| Stroke is the most serious and potentially deadly neurological disease and the most important factor leading to permanent disability in adults,significantly affecting the quality of life of stroke patients.The main type of stroke is ischaemic stroke,which is predominately a result of middle cerebral artery occlusion(MCAO).T-cell death-associated gene 8(TDAG8)is an acid-and psychosine-sensitiveG-protein-coupled receptor(GPCR),and mainly regulates intracellular biological activity in immune cells through the interaction of histidine residues with extracellular protons.Recent studies have shown that TDAG8 is widely expressed in rat brain tissue,especially in the forebrain margin system,such as the hypothalamus,amygdala,hippocampus,frontal cortex and striatum,and activated by specific agonist BTB09089.Although activated TDAG8 is widely involved in the regulation of inflammation,immunity,pain and apoptosis,whether it is involved in cerebral ischemia-reperfusion injury regulation and its potential mechanism of action is not very clear.This study was to establish a rat model of transient cerebral ischemia-reperfusion injury model,first observe the expression of TDAG8 and its downstream protein in the ischemic penumbra region of rat cerebral cortex;followed by intracerebroventricular injection of BTB09089 to stimulate TDAG8 or gene interference to inhibit TDAG8 expression to observe the effect of TDAG8 on the volume,neurobehavior and expression of downstream protein in rats;finally,the possible mechanism of TDAG8 regulation of cerebral ischemia-reperfusion injury was discussed.This study will explore the effect of TDAG8 on ischemic stroke injury and its mechanism,and provide a new therapeutic target and theoretical basis for the treatment of ischemic stroke in rats.Objective(1)To explore the thepression of TDAG8 during the cerebral ischemia reperfusion in rats.(2)To explore the effect of BTB09089 via intracerebrovebtricular injuction on TDAG8 expression.(3)To explore the effect of siRNA via intracerebrovebtricular injuction on TDAG8 expression.(4)To explore the effect of TDAG8 and downstream Akt signaling on neurons apoptosis and post-ischemia inflammation induced by cerebral ischemia reperfusion in rats.Methods(1)Twenty-four healthy male SD rats,270-280 g,were randomly divided into five groups(n=4):sham group,I/R 3 h group,I/R 6 h group,I/R 12 h group and I/R 24 hgroup.Group sham does nothing;other groups were subjected to ischemia 2 h followed reperfussion and harvested according to the definitely time.In addition,primary cortical neurons were subjected OGD and were harvested in 3 h,6 h,12 h and 24 h later.TDAG8 expression in penumbra were detected by western blot and qRT-PCR.(2)Sixteen healthy male SD rats,270-280 g,were randomly divided into five groups(n=4):normal group,BTB09089 5 ?M group(group BTB 5 ?M),BTB 10 ?M group and BTB 20 ?M group.A total of 8 ?L of 5 ?M BTB,10 ?M and 20 ?M BTB was respectively injected into group BTB 5 ?M,group BTB 10 ?M and group BTB 20 ?M;group normal was injected with the same amount of normal saline.The cerebral cortex in the marginal region of the forebrain was harvested 6 h later.The cytotoxicity of BTB was detected by CCK-8 and TDAG8 expression were detected by western blot and qRT-PCR.(3)Twenty healthy male SD rats,270-280 g,were randomly divided into five groups(n=4):normal group,vehicle group,siRNA-control group(group siRNA-c),siRNA group and siRNA positive group(group siRNA-?-actin).A total of 4 ?g of siRNA,siRNA-c and siRNA-?-actin was respectively injected into group siRNA,group siRNA-c and group siRNA-?-actin;group normal was injected with the same amount of normal saline.The cerebral cortex in the marginal region of the forebrain was harvested and TDAG8 expression were detected by western blot and qRT-PCR 24 h later.(4)Forty-six healthy male SD rats,270-280 g,were randomly divided into eight groups(n=6):sham group,vehicle group,BTB 5 ?M group,BTB 10 ?M group,BTB 20 ?M group,siRNA-c group,siRNA group and siRNA+BTB 20 ?M group.Group BTB 5 ?M,group BTB 10 ?M and group BTB 20 ?M was respectively pretreated with total 8 ?L of 5 ?M BTB,10 ?M BTB and 20 ?M BTB via the lateral ventricle 6 h before MCAO;group siRNA-c,group siRNA and group si RNA+BTB 20 ?M was respectively pretreated with 4 ?g(total 8 ?L)of siRNA-c,siRNA and siRNA via the lateral ventricle 6 h before MCAO,in addition,8 ?L 20 ?M BTB was injected into the lateral ventricle 6 h before MCAO in group siRNA+BTB 20 ?M;group sham only surgery without suture-filament;group MCAO was subjected transient MCAO;group vehicle was given appropriate amount of transfection reagent 24 h before MCAO.The penumbra was harvested by decapitating 6 h after reperfusion;the expression of pro-apoptotic factor caspase-3,cleaved caspase-3,anti-apoptotic factor Bcl-2 and TDAG8 downstream-associated protein phosphorylation Akt and CREB was detected by western blot and qRT-PCR.In addition,the expression of TNF-?,MCP-1 and IL-1? was detected by qRT-PCR;and neurological deficit scores and cerebral infarction volume were evaluated at 24 h and 72 h after reperfusion respectively.Results(1)TDAG8 expression was increased begain I/R 3 h and continued to 24 h and reached the peak at about 6 h in MCAO model;while TDAG8 expression was increased begain I/R 3 h and continued to 24 h and reached the peak at 24 h in OGD model.(2)Compared with group normal,TDAG8 expression was increased in group BTB 5 ?M;compared with group BTB 5 ?M,TDAG8 expression was increased in group BTB 10 ?M;compared with group BTB 10 ?M,TDAG8 expression was increased in group BTB 20 ?M.Compared with group normal,the cell viability of each group was greater than 95%.(3)There was no significant difference in the expression of TDAG8 between normal group,group vehicle and group siRNA-c.Compared with group normal,TDAG8 expression was decreased in group siRNA and ?-actin expression was decreased in group siRNA-?-actin.(4)Compared with group sham,infarction volume was enlarged,neurological deficit score was decreased,the expression of TNF-?,MCP-1,IL-1?,cleaved caspase-3,p-Akt and p-CREB was up-regulated and Bcl-2 expression was down-regulated in group MCAO;there are no significant difference in the above between group MCAO and group BTB 5 ?M;compared with group MCAO,infarction volume was reduced,neurological deficit score was increased,the expression of TNF-?,MCP-1,IL-1?,cleaved caspase-3 was down-regulated and Bcl-2,p-Akt and p-CREB expression was up-regulated in group BTB 10 ?M;compared with group BTB 10 ?M,infarction volume was reduced,neurological deficit score was increased,the expression of TNF-?,MCP-1,IL-1?,cleaved caspase-3 was down-regulated and Bcl-2,p-Akt and p-CREB expression was up-regulated in group BTB 20 ?M;there are no significant difference in the above between group MCAO,group vehicle and group siRNA-c;compared with group MCAO,infarction volume was enlarged,neurological deficit score was decreased,the expression of TNF-?,MCP-1,IL-1? and cleaved caspase-3 was up-regulated and the expression of Bcl-2,p-Akt and p-CREB was down-regulated in group siRNA;there are no significant difference in the above between group siRNA,group siRNA+BTB 20 ?M.Conclusion(1)TDAG8 expression was up-regulated during the cerebral ischemia reperfusion in rats.(2)BTB09089 can effectively promote the expression of TDGA8 and without significantly cytotoxicity.(3)BTB09089 can effectively promote the expression of TDGA8 and without significantly cytotoxicity.(4)Activation of TDAG8 may via Akt signalling inhibits neurons apoptosis and post-ischemia inflammation induced by cerebral ischemia reperfusion in rats. |
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