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

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,说明两者之间存在结合作用。流式细胞仪检测结果显示,与OGD/R组比较,6-SH干预组细胞内Ca2+水平明显降低;与6-SH干预组比较,miR-26a-5p抑制剂预处理组Ca2+水平明显升高。Western blotting检测结果显示,与OGD/R组比较,6-SH干预组p-NMDAR2B Ser1303、DAPK1、Beclin1、LC3蛋白表达均明显降低(p-NMDAR2B Ser1303/β-actin:2.34±0.27比4.78±0.39,DAPK1/β-actin:1.40±0.13比2.37±0.21,Beclin1/β-actin:2.61±0.32比4.32±0.29,LC3/β-actin:2.52±0.45比5.09±0.18,均P<0.05),p-DAPK1 Ser308蛋白表达明显升高(p-DAPK1 Ser308/β-actin:0.66±0.09比0.40±0.02,P<0.05);与6-SH干预组比较,miR-26a-5p抑制剂预处理组p-NMDAR2B Ser1303、DAPK1、Beclin1、LC3蛋白表达明显升高(p-NMDAR2B Ser1303/β-actin:4.08±0.14比2.34±0.27,DAPK1/β-actin:1.96±0.15比1.40±0.13,Beclin1/β-actin:3.92±0.31比2.61±0.32,LC3/β-actin:4.33±0.33比2.52±0.45,均P<0.05),p-DAPK1 Ser308蛋白表达明显降低(p-DAPK1 Ser308/β-actin:0.33±0.12比0.66±0.09,P<0.05);免疫荧光法检测显示,与OGD/R组比较,6-SH干预组LC3、Beclin1荧光强度明显降低;与6-SH干预组比较,miR-26a-5p抑制剂预处理组LC3、Beclin1荧光强度明显升高。结论6-SH可通过调控miR-26a-5p/DAPK1减轻细胞自噬及钙超载,从而减轻神经细胞损伤。

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-26a-5p inhibitor pretreatment group was significantly decreased compared to the 6-SH intervention group.Transmission electron microscopy revealed that the number of autophagosomes in the 6-SH intervention group was significantly reduced compared to the OGD/R group,while the number of autophagosomes in the miR-26a-5p inhibitor pretreatment group was significantly increased compared to the 6-SH intervention group.RT-qPCR results showed that compared with the OGD/R group,the expression of miR-26a-5p was significantly upregulated and the expression of DAPK1 mRNA was significantly downregulated in the 6-SH intervention group;compared with the 6-SH intervention group,the expression of miR-26a-5p was significantly downregulated and the expression of DAPK1 mRNA was significantly upregulated in the miR-26a-5p inhibitor pretreatment group.Molecular docking verified the interaction between 6-SH and miR-26a-5p.Dual-luciferase reporter gene assay showed that compared with the negative control group,mmu-miR-26a-5p significantly downregulated the luciferase expression of m-DAPK1-3UTR-WT,indicating a binding interaction between them.Flow cytometry results showed that compared with the OGD/R group,the level of intracellular Ca2+was significantly decreased in the 6-SH intervention group;compared with the 6-SH intervention group,the level of Ca2+was significantly increased in the miR-26a-5p inhibitor pretreatment group.Western blotting results showed that compared with the OGD/R group,the protein expressions of p-NMDAR2B Ser1303,DAPK1,Beclin1,and LC3 were significantly decreased in the 6-SH intervention group(p-NMDAR2B Ser1303/β-actin:2.34±0.27 vs.4.78±0.39,DAPK1/β-actin:1.40±0.13 vs.2.37±0.21,Beclin1/β-actin:2.61±0.32 vs.4.32±0.29,LC3/β-actin:2.52±0.45 vs.5.09±0.18,all P<0.05),while the protein expression of p-DAPK1 Ser308 was significantly increased(p-DAPK1 Ser308/β-actin:0.66±0.09 vs.0.40±0.02,P<0.05);compared with the 6-SH intervention group,the protein expressions of p-NMDAR2B Ser1303,DAPK1,Beclin1,and LC3 were significantly increased in the miR-26a-5p inhibitor pretreatment group(p-NMDAR2B Ser1303/β-actin:4.08±0.14 vs.2.34±0.27,DAPK1/β-actin:1.96±0.15 vs.1.40±0.13,Beclin1/β-actin:3.92±0.31 vs.2.61±0.32,LC3/β-actin:4.33±0.33 vs.2.52±0.45,all P<0.05),while the expression of p-DAPK1 Ser308 protein was significantly decreased(p-DAPK1 Ser308/β-actin:0.33±0.12 vs.0.66±0.09,P<0.05);immunofluorescence staining showed that compared with the OGD/R group,the fluorescence intensity of LC3 and Beclin1 was significantly decreased in the 6-SH intervention group;compared with the 6-SH intervention group,the fluorescence intensity of LC3 and Beclin1 was significantly increased in the miR-26a-5p inhibitor pretreatment group.Conclusion 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.