Mechanism study of 6-shogaol alleviating cerebral ischemia/reperfusion injury by regulating microRNA-26a-5p/death-associated protein kinase 1
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 10 μmol/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.
AutophagyCerebral ischemia/reperfusion injuryCalcium overload6-ShogaolmicroRNA-26a-5p/death-associated protein kinase 1
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