摘要
目的评价miR-20a-5p在M1型小胶质细胞加重氧糖剥夺-复糖复氧(OGD/R)后神经元损伤中的作用及其与线粒体融合蛋白2(MFN2)的关系。方法将生长良好的BV2小胶质细胞(M0型)用脂多糖(100 ng/ml)和干扰素-γ(20 ng/ml)诱导小胶质细胞极化为M1表型,并通过qRT-PCR和免疫荧光法进行鉴定。将生长良好的N2a细胞采用随机数字表法分为6组(n=6):对照组(C组)、OGD/R组(OGD/R组)、M0型小胶质细胞共培养组(M0组)、M1型小胶质细胞共培养组(M1组)、转染miR-20a-5p抑制剂组(I组)和阴性对照组(NC组)。C组细胞常规培养;OGD/R组氧糖剥夺3 h,复糖复氧24 h制备N2a细胞氧糖剥夺-复糖复氧损伤模型;M0组氧糖剥夺3 h,复糖复氧时与M0型小胶质细胞共培养24 h;M1组氧糖剥夺3 h后复糖复氧时与M1型小胶质细胞共培养24 h;I组和NC组分别将转染试剂miR-20a-5p抑制剂和阴性对照miRNA转染至M1型小胶质细胞后,将N2a细胞氧糖剥夺3 h,复糖复氧时与转染后的M1型小胶质细胞共培养24 h。采用CCK-8法测定细胞活力,测定乳酸脱氢酶(LDH)漏出量,采用qRT-PCR法检测miR-20a-5p和MFN2 mRNA的表达,采用Western blot法检测MFN2表达。结果与C组比较,其余5组细胞活力降低,LDH漏出量增加,MFN2及其mRNA表达下调,OGD/R组、M0组和M1组miR-20a-5p表达上调,I组miR-20a-5p表达下调(P<0.05);OGD/R组与M0组细胞活力、LDH漏出量、miR-20a-5p、MFN2及其mRNA表达比较差异无统计学意义(P>0.05)。与OGD/R组和M0组比较,M1组细胞活力下降,LDH漏出量增加,MFN2及其mRNA表达下调,miR-20a-5p表达上调(P<0.05);与M1组比较,I组细胞活力增加,LDH漏出量下降,MFN2及其mRNA表达上调,miR-20a-5p表达下调(P<0.05)。结论M1型小胶质细胞加重N2a细胞OGD/R损伤的机制可能与M1型小胶质细胞miR-20a-5p表达上调抑制N2a细胞MFN2表达有关。
Objective To evaluate the role of miR-20a-5p in M1 microglia aggravating oxygen-glucose deprivation and restoration(OGD/R)-induced injury to neurons and the relationship with mitofusin2(MFN2).Methods The well-growing BV2 microglia(M0 type)were polarized into M1 phenotype by lipopolysaccharide(100 ng/ml)and IFN-γ(20 ng/ml)and identified by quantitative real-time polymerase chain reaction and immunofluorescence.The well-growing N2a cells were divided into 6 groups(n=6 each)by the random number table method:control group(group C),OGD/R group,M0 microglia co-culture group(group M0),M1 microglia co-culture group(group M1),miR-20a-5p inhibitor transfection group(group I)and negative control group(group NC).The cells were routinely cultured in group C,and the cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply to develop the model of OGD/R injury in group OGD/R.The cells were subjected to OGD for 3 h and were co-cultured with M0 microglia for 24 h during restoration of oxygen-glucose supply in group M0.The cells were subjected to OGD for 3 h and were co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply in group M1.In group I and group NC,cells were transfected with miR-20a-5p inhibitor and negative control miRNA into M1 microglia,respectively,and N2a cells were subjected to OGD for 3 h and co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply.The cell viability was determined by cell counting kit-8 assay,amount of lactate dehydrogenase(LDH)released was determined,the expression of miR-20a-5p and MFN2 mRNA was detected by quantitative real-time polymerase chain reaction,and MFN2 expression was detected by Western blot.Results Compared with group C,the cell viability was significantly decreased,the amount of LDH released was increased,and the expression of MFN2 protein and mRNA was down-regulated in the other five groups,miR-20a-5p expression was significantly up-regulated in OGD/R,M0 and M1 groups,and miR-20a-5p expression was significantly down-regulated in group I(P<0.05).There were no significant differences in the cell viability,amount of LDH released,and expression of miR-20a-5p,MFN2 protein and mRNA between group OGD/R and group M0(P>0.05).Compared with group OGD/R and group M0,the cell viability was significantly decreased,the amount of LDH released was increased,and the expression of MFN2 protein and mRNA was down-regulated,and miR-20a-5p expression was up-regulated in group M1(P<0.05).Compared with group M1,the cell viability was significantly increased,the amount of LDH released was decreased,the expression of MFN2 protein and mRNA was up-regulated,and miR-20a-5p expression was down-regulated in group I(P<0.05).Conclusions The mechanism by which M1 microglia aggravates OGD/R-induced damage to N2a cells may be related to the up-regulation of miR-20a-5p expression in M1 microglia and the inhibition of MFN2 expression in N2a cells.
作者
刘文洁
吕海涛
王红
陈静燕
单凯悦
陈怀龙
王明山
张高峰
董瑞
Liu Wenjie;Lyu Haitao;Wang Hong;Chen Jingyan;Shan Kaiyue;Chen Huailong;Wang Ming-shan;Zhang Gaofeng;Dong Rui(Department of Anesthesiology,Qingdao Municipal Hospital,Qingdao University,Qingdao 266071,China;Department of Education and Training,Qingdao Women and Children′s Hospital,Qingdao University,Qingdao 266034,China;Department of Anesthesiology,Qingdao Eighth People′s Hospital,Qingdao 266121,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2022年第8期974-979,共6页
Chinese Journal of Anesthesiology
基金
国家自然科学基金(82001132)
贝朗麻醉科研基金(BBDF-2019-010)
山东省自然科学基金(ZR2021MH365)。
