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

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

doi:10.16098/j.issn.0529-1356.2021.01.001

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Acta Anatomica Sinica ›› 2021, Vol. 52 ›› Issue (1) : 5-13. DOI: 10.16098/j.issn.0529-1356.2021.01.001

Neurobiology

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#

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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

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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 https://doi.org/10.16098/j.issn.0529-1356.2021.01.001

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