Changes Of Calponin And TGFβ1 In Pulmonary Artery Smooth Muscle Cells Of Pulmonary Artery Hypertension Rats

Changes of calponin and TGFβ1 in pulmonary artery smooth muscle cells of pulmonary artery hypertension ratsPrefaceIn the progress of chronic obstructive pulmonary disease(COPD) developed pulmonary heart disease, pulmonary artery hypertension(PAH) played a key role and it was a puzzle around the cadio-vascular surgery period. PAH mechanism was quite complex. Calponin is a particular smooth muscular regulated protein. Its functions included restraining smooth muscle contraction, affecting cell division and differentiation, participating in cell signal transduction, and maintaining cell framework. TGFβ1 was a muti-functional cell active factor, its had extensive activity and modulated important cell functions such as modulated many kinds of cell differentiations and productions of extrocellular matrix. Up to date we have not. read some reports about calponin and TGFβ1 expression changes in pulmonary artery smooth muscle cell(PAMC) of PAH rats induced by MCT. So we will regard them as targets to present evidences for Chinese traditional medicine treating PAH.Materials and Methords1. Animals and Groups36 Male Wistar rats, 12 rats at random in control group, the rest were injected 2% MCT(60 mg·kg^-1).2. MaterialsMCT was from Sigma Co. Rabbit anti-rat calponin multi-clonal antibody was obtained from Beijing boaosen bio-technological Co. Rabbit anti-rat TGFβ1 multi-clonal antibody and coat anti-rabbit IgG were obtained from Beijing Zhongshan bio-technological Co. 3. Example attainments and managementsAfter 21 days, we executed these rats, opened up the thorax, taked out the entire heart and lungs and put them in normal salines. Then we peeled off the pulmonany artery, and separatively weight right ventricules(R), left ventricules(C) and ventricular septum(S).4. Calponin and TGFβ1 detections with immunohistochemistryThe samples in 4% poly-formaldehyde went through dehydration, clarity, routine paraffin embedment, continual 5μm size slice. The first antibody, the second antibody, DAB dyeing. The displaying results were analysed by colorful photo-analytical system. Magnifying the images to 400 times and selecting five visual fields at random to calculate average optical density, then statistical analysis.5. Calponin and TGFβ1 detections with western blotAfter distilled proteins with RIPA and quantified proteins with hydroxybenzen reagent, 10% SDS-PAGE electrophoresis and transferred the membrane via electro-tranference appliance, occluded it. Added the first antibody, the second antibody, dyed the membrane, developed it, scanned it with laser density scanner and calculated average optical density.6. Statistical analysisData were represented as (?)±s. Statistical analysis was performed by t-test. P＜0.05 was considered as statistically significant.Results1. R/L+S0.26±0.03 in control group, 0.42±0.06 in PAH group(P＜0.05, n=12)2. Immunohistochemmistry results of calponin and TGFβ1 expressionCalponin expression was located in cytoplasm of PAMC. Its expression markly decreased in PAH group. Average optical density is 129.5±22.6 in control group whereas 55.2±17.1 in PAH group(P＜0.05, n=6). In comparision to weak TGFβ1 expression in control group, its expression markly increased in PAH group. Average optical density is 28.8±12.5, whereas 69.7±19.4 in PAH group (P＜0.05, n=6). TGFβ1 expression was located in cytoplasma of PAMC and tunica adventitia. 3. Western blot results of calponin and TGFβ1 expressionSingle strip occurred in the same level position both in control group and in PAH group. Calponin expression markly decrease in PAH group. Average optical density decrease from 1.50±0.10 in control group to 0.42±0.16 in PAH group (P＜0.05, n=6). TGFβ1 expression markly increased. Average optical density increased from 0.42±0.03 in control group to 1.30±0.15 in PAH group(P＜0.05, n=6).DiscussionCalponin acted as a kind of particular smooth muscular regulated protein. Winder, etc firstly found calponin could restrain ATPase in constriction system in vitro. They made myosin phoshorate with myosin light chain kinase(MLCK), then added actin into phosphorated myosin, when activity of ATPase increased. If the reactive system was added calponin in advance, activity of ATPase was markly inhibited dosage-depending. These made clear that calponin might block actin combine with myosin when calponin binded with actin, so that activity of ATPase was restrained. Obviously, when smooth muscles accepted outer stimulation to contract, it was destined that there was a mechanism to relieve the inhibition. In previous experiments, decreasion of calponin expression was observed in some animal models with vascular proliferous disease such as cerebrovasallar convulsion and hypertension. TGFβ1 played an important role in embryogenesis, fattiness-formation, fibrin-occurance, muscle-formation, cartilage formation, ostosis, epithelia differentiation and functional regulation of immunocyte. Formation of many diseases might be related to dysfunction of TGFβ1. These disease included scar formation, cancer development, arterioclerosis, osteoporosis, neural recessive disease, etc. Anciently some scholars have respectively discussed calponin expression and TGFβ1 expression in anoxic PAH. In this test, PAH rat was established by injecting MCT. Calponin expression markly decreased in PAH group compared to that in control group. TGFβ1 expression markly increased in PAH group compared to that in control group, whereas if there was a constructive relationship between calponin and TGFβ1 in the condition, the more research need to be done.Conclusion1. Calponin and TGFβ1 are related to PAH formation.2. Calponin expression decreased in PAH group. 3. TGFβ1 expression increased in PAH group.