Division of Neurology, Department of Pediatrics, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Road, Jinan, 250021, China.
Cell Mol Neurobiol. 2013 Apr;33(3):401-10. doi: 10.1007/s10571-013-9906-y. Epub 2013 Jan 18.
The aim of this study was to investigate the role of connexin 43 (Cx43) and its hemichannel (HC1) in the death of astrocytes following ischemia/reperfusion (IR) or oxygen-glucose deprivation/reoxygenation (OGDR) insult. Wistar rats had their bilateral common carotid artery clamped for 1.5 h followed by 0, 4, and 24 h of reperfusion (n = 8 for each time point), respectively. All rats were sacrificed and Cx43, HC1, and caspase 3 (Casp3) in cerebral ischemic tissues were examined by immunohistochemistry and western blotting. Astrocytes cell line, astrocytes transduced with a retroviral empty vector (Psup astrocyte), or a Cx43-specific shRNA construct (shRNA astrocytes) were treated with OGDR insult for various periods. The viability of astrocytes was assessed by MTT assay. The expression of Cx43, HC1, and Casp3 was detected with western blotting. The results showed that the expression of Cx43, HC1, and Casp3 in rats' brain, astrocytes, and Psup astrocytes was significantly increased after 4 h of IR/OGDR and recovered on 24 h of the insult. Cell viability decreased after 4 h of the insult whereas the cell viability increased on 24 h after the insult. In contrast, the expression of Cx43, HC1, Casp3, and cell viability had no statistical differences in the null Cx43 gene-shRNA transfected astrocytes after the treatment of OGDR. The results suggest that Cx43 and HC1 are likely to play the pivotal roles in the mediation of the astrocytic death.
本研究旨在探讨缝隙连接蛋白 43(Cx43)及其半通道(HC1)在缺血/再灌注(IR)或氧葡萄糖剥夺/再复氧(OGDR)损伤后星形胶质细胞死亡中的作用。Wistar 大鼠双侧颈总动脉夹闭 1.5 h,分别再灌注 0、4 和 24 h(每个时间点 8 只)。所有大鼠均处死,采用免疫组织化学和 Western blot 检测脑缺血组织中 Cx43、HC1 和半胱氨酸蛋白酶 3(Casp3)。用 OGDR 损伤星形胶质细胞系、转染逆转录病毒空载体(Psup 星形胶质细胞)或 Cx43 特异性 shRNA 构建体(shRNA 星形胶质细胞)。通过 MTT 测定法评估星形胶质细胞活力。用 Western blot 检测 Cx43、HC1 和 Casp3 的表达。结果显示,IR/OGDR 后 4 h 大鼠脑、星形胶质细胞和 Psup 星形胶质细胞中 Cx43、HC1 和 Casp3 的表达明显增加,24 h 后恢复。损伤后 4 h 细胞活力下降,而损伤后 24 h 细胞活力增加。相反,OGDR 处理后,缺失 Cx43 基因-shRNA 转染的星形胶质细胞中 Cx43、HC1、Casp3 的表达和细胞活力无统计学差异。结果表明,Cx43 和 HC1 可能在介导星形胶质细胞死亡中发挥关键作用。