别孕烯醇酮对6-羟基多巴胺损伤的细胞系SH-SY5Y的保护作用

Allopregnanolone protecting cell line SH-SY5Y against 6-hydroxydopamine-induced lesion

目的明确别孕烯醇酮(APα)对6-羟多巴胺(6-OHDA)损伤的SH-SY5Y细胞系的保护作用,并阐明可能的分子机制。方法向体外培养的SH-SY5Y细胞系中分别加入6-OHDA、APα、γ-氨基丁酸A受体(GABAAR)拮抗剂荷包牡丹碱(Bic)和电压门控L型Ca^(2+)通道拮抗剂硝苯地平(nifedipine),采用免疫荧光细胞化学染色方法观察不同组别酪氨酸羟化酶(TH)阳性细胞的变化,Western blotting检测胞质钙调蛋白(CaM)、钙离子-钙调蛋白依赖性蛋白激酶Ⅱδ3(Ca MKⅡδ3),胞核Ca MKⅡδ3、脑源性神经营养因子(BDNF)和细胞周期蛋白依赖性激酶(CDK1)表达的变化,采用蛋白质免疫共沉淀验证Ca MKⅡδ3与CDK1/BDNF的相互作用。结果APα作用后,6-OHDA损伤的SH-SY5Y细胞TH和5-溴脱氧尿嘧啶核苷(BrdU)阳性细胞数目均明显增加,而TH/Brd U双阳性细胞数目无显著变化;同时Western blotting结果表明,SH-SY5Y细胞胞质和胞核上述蛋白的表达与单纯6-OHDA组相比也明显上升,经Bic处理后各蛋白增加更为明显。免疫共沉淀结果表明,Ca MKⅡδ3与CDK1/BDNF存在相互作用。结论APα对6-OHDA损伤的SH-SY5Y细胞的保护中,GABAAR发挥负性调控作用,通过稳定细胞的内环境达到增加TH阳性神经元数量的目的,其中Ca^(2+)-Ca M-CaMKⅡδ3信号通路和BDNF与CDK1发挥了关键作用。

Objective To clarify the protective effect of allopregnanolone(APα)on cell line SH-SY5Y damaged by 6-hydroxydopamine(6-OHDA)and its possible molecular mechanism.Methods 6-OHDA,APα,γ-aminobutyric acid A receptor(GABAAR)antagonist,voltage-gated L-type Ca^(2+)channel antagonist were added to the in vitro cultured cell line SH-SY5Y.Immunofluorescence cell chemical staining was used to observe the changes of tyrosine hydroxylase(TH)-positive cells.The changes of the expression of calmodulin(CaM),Ca^(2+)/calmodulin-dependent protein kinaseⅡδ3(Ca MKⅡδ3)in the cytoplasm and Ca MKⅡδ3,brain derived neurotrophic factor(BDNF)and cyclin-dependent kinases 1(CDK1)in the nucleus of different groups were detected by Western blotting.The interaction between Ca MKⅡδ3 and CDK1/BDNF was verified by co-precipitation.Results Having treated with APα,the number of TH and 5-Bromo-2-deoxyuridine(Brd U)-positive cells in 6-OHDA-lesioned SH-SY5Y cells increased significantly,but the number of TH/Brd U-double positive cells did not alter significantly.In the cytoplasmic or nucleus fraction of SH-SY5Y cells,the expression of the aforementioned proteins in the APα+6-OHDA group was higher than that in 6-OHDA+DMSO group by Western blotting,in particular,it increased significantly in APα+Bic+6-OHDA group compared with the APα+6-OHDA group.Immunoprecipitation assay showed that there existed an interaction between Ca MKⅡδ3 and CDK1 or BDNF.Conclusion In the neuroprotective effect of APαon 6-OHDA-lesioned SH-SY5Y cells,GABAAR plays a negative regulation.As a result,APαincreases the number of TH-positive neurons by stabilizing the cellular inner environment,in which the Ca^(2+)-Ca M-CaMKⅡδ3 signaling pathway and BDNF,CDK1 plays a key role.