目的:观察BV-2小胶质细胞在氧糖剥夺/复糖复氧(OGD/R)中鞘氨醇激酶2(SphK2)的表达情况,并探讨SphK2是否参与了OGD/R致BV-2小胶质细胞的损伤过程。方法:采用小鼠BV-2小胶质细胞制备OGD/R模型,选取氧糖剥夺时间为2 h,复氧复糖时间为0 h,3 h,6 h,12 h,24 h。(1)设对照组,OGD 2 h组,OGD 2 h/R 3 h组,OGD 2 h/R 6 h组,OGD 2 h/R 12 h组,OGD 2 h/R 24 h组,观察细胞形态变化。细胞经OGD/R处理后CCK-8法检测细胞存活率。(2)设对照组,OGD 2 h/R 12 h组,OGD 2 h/R 12 h+ABC294640(SphK2特异抑制剂)组,用q-PCR及Western blot测定SphK2的基因及蛋白表达变化。结果:与对照组相比,随着OGD/R时间的延长,BV-2活力逐渐下降(P＜0.001),细胞形态发生了不同程度的改变,且有不同程度的损伤;通过观察细胞生存情况确定OGD2 h/R12 h为最适宜的OGD/R时间,与对照组相比,经OGD/R处理后BV-2细胞中SphK2 mRNA表达水平显著升高(P＜0.05),加入ABC294640显著抑制其表达升高(P＜0.001);与对照组相比,经OGD/R处理后BV-2细胞中SphK2蛋白表达水平显著升高(P＜0.001),加入ABC294640后,SphK2蛋白表达明显降低(P＜0.001)。结论:OGD/R损伤后的小胶质细胞可以被SphK2激活,在其损伤过程中发挥着调控作用。
Objective: To investigate the expression of sphingosine kinase 2 (SphK2) in BV-2 microglia during oxygen glucose deprivation/reoxygenation (OGD/R), and to investigate whether SphK2 is involved in the damage process of BV-2 microglia induced by OGD/R. Methods: The OGD/R model was established using mouse BV-2 microglial cells. The oxygen glucose deprivation time was 2 hours, and the reoxygenation and rehydration time was 0, 3, 6, 12, and 24 hours. (1) The control group, OGD2h group, OGD2h/R3h group, OGD2h/R6h group, OGD2h/R12h group and OGD2h/R24h group were set up to observe the changes of cell morphology. Cell viability was detected by CCK-8 method after OGD/R treatment; (2) The control group, OGD2h/ R12h group and OGD2h/R12h+ABC294640 (SphK2 specific inhibitor) group were set up, and the expression of SphK2 gene and protein was determined by q-PCR and Western blot. Results: Compared with the control group, with the prolongation of OGD/R time, the activity of BV-2 gradually decreased (P＜0.001), and the cell morphology changed to varying degrees, with varying degrees of injury By observing cell survival, OGD2h/R12h was determined as the most suitable OGD/R time. Compared with the control group, the expression level of Sphk2 mRNA in BV-2 cells treated with OGD/R was significantly increased (P＜0.05), and the addition of ABC294640 significantly inhibited the increase in its expression (P＜0.001). Compared with the control group, the expression level of SphK2 protein in BV-2 cells treated with OGD/R was significantly increased (P＜0.001), while the expression of SphK2 protein was significantly decreased after adding ABC294640 (P＜0.001). Conclusion: SphK2 activates microglia after OGD/R injury and plays a regulatory role in the process of OGD/R injury.
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