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

doi:10.16098/j.issn.0529-1356.2021.01.001

解剖学报 ›› 2021, Vol. 52 ›› Issue (1): 5-13.doi: 10.16098/j.issn.0529-1356.2021.01.001

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

王彤彤^1,2,3叶鑫^1,3 边维^1,3 陈治池^1,3 杜娟娟^1,4傅维达⁵ 陈梦娇⁵ 李俊楠⁵ 孙臣友^1,3*

1.温州医科大学基础医学院人体解剖学教研室，浙江温州325035; 2.台州职业技术学院医学与制药工程学院护理教研室，浙江台州318000; 3.温州医科大学基础医学院神经科学研究所，浙江温州325035; 4.温州医科大学基础医学院组织学胚胎学教研室，浙江温州325035; 5.温州医科大学第一临床医学院2015、2016级临床医学、医学影像学本科，浙江温州325035

收稿日期:2019-06-24 修回日期:2019-08-07 出版日期:2021-02-06 发布日期:2021-02-06
通讯作者: 孙臣友 E-mail:sunchenyou1972@aliyun.com
基金资助:
别孕烯醇酮促进PD小鼠多巴胺能神经元新生的作用及机制研究;CD133追踪APα诱导的6-OHDA PD小鼠新生多巴胺能神经元起源的应用研究

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

WANG Tong-tong^1,2,3 YE Xin^1,3 BIAN Wei^1,3 CHEN Zhi-chi^1,3 DU Juan-juan^1,4 FU Wei-da⁵ CHEN Meng-jiao⁵ LI Jun-nan⁵ SUN Chen-you^1,3*#br#

#br#

1.Department of Anatomy，School of Basic Medical Sciences of Wenzhou Medical University, Zhejiang Wenzhou 325035, China; 2.Department of Nursing, School of Medicine and Pharmaceutical Engineering of Taizhou Vocational and Technical College, Zhejiang Taizhou 318000, China; 3.Institution of Neuroscience, School of Basic Medical Sciences of Wenzhou Medical University，Zhejiang Wenzhou 325035, China; 4.Department of Histology And Embryology, School of Basic Medical Sciences of Wenzhou Medical University, Zhejiang Wenzhou 325035, China; 5.Bachelor of Clinical Medicine and Medical Imaging, Grade 2015, 2016, the First Clinical Medical College，Wenzhou Medical University，Zhejiang Wenzhou 325035, China

Received:2019-06-24 Revised:2019-08-07 Online:2021-02-06 Published:2021-02-06
Contact: SUN Chen-you E-mail:sunchenyou1972@aliyun.com

摘要/Abstract

摘要：

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Ca²⁺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Ⅱ δ3（CaMKⅡδ3），胞核CaMKⅡδ3、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CaMKⅡδ3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/BrdU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，CaMKⅡδ3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Ca²⁺ -CaM-CaMKⅡδ3信号通路和BDNF与CDK1发挥了关键作用。

关键词: 别孕烯醇酮, γ-氨基丁酸A受体, 多巴胺能神经元, 钙/钙调蛋白依赖性蛋白激酶Ⅱ, 脑源性神经营养因子, 免疫印迹法

Abstract:

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 Ca²⁺ 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), Ca2+/calmodulin-dependent protein kinase Ⅱ δ3 (CaMKⅡδ3) in the cytoplasm and CaMKⅡδ3, 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 CaMKⅡδ3 and CDK1/BDNF was verified by co-precipitation. Results Having treated with APα, the number of TH and 5-Bromo-2-deoxyuridine (BrdU)-positive cells in 6-OHDA-lesioned SH-SY5Y cells increased significantly, but the number of TH/BrdU-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 CaMKⅡδ3 and CDK1 or BDNF. Conclusion In the neuroprotective effect of APα on 6-OHDA-lesioned SH-SY5Y cells, GABAAR plays a negative regulation. As a result, APα increases the number of TH-positive neurons by stabilizing the cellular inner environment, in which the Ca²⁺ -CaM-CaMKⅡδ3 signaling pathway and BDNF, CDK1 plays a key role.

Key words: Allopregnanolone, γ-Aminobutyric acid A receptor, Dopaminergic neuron, Calmodulin-dependent kinase Ⅱ, Brain-derived neurotrophic factor, Western blotting

中图分类号:

Q189

王彤彤叶鑫边维陈治池杜娟娟傅维达陈梦娇李俊楠孙臣友. 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[J]. 解剖学报, 2021, 52(1): 5-13.

WANG Tong-tong YE Xin BIAN Wei CHEN Zhi-chi DU Juan-juan FU Wei-da CHEN Meng-jiao LI Jun-nan SUN Chen-you. Allopregnanolone protecting cell line SH-SY5Y against 6-hydroxydopamine induced lesion[J]. Acta Anatomica Sinica, 2021, 52(1): 5-13.

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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

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