Adenosine Receptors A1 And A2a: Functions And Underlying Mechanisms In Gliosis Induced With Ischemia-like Injury In Vitro

IntroductionGliosis is characterized with astroglial hyperplasia and increasing GFAP expression.Although gliosis is a repair response to various injuries in central nervous system(CNS),excessive gliosis may interfere with neuronal repair and axonal regeneration by glial scar in the CNS.Thus,further understanding of mechanisms of gliosis will facilitate controllable gliosis to attenuate its negative aspects.Among adenosine receptors(A1,A2a,A2b,and A3),A1R and A2aR are two main receptors.They exhibit different effects on astrocytes:A1R induces apoptosis in astrocytoma and its antagonist partly blockades ATP-induced gliosis,suggesting A1R may prevent gliosis.However,reports on A2aR showed GFAP expression was attenuated in A2aR-knockout mice and A2aR inhibitor Sch58261 partly reversed gliosis by bFGF,indicating A2aR may induce gliosis and has reverse roles from A1R. Whether A1R and A2aR are involved in gliosis after ischemia? What mechanisms induced by those two receptors are involved in gliosis after ischemia? Observations in vivo and vitro showed that there were antagonistic functions between the two receptors in CNS.Take ischemia for example:A1R exhibited neuroprotection while A2aR had deleterious role to neural restore.Do the two receptors have antagonistic roles in gliosis after ischemia? Is there interaction of the two receptors? These questions remain elucidation.In this study,we performed oxygen-glucose deprivation(OGD) to mimic ischemic injury in vitro and used molecular biologic technologies including Western blot and transfection to examine the role of A1R and A2aR in gliosis after ischemia/reperfusion and its related signal pathways.Moreover,interaction of A1R and A2aR was investigated by immunoprecipation and Western blot.The purpose of our research is to provide new experimental data for enriching the knowledge on the regulation of gliosis. PartⅠThe role of A1 receptor in gliosisObjective:To explore the role of A1R in the gliosis induced by OGD in vitro and its relative signal pathway.Methods:GFAP expression was used as the biomarker of gliosis and its expression was examined by Western blot after RBA-2 astrocytes were treated with A1R agonist CPA or antagonist DPCPX after exposed to oxygen-glucose deprivation(OGD). Moreover,signal pathways including Akt and Jak2/STAT3 were detected by Western blot.Results:A1R agonist CPA prevented GFAP protein expression,whereas A1R antagonist DPCPX upregulated GFAP expression and resversed the effect of CPA. CPA increased the level of phospho-Akt in dose-dependent manner.The increase in phospho-Akt was attenuated by pretreatment of DPCPX.Akt inhibitor increased GFAP expression and reversed the effect of CPA on GFAP expression.CPA treatment decreased phospho-Jak2 but has no effect on phospho-STAT3.Akt inhibition increased phospho-Jak2 and reversed the effect of CPA on phospho-Jak2.Summary:A1R inhibites gliosis via Akt phosphorylation after OGD.There is crosstalk between Akt and Jak2 signal pathways.PartⅡThe role of A2a receptor in gliosisObjectives:To investigate the role of A2aR in the gliosis after OGD and its related signal pathway.Methods:Eukaryotic expression plasmid pEYFP-A2aR was stably transfected into RBA-2 astrocytes by lipofectin.Western blot and immunofluorescence analysis were performed to verify the transfection.Western blot and fluorescence-activated cell sorter(FACS) were used to analyse the GFAP expression and cell cycles respectively in cells transfected with pEYFP-A2aR(A2aR~+cells) or pEYFP(A2aR~-cells) after OGD.GFAP expression and cell cycles were examined after A2aR~+ cells were treated with A2aR antagonist Sch58261,Akt inhibitor or in combination.Akt/NF-κB pathway was detected by Western blot after A2aR~+ cells were treated with Sch58261,Akt inhibitor or in combination.Results:Both in normal or OGD condition,A2aR~+ cells increased GFAP expression and the percentage of cells in S-phase.The results were more obvious after cells were exposed to OGD.With the increase in phospho-Akt,A2aR antagonist Sch58261 inhibited GFAP expression and decreased the percentage of cells in S-phase.Akt inhibitor reversed the effect of Sch58261 on GFAP expression and cell cycles. Sch58261 downregulated cytoplasmic P65 in A2aR~+ cells while it upregulated nuclear P65.The ratio of phospho-IκBαand IκBαwas decreased after A2aR~+ cells were treated with Sch58261.Akt inhibitor reversed the effect of Sch58261 mentioned above.Summary:A2aR induces gliosis via Akt dephosphorylation after OGD.The procession is associated with P65 translocation regulated by the ratio of phospho-IκBαand IκBα.PartⅢThe interaction of A1 receptor and A2a receptorObjectives:To investigate the interaction of A1R and A2aR and its related signal pathway after OGD.Methods:Immunoprecipitation was used to analyse the interaction of A1R and A2aR in A2aR~+cells.Protein expressions including A2aR,GFAP,Jak2 and Akt signal molecular were detected by western blot after A2aR~+ cells were treated with A1R antagonist DPCPX following OGD.Results:Immunoprecipitation result showed the interaction between A1R and A2aR. A1R antagonist DPCPX induced the expression of A2aR and GFAP in dose-dependent manner after A2aR~+ cells were exposed to OGD.DPCPX downregulated phospho-Akt and phospho-Jak2 in A2aR~+ cells exposed to OGD.Summary:The interaction between A1R and A2aR may be regulated by Akt and Jak2 pathways after OGD. Conclusions1.A1R inhibites gliosis by Akt phosphorylation that can upregulate phospho-JAk2, suggesting Akt mediates A1R-inhibited gliosis via phospho-JAk2 after cells are exposed to OGD.2.A2aR induces gliosis by Akt dephosphorylation,which is associated with P65 translocation regulated by the ratio of phospho-IκBαand IκBα.These results indicate A2aR may induce glosis by Akt/NFκB pathway.3.There is interaction between A1R and A2aR.A1R inhibition can upregulate the expression of A2aR and GFAP but downregulated both phospho-Akt and phospho-Jak2,suggesting interaction of the two receptors may be regulated by Akt and Jak2 pathways.