The Effects And Its Mechanisms Of Dihydroartemisinin Intervention On Pulmonary Arterial Hypertension In Rats Induced By Monocrotaline

BackgroundPulmonary arterial hypertension (PAH) is a progressive disease marked by increased pulmonary artery resistance leading to right heart failure with a high mortality. PAH is histologically characterized by endothelial and smooth muscle cell proliferation, medial hypertrophy, inflammation, and thrombosis in situ. Drugs for the treatment of PAH mainly include calcium channel blockers, prostacyclin, endothelin-receptor-antagonists and phosphodiesterase-5-inhibitors etc, which represent the current therapeutic options. These drugs can block the main pulmonary artery through the neuroendocrine mechanisms of contraction, expansion pulmonary to show treatment effect on patients with early stage. But these drugs can only improve their symptoms and can not reverse the course of the disease. The available treatments for PAH are also quite limited nowadays. Therefore, it is necessary to search for some new drugs to treat pulmonary arterial hypertension which is effective, cheap and suitable to Chinese Domestic Condition.Artemisinin is an endoperoxide sesquiterpene lactone compounds and a highly effective antimalarial drug toxicity which is now mainly used for clinical treatment of malaria and its derivatives are dihydroartemisinin, artesunate, artemether and Artemisiaether. In recent years, further studies have showed, in addition to the role of anti-malaria, Artemisinine possesses effects such as anti-inflammatory, antitumor, radiosensitization, anti-bacterial sensitization, anti- fibrosis and so on. Immunological disease such as pulmonary fibrosis is one of the causes for Secondary PAH. Whether Artemisinin can inhibit pulmonary hypertension during pulmonary artery reconstruction by anti-fibrosis or other mechanisms to prevent pulmonary hypertension have rarely been reported. Dihydroartemisinin is the major metabolites of artemisinin drugs in the body, compared with artemisinin, DHA has a better water solubility, bioavailability higher, more effective antimalarial features. Established pulmonary hypertension models induced by the monocrotaline on rats were treated by DHA in this study, to investigate the effects of dihydroartemisinin on hemodynamics and pulmonary vascular remodeling in rat models of pulmonary hypertension treated by MCT; to investigate the effects of dihydroartemisinin on ultrastructure of right ventricular myocytes, the effects of apoptosis on pulmonary arterial smooth muscle cells and right ventricular myocytes in rat models of pulmonary hypertension treated by MCT; to investigate the effects of dihydroartemisinin on expression of VEGF and ICAM-1 of rat models of pulmonary hypertension treated by MCT and to observe whether DHA has a protective effect on rats with pulmonary hypertension and to explore its possible mechanisms.ObjectiveTo observe the effect of dihydroartemisinin on pulmonary vascular remodeling in rats with pulmonary hypertension, the mechanism of dihydroartemisinin role in the development of pulmonary vascular remodeling were explored.Methods1. To investigate the effects of dihydroartemisinin on hemodynamics and pulmonary vascular remodeling in a rat model of pulmonary hypertension.30 male SD rats were randomly divided into normal control group, pulmonary hypertension group and dihydroartemisinin intervention group (n=10 in each group). Pulmonary hypertension group and experiment group were given monocrotaline (60mg/kg) subcutaneous injection and the normal control group received doses of saline (60mg/kg, about 6ml/kg) subcutaneous injection. After three weeks, the experiment group were given dihydroartemisinin (60mg/kg/d, dissolved in vegetable oil) gavage and the other two groups were administered gavage doses of starch solution for three weeks. Then observe hemodynamic and morphology of each groups; the mean pulmonary artery pressure (mPAP) were observed by Catheterization; neointimal formation were observed by histopathological examination; the right ventricle hypertrophy index that right ventricle/left ventricle plus septum (RV/LV+S) of the change was computed; pulmonary artery wall thickness and lumen area was evaluated; pulmonary arterial without muscle level was evaluated and degree of pulmonary artery smooth muscle cell proliferation, by immunohistochemistry staining; the degree of neointimal proliferation was evaluated by VG staining; collagen formation was evaluated by Sirius red staining in myocardium.2. To investigate the effects of dihydroartemisinin on ultrastructure of right ventricular myocytes, the effects of apoptosis on right ventricular myocytes in a rat model of pulmonary hypertension.30 male SD rats were randomly divided into normal control group, pulmonary hypertension group and dihydroartemisinin experiment group (n=10 in each group), the method of animal model preparation is the same as the first part. Dissociate right ventricular, take the middle 1mm~3 2 to 3 myocardial specimen of right ventricular free wall, and immediately the line 2.5% glutaraldehyde - 1% osmium tetroxide double fixation; conventional ethanol, acetone, dehydrated step by step; epoxy resin embedding, ultra-thin slicing machine slices, uranyl acetate and lead citrate double staining to H-500 transmission electron microscope and photographed to observe the MCT-induced right ventricular cell ultrastructure changes.In situ Cell Death Detection, POD Kit instructions were referenced to carry out TUNEL assay. Paraffin sections, dewaxing to water; 3% hydrogen peroxide methanol blocking POD; protease K 37℃incubation, add TUNEL solution (1:9); POD conversion solution; DAB colouration, hematoxylin afterstain; dry were mounted, microscopic examination. The cardiac muscle cell apoptosis index were observed and calculated by Optical microscope.3. To investigate the effects of dihydroartemisinin on expression of VEGF and ICAM-1 of rat models of pulmonary hypertension treated by MCT.30 male SD rats were randomly divided into normal control group, pulmonary hypertension group and dihydroartemisinin experiment group (n=10 in each group), the method of animal model preparation is the same as the first part. The contents of VEGF and ICAM-1 in blood were determined by radioimmunoassay; the expression of VEGF, ICAM-1 mRNA and protein were determined by RT-PCR and Western blot methods.Results1. Severe pulmonary hypertension and the emergence of neointima can be caused by subcutaneous injection of monocrotaline in rats, compared with other groups,the mPAP, right ventricular weight, gland the degree of muscularization of pulmonary arterioles, pulmonary artery thickness percentage of lung degree of neointimal proliferation of small arteries were significantly higher than other groups (P<0.01). The mPAP was decreased after dihydroartemisinin intervention; The changes of the right ventricular hypertrophy index, the degree of muscularization of pulmonary arterioles, pulmonary artery medial thickness and neointimal proliferation were less after dihydroartemisinin intervention (P <0. 05).2. The right ventricular myocardial ultrastructure of each experimental group shows: Myofilament were arranged in perfect order in normal control groups, sarcomere Z lines, M lines were clearly visible, clear of myocardial nuclear membrane, nuclear structural integrity, mitochondrial full, crest complete. Model group, a large number of broken filaments, dissolved, mitochondrial swelling, part of the ridge fracture accompanied with vacuolar degeneration; myocardial cell cytoplasm condensed, chromatin margination uptake; dihydroartemisinin intervention group compared with myocardial ultrastructural injury model group for the light, still neatly arranged filaments, the sarcomere structure of the basic clear, mild swelling of mitochondria. By TUNEL assay, artemisinin intervention group, the degree of myocardial cell apoptosis compared with model group slightly reduced (P <0.05).3. VEGF, ICAM-1 can be measured in serum of each groups, compared with model group, VEGF, ICAM-1 were significantly lower than model groups in dihydroartemisinintreated groups (P<0.01). The expression of VEGF and ICAM-1 protein can be seen in pulmonary artery of each groups, compared with control groups, the expression of VEGF, ICAM-1 mRNA and protein in model groups were significantly higher than control groups(P<0.01) and there was significant difference between them. Compared with control groups, the expression of VEGF, ICAM-1 mRNA and protein in intervention groups were significantly higher than them and there was difference between them (P<0.01). Compared with pulmonary hypertension model groups, the expression of VEGF, ICAM-1 mRNA and protein in intervention groups were significantly lower than model groups (P<0.05) and there was significant difference between them.Conclusions1. Dihydroartemisinin can directly reduce MCT-induced pulmonary hypertension in pulmonary arterial pressure in rats, significantly inhibited the pulmonary vascular structure remodeling, reducing right ventricular hypertrophy.2. Dihydroartemisinin can inhibit or reduce MCT pulmonary hypertension in rats cardiomyocyte apoptosis and reduce the damage of myocardial ultrastructure. Thickness3. Dihydroartemisinin can inhibit pulmonary vascular structure remodeling by inhibiting the PASMCs proliferation, improving apoptosis of PASMCs and decreasing the pulmonary artery medial thickness in pulmonary hypertension rats induced by MCT. Dihydroartemisinin could directly inhibit the expression of VEGF, ICAM-1 mRNA and protein in the pulmonary artery, thus alleviate or reverse the pulmonary artery reconstruction.