| Objective:The OxyHb released by hemolysis is a key factor to induce cerebral vasospasm (CVS) afterspontaneous subarachnoid hemorrhage (SAH). Likewise, the gap junctions (GJ) proteinremodeling is closely related with the occurrence of a number of cardiovascular andcerebrovascular diseases. Phosphorylation is one of the most important ways of GJ remodeling.As a continuation of the previous study, this experiment was designed to study the expressionchange in the CVS and its signal transduction mechanisms of Connexin43(Cx43) and its Ser368and Ser262phosphorylation levels; to further investigate the molecular mechanisms of CVSinvolved by cerebrovascular GJ channel after SAH, thus providing a neo-target for the medicinalprevention and treatment of CVS.Materials and methods:1. The primary cultured rat basilar artery smooth muscle cells were identified byimmunofluorescence, and observed for the Cx43, Connexin40(Cx40)expression and distributionin the rat basilar artery and smooth muscle cells, and established the CVS cell model: the ratbasilar artery were taken by craniotomy, the rat basilar artery smooth muscle cells were cultivateby tissue explants method, and were observed by the morphological and immunofluorescenceidentification. The Cx43, Cx40expression and distribution in rat basilar artery smooth musclecells were observed by immunofluorescence and confocal microscopy, and10-6MOxyHb-incubated smooth muscle cells were added to establish the CVS cell model.2. The laser confocal microscope was used to observe the P-Cx43-S368, P-Cx43-S262andCx43expression change after adding the PKC, MAPK, and PTK inhibitors in the cell model ofcerebral vasospasm: the solvent, the OxyHb, OxyHb and chelerythrine chloride(PKC inhibitor),the OxyHb and PD98059(MAPK inhibitor), the OxyHb and PP2(PTK inhibitor)were respectivelyadded into each experimental group, and incubated for12,24,48hours. After tripleimmunofluorescence coloring, the confocal microscopy was used to observe the Cx43,P-Cx43-S368and P-Cx43-S262expression changes.3. The Western blotting analysis was conducted for the time-dependent changes of the above-mentioned P-Cx43-S368, P-Cx43-S262and Cx43protein expression.Results:1. The rat basilar artery smooth muscle cells were successfully cultured, the expression ofCx43and Cx40was rich in the basilar artery and smooth muscle cells, and the Cx40expression inthe physiological state was significantly more than that of Cx43;2. After the basilar artery smooth muscle cells having been stimulated by OxyHb for12h,24h,48h, it can be observed by the confocal microscopy and Western blotting technique that theexpression of P-Cx43-S368, P-Cx43-S262and Cx43total protein was significantly increased, andshowed a time-dependent change;(P <0.05)3. The respective application of chelerythrine chloride, a PKC inhibitor, PD98059, a MAPKinhibitor, and PP2, a PTK inhibitor, can significantly inhibit the expression increase ofOxyHb-induced P-Cx43-S368, P-Cx43-S262and Cx43total protein.(P <0.05)Conclusion:1. In the physiological state, the expression of the gap junction protein Cx43and Cx40wasrich in the basilar artery and smooth muscle cells, and the expression of Cx40was significantlymore than that of Cx43;2. In the OxyHb-induced CVS cell model, the spasmogen factor may directly or indirectlyenter into the local vascular smooth muscle cells, activate the intracellular PKC, MAPK, PTK andother signal transduction pathways, increase the total protein expression of Cx43, increase thephosphorylation levels of Cx43Ser368and Ser262, adjust GJ function, make GJ selectivelytransport some electricity, chemical signals, and to ultimately induce diffuse persistent CVSoccurrence and development. Blocking PKC, MAPK, PTK and other signal transductionpathways can reduce the total protein expression of Cx43, block the hyperphosphorylation Ser368and Ser262of Cx43, and significantly relieve the OxyHb-induced CVS, thus providing a novelway for the effective prevention and treatment of CVS. |
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