| Ischemic postconditioning induced by brief cycles of reperfusion/ischemia at the endof ischemia has been shown to salvage brain tissue from ischemia/reperfusion (I/R)injury in several animal models. However, the relationship between ischemicpostconditioning (IPO) and gap junction (GJ) has not yet been elucidated in brain.Now, by an established in vitro astrocytes hypoxia/reoxygenation (H/R) model tomimick the in vivo cerebral I/R, we investigate whether the beneficial effect ofhypoxic postconditioning（HPO） involves a decrease in GJ.Objective:To observe the difference of GJ function on H/R injury and hypoxic HPO in primaryculture astrocytes. To observe the effects of GJ on protection of HPO in primaryculture astrocytes by regulating GJ. To explore the possible mechanisms of theprotection of HPO.Methods:Cerebral cortices from24h neonatal Sprague Dawley rats were isolated andcarefully dissected, then cultured astrocytes, the purity of the astrocytes was assessedby immunochemical staining with glial fibrillary acidic protein (GFAP) after cellpurification. Establishment of astrocyte H/R model and HPO model. Parachute assaywas used to detect the effect of HPO on dye spread of astrocytes. MTT assay was useto detect the effect of HPO on cell viability in astrocytes. Annexin V/PI assay was useto detect the effect of HPO on cell early apoptosis in astrocytes. Hochest33258staining was use to detect the effect of HPO on cell late apoptosis in astrocytes.Western blotting was use to detect the effect of HPO on the expression of Bcl-2, Bax,caspase-3, Cx43and Src kinase. Immunofluorescence assay was used to detect theexpression changes of Cx43on the surface of astrocytes treated with HPO and GJ tools drugs. To explore effect of Cx43-siRNA to H/R injury and HPO byCx43-siRNA transfection.Results:The purity of the astrocytes was>98%, as assessed by immunochemical stainingwith glial fibrillary acidic protein (GFAP). Compared with the control group, thedye-coupling of the H/R group was increased. HPO markedly inhibited the dye spreadfrom donor cells to receiver cells compared with the H/R group. Moreover, comparedwith the HPO group, the dye-coupling of the RA+HPO group was increased and thatof the olea+HPO group was decreased (p<0.01). Those results suggested that HPOcould markedly inhibited GJ function.The MTT assay indicated that the cell viability of the H/R group was decreased0.370±0.027(p<0.01), compared with the control group. Compared with the H/Rgroup, cell viability of HPO was increased0.180±0.096(p<0.05). Moreover,compared with the HPO group, the cell viability of the RA+HPO group wasincreased0.188±0.049(p<0.05) and that of the olea+HPO group was decreased0.186±0.089(p<0.01).Flow cytometric analysis showed that the apoptosis percentages for the differentgroups were0.082±0.009，0.357±0.027，0.217±0.008，0.278±0.021, and0.150±0.019.Compared with the normal control group, the apoptosis percentage of the H/R groupwas increased. Compared with the H/R group, the apoptosis percentage of the HPOgroup was significantly decreased (p<0.01), and that of the RA+HPO group wasincreased and Olea+HPO group was decreased compared with the HPO group(p<0.01).Hochest33258staining showed the late apopotsis. Compared with the control group,the late apoptotic cells of the H/R group was increased. Compared with the H/R group,there were fewer late apoptotic cells in the HPO group, whereas more late apoptoticcells were observed in the RA+HPO group and less late apoptotic cells wereobserved in the olea+HPO group compared with the HPO groupWestern blotting demonstrated that the Bax/Bcl-2expression ratio increased significantly in the H/R group compared with the control group (p<0.01). However,HPO inhibited H/R-induced increase of Bax/Bcl-2expression ratio (p<0.01).Compared with the HPO group, the ratio of Bax/Bcl-2expression was increased in theRA+HPO group and decreased in the olea+HPO group (p<0.05). What’s more, theactivation of caspase-3expression increased significantly in the H/R group comparedwith the control group (p<0.01). However, HPO inhibited H/R-induced increase ofactivation of caspase-3expression (p<0.01). Compared with the HPO group,activation of caspase-3expression was increased in the RA+HPO group anddecreased in the olea+HPO group (p<0.05). Then, the Sic kinase protein expressionincreased in the H/R group compared with the control group (p<0.01). However, HPOinhibited H/R-induced increase of Sic kinase protein expression (p<0.01). Comparedwith the HPO group, Sic kinase protein expression was increased in the RA+HPOgroup (p<0.05) and decreased in the olea+HPO group (p<0.05).The fluorescence intensity of Cx43protein in the H/R group was higher than that inthe normal control group, however, HPO inhibited H/R-induced increase of thefluorescence intensity. Compared with the HPO group, the fluorescence intensity ofCx43protein was increased in the RA+HPO group and decreased in the olea+HPOgroup. Western blotting demonstrated that the Cx43expression increased in the H/Rgroup compared with the control group (p<0.01). However, HPO inhibitedH/R-induced increase of Cx43expression (p<0.01). Compared with the HPO group,Cx43expression was increased in the RA+HPO (p<0.05) group and decreased in theolea+HPO group (p<0.05)Astrocytes were transfected with Cx43-siRNA targeting Cx43. NC-siRNA did nothave any effect on GJ function, cell viability and cell apoptosis (p>0.05). While,Cx43-siRNA reduced H/R-induced GJ function increasing. Cx43-siRNA reducedH/R-induced cell viability decreasing (p<0.01). Cx43-siRNA reduced H/R-inducedearly apoptotic cells increasing (p<0.01). Hochest33258staining showed the lateapopotsis. NC-siRNA did not have any effect on late apoptosis (p>0.05). While,Cx43-siRNA reduced H/R-induced late apoptotic cells increasing (p<0.01). Conclusion:1. HPO could inhibite the increation of GJ function which is induced by H/R.2. The protection of HPO may be related with GJ function.3. Regulating GJ function could affect the protection of HPO.4. Bcl-2/Bax and caspase-3may be involved in the HPO protective effect which maybe related to reduced the GJ function. |
