阿米洛利介导PI3K/AKT信号通路对急性呼吸窘迫综合征大鼠肺损伤的作用机制  

Mechanism of amiloride-mediated PI3K/AKT signaling pathway on the lung injury in acute respiratory distress syndrome rats

作　　者：朱磊[1] 郭鸿[1] 井高静 帅佃奎 熊怀玉 黄蔷茹 刘健[1] ZHU Lei;GUO Hong;JING Gaojing;SHUAI Diankui;XIONG Huaiyu;HUANG Qiangru;LIU Jian(Department of Critical Care Medicine,The First Hospital of Lanzhou University,Lanzhou 730030,China;Department of Endocrinology,The First Hospital of Lanzhou University,Lanzhou 730030,China)

机构地区：[1]兰州大学第一医院重症医学科,甘肃兰州730030 [2]兰州大学第一医院内分泌科,甘肃兰州730030

出　　处：《暨南大学学报（自然科学与医学版）》2021年第1期1-11,共11页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：甘肃省自然科学基金项目(18JR3RA344)。

摘　　要：目的:探讨阿米洛利介导PI3K/AKT信号通路对急性呼吸窘迫综合征(ARDS)大鼠肺损伤的作用机制,为ARDS患者临床诊疗提供新思路.方法:将96只雄性Wistar大鼠随机分为空白对照组、二甲基亚砜(DMSO)组、ARDS组、阿米洛利组,各组24只,空白对照组和DMSO组均不注射油酸,其余各组建立油酸型ARDS大鼠模型,分别在注射油酸4 h、8 h和12 h时取样肺组织和肺泡灌洗液(BALF),ELISA法检测大鼠BALF中肿瘤坏死因子(TNF-α)、白介素-6(IL-6)和白介素-10(IL-10)水平;HE染色观察大鼠肺组织病理变化;免疫组化法检测大鼠肺组织血管内皮细胞生长因子(VEGF)、血管细胞黏附因子(VCAM-1)和内皮素1(ET-1)阳性表达水平;Western blot和RT-PCR检测大鼠肺组织中PI3K/AKT信号通路相关因子PI3K、p-PI3K、AKT和p-AKT蛋白表达水平及mRNA表达水平.结果:注射油酸后的大鼠出现呼吸困难、呼吸频率明显加快、毛发直立、活动减少等急性呼吸窘迫症状,说明大鼠ARDS模型构建成功.ARDS组大鼠BALF中TNF-α和IL-6质量浓度显著上升(P<0.05),IL-10质量浓度显著下降(P<0.01);阿米洛利干预后大鼠BALF中TNF-α和IL-6质量浓度显著降低(P<0.01),IL-10质量浓度升高有统计学差异(P<0.01).HE染色ARDS组大鼠肺组织肺泡壁增厚,肺泡结构被破坏,炎性细胞浸润;免疫组化结果表明,ARDS组大鼠肺组织中VEGF阳性表达显著降低(P<0.01),VCAM-1和ET-1阳性表达显著升高(P<0.01),阿米洛利干预后VEGF表达显著上调(P<0.01),VCAM-1和ET-1阳性表达均显著下调(P<0.01);与ARDS组比较,阿米洛利组中p-PI3K和p-AKT蛋白相对表达降低(P<0.01);RT-PCR检测和Western blot变化趋势相同.结论:阿米洛利干预可以改善ARDS大鼠肺损伤,这可能是通过抑制PI3K/AKT信号通路实现的.Objective:To explore the mechanism of amiloride-mediated PI3K/AKT signaling pathway on lung injury in acute respiratory distress syndrome(ARDS)rats,so as to provide new ideas for the clinical diagnosis and treatment of ARDS patients.Methods:96 male Wistar rats were randomly divided into a blank control group,dimethyl sulfoxide(DMSO)group,ARDS model group and amiloride group,24 rats in each group.Oleic acid was not injected in animals of blank control group and DMSO group.The animals of other groups were intravenously injected with oleic acid to establish the rat model of ARDS.Lung tissue and bronchial alveolar lavage fluid(BALF)were sampled at 4 h,8 h and 12 h after injection of oleic acid,and the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-10(IL-10)in BALF of rats were detected by ELISA.The pathological change was observe using HE staining and the positive expression levels of vascular endothelial growth factors(VEGF),vascular cell adhesion molecule-1(VCAM-1)and endothelin-1(ET-1)were detected by immunohistochemical method in rat lung tissue.The protein and mRNA expression levels of PI3K/AKT signaling pathway-related factors PI3K,p-P13,AKT and p-AKT were measured by Western blot and RT-PCR detection.Results:After the injection of oleic acid,rats showed symptoms of acute respiratory distress,such as dyspnea,significantly increased respiratory frequency,erect hair and reduced activity,indicating the successful construction of ARDS model in rats.The level of TNF-αand IL-6 in BALF of ARDS rats increased significantly(P<0.05),while the level of IL-10 decreased significantly(P<0.01).The TNF-αand IL-6 level in BALF of rats decreased significantly(P<0.01)after amiloride intervention,while the IL-10 level increased statistically(P<0.01).HE staining observation showed increased lung alveolar wall thickness,destroyed alveolar structure and inflammatory cell infiltration in rats of ARDS group.Immunohistochemical results showed that the positive expression of VEGF was reduced significantly

关 键 词：急性呼吸窘迫综合征 油酸 阿米洛利 炎症反应 PI3K/AKT信号通路 

分 类 号：R3[医药卫生—基础医学]