从调控肺泡中性粒细胞凋亡探讨血必净注射液防治体外循环肺损伤的机制  

The mechanism of Xuebijing injection in preventing and treating lung injury induced by cardiopulmonary bypass by regulating the apoptosis of alveolar polymorphonuclear neutrophil

作　　者：徐朝军[1] 张胜康[1] 张怡 周代勇 明润宇 宋岚 Xu Zhaojun;Zhang Shengkang;Zhang Yi;Zhou Daiyong;Ming Runyu;Song Lan(Department of Cardiothoracic Surgery,the First Affiliated Hospital,Hunan University of Chinese Medicine,Changsha 410007,Hunan,China;Department of Biochemistry and Molecular Biology,Hunan University of Chinese Medicine,Changsha 410208,Hunan,China)

机构地区：[1]湖南中医药大学第一附属医院心胸外科,长沙410007 [2]湖南中医药大学医学院生物化学与分子生物学教研室,长沙410208

出　　处：《中华危重病急救医学》2024年第2期166-171,共6页Chinese Critical Care Medicine

基　　金：湖南省教育厅科学研究重点项目(20A364,22A0243);湖南省高等学校"双一流"建设项目(2018-469)。

摘　　要：目的从调控中性粒细胞(PMN)凋亡探讨血必净注射液对体外循环(CPB)所致急性肺损伤(ALI)的防治作用。方法按随机数字表法将30只雄性SD大鼠分为假手术组(Sham组)、CPB模型组(CPB组)、血必净预处理组(XBJ组),每组10只。CPB组和XBJ组CPB 60 min后停机;Sham组不行CPB。XBJ组CPB前2 h腹腔注射4 mL/kg血必净注射液;Sham组和CPB组注射等量生理盐水。CPB后4 h,取动脉血进行血气分析,计算呼吸指数(RI);取肺组织测定肺系数(LI)和肺含水率。收集支气管肺泡灌洗液(BALF)获取PMN,检测天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)活性,并用流式细胞仪检测细胞凋亡率;实时荧光定量聚合酶链反应(RT-qPCR)检测微小RNA-142-3p(miR-142-3p)、FoxO1 mRNA表达;蛋白质免疫印迹试验(Western blotting)检测FoxO1蛋白表达。此外,将HL-60细胞分为对照寡核苷酸转染组、miR-142-3p模拟物转染组和miR-142-3p拮抗物转染组,转染48 h后双荧光素酶报告基因检测miR-142-3p与FoxO1结合的活性。结果与Sham组相比,CPB组大鼠RI、LI及肺含水率显著升高,BALF获取PMN中caspase-3活性和细胞凋亡率显著降低,miR-142-3p表达下降,FoxO1蛋白表达增加。与CPB组比较,XBJ组大鼠RI、LI、肺含水率显著降低〔RI:0.281±0.066比0.379±0.071,LI:4.50±0.26比5.71±0.42,肺含水率:(80.31±3.25)%比(84.59±3.41)%,均P<0.01〕,BALF获取PMN中caspase-3活性和细胞凋亡率显著升高〔caspase-3活性:0.350±0.021比0.210±0.014,凋亡率:(15.490±1.382)%比(8.700±0.701)%,均P<0.01〕,miR-142-3p表达显著上调(2-ΔΔCt:2.61±0.17比0.62±0.05,P<0.01),FoxO1蛋白表达显著降低〔FoxO1/GAPDH(相对表达量):0.81±0.04比1.22±0.06,P<0.01〕。而3组间FoxO1 mRNA表达差异无统计学意义。生物信息学分析结果表明,miR-142-3p可以结合FoxO13’’非翻译区(3’’UTR)。与转染无关对照寡核苷酸序列比较,转染miR-142-3p模拟物可以降低HL-60细胞FoxO1蛋白表达〔FoxO1/GAPDH(相对表达量)Objective To investigate the protective effect of Xuebijing injection on acute lung injury(ALI)associated with cardiopulmonary bypass(CPB)by regulating the apoptosis of polymorphonuclear neutrophils(PMN).Methods Thirty male Sprague-Dawley(SD)rats were randomly divided into sham operation group(Sham group),CPB model group(CPB group)and Xuebijing pretreatment group(XBJ group)according to the random number table method,with 10 rats in each group.Rats in the CPB group and XBJ group undergoing CPB procedures for 60 minutes.Rats in the Sham group did not undergo CPB.Rats in the XBJ group received intraperitoneal injection of 4 mL/kg Xuebijing injection 2 hours before CPB.Rats in the Sham group and CPB group were injected with an equal amount of normal saline.4 hours after CPB,arterial blood was collected for blood gas analysis to calculate respiratory index(RI),and lung tissue of rats was collected for determination of lung index(LI)and pulmonary water containing rate.PMN in bronchoalveolar lavage fluid(BALF)were collected and the activity of caspase-3 was detected.The apoptosis rate was detected by flow cytometry.The expressions of microRNA-142-3p(miR-142-3p)and FoxO1 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The protein expression of FoxO1 was detected by Western blotting.In addition,HL-60 cells were divided into control oligonucleotide transfection group,miR-142-3p mimics transfection group,and miR-142-3p inhibitor transfection group.After 48 hours of transfection,the activity of miR-142-3p binding to FoxO1 was detected using dual luciferase reporter genes.Results Compared with Sham group,RI,LI and pulmonary water containing rate were significantly increased in CPB group.The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly decreased,the expression of miR-142-3p was decreased,and the expression of FoxO1 protein was increased.However,compared with CPB group,RI,LI and pulmonary water containing rate were significantly decreased in XBJ

关 键 词：体外循环 急性肺损伤 血必净注射液 微小RNA-142-3p FOXO1 凋亡 

分 类 号：R285.5[医药卫生—中药学]