The Role Of Cx30/Cx43 And EAAT1 In Astrocyte During Cerebral Ischemia-reperfusion Injury

[Background and Objective]Ischemia-reperfiusion injury(I/R)is caused by recovery of blood supply from brain tissue after ischemia,and excess free radicals cause damage to cells in the blood supply.Brain I/R is the main cause of disability and death in patients.It is very important to reduce brain I/R injury and improve the prognosis of patients with cerebral ischemic diseases.The exact mechanism of brain I/R injury needs further study at present.Astrocytes are astrocytic glial cells unique to the brain and spinal cord.The functions of astrocytes include:biological support for endothelial cells that form the blood-brain barrier,nutrient supply to nerve tissue,maintenance of extracellular ion balance,and post-traumatic brain and spinal cord repair and scar formation.Astrocytes are connected by gap junctions,and the formation of electrically coupled cell bodies and their adjacent cells through gap junctions to affect the activity of other astrocytes away from the original astrocytes.The connexin Cxs,which form the gap junction of the brain,regulate the intercellular spread of ions and small molecules in the brain.The connexin 43(Cx43)is expressed most in astrocytes in brain tissue,and Cx43 is thought to play an important role in regulating the degree of damage to the nervous system.The brain’s main excitatory transmitter,glutamate,may spread through gap junctions,while high concentrations of glutamate can induce neuronal damage and death.The glutamate transporter can affect the extracellular concentration of glutamate receptors to regulate glutamate.Expression of Cx30 affects the level of the glutamate transporter EAAT1 in the brain.The gap junction protein between astrocytes is affected by brain damage,and gap junction protein may be an important factor for regulating glial glutamate under pathological conditions.In this study,we investigated the injury induced by ischemic phase and reperfusion(oxygen glucose deprivation reperfusion OGD/R)in vitro and rat ischemia-reperfusion(middle artery ischemia-reperfusion MCAO/R)in vivo.The mRNA and protein expression of Cx30,Cx43 and glutamate transporter EAAT1 during perfiusion was measured,cell viability,neuronal growth,and malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),Reactive oxygen species(ROS)was explored in vitro and in vivo,and extracellular glutamate content was also detected.The role and mechanism of gap junction protein and glutamate transport protein in ischemia-reperfusion injury was explored in present study.[Methods]1.The role of gap junction protein Cx30/Cx43 and EAAT1 of astrocyte in cerebral ischemia-reperfusion injuryThe astrocytes and neurons of neonatal rats were isolated and identified by immunofluorescence.The astrocyte oxygen glucose deprivation and reperfusion(OGD/R)model was established to investigate the effects of different treatment time on the viability of astrocytes.Neuronal markers of neurons treated with OGD/R astrocyte cell culture medium and astrocyte cells and neurons co-cultured after OGD/R was visualized by immunofluorescence stain.Real-time fluorescent quantitative PCR was used to detect mRNA expression levels of Cx30,Cx43 and EAAT1 after cytoplasmic OGD/R;Western blot analysis was performed to measure protein expression of Cx30,Cx43 and EAAT1;Immunofluorescence was used to further detect expression and localization of Cx30 and Cx43 proteins;The oxidative stress factors as MDA,SOD,CAT,ROS and glutamate content in astrocyte culture supernatants were assayed.2.The role of gap junction protein Cx30/Cx43 and EAAT1 in rat middle arterial ischemia-reperfusion modelA rat model of middle arterial ischemia-reperfusion(MCAO/R)was established.The infarct volume of rat brain tissue,connexin,glutamate transporter and oxidative stress factor and valley in brain tissue were investigated.The mRNA expression of Cx30,Cx43 and EAATI in brain tissue of MCAO/R animals were detected by real-time fluorescent quantitative PCR.The protein expression of Cx30,Cx43 and EAAT1 was detected by Western blot.The expression and localization of Cx30 and Cx43 proteins were further analyzed by immunofluorescence.Oxidative stress factors as MDA,SOD,CAT and ROS levels and glutamate content in brain tissue was detected using kits.[Results]一.The role of gap junction protein Cx30/Cx43 and EAAT1 of astrocyte in cerebral ischemia-reperfusion injury1.Identification of rat astrocytesImmunofluorescence was used to detect the expression and localization of GFAP protein in neonatal rat cortical astrocytes.The red fluorescence(GFAP positive)cells were over 95%,and the cell morphology was in good condition.2.Rat neuron identificationImmunofluorescence was used to detect the expression and localization of Map2 protein in neonatal rat cortical neurons.Green fluorescent(GFAP Map2 positive)neurons with intact cells and good condition.3.Determination of OGD/R cell viability in rat astrocytesP3 neonatal rat cortical astrocytes were deoxygenated for 0,2h,4h,8h and 12h,and reoxygenation cultured for 12h,24h and 48h,CCK8 was used to detect cell viability.The cell viability decreased with the prolongation of oxygen deprivation time.The decline of oxygen glucose deprivation at 12h was not obvious compared with that at 8h.The relative viability(compared with normal cultured cells)decreased with the prolongation of reperfusion time,reperfusion for 24h,48h.The relative viability of the cells decreased compared with 12h.4.Effect of rat astrocyte OGD/R on neuronsAfter neonatal rat astrocyte P2 was subjected to oxygen glucose deprivation and reperfusion(OGD/R),the supernatant of culture supernatant was added to DIV13 neurons for 24 hours,and the map2 protein-labeled neurons were detected by immunofluorescence.The effects of astrocyte oxygen deprivatization on the 8h reperfusion for 12h compared with the oxygen-only deprivation of 8h on the growth state and morphology of neurons were more significant.Neonatal rat astrocytes P2 were co-cultured with neurons(DIVl1)for 8h after OGD.After reperfusion for 12h,the expression of Map2 protein was detected by immunofluorescence.Similar to the neuron results of astrocyte OGD/R culture supernatant treatment,the cell state of oxygen glucose deprivation for 8h reperfusion for 12h was more significant than that of oxygen only deprivation for 8h.5.qPCR detection of Cx30,Cx43,EAAT1 mRNA expression in rat astrocytes OGD/RqPCR was used to detect Cx30,Cx43,EAAT1 mRNA expression of Neonatal rat astrocytes(P3)after OGD8h/R12h.Compared with cells cultured under normal conditions,the expression of Cx43 and EAAT1 mRNA in cells with oxygen glucose deprivation(OGD)for 8 h and reperfusion for 12h were significantly increased(P<0.05),and the difference in Cx30 was not significant;despite oxygen deprivation The expression of Cx43 and EAAT1 mRNA in the reperfusion(OGD8h/R12h)group were not significantly different from those in the oxygen glucose deprivation group(OGD8h)(P>0.05),but there was an upward trend.6.Western blot analysis of Cx30,Cx43,EAAT1 protein expression in rat astrocytes OGD/RWestern blot was used to analyzed Cx30,Cx43,EAAT1 protein expression of neonatal rat astrocytes(P3)after OGD8h/R12h.Compared with the cells cultured under normal conditions,the Cx43 and EAAT1 protein in the cells of oxygen glucose deprivation(OGD)for 8h and reperfusion for 12h were significantly increased(P<0.05),and the expression of Cx30 was significantly decreased.Oxygen sugar deprivation The expression of Cx43 and EAAT1 in the perfused(OGD8h/R12h)group were significantly higher than those in the oxygen glucose deprivation group(OGD8h)(P>0.05).7.Immunofluorescence detection of Cx30 and Cx43 expression in rat astrocytes after OGD/RImmunofluorescence was used to analyze Cx30,Cx43 protein expression and localization in neonatal rat astrocytes(P3)after OGD8h/R12h.Compared with the cells cultured under normal conditions,the expression of Cx30 was significantly decreased and the expression of Cx43 protein was significantly increased in the astrocytes of OGD for 8 h.The expression of Cx30 was significantly decreased in OGD8h/R12h compared with OGD 8h,and the expression of Cx43 protein was significantly decreased.8.Detection of MDA,SOD,CAT,ROS and glutamate in rat astrocytes after OGD/RIschemia-reperfusion induces increased levels of MDA and ROS in astrocytes;induces decreased activity of CAT and SOD;induces an increase in glutamate release from astrocytes;The effects of reperfusion on cell viability and intracellular connexins,glutamate transporters,and oxidative stressors(MDA,SOD,CAT,ROS)are more pronounced compared to ischemic treatment.二.The role of gap junction protein Cx30/Cx43 and EAAT1 in rat middle arterial ischemia-reperfusion model1.Neurobehavioral score and TTC staining of rat of middle arterial ischemia-reperfusion(MCAO/R)modelAfter 12h of reperfusion(R)in SD rats,the longan scores of ischemia group(MCAO)and ischemia-reperfusion group(MCAO/R)were significantly higher than that of sham operation group;The difference of rats in MCAO/R group and the ischemic group was not significant.The results of TTC staining showed that the infarcted area in the ischemic group(MCAO)and ischemia-reperfusion group(MCAO/R)was significantly larger than that in the sham operation group;the infarct area in the MCAO/R group was not significantly different from that in the ischemic group.2.Detection of Cx30,Cx43 and EAAT1 mRNA in brain tissue of SD rats after ischemia and reperfusionAfter SD rats were undergone reperfusion(MCAO/R),the expression of Cx43 and EAAT1 mRNA in MCAO 2h animals and MCA02h/R12h animals were significantly higher than those in sham-operated animals(P<0.05),Cx30 was significantly decreased;the expression of Cx43 and EAATI mRNA in MCA02h/R12h group were significantly higher than those in MCAO2h group(P>0.05),and Cx30 mRNA expression was significantly lower than MCAO2h group(P>0.05).3.Detection of Cx30,Cx43,EAAT1 protein in brain tissue of SD rats after ischemia and reperfusionAfter SD rats were undergone reperfusion(MCAO/R),the expression of Cx43 and EAAT1 in MCAO 2h animals and MCAO2h/R12h animals were significantly higher than those in sham-operated animals(P<0.05),Cx30 was significantly decreased;the expression of Cx43 and EAAT1 protein in MCAO2h/R12h group was significantly higher than that in MCA02h group(P>0.05),and Cx30 protein expression was significantly lower than MCAO2h group(P>0.05).4.Immunofluorescence detection of Cx30 and Cx43 expression in brain tissue of SD rats after ischemia and reperfusionAfter SD rats were subjected to ischemia-reperfusion(MCAO/R),the expression of Cx30 and Cx43 protein were detected by immunofluorescence in hippocampus of ischemic hemisphere.Compared with the sham operation group,the expression of Cx30 was significantly decreased in MCAO 2h animals and MCAO2h/R12h animal brain tissues,and the expression of Cx43 protein was significantly increased.The expression of Cx30 was significantly decreased in MCAO2h/R12h compared with MCAO 2h group,and Cx43 protein expression was significantly increased.Raise.5.Detection of MDA,SOD,CAT,ROS and glutamate in brain tissue of SD rats after middle cerebral ischemia reperfusion(MCAO/R)MCAO/R induced increased MDA and ROS levels in brain tissue,decreased CAT and SOD activity,and increased glutamate content in brain tissue of rats;The effects of reperfusion on connexin,glutamate transporter and oxidative stress factors(MDA,SOD,CAT,ROS)in animal brain tissue are more pronounced compared with ischemic treatment.[Conclusion]1.OGD/R can induce the decrease of astrocyte viability;OGD/R-induced astrocyte release substance(astrocyte culture medium)affects neuronal growth;Neurons co-cultured with glial cells after OGD/R affects neuronal growth;2.Ischemia-reperfusion induced the up-regulation of Cx43 and EAAT1 expression in astrocytes and animal brain tissues;induced down-regulation of Cx30 expression.3.Ischemia-reperfusion induced astrocyte and animal brain tissue MDA,ROS content increased;induced CAT and SOD activity decreased;induced astrocyte releasing glutamate and increased glutamate content in animal brain tissue;4.Compared with ischemia,reperfusion has a more significant effect on cell viability and connexin,glutamate transporter and oxidative stress factor(MDA,SOD,CAT,ROS)in astrocyte and animal brain tissue;5.Cerebral ischemia-reperfusion injury affect astrocytes in the brain through regulation connexin and glutamate transporter in response to oxidative stress and glutamate.