白藜芦醇预处理对星形胶质细胞氧糖剥夺/再复氧损伤后活化及炎症反应的影响

doi:10.16098/j.issn.0529-1356.2020.03.001

摘要/Abstract

摘要：

目的探讨白藜芦醇预处理对体外氧糖剥夺/再复氧(OGD/R) 损伤后星形胶质细胞活化及炎症反应的影响。方法征集20只新生SD大鼠（生后24 h内），将其原代分离培养的大脑皮层星形胶质细胞行氧糖剥夺150 min，再复氧培养24 h。实验分为正常组(Nor)、对照组(Ctrl) 和白藜芦醇预处理组(Res)。 CCK-8法检测细胞活力，5-乙炔基-2’脱氧尿嘧啶核苷（EdU）法检测细胞增殖，免疫荧光染色法检测胶质纤维酸性蛋白（GFAP）、S100β蛋白表达，Western blotting 检测GFAP、S100β、波形蛋白(vimentin)、白细胞介素（IL）-10、β干扰素（IFN-β）、肿瘤坏死因子α(TNF-α）和 IL-1β蛋白表达，ELISA法检测IL-10、IFN-β、TNF-α和IL-1β蛋白含量。结果 CCK-8法检测结果显示，随着白藜芦醇浓度（5、20、40 μmol/L）的增高，细胞活力逐渐增高，且与对照组差异有显著性（P<0.01），以40 μmol/L组细胞活力最强。之后随白藜芦醇浓度(80、100 μmol/L) 的增高，细胞活力显著降低，以100 μmol/L组细胞活力最弱 (P<0.05)。EdU检测显示，在OGD/R损伤后，对照组和白藜芦醇组显著高于正常组EdU阳性细胞比例(P<0.01)，而白藜芦醇组显著低于对照组 (P<0.01)。免疫荧光染色、Western blotting和ELISA法显示，对照组和白藜芦醇组GFAP、S100β、vimentin、IL-10、IFN-β、TNF-α和IL-1β 蛋白表达或含量显著高于正常组(P<0.05)，但白藜芦醇组除 IL-10 和IFN-β蛋白表达或含量较对照组增高外，其余均低于对照组(P<0.05)。结论白藜芦醇预处理可抑制 OGD/R 后体外星形胶质细胞的活化，并减轻炎症反应。

关键词: 白藜芦醇, 星形胶质细胞, 活化, 炎症反应, 免疫印迹法, 酶联免疫吸附测定, 免疫荧光, 大鼠

Abstract:

Objective To investigate the effects of resveratrol pretreatment on activation and inflammation of astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. Methods Twenty newborn SD rats within 24 hours were recruited,and their primary cultured astrocytes were deprived of oxygen glucose fpr 150 minutes and reoxygenation for 24 hours.The experment was divided into the normal (Nor),the control (Ctrl) and resveratrol pretreatment (Res) groups.Cell viability was detected with CCK-8 assay.Cell proliferation was measured with 5-ethynyl-2,-deoxyuridine (EdU).The expressions of glial fibrillary acidic protein (GFAP) and S100βproteins were determined with immunofluorescence.Theexpressions of GFAP,S100β,vimentin,interleukin (IL)-10,β-interferon (IFN-β),tumor necrosis factor α(TNF-α)and IL-1 β proteins were detected with Western blotting.The levels of IL-10,IFN-β,TNF-αand IL-1βproteins in the supernatant were measured by ELISA. Results CCK-8 assay showed that with the increase of resveratrol concentration (5, 20 and 40 μmol/L), the cell viability increased gradually, which was significantly lower than that of the control group (P<0.01). After that, with the increase of resveratrol concentration (80 and 100 μmol/L), the cell viability decreased significantly, and the cell viability of 100 μmol/L resveratrol group was significantly lower than in the control group (P<0.02). EdU testing showed that after OGD/R injury, the percentage of EdU positive cells in the control and resveratrol group was significantly higher than that of in the normal group (P<0.01), while it was significantly lower in the resveratrol pretreatment group than that of the control group (P<0.01).Immunofluorescence, Western blotting and ELISA method showed that the expressions or levels of GFAP, S100β, vimentin, IL-10, IFN-β, TNF-α, IL-1β proteins in the control and resveratrol group were significantly higher than that of the normal group (P<0.05), but the expresions or levels of IL-10 and IFN-β proteins in the resveratrol group were higher than those of the control group, and the others were lower than the control group (P<0.05).However，the expressions or levels of IL-10 and IFN-β proteins in the resveratrol group were higher than those of the control group, and the others in the resveratrol group were lower than those of the control group (P<0.05). Conclusion Resveratrol pretreatment can inhibit the activation of astrocytes and reduce the inflammation after OGD/R injury in vitro.

Key words:

Resveratrol, Astrocyte, Activation, Inflammation, Western blotting, Enzyme-linked immunosobent assay, Immunofluorescence, Rat

中图分类号:

R322
R965

刘菁刘杰廖鸿雁陈月向勤杨琴 . 白藜芦醇预处理对星形胶质细胞氧糖剥夺/再复氧损伤后活化及炎症反应的影响[J]. 解剖学报, 2020, 51(3): 313-319.

LIU Jing LIU Jie LIAO Hong-yan CHEN Yue XIANG Qin YANG Qin. Effects of resveratrol pretreatment on activation and inflammation of astrocytes after oxygen-glucose deprivation/reoxygenation injury in vitro[J]. Acta Anatomica Sinica, 2020, 51(3): 313-319.

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