| 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 expressionDetect 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 hypertension2.Effect of FXS on the pulmonary hypertension rats lung tissue VEGF, TGF-β1/Smad4 pathway molecule expressionImmunohistochemistry 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. |
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