Comparative Study Of Several Drugs On Pulmonary Artery Smooth Muscle Cell Proliferation

IntroductionPulmonary artery hypertension (PAH) is one of the commonly encountered diseases. Both the morbidity and mortality are rather high. Up to now, the pathogenesis of PAH is not quite clear. So far none of the satisfied therapy has been found. The pathologic changes of PAH compromise two essential changes, i.e. pulmonary vascular remodeling and pulmonary vasoconstriction. Therefore the key point to cure PAH is to improve the pulmonary circulation, inhibit pulmonary vasoconstriction and pulmonary vascular smooth muscle cell. It had been reported that vascular smooth muscle cell ( VSMC) proliferation, no matter it is stimulated by inside or outside factors, can be inhibited by certain drugs.It is also reported that nifedipine, captopril and enalapril could reverse structural remodeling of pulmonary vasculature during PAH. This effect had been proved by many studies. Further more, diltiazem has been frequently used to treat PAH, but it is not clear whether this agent has an effect against pulmonary vascular remodeling, during PAH. Few researches had been done to compare different calcium antagonists and angiotensin converting enzyme inhibitors (ACEI). So in this study, the effects of diltiazem on PASMC proliferation was undertaken , followed by the comparative study of effects of nifedipine, cap-topril, diltiazem and enalapril on PASMC proliferation. The purpose of this paper was to construct pre-clinical basis to improve the drug therapies for pulmonary hypertension.Experiment materials1. Drugs and agentsDiltiazem; made in Tianjin Tianbian Pharmacy Limited Company.Captopril and Enalapril: made in Changzhou Pharmaceutical Factory.FBS; Tianjin TBD Biology Technique Development CenterDMEM and Tyrosin; American Gibco productsMTT; Shanghai Chemical Reagent Station3H-TdR; Isotope Research Institute of China Atom Science Research Institute.POPOP, PPO: flicker pure2. Instrumentscell culture flasks (25cm2 ) , 96-well or 24-well cell culture plates; Nunclon Company Products, Denmark.C02 incubator; Forma Scientific Products American.Microplatereader; Model 550, Japanese BIO-RAD products.Liquid Scintillator, LS 3801, BECKMAN Company products, USA.3. Experiment animalsMale Wistar rats, 8 weeks old, weighing 274 + 16g, provided by Animal Laboratory Center, China Medical University. Certificate of Quality No: Liaoning Experiment Animal No. 034Experiment Method1. Rat PASMC primary culture and transfer cultureRat PASMC primary culture and transfer culture were made as described elsewhere. Briefly, WIstar rats, the 8-week-old were used. The main pulmonary artery media was taken off using pasting pieces and were cultured under sterile condition, on the culture plates. A-bout 10 days later PASMC growth was seen, the cells were in the shape of shuttles, typically in the shape of peak and valley. After confluence of fifth generation, the cells were transfered into 96- well or 24-well culture plates for study.2. Drug effects on PASMC growth curvesNifedipine, diltiazem, captopril, enalapril were added into the 96-well culture plates in the concentration of 10 mol L-1 respectively. The cells were cultured 20 hours. The value of light absorption was measure under the wave length of 570 nm using MTT method. The measurement was kept on for 7 days and the growth curves of PASMC proliferation were established.3. Drug effects on PASMC proliferationThe Comparative study of these drugs under different concentration on PASMC proliferation was conducted. Nifedipine, diltiazem, captopril and enalapril from the concentration of 10" tO 10 ~ mol L~ were added into the 96-well plates respectively and kept on culturing for 20 hours. The value of light absorption was measured under the wave length of 570 nm using MTT method.4. Effects on PASMC proliferation by combination of different drugsDrugs in combination of nifedipine plus captopril or diltiazem plus enalapril were administered into different wells. Using methods as above mentioned, the inf...