The Change And Mechanism Of Cx43in Rat Astrocytes Under Ischemia/Reperfusion Condition

Background:Neurovascular unit, including neurons, glial cells and vascular endothelial cells,which emphasizes the importance of maintaining normal brain function as a whole.Astrocytes are the key to maintain neurovascular unit function, and gap junctions areuniversally found between astrocytes, and are made up of connexin43(Cx43). As animportant channel of information transmission between cells, and ischemic stroke isdue to cerebral blood flow stopped to interrupt the energy supply and destroy braintissues, leading to vascular dysfunction and inevitable change of astrocytes of Cx43.Cx43is affected by many factors in its life cycles, and the internalization anddegradation pathways are not clear. However, as membrane protein, it’s not clear thatwhether the transportation of Cx43into the cytoplasm is associated with traditionalendocytosis. Cx43is moved to cell membrane by means of microtube transportsystem in its synthesis process, then the matter that whether its internalization is alsoassociated with microtubule remains to be studied. Intracellular degradation of proteinis mainly through the lysosomal autophagy pathway and the ubiquitin-proteasomesystem pathway. The transportation of Cx43into cytoplasm can be activated byischemia, change the phosphorylation state, lead to degradation via activating proteinkinase signaling pathways and affect the function of gap junctional intercellularcommunication. However, the exact mechanism is not clear till now, which becomesthe focus of this field.Object:To observe the changing regulation of the quantity and quality of Cx43in ratastrocytes under ischemia/reperfusion (I/R), and to analyze the mechanism in it.Methods:The cultured rat astrocytes in vitro with continuous passage culture methods,established cell I/R model by adjusting the incubator parameters and replacingnutrient solution. Oxygen glucose deprivation at different time points after2h,respectively with oxygen and glucose,0.5h,1h,3h,6h,12h,24h,72h, using theWestern blotting method for the quantitative analysis for Cx43. By using the Westernblotting and RT-PCR method, we analyzed the change of EB1expression quantity in the aspects of protein and gene. We selected the time points by using MTTcolorimetric method, when cell injury was most serious to observe the change ofCx43distribution: setting normal culture group, I/R Saline group, the I/R ofcarbenoxolone intervention group, observation was carried out by using confocal laserscanning microscope. After blocking endocytosis by dynasore, observe the change ofthe distribution of Cx43again in the confocal laser scanning microscope. Meanwhile,the experiments were divided into5groups: normal group, I/R group, I/R TPA group,I/R MG132group, I/R group TPA+MG132, using the Western blotting method for thequantitative analysis of p-Cx43, p-ERK1/2, Beclin protein.Results:(1) We successfully established purification system of rat astrocytes and I/R model.(2) The Western blotting results suggested that total Cx43each time point after I/R didnot change (P>0.05).(3) The MTT method suggested that the most serious injury timeof cell is at6h after I/R, at this time using the confocal microscopy, Cx43internalization could be observed, and less on cell membrane while more oncytoplasm distribution (P<0.05).(4) CBX blocked gap junction and did not affect theexpression of Cx43(P<0.05).(5) Dynasore could partly inhibitted the transmission ofCx43into the cytoplasm, and the internalization of Cx43was reduced by29.2%compared with the control group (P>0.05).(6) There was no difference in protein andgene relative expression level of EB1among each group (P>0.05).(7) After oxygenischemia/reperfusion6h: the protein expression of p-Cx43(Ser368) decreasedcompared to normal culture group (P<0.01); After the activity of PKC being a longtime influenced by TPA, the protein expression level of p-ERK1/2and Beclin1weredecreased, while p-Cx43(Ser368) was increased (P<0.01); MG132as a proteasomeinhibitor, which made the protein expression level of p-Cx43, p-ERK1/2and Beclin1were increased (P<0.01).Conclusion:(1) We successfully established purification system of rat astrocytes and I/R model.(2) Cx43internalization and endocytosis are related, while there was nothing to dowith the microtubule transportation.(3) After oxygen glucose deprivation/reperfusion, Cx43occurred phosphorylation in the Ser368site through ERK1/2pathway, and Cx43degraded by lysosomal pathway.(4) The degradation of Cx43process and the ubiquitin proteasome pathway were related.