Study On The Relationship Of Connexin43Gap Junction Communication And Astrocytes Activation, Proliferation And Secretion In Vitro

Objective: To study the expression of connexin43gap junction protein on primarycultured astrocytes under normal and OGD conditions.Methods: The cortex astrocytes were cultured from newly born Wista rats according topreviously description and the purity were identified by immunofluorescence staining ofGFAP. The expression of connexin43gap junction protein on astrocytes was analyzed bydouble immunofluorescence staining with GFAP and Cx43. The primary culturedastrocytes were randomly divided into two groups: control group (medium is EBSS withinsugar, cultured in37oC incubator), OGD group (medium is EBSS without sugar, culturedin the hypoxia incubator). After oxygen glucose deprivation (OGD)1h, at different timepoints reperfusion (0h,3h,6h,12h, and24h), The temporal changes of connexin43expression in cultured astrocytes was analyzed with immunofluorescence staining andWestern Blot.Results：The primary astrocytes culture of rat cortex was prepared and indentified. Theconnexin43gap junction protein was expressed on the border and membrane of astrocytes.Compared with control group, the cell viability was not affected by OGD treatment within1hour. OGD treatment up-regulated the expression of the Cx43gap junction total proteinand the phosphorylated protein (P-Cx43) on cultured astrocytes in vitro.Conclusion：Connexin43gap junction protein was abundantly expressed on the border ofthe astrocytes. After OGD treatment, the expression of the Cx43gap junction total proteinand the phosphorylated protein (P-Cx43) was increased. Objective: To study the effect of connexin43blocker on cultured astrocytes activation,proliferation and secretion following OGD in vitro.Methods: Primary cultured rat cortex astrocytes were randomly divided into four groups:control group(medium is EBSS within sugar), OGD group(medium is EBSS without sugar),CBX group(medium is EBSS without sugar but contain broad-spectrum gap junction channelblockers CBX) and MFA group (medium is EBSS without sugar but contain the relativespecificity Cx43channel blockers MFA), normal group was cultured in37oC incubator,OGD group, CBX group and MFA group were cultured culture in the hypoxia incubator.After oxygen glucose deprivation (OGD)1h, at different time points reperfusion (0h,3h,6h,12h, and24h), the toxic effects of CBX and MFA were detected by LDH release assay kit.The astrocytes activation were evaluated by immunofluorescence staining and Western blotof GFAP, the proliferation rates were measured by BrdU incorporation assay. The changes ofglutamic release in the cultured media were analyzed by glutamic acid assay kit.Results：Compared with control group, the cell viability was not affected by the drugs of theconnexin43gap junction blockers CBX and MFA. OGD treatment increased the expressionof GFAP protein, the percentage of BrdU+cell and the glutamic release on culturedastrocytes in vitro, which were inhibited by the CBX and MFA.Conclusion：Blockers of connexin43gap junction can be inhibite astrocyte activation,proliferation and glutmate release after OGD, suggesting that it may be a potential thraputictarget of cerebral ischemia based on the astrocytes.