肺动脉平滑肌细胞凋亡在大鼠低氧性肺动脉重构自然逆转中的作用及分子机制

Effect of PASMC apoptosis on reversal of hypoxic pulmonary arterial remodeling during reoxygenation and its related molecular mechanism

目的:研究肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)凋亡在低氧性肺动脉重构自然逆转中的作用,并探讨其可能机制。方法:24只SD大鼠随机均分为常氧4周组、低氧4周组、低氧4周后复氧1周组及复氧6周组。分别检测右室收缩压(right ventricular systolic pressure,RVSP)、肺动脉中膜厚度(medial thickness,MT)和中膜面积(medial area,MA),以及肺动脉中膜自噬、凋亡等在低氧-复氧中的变化。大鼠原代PASMCs分为常氧48 h组、低氧48 h组、低氧48 h后复氧24 h组及常氧72 h组,观察PASMCs凋亡和自噬在低氧-复氧中的变化。再将PASMCs分为常氧72 h组、低氧48 h后复氧24 h组及低氧48 h+氯喹(自噬抑制剂)干预后复氧24 h组,观察PASMCs低氧阶段的自噬对其复氧阶段凋亡的影响。结果:(1)低氧使大鼠RVSP、右室肥厚指数、MT及MA显著升高(P<0.05);复氧后上述指标逐渐降低。(2)低氧使肺动脉中膜LC3表达升高,P62表达降低,复氧后上述分子的表达逐步恢复正常。低氧显著降低了中膜cleaved caspase-3的表达,复氧1周其表达显著高于低氧组。(3)低氧期原代PASMCs cleaved caspase-3/PARP的表达显著低于常氧组,复氧后其表达明显升高(P<0.05);PASMCs LC3和P62的表达在低氧期显著降低(P<0.05)。(4)抑制了PASMCs低氧阶段的自噬后,其复氧阶段cleaved caspase-3/PARP表达显著降低(P<0.05)。结论:PASMCs的凋亡参与了低氧性肺动脉重构的自然逆转;复氧期PASMCs凋亡的发生可能与其低氧期的自噬有关。

AIM： To explore the effect of pulmonary arterial smooth muscle cell（ PASMC） apoptosis on the reversal of hypoxic pulmonary arterial remodeling during reoxygenation and its possible mechanism. METHODS： Male SD rats（ n = 24） were randomly divided into normoxia for 4 weeks group,hypoxia for 4 weeks group,reoxygenation for 1 week after hypoxia for 4 weeks group and reoxygenation for 6 weeks after hypoxia for 4 weeks group. Right ventricular systolic pressure（ RVSP）,right ventricular hypertrophy index,pulmonary arterial medial thickness（ MT） and medial area（ MA）as well as autophagy and apoptosis in the pulmonary arterial medial layer were examined during hypoxia-reoxygenation. The rat primary PASMCs were divided into normoxia for 48 h group,hypoxia for 48 h group,reoxygenation for 24 h after hypoxia for 48 h group and normoxia for 72 h group to explore the changes of PASMC autophagy and apoptosis following hypoxiareoxygenation. Finally,primary PASMCs were divided into normoxia for 72 h group,reoxygenation for 24 h after hypoxia for 48 h group and reoxygenation for 24 h after hypoxia for 48 h＋chloroquine（ inhibitor of autophagy） group to investigate the effect of PASMC autophagy during hypoxia on the apoptosis during reoxygenation. RESULTS： After hypoxia for 4weeks,the RVSP,during right ventricular hypertrophy index,MT and MA increased significantly compared with normoxia group（ P〈0. 05）,and gradually decreased during reoxygenation. The expression of LC3 in the pulmonary arterial medial layer increased evidently after hypoxia and gradually reversed during reoxygenation. Moreover,the P62 and cleaved caspase-3 expression decreased after hypoxia compared with normoxia group,and increased markedly following reoxygenation. The expression of cleaved caspase-3/PARP in rat primary PASMCs decreased significantly under hypoxia（ P〈0. 05）,and increased evidently during reoxygenation. The expression of P62 and LC3-II decreased markedly under hypoxia（ P〈0. 05）. After inhibition of PASMC autophagy under hypoxia,the expression of cleaved caspase-3/PARP decreased remarkably during reoxygenation（ P〈0. 05）. CONCLUSION： The PASMC apoptosis participates in the reversal of hypoxic pulmonary arterial remodeling,and the PASMC autophagy under hypoxia might facilitate its apoptosis during reoxygenation.