Effect Of Reactive Oxygen Species And ERK1/2 Signaling Pathway On The Regulation Of Cell Proliferation And Apoptosis In Hypoxic Pulmonary Arterial Smooth Muscle Cells Of Rats

ObjectiveThis work was designed to investigate the change of reactive oxygen species(ROS) production in hypoxic pulmonary arterial smooth muscle cells(PASMCs) of rats, the effect of ROS on the proliferation and apoptosis of rat PASMCs under hypoxic condition. In addition, this work to explore the role of extracellular signal-regulated kinase(ERK)1/2 signaling pathway in the imbalance of proliferation/apoptosis of PASMCs by ROS.Methods and MaterialsPrimary cultures of PASMCs were established and cells between passages 2 to 4 were used for experiments.PASMCs were treated with ROS scavenger Tiron and ERK inhibitor PD98059 under normoxia or hypoxia condition.The ROS production was measured by DCFH-DA and NBT reduction.The expression of phosphorylated-ERK1/2(p-ERK1/2)protein were detected by immunofluorescence.Cells proliferation were examined by MTT colorimetric assey and the expression of PCNA.Cells apoptosis were detected by TUNEL. The expression of Bcl-2 and Bax protein was detected by immunocytochemical.Results1. Compared with control group(NBT:0.114±0.017,DCFH-DA:15.84±2.86),the ROS levels of hypoxia group(NBT:0.214±0.024,DCFH-DA:39.33±3.27) were significantly increased(n=6,P<0.01). 2. the proliferative capacity of hypoxia group (MTT:0.325±0.035,PCNA:53.83±4.48)was higher than control group(MTT: 0.228±0.019,PCNA:42.58±3.71) (n=6,P<0.01) and the apoptosis index of hypoxia group (2.42±0.74)was lower than control group(4.50±0.45) (n=6,P<0.01),Tiron significantly attenuated hypoxia-induced cells proliferation(MTT: 0.281±0.029,PCNA:47.82±3.41)(n=6,P<0.05)and it also significantly raised the apoptosis index of hypoxia cells(5.75±0.94)(n=6,P<0.01).3. the expression of p-ERK1/2 of hypoxia group(63.50±6.16) was higher than control group(26.17±3.76)(n=6,P<0.01),which was significantly suppressed by Tiron (31.00±4.15)(n=6,P<0.01).4. PD98059 significantly attenuated hypoxia-induced cells proliferation(MTT:0.292±0.028,PCNA:49.75±2.66)(n=6,P<0.05)and it also significantly raised the apoptosis index of hypoxia cells(4.83±0.82)(n=6,P<0.01),but the proliferative capacity(MTT:0.284±0.032,PCNA:48.41±3.25)and apoptosis index(5.33±0.75) in hypoxia+Tiron+PD98059 group were not significantly different as compared with hypoxia+Tiron group(n=6,P>0.05).5. Compared with control group (Bcl-2:0.098±0.017, Bax:0.210±0.031, Bcl-2/Bax:0.465±0.021),the expression of Bcl-2 protein of hypoxia group (0.141±0.024)was markedly increased ( n=6,P<0.05 ) , the expression of Bax protein(0.074±0.020) was markedly decreased (n=6,P<0.05),the ratio of Bcl-2/Bax (1.972±0.283) was increased (n=6,P<0.01).6. the expression of Bcl-2 protein of hypoxia+Tiron group(0.124±0.018) was lower than that of hypoxia group(n=6,P<0.05), meanwhile the expression of Bax protein (0.182±0.019)was higher than that of hypoxia group, the ratio of Bcl-2/Bax(0.678±0.071)was decreased (n=6,P<0.01).ConclusionThis work has shown that the hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2,in addiation, the ROS-mediated decrease of apoptosis in hypoxic is related to the rising of the ratio of Bcl-2/Bax.ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.