靶向激活miR-148b-3p对缺血性脑卒中小鼠脑功能的影响及机制  

Effect and Mechanism of Targeted Activation of mir-148b-3p on Brain Function in Mice with Ischemic Stroke

作　　者：李明 刘勇[1] 董海[1] 王彪[1] 阎登富[1] 刘磊 何仲春 Li Ming;Liu Yong;Dong Hai;Wang Biao;Yan Dengfu;Liu Lei;He Zhongchun(Department of Neurology, The First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China)

机构地区：[1]成都医学院第一附属医院神经内科,成都610500

出　　处：《成都医学院学报》2020年第4期432-437,共6页Journal of Chengdu Medical College

基　　金：成都医学院校基金(No:CYZ18-33)。

摘　　要：目的观察激活miR-148b-3p对小鼠缺血性脑卒中的影响,探讨硫氧还蛋白互作蛋白(TXNIP)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)轴在该影响中的作用。方法将50只健康雄性C57BL/6小鼠按照随机数字表法分为假手术组(Sham组)、栓塞模型组(MCAO组)、NLRP3抑制剂组(MCC950组)、miR阴性对照转染组(NC agomiR组)、miR-148b-3p激动剂转染组(miR-148b-3p agomiR组),每组10只。假手术组仅做手术损伤;其他各组小鼠采用线栓致大脑中动脉阻塞(MCAO)法建立小鼠缺血性脑卒中模型,使小鼠接受脑缺血1 h和再灌注72 h。MCC950组于手术后即刻腹腔注射50 mg/kg体重的NLRP3抑制剂MCC950。Sham组和MCAO组于手术后即刻腹腔注射同等容积的磷酸缓冲盐溶液(PBS)。NC agomiR组、miR-148b-3p agomiR组在手术前60 min通过侧脑室注射30 pmol/g体重的agomiR阴性对照品或miR-148b-3p agomiR。再灌注72 h后评估小鼠脑功能缺陷程度,氯化三苯基四氮唑染色法检测脑梗死灶体积百分数,RT-PCR法检测脑组织miR-148b-3p表达量,蛋白质印迹技术检测脑组织TXNIP、NLRP3蛋白表达量。另外,在小鼠神经瘤母细胞N2a细胞系上,采用双荧光素酶实验验证miR-148b-3p对TXNIP mRNA的靶向调节作用。结果与Sham组比较,MCAO组小鼠脑功能缺陷评分和脑梗死灶体积百分数均明显增大,缺血区脑组织miR-148b-3p表达量减少,TXNIP、NLRP3蛋白表达量增多;与miR转染阴性对照组比较,miR-148b-3p激动剂转染组小鼠脑功能缺陷评分和脑梗死灶体积百分数均明显降低,缺血区脑组织miR-148b-3p表达量增多,TXNIP、NLRP3蛋白表达量减少;与MCAO组比较,MCC950组小鼠脑功能缺陷评分和脑梗死灶体积百分数均明显降低。荧光素酶实验显示,TXNIP是miR-148b-3p的1个直接调节靶。结论内源性miR-148b-3p的表达下调可能与小鼠缺血性脑卒中发生相关;激活miR-148b-3p则对小鼠缺血性脑卒中有一定保护性作用,其机制与其抑制�Objective To observe the effect of miR-148b-3p activation on ischemic stroke in mice and explore the role of the thioredoxin-interacting protein(TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)axis in this effect.Methods 50 healthy male C57BL/6 mice were randomly divided into 5 groups sham group,with 10 mice in each group,middle cerebral artery occlusion(MCAO)model group,NLRP3 inhibitor(MCC950)group,with to mice in each group,miR negative control transfection(NC agomiR)group,and miR-148b-3p agonist transfection(miR-148b-3p agomiR)group.Sham group only received surgical trauma.For other groups,all the mice were subjected to middle cerebral artery occlusion(MCAO)for 1 hour and followed by 72-h reperfusion.MCC950 group was administrated intraperitoneally with the NLRP3 inhibitor MCC950(50mg/kg)right after MCAO.Sham group and MCAO group were administrated intraperitoneally with an equal volume of phosphate buffer saline(PBS).For miR-148b-3p agomiR group and NC agomiR group,miR-148b-3p agomiR(30pmol/g weight)or agomiR negative control(30 pmol/g weight)were administered by left intracerebroventricular injection 60 min before MCAO.After 72-h reperfusion,neurological deficit scores were examined and percents of infarct volume were calculated by triphenyltetrazolium chloride(TTC)staining.RT-PCR was used to determine the expression of miR-148b-3p.Western blot was applied to measure protein expression of TXNIP and NLRP3.Additionally,Dual-luciferase reporter assay was used in N2a cells to investigate the targeted modulation of TXNIP by miR-148b-3p.Results When compared with the sham group,MCAO group showed increased neurological deficit scores,elevated percents of infarct volume,reduced miR-148b-3p expression,and enhanced protein expressions of TXNIP and NLRP3.In contrast,when compared with NC agomiR group,miR-148b-3p agomiR group showed decreased neurological deficit scores,reduced percents of infarct volume,increased miR-148b-3p expression,and reduced protein expressions of TXNIP and NLRP3.Wh

关 键 词：miR-148b-3p 缺血性脑卒中 硫氧还蛋白互作蛋白 核苷酸结合寡聚化结构域样受体蛋白3 

分 类 号：R743.3[医药卫生—神经病学与精神病学]