二十二碳六烯酸通过活化T细胞核因子1调控低氧诱导的大鼠肺动脉平滑肌细胞表型转化  

Docosahexaenoic acid inhibits hypoxia-induced pulmonary arterial smooth muscle cells phenotype switching by inhibiting NFATcl signaling

作　　者：谢其非 陈蕊[1] 卢毅[1] 严金川[1] 刘朔[1] 李梅[1] 宋娟[1] 邵晨[1] 王中群[1] 刘培晶[1] 

机构地区：[1]江苏大学附属医院心内科,镇江212001

出　　处：《中华心血管病杂志》2017年第2期148-153,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金（81400208）;江苏省自然科学基金（BK2010335,BK20160549）;镇江市科技支撑计划（SH2014025）

摘　　要：目的探讨二十二碳六烯酸（DHA）调控低氧诱导的大鼠肺动脉平滑肌细胞（PASMC）表型转化的分子机制。方法采用组织贴块法培养大鼠PASMC。将PASMC分为常氧组、低氧组（1%O2、94%N2、5%CO2，低氧刺激12 h）、低氧（1%O2、94%N2、5%CO2）＋DHA（10 μmol/L）组（给予DHA处理后低氧孵育12 h），低氧（1%O2、94%N2、5%CO2）＋DHA（10 μmol/L）＋过表达NFATc1组（细胞转染过表达NFATc1载体24 h，然后再给予DHA及低氧刺激12 h）及低氧（1%O2、94%N2、5%CO2）＋DHA（10 μmol/L）＋敲低NFATc1组（细胞转染NFATc1小干扰RNA 24 h，然后再给予DHA及低氧刺激12 h）。使用三气培养箱制造低氧环境加以干预。分别采用实时定量PCR和Western blot法检测各组细胞的NFATc1mRNA和蛋白表达水平，分别采用免疫荧光染色、Western blot和实时定量PCR法检测α-平滑肌肌动蛋白（α-SMA）表达水平，采用实时定量PCR法检测平滑肌肌动蛋白相关蛋白22（SM22）mRNA表达水平，采用5-乙炔基-2′-脱氧尿苷（EDU）法检测PASMC增殖情况。 结果低氧刺激12 h，低氧组大鼠PASMC NFATc1的mRNA及蛋白表达水平均明显高于常氧组（P均〈0.05），而低氧＋DHA组则均明显低于低氧组（P〈0.05）。转染NFATc1过表达病毒及小干扰RNA 24 h后，低氧刺激大鼠PASMC 12 h，低氧组大鼠PASMC α-SMA阳性的细胞数、α-SMA蛋白及mRNA以及SM22 mRNA表达水平均明显低于常氧组（P均〈0.05），低氧＋DHA组则高于低氧组（P〈0.05），低氧＋DHA＋过表达NFATc1组则明显低于低氧＋DHA组（P〈0.05），而低氧＋DHA＋敲低NFATc1组又明显高于低氧＋DHA＋过表达NFATc1组（P〈0.05）。低氧组大鼠PASMC增殖率明显高于常氧组（P〈0.05），低氧＋DHA组则明显低于低氧组（P〈0.05），低氧＋DHA＋过表达NFATc1组则明显高于低氧＋DHA组（P〈0.05），而低氧＋DHA＋敲低NFATc1组又明显低于低氧＋DHA＋过表达NFATc1组�ObjectiveTo explore the molecular mechanism of docosahexaenoic acid （DHA） on regulating the phenotype switching of hypoxia-induced pulmonary arterial smooth muscle cells （PASMCs）.MethodsThe PASMCs were isolated from Sprague Dawley rats. PASMCs were divided into five groups： normal control group, hypoxia group （1%O2, 94%N2, 5% CO2 stimulation for 12 hours）, hypoxia＋ DHA group （10 μmol/L DHA pretreatment followed by 12 hours hypoxia）, hypoxia＋ DHA＋ NFATc1 overexpression group （transfection of the NFATc1 lentivirus for 24 hours, followed by hypoxia stimulation for 12 hours after 10 μmol/L DHA treatment）, and hypoxia＋ DHA＋ siNFATc1 group （transfection the siNFATc1 for 24 hours, followed by hypoxia stimulation for 12 hours after 10 μmol/L DHA treatment）. The hypoxia stimulation was achieved by use of a special hypoxia incubator （1%O2, 94%N2, 5%CO2）. The expressions of NFATc1 of various groups were determined by qRT-PCR and Western blot. The expression of α-SMA was determined by immunofluorescence staining, qRT-PCR and Western blot. The expression of SM22 was determined by qRT-PCR. The proliferation of PASMC was determined by EDU staining.ResultsThe mRNA and protein expression levels of NFATc1 were significantly upregulated in hypoxia group compared with the normal control group （P〈0.05）, while hypoxia-induced upregulation of NFTAc1 could be significantly downregulated by DHA treatment （P〈0.05）. The α-SMA positive cell number, protein and mRNA levels of α-SMA and the mRNA level of SM22 were significantly lower in the hypoxia group than in normal control group, which could be significantly reversed by DHA, the protective effects could then be abolished by NFATc1 overexpression. Above indices were significantly lower in the hypoxia＋ DHA＋ siNFATc1 group than in hypoxia＋ DHA＋ NFATc1 overexpression group （P〈0.05）. The proliferation of PASMCs was significantly higher in the hypoxia group than in the control group （P〈0.05）, and which could be signif

关 键 词：肺动脉 肌细胞 平滑肌 二十二碳六烯酸类 

分 类 号：R544.1[医药卫生—心血管疾病]