微RNA-146a通过抑制NLRP3表达缓解呼吸机所致肺损伤的机制研究  

chanism study of microRNA-146a alleviating ventilator induced lung injury by inhibiting nucleotide-binding dom?cine-rich repeat and pyrin domain-containing receptor 3 expression

作　　者：秦晓枫 张明茹 吴海峰 谷长平 王月兰 Qin Xiaofeng;Zhang Mingru;Wu Haifeng;Gu Changping;Wang Yuelan(Department of Anesthesiology,the Shandong Provincial Hospital Affiliated to Shandong First Medical University,Jinan 250021,China;Department of Anesthesiology,Beijing Tongren Hospital,Capital Medical University,Bejing 10004l,China;Department of Anesthesiology and Perioperative Medicine,the First Afiliated Hospital of Shandong First Medical University,Jinan 250014,China)

机构地区：[1]山东第一医科大学附属省立医院麻醉科,济南250021 [2]首都医科大学附属北京同仁医院麻醉科,北京100041 [3]山东第一医科大学第一附属医院麻醉与围术期医学科,济南250014

出　　处：《国际麻醉学与复苏杂志》2024年第11期1127-1134,共8页International Journal of Anesthesiology and Resuscitation

基　　金：国家自然科学基金(82270093,82070078);山东第一医科大学学术提升计划(2019QL015);泰山学者项目(ts20190981,tsqn20181244)。

摘　　要：目的探讨微RNA-146a(miR-146a)对核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(NLRP3)的调节作用及其在呼吸机所致肺损伤(VILI)中的作用及其机制。方法①MLE-12细胞按照随机数字表法分成3组(每组3孔):机械牵张(CS)0h组、CS2h组、CS4h组,分别进行幅度为20%、频率为0.5Hz的CS0、2、4h。②MLE-12细胞按照随机数字表法分为4组(每组3孔):Negativecontrol(NC)对照组(NC+CS0h组)、miR-146a过表达组(Mimic+CS0h组)、NC+CS4h组、miR-146a过表达+CS4h组(Mimic+CS4h组),分别转染序列NC或miR-146a-5pMimic后进行CS0或4h。③MLE-12细胞按照随机数字表法分成4组(每组3孔):微RNAInhibitorN.C.(N.C.)对照组(N.C.+CS0h组)、miR-146a抑制组(Inhibitor+CS0h组)、N.C.+CS4h组、miR-146a抑制+CS4h组(Inhibitor+CS4h组),分别转染N.C.或miR-146a-5pInhibitor后进行CS0或4h。④MLE-12细胞按照随机数字表法分成8组(每组3孔):对照组(NC组)、miR-146a过表达组(Mimic组)、自噬溶酶体途径抑制剂组(NC+CQ组)、miR-146a过表达+自噬溶酶体途径抑制剂组(Mimic+CQ组)、泛素蛋白酶体抑制剂组(NC+MG132组)、miR-146a过表达+泛素蛋白酶体抑制剂组(Mimic+MG132组)、蛋白合成酶抑制剂组(NC+CHX组)、miR-146a过表达+蛋白合成酶抑制剂组(Mimic+CHX组),分别转染序列NC或miR-146a-5pMimic后加入自噬溶酶体途径抑制剂氯喹(CQ)、泛素蛋白酶体抑制剂MG132或蛋白合成酶抑制剂放线菌酮(CHX)。免疫印迹法(Westernblot)检测上皮钙黏素(E-cadherin)、闭合蛋白(Occludin)、NLRP3蛋白水平,实时荧光定量聚合酶链反应检测miR-146a水平。结果①与CS0h组比较,CS2h组和CS4h组miR-146a、E-cadherin、Occludin水平较低,NLRP3蛋白水平较高(均P<0.05);与CS2h组比较,CS4h组miR-146a、E-cadherin、Occludin蛋白水平较低,NLRP3蛋白水平较高(均P<0.05)。②与NC+CS0h组比较,NC+CS4h组NLRP3蛋白水平较高,E-cadherin及Occludin水平较低(均P<0.05);Mimic+CS0h组NLRP3蛋白水平较�Objective To investigate the regulatory effect of microRNA-146a(miR-146a)on nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3(NLRP3)and its role in the ventilator-induced lung injury(VILI)and its mechanism.Methods①MLE-12 cells were divided into 3 groups(n=3)according to the random number table method:mechanical stretch(CS)0 h group,CS 2 h group,and CS 4 h group.CS with an amplitude of 20%and a frequency of 0.5 Hz was performed for 0,2 h,and 4 h,respectively.②MLE-12 cells were divided into 4 groups(n=3)according to the random number table method:control group(NC+CS 0 h group),miR-146a overexpression group(Mimic+CS 0 h group),CS 4 h group(NC+CS 4 h group),miR-146a overexpression+CS 4 h group(Mimic+CS 4 h group).After transfecting Negative control(NC)/miR-146a-5p Mimic,CS was administered for O or 4 h.③MLE-12 cells were divided into 4 groups(n=3)according to the random number table method:control group(N.C.+CS 0 h),miR-146a inhibition group(Inhibitor+CS O h group),CS 4 h group(N.C.+CS 4 h group),miR-146a inhibition+CS 4 h group(Inhibitor+CS 4 h group),CS O or 4 h after transfection with mircoRNA Inhibitor N.C.(N.C.)/miR-146a-5p Inhibitor,respectively.MLE-12 cells were divided into 8 groups(n=3)according to the random number table method:control group(NC group),miR-146a overexpression group(Mimic group),autophagy lysosomal pathway inhibition agent group(NC+CQ group),miR-146a overexpression+autophagy lysosomal pathway inhibitor group(Mimic+CQ group),ubiquitin proteasome inhibitor group(NC+MG132 group),miR-146a overexpression+ubiquitin protease body inhibitor group(Mimic+MG132 group),protein synthase inhibitor group(NC+CHX group),and miR-146a overexpression+protein synthase inhibitor group(Mimic+CHX group).After transfecting sequence NC/miR-146a-5p Mimic,autophagic lysosomal pathway inhibitor chloroquine(CQ),ubiquitin proteasome inhibitor MG132 or protein synthetase inhibitor cycloheximide(CHX)were added respectively.The protein expression levels of E-cadherin,Occludin and NLRP3

关 键 词：呼吸机所致肺损伤 微RNA-146a 核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3 

分 类 号：R614[医药卫生—麻醉学]