间充质干细胞外泌体对海马神经元氧化应激的调控作用

目的 探讨间充质干细胞外泌体(Mesenchymal stem cell derived-exosomes,MSC-Exo)对海马神经元氧化应激的作用。方法 首先体外培养原代小鼠海马神经元,用H₂O₂刺激建立氧化应激模型,细胞计数试剂盒(Cell counting kit-8,CCK8)筛选最佳H₂O₂水平并检测不同水平MSC-Exo对细胞活力的作用,试剂盒检测超氧化物歧化酶(Superoxide dismutase,SOD)的活性,酶联免疫吸附测定法(Enzyme linked immunosorbent assay,ELISA)检测DNA氧化损伤分子8-羟基脱氧鸟苷(8-hydroxy-2 deoxyguanosine,8-OHdG)的表达水平,然后免疫荧光和免疫印迹检测应激和损伤分子一氧化氮合成酶(Inducible nitric oxide synthase,iNOS)、高迁移率族蛋白B1(High mobility group box 1,HMGB1)的表达水平。结果 CCK8细胞活力实验显示,10 μg/mL及以上水平的MSC-Exo对海马神经元氧化损伤具有明显的保护作用; 与对照组比较,H₂O₂组SOD的活性显著下降,而在MSC-Exo+H₂O₂组有所提高,趋于正常; 与对照组比较,H₂O₂作用后iNOS,HMGB1,8-OHdG等分子的表达水平显著上调(P<0.01),MSC-Exo作用于模型后与H₂O₂组比较,这些分子的表达水平显著下调(P<0.01)。结论 MSC-Exo作用于H₂O₂诱导的海马神经元氧化应激模型后能够增加SOD的活性,抑制海马神经元iNOS,HMGB1,8-OHdG等分子的表达,表明MSC-Exo对海马神经元氧化应激有一定的调控作用。

Regulation of mesenchymal stem cell derived-exosomes on H2O2 induced oxidative stress in hippocampal neurons

Objective To study the effect of mesenchymal stem cell derived-exosomes(MSC-Exo)on H₂O₂ induced oxidative stress of hippocampal neurons.Methods Primary mouse hippocampal neurons were cultured in vitro and stimulated with H₂O₂ to establish an oxidative stress model. The CCK8 experiment was applied to screen the optimal H₂O₂ concentration and test the effect of different concentrations of MSC-Exo on cell viability. The activity of superoxide dismutase(SOD)was detected by commercial kit. The expression of DNA oxidative damage molecule 8-OHdG was detected by ELISA assay. Then the stress and damage molecules iNOS and HMGB1 were detected by immunofluorescence and Western blotting.Results Cell viability experiments show that MSC-Exo at a concentration of 10μg/ml and above has a significant protective effect on hippocampal neurons from oxidative damage. Compared with the control group, the activity of SOD decreased significantly in the H₂O₂ group, but increased in the MSC-Exo+H₂O₂ group. Compared with the control group, the expression of iNOS, HMGB1 and 8-OHdG other molecules were significantly up-regulated(P<0.01)after the H₂O₂ treatment. In addition, compared with the H₂O₂ group, the expression of these molecules after MSC-Exo administration is significantly down-regulated(P<0.01).Conclusion MSC-Exo can increase the activity of SOD and inhibit the expression of iNOS, HMGB1 and 8-OHdG in hippocampal neurons after it acts on the oxidative stress model of hippocampal neurons induced by H₂O₂, which indicates that MSC-Exo can regulate the oxidative stress of hippocampal neurons to a certain extent and has a great application prospect in the treatment of nervous system diseases.