The Wechanism Of Survivin Inhibiting Apoptosis Of Hypoxic Human Pulmonary Arterial Smooth Muscle Cells

Hypoxic pulmonary hypertension（HPH）is a disease caused by multiple factors,the current pathogenesis is not clear,the early lack of specific clinical symptoms,with arterial pressure,can lead to right heart failure and death.The treatments for pulmonary hypertension（PH）is to decrease the pulmonary artery pressure,increase the cardiac output,relieve the symptoms,and enhance the body constitution.Ideal pulmonary vasodilator drugs are selective relaxation of pulmonary vascular smooth muscle,improve ventilation and PaO₂.However,such treatment is not specific on the pulmonary circulation and has a stronger role in systemic circulation,thus affecting arterial blood pressure,and even decreased PaO₂.The main pathological physiology of PH is the imbalance between the proliferation and apoptosis of PASMCs.The effect of vasodilator drugs on pulmonary hypertension in pulmonary vascular remodeling is relatively small,leading to the poor treatment effect and rates of mortality remain unacceptably high.Clearly,alternative therapies are needed to combat pulmonary hypertension,including HPH.So the fundamental inhibition of PASMCS proliferation or promote its apoptosis is the key to the current treatment of PH and research hot spots,thereby reducing pulmonary vascular remodeling.Survivin has the strongest apoptosis in the inhibitor of apoptosis proteins（IAP）family,which has the dual functions of promoting cell proliferation and inhibiting apoptosis.Survivin is not expressed in healthy human tissue.A large number of human tumors have been found to express survivin,suggesting that survivin plays an important role in the inhibition of apoptosis in tumors.Mitochondrial is not only a very important energy-generating site in the cell,but also plays an important role in cell apoptosis.A previous study on the mechanism of survivin inhibiting tumor cells apoptosis.Survivin combine with Caspase-9 prevent the formation of apoptotic bodies,and then block the mitochondria-regulated apoptosis pathway.The present study shows that mitochondria are closely related to cell apoptosis and cell hypoxia receptors,also play a very important role in the pathogenesis of pulmonary artery.Our previous study found Survivin was expressed in hypoxic human pulmonary arterial smooth muscle cells（HPASMCs）,but not in normal HPASMCs.However,the mechanism of survivin inhibiting the apoptosis of HPAMSCs is still unclear.In this study,we investigated the mechanism of survivin in inhibiting the apoptosis of HPASMCs.HPASMCS were divided randomly into 6 groups : Normoxia（N group）;Hypoxia（H group）;Normoxia﹢YM155（NY group）;Hypoxia ﹢ 1nmol/L YM155（HY1 group）;Hypoxia ﹢10nmol/l YM155（HY10 group）;Hypoxia﹢100nmol/L YM155（HY100 group）.Mitochondrial membrane potential was measured by staining cells with tetramethylrhodamine ethyl ester（TMRE）.The level of ROS in HPASMCS were tested with 2,7-dichlorfluorescein-diacetate（DCFH-DA）staining and photofluorography.Western blot technique was used to detect the expression of cytochrome C protein in cell plasma and mitochondrial and Caspase-9 protein respectively.The mitochondrial membrane potentials（expressed by intensity of TMRE）of H group（60.453±6.008）was significantly increased compared with that of N group（47.831±4.550,q=5.199,P<0.05）.The level of ROS of H group（17.662±3.116）was significantly decreased compared with that of N group（26.160±3.500,q=5.3.248,P<0.05）.As compared with the H group,the mitochondrial membrane potentials in hypoxia plus 1nmol/L,10nmol/L,100nmol/L YM155 groups（49.183±1.007、37.180±1.047、17.568±5.836,q=4.642、9.585、17.663,all P﹤0.05）significant decreased,but the level of ROS in hypoxia plus 1nmol/L,10nmol/L,100nmol/L YM155 groups（34.825±3.225、52.225±5.794、71.744±7.050,q=6.561、13.212、20.674,all P﹤0.05）enhanced in a dose-dependent manner.The expression of cytochrome C protein in cell plasma to that in cell mitochondrial and Caspase-9 protein of H groups（0.173,0.124±0.113）were significantly decreased as compared with N group（0.733,0.531±0.102,all P<0.05）.As compared with the H group,the expression of cytochrome C protein in cell plasma to that in cell mitochondrial and Caspase-9 protein in hypoxia plus 1nmol/L,10nmol/L,100nmol/L YM155 groups（0.846、1.908、3.258;1.016±0.225、1.732±0.156、1.732±0.156,q=9.389、14.400、29.042,all P﹤0.05）as significant increased in a dose-dependent manner.In conclusion,the expression of survivin might contribute to increase of the mitochondrial membrane potential（ΔΨm）and decrease the level of ROS followed by depolarization of ΔΨm in hypoxic HPASMCs,then surpress the release of cytochrome C from cell mitochondria to cell plasma in HPASMCs and reactive Caspase-9.This might be a mechanism of survivin inhibiting HPASMCs apoptosis.