Abstract：
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.
CONCLUSIONS: 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.
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.
CONCLUSIONS: 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.
摘要：
目的：探讨6-shogaol（6-SH）是否通过促进microRNA-26a-5p（miR-26a-5p）表达和抑制死亡相关蛋白激酶1（DAPK1）减轻氧糖剥夺/复氧（OGD/R）诱导的神经元自噬和钙超载。并探索其潜在机制。
方法：取原代培养的对数生长期小鼠海马神经元HT22细胞，用细胞计数试剂盒-8（CCK-8）检测细胞活力，寻找Na2S2O4的最佳浓度。将HT22细胞分为空白对照组(NC组),OGD/R组（无糖培养基+10mmol/LNa2S2O4处理1.5小时，然后正常培养基处理4小时），6-SH干预组(OGD后10μmol/L6-SH培养4小时),阴性对照抑制剂预处理组（用阴性对照抑制剂转染48小时，然后进行OGD，然后用6-SH培养4小时)，和miR-26a-5p抑制剂预处理组（用miR-26a-5p抑制剂转染48小时，然后进行OGD，然后用6-SH培养4小时)。CCK-8法检测各组细胞活力;透射电镜下观察细胞超微结构;实时定量聚合酶链反应(RT-qPCR)检测DAPK1和miR-26a-5p的基因表达;分子对接法验证6-SH与miR-26a-5p的相互作用;双荧光素酶法验证DAP5K1与细胞内磷酸化相关蛋白的表达;Western-流式细胞术检测DAP52B与Cablo3的表达;和p-DAPK1Ser308；免疫荧光法检测LC3和Beclin1的表达。
结果：CCK-8测定结果表明，与OGD/R组相比，6-SH干预组的细胞活力显着增加，而与6-SH干预组相比,miR-26a-5p抑制剂预处理组的细胞活力显著下降.透射电镜显示,与OGD/R组相比,6-SH干预组的自噬体数量显著减少,而与6-SH干预组相比,miR-26a-5p抑制剂预处理组的自噬体数量显著增加.RT-qPCR结果显示,与OGD/R组相比,6-SH干预组miR-26a-5p表达显著上调,DAPK1mRNA表达显著下调;与6-SH干预组相比,miR-26a-5p抑制剂预处理组miR-26a-5p表达显著下调,DAPK1mRNA表达显著上调.分子对接验证了6-SH和miR-26a-5p之间的相互作用。双荧光素酶报告基因检测显示,与阴性对照组相比,mmu-miR-26a-5p显著下调m-DAPK1-3UTR-WT的荧光素酶表达,表示它们之间的绑定相互作用。流式细胞仪检测结果显示,与OGD/R组相比,6-SH干预组细胞内Ca2+水平显著降低;与6-SH干预组相比,miR-26a-5p抑制剂预处理组的Ca2+水平显著升高.Westernblotting结果显示,与OGD/R组相比,6-SH干预组p-NMDAR2BSer1303，DAPK1，Beclin1和LC3的蛋白表达显着降低（p-NMDAR2BSer1303/β-actin：2.34±0.27vs.4.78±0.39，DAPK1/β-肌动蛋白：1.40±0.13vs.2.37±0.21，Beclin1/β-肌动蛋白：2.61±0.32vs.4.32±0.29，LC3/β-肌动蛋白：2.52±0.45vs.5.09±0.18，均P<0.05），而p-DAPK1Ser308的蛋白表达显着增加（p-DAPK1Ser308/β-肌动蛋白：0.66±0.09vs.0.40±0.02,P<0.05);与6-SH干预组相比,在miR-26a-5p抑制剂预处理组中，p-NMDAR2BSer1303，DAPK1，Beclin1和LC3的蛋白表达显着增加（p-NMDAR2BSer1303/β-actin：4.08±0.14vs.2.34±0.27，DAPK1/β-肌动蛋白：1.96±0.15vs.1.40±0.13，Beclin1/β-肌动蛋白：3.92±0.31vs.2.61±0.32，LC3/β-肌动蛋白：4.33±0.33vs.2.52±0.45，均P<0.05），而p-DAPK1Ser308蛋白的表达明显降低(p-DAPK1Ser308/β-肌动蛋白：0.33±0.12vs.0.66±0.09,P<0.05);免疫荧光染色显示,与OGD/R组相比,6-SH干预组LC3和Beclin1的荧光强度明显下降;与6-SH干预组相比,miR-26a-5p抑制剂预处理组LC3和Beclin1的荧光强度显著升高.
结论：6-SH可通过调控miR-26a-5p/DAPK1减轻细胞自噬和钙超载来减轻神经元损伤。
方法：取原代培养的对数生长期小鼠海马神经元HT22细胞，用细胞计数试剂盒-8（CCK-8）检测细胞活力，寻找Na2S2O4的最佳浓度。将HT22细胞分为空白对照组(NC组),OGD/R组（无糖培养基+10mmol/LNa2S2O4处理1.5小时，然后正常培养基处理4小时），6-SH干预组(OGD后10μmol/L6-SH培养4小时),阴性对照抑制剂预处理组（用阴性对照抑制剂转染48小时，然后进行OGD，然后用6-SH培养4小时)，和miR-26a-5p抑制剂预处理组（用miR-26a-5p抑制剂转染48小时，然后进行OGD，然后用6-SH培养4小时)。CCK-8法检测各组细胞活力;透射电镜下观察细胞超微结构;实时定量聚合酶链反应(RT-qPCR)检测DAPK1和miR-26a-5p的基因表达;分子对接法验证6-SH与miR-26a-5p的相互作用;双荧光素酶法验证DAP5K1与细胞内磷酸化相关蛋白的表达;Western-流式细胞术检测DAP52B与Cablo3的表达;和p-DAPK1Ser308；免疫荧光法检测LC3和Beclin1的表达。
结果：CCK-8测定结果表明，与OGD/R组相比，6-SH干预组的细胞活力显着增加，而与6-SH干预组相比,miR-26a-5p抑制剂预处理组的细胞活力显著下降.透射电镜显示,与OGD/R组相比,6-SH干预组的自噬体数量显著减少,而与6-SH干预组相比,miR-26a-5p抑制剂预处理组的自噬体数量显著增加.RT-qPCR结果显示,与OGD/R组相比,6-SH干预组miR-26a-5p表达显著上调,DAPK1mRNA表达显著下调;与6-SH干预组相比,miR-26a-5p抑制剂预处理组miR-26a-5p表达显著下调,DAPK1mRNA表达显著上调.分子对接验证了6-SH和miR-26a-5p之间的相互作用。双荧光素酶报告基因检测显示,与阴性对照组相比,mmu-miR-26a-5p显著下调m-DAPK1-3UTR-WT的荧光素酶表达,表示它们之间的绑定相互作用。流式细胞仪检测结果显示,与OGD/R组相比,6-SH干预组细胞内Ca2+水平显著降低;与6-SH干预组相比,miR-26a-5p抑制剂预处理组的Ca2+水平显著升高.Westernblotting结果显示,与OGD/R组相比,6-SH干预组p-NMDAR2BSer1303，DAPK1，Beclin1和LC3的蛋白表达显着降低（p-NMDAR2BSer1303/β-actin：2.34±0.27vs.4.78±0.39，DAPK1/β-肌动蛋白：1.40±0.13vs.2.37±0.21，Beclin1/β-肌动蛋白：2.61±0.32vs.4.32±0.29，LC3/β-肌动蛋白：2.52±0.45vs.5.09±0.18，均P<0.05），而p-DAPK1Ser308的蛋白表达显着增加（p-DAPK1Ser308/β-肌动蛋白：0.66±0.09vs.0.40±0.02,P<0.05);与6-SH干预组相比,在miR-26a-5p抑制剂预处理组中，p-NMDAR2BSer1303，DAPK1，Beclin1和LC3的蛋白表达显着增加（p-NMDAR2BSer1303/β-actin：4.08±0.14vs.2.34±0.27，DAPK1/β-肌动蛋白：1.96±0.15vs.1.40±0.13，Beclin1/β-肌动蛋白：3.92±0.31vs.2.61±0.32，LC3/β-肌动蛋白：4.33±0.33vs.2.52±0.45，均P<0.05），而p-DAPK1Ser308蛋白的表达明显降低(p-DAPK1Ser308/β-肌动蛋白：0.33±0.12vs.0.66±0.09,P<0.05);免疫荧光染色显示,与OGD/R组相比,6-SH干预组LC3和Beclin1的荧光强度明显下降;与6-SH干预组相比,miR-26a-5p抑制剂预处理组LC3和Beclin1的荧光强度显著升高.
结论：6-SH可通过调控miR-26a-5p/DAPK1减轻细胞自噬和钙超载来减轻神经元损伤。
