梓葛冻干粉针对体外氧糖剥夺/再灌注损伤大鼠大脑皮层星形胶质细胞的保护作用

Neuroprotective Effect of Lyophilized Powder of Catalpol and Puerarin on Oxygen-glucose Deprivation/reperfusion Injury in Astrocytes of Rat Cerebral Cortex in Vitro

目的探讨梓葛冻干粉针(C-P)对体外氧糖剥夺/再灌注(OGD/R)损伤大鼠大脑皮层星形胶质细胞的保护作用及可能的机制。方法原代分离培养大鼠大脑皮层星形胶质细胞,神经胶质纤维酸性蛋白(GFAP)免疫荧光鉴定星形胶质细胞的纯度。实验分正常组、模型组、辅料组、C-P:12.25、24.50、49.00μg·m L-1组。对星形胶质细胞进行OGD 6h/R 12h损伤并同时给予C-P。噻唑蓝(MTT)法检测细胞存活率、比色法测定乳酸脱氢酶(LDH)漏出率、TUNEL荧光染色法检测细胞凋亡率。利用试剂盒的方法分别检测细胞中超氧化物歧化酶(SOD)的活性,谷胱甘肽(GSH)、活性氧(ROS)和丙二醛(MDA)的含量以及一氧化氮(NO)的释放量,ELISA法测定细胞培养液中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和前列腺素(PG)E2的含量,Western blot检测细胞内caspase-3,诱导型一氧化氮合酶(i NOS),环氧酶(COX)-2,核因子(NF)-κB p65,p-NF-κB p65,IκB-α,p-IκB-α蛋白表达量。结果原代培养的星形胶质细胞纯度在97%以上。与正常组相比,模型组细胞存活率显著降低,LDH漏出率及细胞凋亡率明显增高;细胞内SOD活性和GSH含量明显降低,MDA和ROS含量显著升高;细胞培养液中NO、TNF-α、IL-1β和PGE2的释放量也显著增加。而以上相关生化指标均可被不同剂量的C-P所抑制。辅料组与模型组无显著差异。进一步研究发现,C-P能显著抑制OGD/R损伤引起的细胞中caspase-3、i NOS、COX-2蛋白表达量及NF-κB p65和IκB-α的磷酸化水平的增高。结论 C-P对体外OGD/R损伤的星形胶质细胞具有保护作用,其保护机制与其抗炎、抗氧化以及抑制细胞凋亡和NF-κB信号通路的激活有关。

OBJECTIVE To investigate the protection of lyophilized powder of catalpol and puerarin （C-P） on oxygen-glucose deprivation/reperfusion（OGD/R） -injured astrocytes and the possible mechanism in vitro. METHODS Primary astrocytes were isola- ted from the cerebral cortex of neonatal rats. The purity of astrocytes was identified by GFAP immunofluorescence. Astrocytes were di- vided into 6 groups：normal group, model group, excipients group, and three groups with gradient doses of C-P （ 12.25, 24. 50, 49.00μg·mL-1 ）. After astrocytes suffered from OGD/R （OGD 6 h/R 12 h） with or without C-P, cell survival, LDH leakage, and apoptosis were analyzed by MTT, colorimetry, and TUNEL respectively. The apoptotic protein, caspase-3, was evaluated by Western blot. Meanwhile, oxidative indexes including SOD, GSH, ROS, MDA, and NO were detected by assay kits. In terms of inflammation, TNF-α, IL-1β, and PGE2 in medium were evaluated via ELISA. iNOS, COX-2, NF-κB p65, p-NF-κB p65, IκB-α, and p-IκB-α were examined by Western blot. RESULTS The purity of primary astrocytes was more than 97%. Compared with normal group, the cell survival rate of model group decreased significantly, while the LDH leakage rate and cell apoptosis rate markedly increased ＇after OGD/R injury in model group. Meanwhile, SOD activity and GSH level in astrocytes markedly decreased. The level of MDA and ROS significantly increased. The content of NO, TNF-α, IL-1 β, and PGE2 released in medium also sharply increased. However, those changes of related biochemical indexes above could be reversed by different gradient doses of C-P. There was no significant difference between ex- cipients group and model group. Further study found that C-P could inhibit the protein expression of caspase-3, iNOS, and COX-2, as well as the increase of phosphorylation level of NF-KB p65 and IKB-ot significantly. CONCLUSION C-P shows a protective effect on the OGD/R injured astrocytes in vitro, and its protection mechanism involves in anti-inflammation, antioxidant, inhibiting the cell apoptosis and activation of NF-KB signaling pathway.