mir-375调节notch通路对高氧诱导新生鼠肺上皮细胞凋亡的影响研究

Effects of mir-375 regulating notch pathway on hyperoxia-induced apoptosis of epithelial cells from lung tissues of neonatal rat

目的探讨微小RNA(mir)-375调节notch通路对高氧诱导支气管肺发育不良(BPD)新生鼠肺上皮细胞凋亡的影响。方法80只出生后3 d大鼠随机分为对照组、模型组、mir-375阴性对照组和mir-375拮抗剂组,每组20只。除对照组外,其他3组置于氧浓度(70±5)%氧箱中24 h,对照组置于空气中正常饲养,24 h后mir-375阴性对照组注射2μl(浓度为0.5μg/μl)mir-375拮抗剂阴性对照和纳米转染复合物的混合物;mir-375拮抗剂组注射2μl(浓度为0.5μg/μl)mir-375拮抗剂和纳米转染复合物的混合物;对照组和模型组注射2μl无菌0.9%氯化钠溶液,大鼠建模7 d和14 d分别每组取10只,称重检测大鼠体重变化;实时荧光定量PCR(qRT-PCR)检测大鼠肺组织中mir-375水平;苏木素-伊红(HE)染色观察肺组织形态学;TUNEL检测肺组织凋亡情况;免疫组化检测大鼠肺组织中notch1表达水平;硫代巴比妥酸法测定丙二醛(MDA)水平、氮蓝四唑光还原法测定超氧化物歧化酶(SOD)水平;蛋白免疫印迹(WB)检测肺组织B淋巴细胞瘤-2基因(Bcl2)、Bcl2相关X蛋白(Bax)蛋白表达情况。结果建模7、14 d,对照组大鼠肺组织支气管完整且排列整齐,肺泡排列整齐;模型组与mir-375阴性对照组肺组织结构不完整,肺泡数量减少、体积增大,结构简单化;mir-375拮抗剂组肺组织结构有所改善,与模型组相比肺泡数量升高、体积减少(P<0.05)。与对照组相比,模型组、mir-375阴性对照组体重、肺组织中notch1水平、SOD水平、Bcl2蛋白水平降低(P<0.05),肺组织中mir-375水平、肺泡上皮细胞凋亡指数、MDA水平、Bax蛋白升高(P<0.05);分别与模型组、mir-375阴性对照组相比,mir-375拮抗剂组体重、肺组织中notch1水平、SOD水平、Bcl2蛋白水平升高(P<0.05),肺组织中mir-375水平、肺泡上皮细胞凋亡指数、MDA水平、Bax蛋白降低(P<0.05)。结论抑制mir-375可上调notch通路减轻氧化应激实现对BPD新生鼠肺上皮细胞凋亡的缓解,从而实现对大鼠的保护。

Objective To investigate the effects of microRNA(mir)-375 regulating notch pathway on the apoptosis of lung epithelial cells in neonatal rats with bronchopulmonary dysplasia(BPD)induced by hyperoxia.Methods A total of 803-day-old rats were randomly divided into control group,model group,mir-375 negative control group,and mir-375 antagonist group,with 20 rats in each group.Except the control group,the other groups were placed in an oxygen box with an oxygen concentration of(70±5)%for 24h,and the control group was placed in the air for normal feeding.After 24h,the mir-375 negative control group was injected with a 24μl(at a concentration of 0.5μg/μl)mixture of mir-375 antagonist and nanotransfection complex;the mir-375 antagonist group was injected with a 24μl(at a concentration of 0.5μg/μl)mixture of mir-375 antagonist and nanotransfection complex;the control group and the model group were injected with 24μl sterile 0.9%sodium chloride solution.Ten rats were taken from each group at 7d and 14d after modeling,and the weight changes of rats were weighed.Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the level of mir-375 in rat lung tissue;hematoxylin-eosin(HE)staining was used to observe morphology of lung tissue;TUNEL was used to detect lung tissue apoptosis;immunohistochemistry was used to detect notch1 expression in rat lung tissue.The levels of malondialdehyde(MDA)were detected by thiobarbituric acid method,and the levels of superoxide dismutase(SOD)were determined by nitroblue tetrazolium photoreduction method.Western Blot(WB)was used to detect the expression levels of B lymphocyte tumor 2 gene(Bcl2)and Bcl2-related X protein(Bax).Results At 7d and 14 d after modeling,the lung tissues in control group were complete and neatly arranged,and the alveoli were arranged neatly.The lung tissue structure in model group and mir-375 negative control group was incomplete,the number of alveoli was reduced,the volume was increased,and the structure was simplified.The lung tissue structure in mir 375 antagonist group was improved,the number of alveoli was increased and the volume was decreased,as compared with that in model group.Compared with those in control group,the body weight,notch1 level,SOD level,and Bcl2 protein level were significantly decreased in model group and mir-375 negative control group(P<0.05),whereas the mir-375 levels,and apoptosis index of alveolar epithelial cell,MDA level,and Bax protein were increased(P<0.05).Compared with the model group and the mir-375 negative control group,the weight,the level of notch1 in the lung tissue,the level of SOD,and the Bcl2 protein level were significantly increased in mir-375 antagonist group(P<0.05),while the mir-375 level,apoptosis index of alveolar epithelial cell,MDA level,and Bax protein were significantly decreased(P<0.05).Conclusion To inhibit mir 375 can up-regulate the notch pathway to reduce oxidative stress and achieve alleviation of lung epithelial cell apoptosis in neonatal rats with BPD,thereby protecting rats.