Activation of γ-Aminobutyric Acid Type A Receptor Attenuates Oxygen-Glucose Deprivation/Reoxygenation-Induced Astrocyte Injury
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摘要:
目的 探讨γ-氨基丁酸A型受体（γ-aminobutyric acid type A receptor，GABA_AR）对氧糖剥夺/复氧（oxygen-glucose deprivation/reoxygenation，OGD/R）诱导的星形胶质细胞损伤的保护作用及潜在机制。 方法 采用C8-D1A星形胶质细胞建立OGD/R损伤模型，随机分为正常对照组（NC）、OGD/R模型组、GABA_AR激动剂CL218872干预组（OGD/R+CL218872）及抑制剂MRK-016干预组（OGD/R+MRK-016）。通过EdU染色法检测细胞增殖；流式细胞术分析细胞凋亡；免疫荧光染色检测GABA_AR及胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）的表达；Western blot法检测脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）的表达。 结果 与OGD/R组比较，OGD/R+CL218872组细胞增殖率显著增强（P < 0.05），凋亡率明显降低（P < 0.01），GABA_AR与GFAP表达均上调（P < 0.05），BDNF蛋白表达水平显著升高（P < 0.05），炎症因子IL-6（P < 0.01）与TNF-α（P < 0.05）水平显著减少。而OGD/R+MRK-016组各指标均呈相反变化。 结论 GABA_AR激活可通过促进星形胶质细胞增殖、抑制其凋亡与炎症反应，并上调BDNF与GFAP的表达，从而减轻OGD/R诱导的细胞损伤。 -
关键词:
- γ-氨基丁酸A型受体 /
- 星形胶质细胞 /
- 缺糖缺氧/复糖复氧 /
- 突触重塑 /
- 细胞凋亡
Abstract:Objective To investigate the protective role and potential mechanisms of γ-aminobutyric acid type A receptor （GABA_AR） against oxygen-glucose deprivation/reoxygenation （OGD/R）-induced astrocyte injury. Methods C8-D1A astrocytes were used to establish an OGD/R injury model, which was randomly divided into four groups: the normal control group （NC）, the OGD/R model group, the GABA_AR agonist CL218872 intervention group （OGD/R+CL218872）, and the inhibitor MRK-016 intervention group （OGD/R+MRK-016）. Cell proliferation was assessed by EdU staining, apoptosis was analyzed by flow cytometry, GABA_AR and glial fibrillary acidic protein （GFAP） expression were examined by immunofluorescence staining, and brain-derived neurotrophic factor （BDNF） expression was determined by Western blotting. Results Compared with the OGD/R group, the OGD/R+CL218872 group showed significantly increased cell proliferation rate （P < 0.05） , markedly decreased apoptosis rate（P < 0.01）, upregulated expression of both GABA_AR and GFAP（P < 0.05） significantly elevated BDNF protein expression level（P < 0.05）, and significantly reduced levels of inflammatory factors IL-6 （P < 0.01） and TNF-α （P < 0.05）. The OGD/R+MRK-016 group showed opposite changes in all parameters. Conclusion GABA_AR activation can attenuate OGD/R-induced astrocyte injury by promoting astrocyte proliferation, inhibiting apoptosis and inflammatory response, and upregulating the expression of BDNF and GFAP. -
图 1 EdU染色与流式细胞术分析AS的增殖与凋亡水平[（$ \bar x \pm s $），n = 3]
A：EdU染色的代表性图片（100×，比例尺：100 μm）；B：流式细胞术检测细胞凋亡的散点图；C：细胞增殖率统计结果；D：细胞凋亡率统计结果；蓝色荧光为细胞核，红色荧光为增殖细胞；***P < 0.001，**P < 0.01，*P < 0.05。
Figure 1. Analysis of proliferation and apoptosis levels in AS by Edu staining and flow cytometry [（$ \bar x \pm s $），n = 3]
图 2 免疫荧光检测AS中GABA_AR的表达[（$ \bar x \pm s $），n = 5]
A：免疫荧光检测AS中GABA_AR的定位和表达（100×，比例尺：100 μm），蓝色代表细胞核，红色代表GABA_AR，；B：GABA_AR荧光强度的统计结果；*P < 0.05。
Figure 2. Immunofluorescence detection of GABA_AR expression in AS[（$ \bar x \pm s $），n = 5]
图 3 GABA_AR调节对AS中GFAP表达及炎症因子释放的影响[（$ \bar x \pm s $），n = 5]
A：GFAP免疫荧光染色图像（100×，比例尺：100 μm），蓝色为细胞核，绿色为GFAP；B：GFAP表达的统计分析；C：AS中TNF-α水平；D：AS中IL-6水平；***P < 0.001，**P < 0.01，*P < 0.05。
Figure 3. Effects of GABA_AR modulation on GFAP expression and inflammatory factor release in AS [（$ \bar x \pm s $），n = 5]
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