Dexmedetomidine ameliorates ischemia-induced nerve injury by up-regulating Sox11 expression

Abstract

Background: Dexmedetomidine (Dex) is associated with several biological processes. Ischemic stroke has the characteristics of high morbidity and mortality. Herein, we aimed to explore whether Dex ameliorates ischemia-induced injury and determine its mechanism.

Methods: Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to measure gene and protein expression. Cellular viability and proliferation were assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, respectively. Cell apoptosis was detected by flow cytometry. An oxygen-glucose deprivation/reoxygenation model of SK-N-SH and SH-SY5Y cells was constructed. A middle cerebral artery occlusion (MCAO) model was also built to assess Dex function in vivo. Neuronal function was assessed using the Bederson Behavior Score and Longa Behavior Score.

Results: We found that Dex positively and dose-dependently regulated Sox11 expression and prevented damage caused by oxygen-glucose deprivation/reoxygenation (OGD/R), enhancing cell viability and proliferation and reducing apoptosis in SK-N-SH and SH-SY5Y cells. The overexpression of Sox11 antagonized OGD/R-induced SK-N-SH and SH-SY5Y cell apoptosis and promoted cell growth in vitro. Furthermore, cell proliferation was decreased and cell apoptosis was increased after Sox11 knockdown in Dex-treated SK-N-SH and SH-SY5Y cells. We demonstrated that Dex prevented OGD/R-induced cell injury by up-regulating Sox11. Furthermore, we also confirmed that Dex protected rat from ischemia-induced injury in the MCAO model.

Conclusions: The role of Dex in cell viability and survival was verified in this study. Moreover, Dex protected neurons from MCAO-induced injury by up-regulating the expression of Sox11. Our research proposes a potential drug to improve the functional recovery of stroke patients in the clinic.

Keywords: Ischemic stroke; Sox11; dexmedetomidine (Dex); middle cerebral artery occlusion (MCAO); oxygen-glucose deprivation/reoxygenation (OGD/R).

Figure 1

Dex positively regulates the expression of Sox11 under OGD/R conditions. (A) The relative Sox11 mRNA expressions in the different groups measured by qRT-PCR. (B) Sox11 protein levels evaluated by western blotting. (C) Quantitation of Sox11 protein levels in the different groups. For statistical analysis, different letters showed significant differences between groups in SH-N-SH or SH-SY5Y cells. OGD/R, oxygen-glucose deprivation/reoxygenation; Dex, dexmedetomidine; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2

Dex rescues the reduced viability and proliferation of cells after OGD/R. (A) Viability measured by CCK-8 assays. (B) Cell proliferation measured by EdU staining assay (scale bar, 50 µm). (C) Quantitative analysis of the EdU assay results for the different groups. For statistical analysis, different letters showed significant differences between groups in SH-N-SH or SH-SY5Y cells. OGD/R, oxygen-glucose deprivation/reoxygenation; Dex, dexmedetomidine; CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride.

Figure 3

Dex ameliorated cellular apoptosis induced by OGD/R. (A) Apoptosis ratios determined by flow cytometry in the different groups. (B) Quantitation of apoptosis measured by flow cytometry. (C) Levels of apoptosis-related proteins shown by western blotting. (D) Quantitative analysis of apoptosis-related protein levels. For statistical analysis, different letters showed significant differences between groups in SH-N-SH or SH-SY5Y cells. OGD/R, oxygen-glucose deprivation/reoxygenation; Dex, dexmedetomidine; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4

Sox11 ameliorates reduced cellular viability and proliferation induced by OGD/R. (A) Verification of Sox11 overexpression, as shown by qRT-PCR. (B) Verification of Sox11 overexpression, as shown by western blotting. (C) Quantitation of Sox11 protein levels in the different groups. (D) Cell viabilities in the different groups, as shown by the CCK-8 assays. (E) Cell proliferation in the different groups, shown by the EdU staining assay (scale bar, 50 µm). For statistical analysis, different letters showed significant differences between groups in SH-N-SH or SH-SY5Y cells. OGD/R, oxygen-glucose deprivation/reoxygenation; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; OE, overexpression; EdU, 5-ethynyl-2'-deoxyuridine; DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride.

Figure 5

Sox11 reduces apoptosis induced by OGD/R. (A) Apoptosis ratios for the different groups, as measured by flow cytometry. (B) Quantitation of apoptosis in the different groups, as measured by flow cytometry. (C) Apoptosis-related proteins in the different groups, as shown by western blotting. (D) Quantitation of apoptosis-related in the different groups. For statistical analysis, different letters showed significant differences between groups in SH-N-SH or SH-SY5Y cells. OGD/R, oxygen-glucose deprivation/reoxygenation; OE, overexpression; FITC, fluorescein isothiocyanate; PI, propidium iodide; CTRL, control.

Figure 6

Dex ameliorates OGD/R-mediated cell damage via up-regulation of Sox11. (A) The efficacy of Sox11 knockdown verified by western blotting. (B) Cell viability in the different groups, as determined by CCK-8 assays. (C) Quantitation of proliferation in the different groups, as determined by EdU assays. (D) Quantitation of apoptosis in the different groups, as measured by flow cytometry. (E) Levels of apoptosis-related proteins in the different groups, as shown by western blotting. For statistical analysis, different letters showed significant differences between groups in SH-N-SH or SH-SY5Y cells. Dex, dexmedetomidine; OGD/R, oxygen-glucose deprivation/reoxygenation; CCK-8, Cell Counting Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; sh, short hairpin RNA; CTRL, control.

Figure 7

Dex protects rat from ischemia-induced injury in the MCAO model. (A) Representative images of rat brains after MCAO for the different groups. (B) Infarct areas in the rat brains. (C) Bederson behavioral scores for the different groups. (D) Longa behavioral scores for the different groups. (E) Levels of apoptosis-associated proteins in the brain tissue of the different groups, as shown by western blotting. ***, P<0.001 vs. sham group; and ^##, P<0.01 vs. MCAO group. Dex, dexmedetomidine; MCAO, middle cerebral artery occlusion.