Effects Of Cx43 And Its Hemichannels Of Astrocytes On Cerebral Ischemia Reperfusion Injury

BackgroundIschemic cerebrovascular disease is a common clinical neurological disease. Ischemia reperfusion injury (IRI) which occurs in the treatment of developmental processing is the key to decide the prognosis and considered to be more destructive than the disease itself. Brain neurons and glial cells, their apoptosis is the major pathological manifestation in cerebral ischemia reperfusion injury. In this area, researches on neurons are more than on glial cells.Astrocyte is the largest glial cell. It is the most generally distributed in the mammalian brain and has more gap junctions than any other cells in the brain. Gap junction is a material pipe which passes information between adjacent cells and formed by two hemichannels. Hemichannel is formed by gap junction protein. Connexin 43(Cx43) is the most and the most widely distributed protein. Gap junctions have been confirmed that they are closely related to all sorts of craniocerebral injury and cell apoptosis signal transduction.Our experiment used an ischemia-reperfusion injury model in vitro (cells in oxygen-glucose deprivation/reoxygenation).We identified the effects of Cx43 and its hemichannels on cell apoptosis in cerebral ischemia reperfusion injury by testing the expression of Cx43, hemichannel antibodies 1 (HC1) and cell apoptosis marker-caspase3 (Casp3).ObjectiveTo investigate the effects of Connexin 43 and its hemichannels on the astrocytes' activity and apoptosis after the treatment of oxygen-glucose deprivation reoxygenation (OGDR), then provide the new theoretical basis and guiding for the treatment of ischemic cerebrovascular disease.MethodsHuman brain cortex astrocytic cell lines for cell culture were identified by using immunocytochemistry method to detect glial fibrillary acidic protein (GFAP), Cx43 and HCl.The cells were divided into three groups after transfection—cell line, empty vector transfection (Psup) and Cx43-specific shRNA transfection group (shRNA). The expression of Cx43 and HC1 was identified by western blot after the transfection. Cells in each group were treated by OGDR respectively, untreated cells as the control. The viability of astrocytes was assessed by MTT assay and the expression of Cx43, HC1 and Casp3 was detected with western blotting methods at different reoxygenation time (0 h,4 h,24 h).Results1. The expression of GFAP+Cx43 and GFAP+HC1 in experimental cells was showed. It was proved those cells were astrocytes and Cx43 and its hemichannels were existed.2. After the transfection, Cx43 and HC1 were detected in cell line and Psup, but not in shRNA cells, which means the transfection was successful.3. Compared with control group after OGDR, the activity of cell line and Psup cells was decreased significantly in reoxygenation 4 h (P< 0.05), there was no statistical difference in reoxygenation 0 h and 24 h; the activity of shRNA cells had no statistically significant difference to control.4. Compared with control after OGDR, the expression of Cx43, HC1 and Casp3 in cell line and Psup cells was increased significantly in reoxygenation 4 h (P< 0.05), there were no statistical differences in 0 h and 24 h; the expression of Cx43, HC1 and Casp3 in shRNA cells had no statistically significant difference to control.Conclusions1. The activity of astrocytes is decreased in the early stage of brain ischemia reperfusion injury, Cx43 and its hemichannels may play a promoting effect.2. The apoptosis in astrocytes was increased in the early stage of brain ischemia reperfusion injury, Cx43 and its hemichannels may play a pivotal role to promote this process which could be an index for the prognosis.3. As an analysis tool, but Cx43-specific transfection provides a promising method of treatment for improving the activity of brain cells after IRI.