| Background:Excitatory amino acid toxicity is one of the important mechanisms of cerebral ischemia reperfusion injury.Cerebral ischemia-reperfusion injury is a multi-target and multi-process cascade reaction.At present,it is difficult to reverse ischemia-reperfusion injury with single target and single drug.Therefore,the method of mobilizing endogenous protective mechanism through ischemic preconditioning emerges as the times require.Ischemic preconditioning refers to one or repeatedly short sublethal ischemia that can mobilize endogenous protective mechanisms and thus protect neurons and glias from subsequent lethal ischemia.That is,preconditioning can induce ischemic tolerance.Studies have shown that many neurotransmitters and proteins may be involved in the formation of protective effects of pretreatment,but its mechanism has not been clarified.Studies have shown that excitatory amino acid transporter-2 and gap junction and half-channel composed of connexin-43 play an important role in regulating glutamate content in extracellular space after ischemia.Object:To observe whether in situ cerebral ischemic preconditioning can play an important protective role by regulating the expression and function of EAAT2 and gap junctions and hemichannels composed of Cx43 to alleviate excitotoxicity injury.Methods:In vivo,the rat model of focal cerebral ischemia-reperfusion injury(ischemia for 2 hours,reperfusion for 12 hours)was established by middle cerebral artery embolization(MCAO).250±20g male Wistar rats were randomly divided into sham group(Sham),ischemia/reperfusion injury group(IR)and ischemic preconditioning group(IPC,Cerebral blood flow was blocked in situ for 10 minutes 72 hours before ischemia).Longa scoring method was used to evaluate the neurological deficit after MCAO.TTC staining was used to calculate the relative infarct volume percentage of rat brain.HE pathological examination of brain tissue was carried out.Western blotting was used to analyze the expression changes of Cx43 and EAAT2 proteins.In vitro,primary astrocytes were extracted from neonatal rats within 72 hours and cultured until the third generation.They were divided into Sham group,oxygen and glucose deprivation / reoxygenation group(OGD/R,OGD 12 h followed by reoxygenation 6h),ischemic preconditioning group(IPC,30 min OGD 24 hours before OGD/R),ischemic preconditioning with gap junction blocker(CBX)group(IPC+CBX),ischemic preconditioning with selective half-channel blocker Gap19 group(IPC+Gap19),ischemic preconditioning with selective EAAT2 blocker THA group(IPC + THA).CCK-8 was used to detect the survival and apoptosis of astrocytes.The concentration of glutamate in the supernatant was measured.The expression and distribution of Cx43 and EAAT2 in astrocytes were observed by Western blotting and co-focal microscopy.Results:1.Compared with Sham group,the Longa score in IR group was 2 points,and the symptoms of neurological impairment were obvious.The relative cerebral infarction volume was 54.83%(P < 0.001).Compared with I/R group,the Longa score in IPC group was 1.57 points,and the relative infarction volume of brain was 40.29%(P < 0.01).The neurological impairment and the volume of cerebral infarction were significantly improved.2.The expression of Cx43 and EAAT2 protein in rat brain tissue showed that compared with Sham group,the expression of Cx43 protein in I/R group increased(P < 0.05),and the expression of EAAT2 protein decreased significantly(P < 0.01);compared with I/R group,the expression of Cx43 protein in IPC group decreased significantly(P < 0.05),and the expression of EAAT2 protein increased(P < 0.05).3.Relative cell viability test results showed that compared with the control group,the cell viability of OGD/R group was significantly lower(P < 0.001);compared with OGD/R group,the cell viability of IPC group was significantly higher(P < 0.05).After half-channel specific blocker Gap19 was administered,the relative survival rate of cells in IPC group decreased,but the difference was not statistically significant(P > 0.05).However,after THA and EAAT2 specific blocker THA were administered,the relative survival rate of cells in IPC group decreased significantly(P < 0.05).4.Western blotting and confocal microscopy were used to observe the expression and distribution of Cx43 and EAAT2 in astrocytes.Compared with the control group,the expression level of Cx43 protein in astrocytes in OGD/R group had no significant difference(P > 0.05),the expression level of Cx43 protein in cytoplasm increased significantly(P < 0.001),and the expression level of Cx43 protein in membrane decreased significantly(P < 0.05);compared with the OGD/R group,the expression level of Cx43 protein in astrocytes in IPC group decreased significantly(P < 0.01).The expression of Cx43 protein was significantly decreased(P < 0.001)and the expression of Cx43 protein was significantly increased(P < 0.01).Compared with the control group,the expression of EAAT2 protein in astrocytes in OGD/R group was significantly lower(P < 0.001),while the expression of EAAT2 protein in astrocytes in IPC group was significantly higher(P < 0.05)than that in OGD/R group.5.Glutamate content in cell supernatant of OGD/R group was significantly higher than that of Control group(P < 0.01).Compared with OGD/R group,glutamate content in IPC group was lower(P < 0.05),suggesting that pretreatment could significantly reduce glutamate content in extracellular space.After half channel specific blocker Gap19 was administered,the glutamate content was significantly lower than that in IPC group(P < 0.05).It is worth noting that after EAAT2 specific blocker THA was given,the glutamate content in cell supernatant tended to increase(P < 0.01).Conclusion:1.IPC can play a protective role in focal cerebral ischemia reperfusion injury in rats.2.After OGD/R injury,the expression of Cx43 in the cytoplasm and Cx43 in the cytoplasm of astrocytes decreased;After IPC treatment,the expression of Cx43 in astrocytes increased,while that in cytoplasm decreased.3.After OGD/R,the half-channel release of glutamate increased in astrocytes;The release of half-channel glutamate in astrocytes was decreased after IPC treatment.4.After OGD/R injury,the expression level of total protein EAAT2 in astrocytes was significantly. |
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