Role And Mechanisms Of Gap Junctions In Cerebral Ischemic Postconditioning

Cerebral ischemia is the third cause of death worldwide following cardiovasculardiseases and cancer. Reperfusion has the potential to produce ischemia/reperfusioninjury, although rapid initiation of reperfusion is the most effective treatment for thedisease. The mechanisms of ischemia-reperfusion injury include oxidative stress, freeradical damage, excitatory amino acid toxicity, inflammation, calcium overload andapoptosis. Exploring effective measure to anti-ischemia-reperfusion injury has been aresearch focus. The experimental research focused on internal protective mechanisms ofischemia-reperfusion injury and the discovery of effective drugs for cerebralischemia/reperfusion injury.Ischemic preconditioning and ischemic postconditioning are internal protectionmechanisms of ischemia-reperfusion injury. Postconditioning has inherent advantages in practical applications, thus attracting much attention. Studies have shown that gapjunction inhibiors have protective effect against cerebral ischemia/reperfusion.Whether gap junction is involved in cerebral ischemic postconditioning, and thepossible mechanism is still unclear.Objective:To explore the role and possible mechanisms of gap junctions in cerebral ischemicpostconditioning.Method:1. The middle cerebral artery occlusion rat models were established, and the middlecerebral artery was occluded for2hours following with24hours reperfusion. Adultmale SD rats were randomly divided into five groups, including sham group,ischemia-reperfusion group,‘ischemic postconditioning’ group,‘ischemia-reperfusion+carbenoxolone’ group and ‘ischemic postconditioning+carbenoxolone’ group.Carbenoxolone (a gap junction inhibitor) was administered intravenously in1minutebefore the initiation of reperfusion, the other groups were injected with proportionabledose of saline. Neurological deficit scores of different group were evaluated with5-point scoring method. TTC stain method was used to calculate the percentage ofinfarct volume, and H-E stain method was used to detect the pathology of brain tissue.Western blotting was used to detect the expressionCx43protein. Src kinases andapoptosis-related protein Bax/Bcl-2were also detected by Western blotting.2. The primary cultured astrocytes went with hypoxia-reoxygenation injury (hypoxia8h/reoxygenation24h). Astrocytes of the study were divided into normal control group, hypoxia-reoxygenation group, hypoxic postconditioning group,‘hypoxicpostconditioning+retinoic acid’ group and ‘hypoxic postconditioning+oleamide’group. MTT assay was used to measure the survival fraction of astrocytes;Hoechst33258staining and Annexin V/PI double staining following with flowcytometry were applied to detect apoptosis of astrocytes; Parachute assay was used todetect the function of gap junction of astrocytes; Cx43membrane protein was observedby cellular immune fluorescence; Western blotting was used to detect the expression ofCx43protein, Src kinase protein, Bax protein and Bcl-2protein.Results:1. In order to observe the protective effect of iscehmic postconditioning,ischemia-reperfusion injury model of rats was preparaed. Compared with the shamgroup, rats in ischemia-reperfusion group had significant neurological dysfunction,brain tissue pathological changes and brain tissue infarction; compared withischemia-reperfusion group, the ischemic postconditioning can significantly reduce theneurological deficit scores score, reduce histopathological changes of necrosis andpyknosis of neurons and congestion of blood vessels, and reduce the percentage of theinfarct volume of brain tissue. The results suggest that the preparation of rat model oflocal cerebral ischemia and reperfusion injury is successful, and ischemicpostconditioning has the protective effect on cerebral ischemia-reperfusion injury.Results of Cx43total protein showed that Cx43protein expression had differencesbetween the groups. Compared with sham group, Cx43protein expression in theischemia group was significantly increased (P <0.05); Compared withischemia-reperfusion group, the expression of Cx43protein of rat brains in ischemicpostconditioning group was significantly decreased (P <0.05). The results indicate that the potective effect of cerebral ischemic postconditioning may related with theinhibition of gap junctions.In order to realize the role of gap junctions in cerebral ischemic postconditioning, gapjunction inhibitor carbenoxolone was used in the study. Compared withischemia-reperfusion group, neurological function disability scores and the percentageof cerebral infarction of rat in ‘ischemia-reperfusion+carbenoxolone’ group wassignificantly decreased (P <0.05), and brain tissue pathological change wassignificantly reduced; Results of Cx43total protein showed that Cx43proteinexpression had differences between the groups. Compared with the ischemicpostconditioning group, Cx43protein expression of rat brain in ‘ischemicpostconditioning+carbenoxolone’ group was significantly reduced (P <0.05). Theresults suggest that the protective effect of postconditioning in cerebral ischemia isrelated to the functional inhibition of gap junctions, and gap junction inhibitors canenhance the protective effect of cerebral ischemic postconditioning.In order to further clarify the mechanism of the protective effect of ischemicpostconditioning by inhibiting the gap junction, expression of Src kinase protein and theratio of Bax/Bcl-2were detected.Compared with sham group, Src kinase protein expression in the ischemia-reperfusiongroup was significantly increased (P <0.01); Compared with ischemia-reperfusiongroup, expression of Src kinase protein of rat brains in ischemic postconditioning groupwas significantly decreased (P <0.05); Compared with ischemic postconditioning group,expression of Src kinase protein in ‘ischemia-reperfusion+carbenoxolone’ group wassignificantly decreased (P <0.01). Compared with sham group, ratio of Bax/Bcl-2protein in the ischemia/reperfusiongroup was significantly increased (P <0.01); Compared with ischemia-reperfusiongroup, expression of ratio of Bax/Bcl-2protein of rat brains in ischemicpostconditioning group was significantly decreased (P <0.01).2. In order to observe the protective effect of hypoxic postconditioning, hypoxiareoxygenation injury model of astrocytes was preparaed. Compared with the controlgroup, survival rate of astrocyte in hypoxia reoxygenation was significantly decreased(P <0.01), and the apoptosis rate was significantly increased (P <0.01); Compared withhypoxia reoxygenation group, survival of astrocytes in postconditioning group wasincreased (P <0.01), and apoptosis rate was significantly decreased (P <0.01). Theresults suggest that hypoxia reoxygenation model of astrocytes was preparaedsuccessfully, and hypoxic postconditioning has protective effect on hypoxiareoxygenation injury.In order to realize the role of gap junctions in hypoxic postconditioning, gap junctionpotentiator (retinoid acid) and inhibitor (oleamide) were used in the study.Compared with hypoxic postconditioning group, the survival fraction of astrocytes in‘hypoxic postconditioning+retinoic acid’ group was reduced (P <0.01), and theapoptosis rate of astrocytes was significantly increased (P <0.01). The survival fractionof astrocytes in ‘hypoxic postconditoning+oleamide’ group was increased (P <0.01),and the apoptosis rate of astrocytes was significantly decreased (P <0.01).Compared with the control group, astrocyte gap junctional intercellular communicationin hypoxia/reoxygenation injury group was significantly increased (P <0.01);Compared with hypoxia/reoxygenation group, gap junctional intercellular communication of astrocytes in hypoxic postconditioning group was decreasedsignificantly (P <0.01); Compared with the hypoxic postconditioning group, gapjunctional intercellular communication of astrocytes in ‘hypoxic postconditioning+oleamide’ group was decreased (P <0.01), which in ‘hypoxic postconditioning+retinoid acid’ group was increased (P <0.01).Compared with normal control group, Cx43membrane protein immunofluorescence ofastrocytes in the hypoxia/reoxygenation group was increased; Compared withhypoxia/reoxygenation group, fluorescence intensity of astrocytes in hypoxicpostconditioning group was decreased; Compared with hypoxic postconditioning group,fluorescence intensity of astrocytes in ‘hypoxic postconditioning+retinoid acid’ wasincreased, and which in ‘hypoxic postconditioning+oleamide’ group was decreased.Compared with normal control group, Cx43total protein of astrocyte in the hypoxiareoxygenation group was increased (P <0.01); Compared with hypoxia reoxygenationgroup, Cx43total protein in hypoxic postconditioning group was decreased(P <0.01);Compared with hypoxic postconditioning group, Cx43total protein in ‘hypoxicpostconditioning+retinoid acid’ group increased significantly (P <0.05), which in‘hypoxic postconditioning+oleamide’ group decreased significantly (P <0.05).The results suggest that the protective effect of postconditioning in cerebral ischemia isrelated to the functional inhibition of gap junctions. Enhancement of gap junctiondecrease the protective effect of the cerebral ischemic postconditioning, and inhibitionof gap junction increase the protective effect.3. In order to further clarify the mechanism of the protective effect of ischemia andhypoxic postconditioning by reducing the gap junction function processing, expression of Src kinase protein which related to phosphorylation of connexin and the ratio of Baxand Bcl-2which related to apoptosis were detected.Compared with normal control group, Src kinase of astrocyte in the hypoxia/reoxygenation group was increased (P <0.01); Compared with hypoxia/reoxygenationgroup, expression of Src kinase in hypoxic postconditioning group was decreased (P <0.01); Compared with hypoxic postconditioning group, Src kinase of astrocyte in‘hypoxic postconditioning+retinoid acid’ group was significantyly increased (P <0.05), and which in ‘hypoxic postconditioning+oleamide’ group was significantylydecreased (P <0.05).Compared with normal control group, ratio of Bax/Bcl-2protein of astrocytes inhypoxia/reoxygenation group was increased (P <0.01); Compared withhypoxia/reoxygenation group, ratio of Bax/Bcl-2protein of astrocytes in hypoxicpostconditioning group was decreased (P <0.01); Compared with hypoxicpostconditioning group, ratio of Bax/Bcl-2protein of astrocytes in ‘hypoxicpostconditioning+retinoid acid’ group was increased(P <0.05), and which in ‘hypoxicpostconditioning+oleamide’ group was decreased (P <0.05).Conclusion:(1) The expression of Cx43protein increased during cerebral ischemia-reperfusioninjury，which suggests that cerebral ischemia/reperfusion injury may related with thefunctional enhancement of gap junctions.(2) The protective effect of cerebral ischemic postconditioning or hypoxicpostconditiong is probably related to the inhibition of gap junctions.(3) Gap junction inhibition in the ischemic postconditioning is probably related to the reduced src kinase expression.(4) Decreased apoptosis in ischemic postconditioning through gap junction inhibition isprobably related to the reduced ratio of Bax/Bcl-2.