Effects of early digoxin treatment on hypoxia-induced pulmonary artery hypertension

目的 探讨地高辛早期干预对低氧诱导肺动脉高压(PAH)大鼠肺血管重构的影响及其机制.方法 48只SD大鼠按随机数字表法随机均分为4组并分别进行如下处理:(1)常氧对照组,(2)常氧+地高辛组,(3)低氧对照组,(4)低氧+地高辛组.常氧干预大鼠正常氧浓度(21％)仓内饲养,低氧干预大鼠则置于低氧仓内,氧浓度控制(10.5±0.5)％范围内,每日间断低氧8h(8:00～16:00);地高辛干预大鼠每次入仓前腹腔注射地高辛注射液1.0 mg·kg-1·d-1,对照组大鼠每次入仓前腹腔注射等体积注射用水.21 d后测定各组大鼠肺动脉平均压(mPAP),计算右室肥厚指数[RV/(LV+S)],测定管壁厚度占血管外径的百分比(WT％)和管壁面积占血管总面积的百分比(WA％).肺动脉平滑肌细胞培养采用组织贴块法,细胞增殖及迁移检测分别采用噻唑蓝比色法和Transwell小室法.肺动脉平滑肌细胞收缩表型标志物α肌动蛋白、调宁蛋白和肌动蛋白相关蛋白mRNA水平检测采用实时定量PCR法.肺动脉平滑肌细胞基质金属蛋白酶(MMPs)分泌水平检测采用Western印迹法.结果 低氧对照组大鼠mPAP、RV/(LV+S)、WT％、WA％均显著高于常氧对照组[(38.5±2.3)比(21.0±1.5)mmHg(1 mmHg =0.133 kPa)、(42.8±2.6)％比(26.7±1.5)％、(39.3±2.0)％比(17.5±3.3)％、(79.5±5.7)％比(45.7±4.9)％,均P＜0.05].而低氧+地高辛组大鼠上述指标均显著低于低氧对照组[(27.3±2.7)比(38.5 ±2.3)mmHg、(30.9±3.3)％比(42.8 ±2.6)％、(21.7±3.6)％比(39.3±2.0)％、(56.3±4.7)％比(79.5±5.7)％,均P＜0.05].地高辛(100 nmol/L)显著抑制低氧诱导的肺动脉平滑肌细胞增殖(吸光度值:0.575±0.030比0.747±0.039,P＜0.05),减少低氧诱导的肺动脉平滑肌细胞迁移31.9％ (P ＜0.05),并抑制低氧引起的收缩表型标志物mRNA表达的下降和MMP-2、MMP-9蛋白表达的升高.结论 地高辛早期应用可延缓低氧诱导的大鼠PAH形成及肺血管重构,该作用可能是通过抑制肺动脉平滑肌细胞增殖、迁移、表型转换和MMPs分泌而起作用.Objective To explore the effects of digoxin on hypoxia-induced pulmonary artery hypertension (PAH) and the possible mechanisms.Methods A total of 48 Sprague-Dawley rats were randomly divided into 4 groups:normoxia control,normoxia + digoxin,hypoxia control and hypoxia +digoxin.The animals were exposed to chronic intermittent hypoxia (PO2:(10.5 ±0.5) ％,8:00-16:00)or room air for 21 days.Each rat received a daily intraperitoneal injection of either digoxin (1.0 mg· kg-1 ·d-1) or an equal volume of vehicle,starting at the first day of hypoxia or normoxia.At Day 21,mean pulmonary arterial pressure (mPAP),right ventricular hypertrophy (RV/(LV + S)) and index of wall thickness of small pulmonary artery (WT％ and WA％) among groups were compared.And in vitro the changes of pulmonary artery smooth muscle cells (PASMCs) proliferation were determined by methyl thiazolyl tetrazolium (MTT) assay.Migration assay was performed with a Transwell chamber.Real-time quantitative polymerase chain reaction (PCR) was performed to quantify the mRNA levels of smooth muscle cell phenotype markers such as smooth muscle-α-actin,calponin and smooth muscle 22α under normoxic or hypoxic conditions in the absence or presence of digoxin.And the protein expressions of matrix metalloproteinase (MMPs)were determined by Western blot.Results Digoxin treatment significantly lowered mPAP,reduced WT％ and WA％ and right ventricular hypertrophy compared with those of the hypoxic group (mPAP:(27.3 ± 2.7) vs (38.5 ± 2.3) mmHg(1 mmHg =0.133 kPa); RV/(LV + S):(30.9±3.3)％ vs (42.8±2.6)％,WT％:(21.7±3.6)％ vs (39.3±2.0)％; WA％:(56.3±4.7)％ vs (79.5 ±5.7)％,all P ＜0.05).And in vitro,digoxin restored the hypoxia-induced inhibition of the expression of smooth muscle cell phenotype markers and prevented the hypoxia-induced activation of MMPs in PASMCs.Conclusion Early digoxin therapy reduces pulmonary artery remodeling in hypoxiainduced PAH rat model and this effect is probably correlated with the inhibitions of proliferation,migration,phenotype switching and expression of MMPs induced by hypoxia in PASMCs.国家自然科学基金中文核心期刊要目总览(PKU)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)02139-1439