6-姜烯酚通过调控微小RNA-26a-5p/DAPK1减轻脑缺血/再灌注损伤的机制研究  

Mechanism study of 6-shogaol alleviating cerebral ischemia/reperfusion injury by regulating microRNA-26a-5p/death-associated protein kinase 1

作　　者：李世欣 饶欧阳 朱宁 周航向 陶浚泠 李叶红 刘颖 Li Shixin;Rao Ouyang;Zhu Ning;Zhou Hangxiang;Tao Junling;Li Yehong;Liu Ying(Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Critical Care Medicine,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学,贵阳550004 [2]贵州医科大学附属医院重症医学科,贵阳550004

出　　处：《中华危重病急救医学》2024年第6期616-623,共8页Chinese Critical Care Medicine

基　　金：贵州省科技计划项目(2023-401)。

摘　　要：目的探讨6-姜烯酚(6-SH)是否通过促进微小RNA-26a-5p(miR-26a-5p)表达并抑制死亡相关蛋白激酶1(DAPK1),减轻氧糖剥夺/复氧(OGD/R)神经细胞自噬及神经细胞钙超载,并探究其潜在机制。方法取体外培养的对数生长期小鼠海马神经元HT22细胞,用细胞增殖与毒性检测试剂盒(CCK-8)检测细胞活性,寻找Na2S2O4的最佳制模浓度。将HT22细胞分为空白对照组(NC组)、OGD/R组(无糖培养基+10 mmol/L Na2S2O4处理1.5 h后换正常培养基培养4 h)、6-SH干预组(OGD后用10μmol/L 6-SH培养4 h)、阴性对照抑制剂预处理组(阴性对照抑制剂转染48 h后行OGD,再用6-SH培养4 h)、miR-26a-5p抑制剂预处理组(miR-26a-5p抑制剂转染48 h后行OGD,再用6-SH培养4 h)。采用CCK-8法检测各组细胞活性;透射电镜下观察细胞超微结构;实时荧光定量聚合酶链反应(RT-qPCR)检测DAPK1、miR-26a-5p基因表达;分子对接验证6-SH与miR-26a-5p的相互作用;双荧光素酶验证DAPK1与miR-26a-5p的靶向关系;流式细胞仪测定细胞内Ca2+水平;蛋白质免疫印迹试验(Western blotting)检测磷酸化谷氨酸受体2B(p-NMDAR2B)Ser1303、DAPK1、自噬相关蛋白Beclin1、微管相关蛋白1轻链3(LC3)、p-DAPK1 Ser308蛋白表达;免疫荧光法检测LC3和Beclin1的表达。结果CCK-8法检测结果显示,6-SH干预组细胞活性较OGD/R组明显升高,miR-26a-5p抑制剂预处理组细胞活性较6-SH干预组明显降低。透射电镜下显示,6-SH干预组自噬小体较OGD/R组明显减少,miR-26a-5p抑制剂预处理组自噬小体较6-SH干预组明显增多。RT-qPCR检测结果显示,与OGD/R组比较,6-SH干预组miR-26a-5p表达明显上调,DAPK1 mRNA表达明显下调;与6-SH干预组比较,miR-26a-5p抑制剂预处理组miR-26a-5p表达明显下调,DAPK1 mRNA表达明显上调。分子对接验证6-SH与miR-26a-5p可互相作用。双荧光素酶报告基因检测显示,与阴性对照组比较,mmu-miR-26a-5p显著下调m-DAPK1-3UTR-WT的荧光素酶表达,说明两者之�Objective To investigate whether 6-shogaol(6-SH)alleviates oxygen-glucose deprivation/reoxygenation(OGD/R)-induced neuronal autophagy and calcium overload by promoting the expression of microRNA-26a-5p(miR-26a-5p)and inhibiting death-associated protein kinase 1(DAPK1),and to explore its potential mechanisms.Methods Primary cultured logarithmic growth phase mouse hippocampal neurons HT22 cells were taken and cell counting kit-8(CCK-8)was used to detect cell viability,searching for the optimal concentration of Na2S2O4.HT22 cells were divided into blank control group(NC group),OGD/R group(sugar-free culture medium+10 mmol/L Na2S2O4 treatment for 1.5 hours followed by normal culture medium for 4 hours),6-SH intervention group(cultured with 10μmol/L 6-SH for 4 hours after OGD),negative control inhibitor pretreatment group(transfected with negative control inhibitor for 48 hours followed by OGD,then cultured with 6-SH for 4 hours),and miR-26a-5p inhibitor pretreatment group(transfected with miR-26a-5p inhibitor for 48 hours followed by OGD,then cultured with 6-SH for 4 hours).Cell viability of each group was detected by CCK-8 method;cell ultrastructure was observed under transmission electron microscopy;real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the gene expressions of DAPK1 and miR-26a-5p;molecular docking were used to verify the interaction between 6-SH and miR-26a-5p;dual-luciferase assay was used to verify the targeting relationship between DAPK1 and miR-26a-5p;flow cytometry was used to determine the levels of intracellular Ca2+;Western blotting was used to detect the protein expressions of phosphorylated-glutamate receptor 2B(p-NMDAR2B)Ser1303,DAPK1,autophagy related protein Beclin1,light chain 3(LC3),and p-DAPK1 Ser308;immunofluorescence was used to detect the expression of LC3 and Beclin1.Results The results of the CCK-8 assay showed that the cell viability of the 6-SH intervention group was significantly increased compared to the OGD/R group,while the cell viability of the miR

关 键 词：自噬 脑缺血/再灌注损伤 钙超载 6-姜烯酚 微小RNA-26a-5p/死亡相关蛋白激酶1 

分 类 号：R743.31[医药卫生—神经病学与精神病学]