| ObjectiveTo study the effect and mechanism of?-asarone on Oxygen and glucose deprivation/reoxygenation-induced SH-SY5Y cells damage.Methods1 Model preparation:Na2S2O4 was dissolved in sugar-free Earle's solution to prepare a solution with a concentration of 15 mmol/L.SH-SY5Y cells were given to oxygen-sugar deprivation for 90 min,and then complete medium was added to continue culturing for 24 h to achieve reoxygenation.Oxygen and glucose deprivation/reoxygenation?OGD/R?injury cell model.Screening the best dosing concentration by CCK-8 method and LDH release rate determination method;2 Administration treatment:take the long-term SH-SY5Y cells,with a density of5x104mL-11 inoculated in 96-well plate,the cell is divided into 7 groups,each group of5 compound holes,respectively,blank control group,OGD/R model group,OGD/R+?-asarone group concentration respectively 10?g·mL-1?20?g·mL-1?40?g·mL-1?80?g·mL-11 OGD/R+15?g·mL-11 Edaravon-positive group,respectively;3 ELISA method was used to detect the effect of the optimal concentration of?-asarone on inflammation factor?TNF-??after OGD/R-induced SH-SY5Y cell injury;4 Immunoblotting assay to detect the effect of the optimal concentration of?-asarone on the expression of TNF-R1 and Glu R2 protein after OGD/R-induced SH-SY5Y cell injury;5 Calcium ion imaging to detect the effect of the optimal concentration of?-asarone on calcium concentration in SH-SY5Y cells induced by OGD/R;6 ELISA method was used to detect the activity of superoxide dismutase,catalase,glutathione peroxidase and malondialdehyde after?-asarone administration at a good concentration in SH-SY5Y cells induced by OGD/R The effect of MDA content;fluorescent probe method?DCFH-DA?to detect the content of reactive oxygen species?ROS?.Results:1 The test result of CCK-8 method shows:The OGD/R group cell viability was reduced to?64.83±2.2?%?P<0.001?Compared to the control group,while?-asarone(10?20?40?80?g·mL-1)and Edaravon?P<0.001?were given(10?20?40?80?g·mL-1)and Edaravon?P<0.001?15?g·mL-1can increase cell survival to?72.65±8.85?83.39±11.10?83.63±5.83?80.87±2.92?%??80.28±4.76?%?P<0.001or P<0.01?indicating that?-asarone(20,40,80?g·mL-1)can improve the survival of SH-SY5Y cells after OGD/R injury,with statistical differences.2 Cell LDH release test results indicate:the OGD/R group leakage rate increased to?1895±115.2?%?P<0.001?Comparedto the control group,while the yield of?-asarone(10,20,40,80?g·mL-1)and Edaravon(15?g·mL-1)was reduced by LDH?1171±131.21?1221±248.3?1142±124.8?1146±71.44?%??1007±142.6?%?P<0.01or P<0.001?,indicating?-asarone(20,40,80?g·mL-1)There were statistical differences in the release of LDH in THE SH-SY5Y cells that were able to reduce OGD/R.3 ELISA detection of inflammatory factors showed that:compared with the control group,the OGD/R group caused an increase in the concentration of inflammatory factor TNF-??P<0.001?.Compared with the OGD/R model group,the inflammatory content of?-asarone(20?g·mL-1)and Edaravone(15?g·mL-1)showed a decreasing trend,indicating that?-asarone(20?g·mL-1)and Edaravone(15?g·mL-1)group had a certain improvement effect,and the results were statistically significant?P<0.05 or P<0.001?.4 Immunoblotting results showed that:compared with the control group,OGD/R induced SH-SY5Y cells and found that TNF-R1 protein expression was significantly increased?P<0.001?,while?-asarone(20?g·mL-1)After treatment with Edaravone(15?g·mL-1),TNF-R1 protein expression significantly decreased?P<0.05 or P<0.001?;compared with the control group,the OGD/R group induced SH-SY5Y When cells were found,the expression of Glu R2 protein was significantly reduced,but after treatment with?-asarone(20?g·mL-1)and Edaravone(15?g·mL-1),the expression of Glu R2 protein obviously increased,and there was statistical significance.5 Calcium ion imaging test results show that:compared with the control group,the fluorescence intensity of calcium ions was significantly increased after OGD/R induction of SH-SY5Y cells,while the fluorescence intensity of calcium ions was significantly decreased after?-asarone(20?g·mL-1)and Edaravone(15?g·mL-1)intervention.6 ELISA detection of cellular oxidative stress showed that:compared with the control group,OGD/R induced SH-SY5Y cells showed decreased activity of SOD,CAT and GSH-PX?P<0.001 or P<0.01?and increased MDA and ROS content,while intervention with?-asarone(20?g·mL-1)and Edaravone(15?g·mL-1)increased activity of SOD,CAT and GSH-PXin cells?P<0.05 or P<0.01?and decreased MDA and ROS content?P<0.05?.Conclusion1 Each dose of?-asarone(10?g·mL-1,20?g·mL-1,40?g·mL-1,and 80?g·mL-1)can improve the OGD/R-induced SH-SY5Y cell damage;2?-asarone(20?g·mL-1)can significantly improve the oxidative stress and inflammatory response after OGD/R induced SH-SY5Y cell injury;3?-asarone(20?g·mL-1)can significantly increase the expression of TNF-R1protein and decrease the expression of Glu R2 protein after OGD/R-induced SH-SY5Y cell injury;4?-asarone(20?g·mL-1)can reduce the excessive increase of calcium concentration in SH-SY5Y cells induced by OGD/R. |
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