著性差异;与模型组比较各治疗组VEGFmRNA、TGF-β1mRNA、Smad4mRNA表达水平都有不同程度的降低,说明各治疗组大鼠肺组织VEGFmRNA、TGF-β1mRNA、Smad4mRNA表达水平均受不同程度抑制,与西药组比较,预防组与肺心舒高、低剂量组和中西药联合组其表达水平降低明显,而以预防组与中西药联合组有更显著降低;肺心舒高剂量组略优于低剂量组,但中药高、低剂量组间比较无显著性差异。
结果表明,VEGF、TGF-β1及Smad4参与了MCT诱导大鼠肺心病肺动脉高压的发生,并且VEGF和TGF-β1、Smad4相互作用参与肺动脉高压及肺血管重塑,肺心舒能明显抑制肺心病肺动脉高压大鼠肺组织、血管中VEGF、TGF-β1、Smad4蛋白含量及mRNA的表达,从免疫组化和分子生物学层面显示了肺心舒降低肺心病肺动脉高压的发生和发展程度的部分机制。
结论:
1腹腔一次性注射野百合碱(60mg/kg)成功复制肺心病肺动脉高压模型,并模拟肺血管重塑的病理改变。模型大鼠内源性内皮素-1(ET-1)表达增加、一氧化氮(NO)表达减少并与肺动脉血压增高和右心肥厚并存。VEGF、TGF-β1及Smad4参与了MCT诱导大鼠肺心病肺动脉高压的发生与发展,并且VEGF和TGF-β1、Smad4相互作用参与了肺动脉高压及肺血管重塑,TGF-β1促进VEGF的表达,且受Smad4的调控。
2肺心舒对MCT诱导肺心病肺动脉高压大鼠有一定预防保护作用。其可能的作用机制与降低大鼠血浆ET-1,增加NO表达,保护血管内皮功能,减少细胞外基质中转化生长因子β的生成,从而防止肺血管重构,改善肺血流动力学状态,改善右心功能有关;可能通过对VEGF和TGF-β1的表达及其Smad4蛋白信号转导通路的抑制调节,阻止肺血管重塑,降低肺动脉高压,延缓病情进展。
3肺心舒预防治疗和中西药联合治疗肺心病肺动脉高压效果较显著。体现了中医药预防治疗及中西药联合治疗肺心病肺动脉高压的优势,充分展示了中医“治未病”思想在肺心病防治过程中应用的广阔前景。为中医多角度、多中心、多靶点防治肺心病肺动脉高压提供思路和方法。
Purpose:
Chronic pulmonary heart disease is a serious hazard to human health common disease, the Frequently occurring disease, and its incidence is upward trend, of which 80% to 90% is caused by chronic obstructive pulmonary disease (COPD), which has attracted extensive attention of the medical profession. Moreover pulmonary hypertension is an important part of the process of a chronic pulmonary heart disease, of which the generation and severity affect significantly the course and prognosis of COPD and pulmonary heart disease. And pulmonary vascular structural remodeling is the main reason of causing sustained increasing in pulmonary vascular resistance and the development of pulmonary hypertension. How to reduce pulmonary hypertension, and reverse pulmonary vascular remodeling are one of the important elements of the current research about pulmonary heart disease or lung disease.
Pulmonary hypertension involve abnormal cellular、molecular and genetic factors, and other media channels; And that endothelial cells, smooth muscle cells, fibroblasts and platelets, and other abnormal cells involve in the formation of it; And the imbalance of multiple vasoactive substances promote its occurrence; Further more genetic factors also play the important role in the pathogenesis of it.
The current mechanism of vascular remodeling in pulmonary hypertension research have been deep into the level of cell factor and signal transduction, but these studies of cytokines signal transduction mostly is the level of a single factor, moreover the studies of cascade pathway between factors is not yet fully understood. Therefore, the role of various cell growth factors in the process of pulmonary hypertension and pulmonary vascular remodeling are having got increasing number of researchers’attention.
In the treatment of pulmonary hypertension there is not more mature treatment drug and measures, while the TCM treatment of pulmonary hypertension demonstrated the unique advantages in clinical work. On the basis of Chinese medical theory ,it is argued that macronosia deficiency syndrome of the lung as an important prerequisite for the pathogenesis of pulmonary heart disease, invasion of exogenous pathogen as the main predisposing factor for the pathogenesis of pulmonary heart disease. The occurrence and development of pulmonary heart disease is closely linked to lung, spleen, kidney, heart. Phlegm and blood stasis obstructing cross is a major pathological basis of pulmonary hypertension, deficiency of Ben and repletion of Biao is actually the main features of its pathogenesis.
In this study, the therapies of tonifying qi, invigorating the circulation of blood and reducing phlegm were used to protocol a compound preparation named“Fei Xinshu”(FXS). It used to treat the chronic pulmonary heart disease caused by pulmonary hypertension. Discuss parts of the pathogenesis mechanism of pulmonary hypertension of the cor pulmonale rats induced by monocrotaline,and the possible means and targets in effect of FXS intervention in pulmonary hypertension, and fully reflect the predominance of Chinese medicine by observing the two levels of FXS on pulmonary vascular endothelial function, pulmonary vascular remodeling effect and on molecules expression of VEGF,TGF-β1/Smad4 conducting pathway in lung vascular effect of the rats of pulmonary hypertension induced by monocrotaline, from the morphology, function, metabolism and other aspects and from whole,organs,molecular and different levels. Provide the academic and experimental basics for that Traditional Chinese Medicine Compound cure pulmonary hypertension and exploitation of new drugs which against pulmonary hypertension.
Material and method:
1 The effect of Pulmonary diastolic on endothelial function and pulmonary vascular remodeling of the rats which with Pulmonary hypertension induced by monocrotaline.
The subject simulates pulmonary of acute chronic cor pulmonale with animal model, made with rats, in chronic and phylogenic pulmonary hypertension induced by monocrotaline. Select 105 health Wister rats of male, and then randomly detach in average 7 groups,15 rats in every group, which are control group and model group and the group with high-dose FXS and group with low-dose FXS and group with prevention of FXS and WM controlled group and TCM and WM combined group. On the first day injected saline, 2ml/kg, beneath the skin to the control group and the others received injection of monocrotaline, 60mg/kg, to their abdominal cavity to made model.
10ml/kg distilled water have been instilled into both control group and model group’s stomach once every day in the 2nd-30th days; 10ml/kg the FXS decoction (raw dose 1.4g/ml) have been instilled into prevention group’s stomach once a day for the 2nd-30th days. High and low dose group of Chinese medicine, WM controlled group, and TCM and WM combined group have been injected with 10ml/kg of distilled water into the stomach once a day for 2nd-15th days; High and low dose group of Chinese medicine have been separately injected with high-dose FXS (raw dose 5.6g/ml)and low-dose FXS(raw dose 1.4g/ml),both with 10ml/kg of decoction once-daily for 16th-30th days. WM controlled group have been injected with 20mg/kg Nifedipine of 10ml/kg into the stomach once-daily for 16th-30th days. TCM and WM combined group have been injected with Nifedipine 20mg/kg + the decoction of FXS (raw dose 1.4g/ml) into the stomach, 10ml/kg once-daily for 16th-30th days.
Through the study of the general case and survival rate of pulmonary hypertension rats induced by MCT,Weight changes, Pulmonary artery pressure (mPAP) and right ventricular pressure (RSVP),Right ventricular index [RV / (LV + S)] and right ventricle / body weight (RV / BW) ratio, Blood levels of ET-1 and NO, And morphological changes of Pulmonary blood vessel(Analysis of the HE staining pathology images: systematically observing morphological changes in pulmonary artery,and statistically analyzing the pulmonary artery wall thickness which is accompanying terminal bronchiole ), FXS’s effects on the rats of pulmonary hypertension were observed.
Data was symbolized by "mean±standard deviation" ( X±S). SPSS 15.0 statistical software was used for one-way ANOVA. LSD method was used for pairwise comparison of groups, with P <0.05 as statistically significant. Effect of FXS on the pulmonary hypertension rats lung tissue VEGF, TGF-β1/Smad4 pathway molecule expression
Detect VEGF,TGF-β1 and Smad4 in the lung vasculature which is belong to the pulmonary hypertension rats induced by MCT expression, through the observation of immunohistochemistry, and combining Semi-quantitative analysis. RT-PCR is used for detect the mRNA levels of VEGF, TGF-β1, Smad4 expression in lung tissue, and effects of Fei xinshu on VEGF, TGF-β1 and Smad4; Then further explore the mechanism of the Fei xinshu effect on pulmonary hypertension from immunohistochemical and molecular aspects.
Results:
1. Effect of FXS on rats induced by MCT pulmonary vascular endothelial function and vascular remodeling.
The observations on the rats general case showed that: the MCT model group after intraperitoneal injection of 1 week compared with the control group began to emerge in varying degrees reduced activities, tired of lying, food intake, dry coarse fur appeared after 2 weeks, decreased activity, weight loss, even wheezing, the 3rd weekend more serious, nose and lip cyanosis, severe right heart failure and even death. Each drug group could improve to the spirit of MCT rats, the fur color, breathing, activity, appetite, weight and other conditions in varying degrees; the prevention group and high-dose group were significantly better than WM controlled group. Particularly TCM prevention group acquired obvious effect, indicating that Fei xinshu can significantly improve overall state of rats and the survival rate.The results of Hemodynamic indexes , Pathological observation and ET-1 and NO blood test indicator show that: The statistics of the model group, compared with the control group’s, the mPAP, RVSP, RV/(LV + S) and RV/BW were significantly higher; Combined with HE staining image analysis, light microscopy of pulmonary artery wall thickening, smooth muscle hypertrophy significantly, endometrial hyperplasia and inflammatory cell infiltration, luminal stenosis, small intra-acinar pulmonary arterial muscularization, perivascular inflammatory cell infiltration, marked pulmonary vascular injury, endothelial cell degeneration and swelling, convex to endovascular,or even necrotic; wall thickness and vessel radius percentage (T/D), wall area and vascular area (W/V) percentage significantly higher ,blood levels of ET-1 significantly increased, NO level was significantly lower, all of the results indicated that pulmonary hypertension model reproduced successfully. Compared with the model group, as indicators all above of the treatment groups decreased significantly, indicate the drug of treatment group can significantly lower pulmonary artery pressure, right ventricular pressure, right ventricular weight, and improve endothelial function, reverse pulmonary vascular remodeling. TCM groups’and TCM and WM combined group’s were better than WM group’s. With the comparison between two groups, Prevention group and TCM and WM combined group had better effect than the others. There were no significant differences between TCM high and low-dose group. The results showed: FXS shows a certain protective effect on MCT rats; Its mechanism is relevant to function of Protecting vascular endothelial, reverse pulmonary vascular remodeling, improve pulmonary hemodynamic in order to reduce the right ventricular after-load, and improve right ventricular function; The protective effect was no significant dose-dependent, Combined treatment of traditional Chinese medicine and western medicine is more significant effect. The mechanism may be that: protection of pulmonary endothelial cell function, and to reduce the formation of growth factors in extracellular matrix (such as transforming growth factorβ)can prevent pulmonary vascular from remodeling and reversing pulmonary hypertension
2.Effect of FXS on the pulmonary hypertension rats lung tissue VEGF, TGF-β1/Smad4 pathway molecule expression
Immunohistochemistry results show that: the brown granules can be positively expressed under the Microscope. In control group’s lungs pulmonary artery endothelial cells, part of the arterial wall smooth muscle cells, capillary endothelial cells, macrophages exuding from alveolus and bronchial epithelium showed weak positive expression, TGF-model groupβ1, VEGF, Smad4 expression in a wide range, showing very strongly positive expression ; compare with model group, TGF-β1, VEGF, Smad4 expression have different-levels inhibition of the each treatment group, combine with semi-quantitative analysis of protein shows that: TGF-β1, VEGF, Smad4 in the pneumonic blood vessel of the model group whose expression was significantly higher than the control group; TGF-β1, VEGF, Smad4 expression of the treatment group have reduction with different levels, and TGF-β1,VEGF ,Smad4 of that which is prevention of FXS group, FXS high and low dose group and Chinese and Western medicine combined group whose expressions were significantly reduced compare with the single western medicine group’s; but comparison between the each two groups , prevention of FXS group and pulmonary diastolic high and low dose group and western medicine combined group have no significant difference. Practically, the expression of prevention group and Chinese and western medicine combined group were significantly lower, effectiveness of that is more effective than other treatment groups, no significant difference between the two groups. TGF-β1, VEGF, Smad4 of, high and low dose of FXS, the group’s expressional level lower than western medicine groups’, but no significant difference between the two groups. Explained that the effectiveness of prevention is remarkable, which with Chinese medicine. And Western medicine combines with Chinese medicine has significant effectiveness.
Results of analytical method, which with mRNA-PCR showed that: through the results of VEGF, TGF-β1, Smad4mRNA whose semi-quantitative analysis of protein could understand that VEGF, TGF-β1, Smad4 whose genetic- transcription expressive level in lung tissue of the control group was very low, the expressive level of VEGF, TGF-β1, Smad4 in model group was significantly increased. The two groups’results were significantly different. Compared with model group, VEGF, TGF-β1, Smad4 of treatment groups whose genetic-transcription level, which was expressed, in the lower has a different degree. The phenomenon indicate that VEGF, TGF-β1, Smad4m in the lung tissue of rats, which of the treatment group ,whose expressive level of RNA was restrained in varying degrees. Prevention group, Chinese and western medicine group whose expressive level decreased significantly, but there were no differences between the two groups. Compare with Western group, gene expressive level of high and low dose FXS group also have significant reduce, but high and low doses of Chinese medicine groups were no significant differences.
The results showed that, VEGF, TGF-β1 and Smad4 participate in the pulmonary hypertension which is induced by MCT occurring; and VEGF and TGF-β1, Smad4 interaction involves in pulmonary hypertension and pulmonary vascular remodeling, FXS can inhibit significantly VEGF,TGF-β1 and Smad4 protein content and mRNA expression in the pulmonary hypertension rats’pulmonary vessels. From the immunohistochemical and molecular aspects of pulmonary hypertension reduce the incidence and the degree of development.
Conclusion:
1. Intraabdominal injection of monocrotaline, 60mg/kg, successfully replicated model of pulmonary hypertension and simulated patho- logical changes of pulmonary vascular remodeling. Expression of model rats’endothelin-1 (ET-1) has increased and expression of nitric oxide (NO) has decreased and coexists with both pulmonary artery blood pressure increasing and right ventricular hypertrophy. VEGF, TGF-β1 and Smad4 participate in the pulmonary hypertension which is induced by MCT occurring and developing; and VEGF and TGF-β1, Smad4 interaction involves in pulmonary hypertension and pulmonary vascular remodeling, TGF-β1 could promote the expression of VEGF, and be controlled by Smad4.
2. FXS have some protective effect on the pulmonary hypertension rats induced by MCT. The possible mechanism has to do with the function of protecting vascular endothelial, reducing the generation of transforming growth factorβin the extracellular matrix, thereby preventing pulmonary vascular remodeling, improving the status of pulmonary hemodynamic and improving the function of right ventricular; Maybe through the expression of VEGF and TGF-β1, and regulating inhibition in Smad protein signaling pathway, prevent pulmonary vascular remodeling, improve pathological changes of pulmonary artery, reduce pulmonary hypertension and delay the progression of the disease.
3.Both FXS prophylactic curative effect and combination therapy effect with TCM and WM on pulmonary hypertension are significant. So reflects the advantages of the TCM prevention and treatment, and the advantages of combination therapy with TCM and WM about pulmonary hypertension; fully demonstrate broad prospects for the TCM“prevention of disease”thought in the applying of pulmonary heart disease prevention and control. The advantages offer ideas and methods for the TCM multi-angle, multi-center, multi-target controlling pulmonary hypert- ension.
引文
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