T细胞死亡相关基因8对大鼠局灶性脑缺血再灌注损伤的影响
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摘要
目的:探讨活化T细胞死亡相关基因8(T cell death associated gene 8,TDAG8)对大鼠局灶性脑缺血再灌注损伤的影响。方法:将健康成年雄性SD大鼠132只采用随机数字表法分为6组:对照组(n=18),缺血再灌注组(I/R组,n=42),TDAG8激动剂BTB09089组(BTB组,n=18),空白载体组(空载体组,n=18),TDAG8-siRNA阴性对照组(siRNA阴性组,n=18)和TDAG8-siRNA组(siRNA组,n=18)。采用线栓法短暂性阻塞大鼠大脑中动脉(middle cerebral artery occlusion,MCAO)制备大鼠局灶性脑I/R模型。BTB组、空载体组、siRNA阴性组和siRNA组于缺血前72 h、48 h和24 h分别经侧脑室注射20μmol/L的BTB09089、空白转染试剂jetPEI、4μg siRNA阴性对照和4μg TDAG8-siRNA各8μL,其中空载体组、siRNA阴性组和siRNA组同时均注入等量转染试剂jetPEI。于再灌注6 h时每组取6只大鼠断头取脑,蛋白质印迹法检测缺血半暗带TDAG8、cleaved caspase-3、Bcl-2和TDAG8下游相关蛋白p-Akt、p-CREB的表达水平;于再灌注24 h和72 h时每组分别取6只大鼠评价神经功能并测定脑梗死体积百分比。结果:与对照组比较,其他5组再灌注6 h时缺血半暗带TDAG8、cleaved caspase-3、p-Akt和p-CREB表达均上调(均P <0. 05),Bcl-2表达下调(P <0. 05),再灌注24 h和72 h时脑梗死体积百分比明显升高、神经功能缺陷评分显著降低(均P <0. 05);与I/R组比较,BTB组再灌注6 h时TDAG8、Bcl-2、p-Akt和p-CREB水平上调(均P <0. 05),cleaved caspase-3表达下调(P <0. 05),再灌注24 h和72 h时脑梗死体积百分比显著降低,神经功能缺陷评分明显升高(均P <0. 05);与siRNA阴性组比较,siRNA组再灌注6 h时TDAG8、Bcl-2、p-Akt和p-CREB水平下调(P <0. 05),cleaved caspase-3表达上调(P <0. 05),再灌注后24 h和72 h脑梗死体积百分比显著升高,而神经功能缺陷评分明显降低(P <0. 05)。结论:活化TDAG8可能通过Akt信号通路调控短暂性大鼠MCAO诱导的脑缺血再灌注损伤。
Objective: To investigate the effect of activated T cell death associated gene 8(TDAG8 on focal cerebral ischemia-reperfusion injury in rats. Methods: One hundred and thirty-two healthy adult male Sprague-Dawley rats were divided into 6 groups using a random number table: control group(Group C,n = 18),ischemia-reperfusion group(Group I/R,n = 42),TDAG8 agonist BTB09089 group(Group BTB,n = 18),blank vector group(Group V,n = 18),TDAG8-siRNA negative control group(n = 18)and TDAG8-siRNA group(Group siRNA,n = 18). Middle cerebral artery occlusion(MCAO) was used to establish rat focal cerebral ischemia-reperfusion model. Group BTB,Group V,Group siRNA(-)and Group siRNA were injected into the lateral ventricle with 8 μL of 20 μmol/L BTB09089,8 μL blank transfection reagent jetPEI,4 μg siRNA negative control and 4 μg siRNA,respectively,at 72 h,48 h and 24 h before ischemia. The Group V,Group siRNA(-) and Group siRNA contained the same amount of transfection reagent jetPEI. At 6 h after reperfusion,the brain was decapitated,the expression of TDAG8,pro-apoptotic factor cleaved caspase-3,anti-apoptotic factor Bcl-2,p-Akt and p-CREB in ischemic penumbra was detected by Western blotting. The neurological deficit score and infarct volume percentage were respectively measured at 24 h and 72 h after reperfusion. Results: Compared with Group C,at 6 h after reperfusion,the expression of TDAG8,cleaved caspase-3,p-Akt and p-CREB was up-regulated(P < 0. 05) and Bcl-2 expression was down-regulated(P < 0. 05) in Group I/R,Group BTB,Group V,Ggroup siRNA(-) and Group siRNA,and at 24 h and 72 h after reperfusion,the infarct volume percentage increased and the neurological deficit score reduced(P < 0. 05). Compared with Group I/R,at 6 h after reperfusion,the expression of TDAG8,Bcl-2,p-Akt and p-CREB was up-regulated(P< 0. 05) and cleaved caspase-3 expression was down-regulated(P < 0. 05) in Group BTB,and at 24 h and 72 h after reperfusion,the infarct volume percentage reduced and the neurological deficit score increased(P < 0. 05). Compared with Group I/R,at the 6 h after reperfusion,the expression of TDAG8,Bcl-2,p-Akt and p-CREB was down-regulated(P < 0. 05) and cleaved caspase-3 expression was up-regulated(P < 0. 05) in Group siRNA,and at 24 h and 72 h after reperfusion,the infarct volume percentage increased and the neurological deficit score reduced(P < 0. 05). Conclusion: Activation of TDAG8 may be involved in focal cerebral ischemia-reperfusion injury induced by transient middle cerebral artery occlusion in rats by the Akt signaling pathway.
Objective: To investigate the effect of activated T cell death associated gene 8(TDAG8 on focal cerebral ischemia-reperfusion injury in rats. Methods: One hundred and thirty-two healthy adult male Sprague-Dawley rats were divided into 6 groups using a random number table: control group(Group C,n = 18),ischemia-reperfusion group(Group I/R,n = 42),TDAG8 agonist BTB09089 group(Group BTB,n = 18),blank vector group(Group V,n = 18),TDAG8-siRNA negative control group(n = 18)and TDAG8-siRNA group(Group siRNA,n = 18). Middle cerebral artery occlusion(MCAO) was used to establish rat focal cerebral ischemia-reperfusion model. Group BTB,Group V,Group siRNA(-)and Group siRNA were injected into the lateral ventricle with 8 μL of 20 μmol/L BTB09089,8 μL blank transfection reagent jetPEI,4 μg siRNA negative control and 4 μg siRNA,respectively,at 72 h,48 h and 24 h before ischemia. The Group V,Group siRNA(-) and Group siRNA contained the same amount of transfection reagent jetPEI. At 6 h after reperfusion,the brain was decapitated,the expression of TDAG8,pro-apoptotic factor cleaved caspase-3,anti-apoptotic factor Bcl-2,p-Akt and p-CREB in ischemic penumbra was detected by Western blotting. The neurological deficit score and infarct volume percentage were respectively measured at 24 h and 72 h after reperfusion. Results: Compared with Group C,at 6 h after reperfusion,the expression of TDAG8,cleaved caspase-3,p-Akt and p-CREB was up-regulated(P < 0. 05) and Bcl-2 expression was down-regulated(P < 0. 05) in Group I/R,Group BTB,Group V,Ggroup siRNA(-) and Group siRNA,and at 24 h and 72 h after reperfusion,the infarct volume percentage increased and the neurological deficit score reduced(P < 0. 05). Compared with Group I/R,at 6 h after reperfusion,the expression of TDAG8,Bcl-2,p-Akt and p-CREB was up-regulated(P< 0. 05) and cleaved caspase-3 expression was down-regulated(P < 0. 05) in Group BTB,and at 24 h and 72 h after reperfusion,the infarct volume percentage reduced and the neurological deficit score increased(P < 0. 05). Compared with Group I/R,at the 6 h after reperfusion,the expression of TDAG8,Bcl-2,p-Akt and p-CREB was down-regulated(P < 0. 05) and cleaved caspase-3 expression was up-regulated(P < 0. 05) in Group siRNA,and at 24 h and 72 h after reperfusion,the infarct volume percentage increased and the neurological deficit score reduced(P < 0. 05). Conclusion: Activation of TDAG8 may be involved in focal cerebral ischemia-reperfusion injury induced by transient middle cerebral artery occlusion in rats by the Akt signaling pathway.
引文
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[14]马晓冬,邵东华,杭黎华,等.TDAG8与氧糖缺失-复糖复氧诱发大鼠神经元凋亡时内源性保护机制的关系[J].中华麻醉学杂志,2016,36(9):1080-1084.
[15]Osuka K,Watanabe Y,Usuda N,et al.Modification of endothelial NO synthase through protein phosphorylation after forebrain cerebral ischemia/reperfusion[J].Stroke,2004,35(11):2582-2586.
[2]Mcguire J,Herman JP,Ghosal S,et al.Acid-sensing by the T cell death-associated gene 8(TDAG8)receptor cloned from rat brain[J].Biochem Biophys Res Commun,2009,386(3):420-425.
[3]Gaublomme JT,Yosef N,Lee Y,et al.Single-cell genomics unveils critical regulators of Th17 cell pathogenicity[J].Cell,2015,163(6):1400-1412.
[4]An C,Sato K,Wu T,et al.Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner[J].Biochem Biophys Res Commun,2015,460(2):191-197.
[5]濮健峰,邵东华,杭黎华,等.T细胞死亡偶联基因8在大鼠局灶性脑缺血-再灌注损伤中的表达[J].临床麻醉学杂志,2012,28(5):501-503.
[6]Ma Q,Chen S,Hu Q,et al.NLRP3 inflammasome contributes to inflammation after intracerebral hemorrhage[J].Ann Neurol,2014,75(2):209-219.
[7]Liang X,Hu Q,Li B,et al.Follistatin-like 1 attenuates apoptosis via disco-interacting protein 2 homolog A/Akt pathway after middle cerebral artery occlusion in rats[J].Stroke,2014,45(10):3048-3054.
[8]陈晓娟,肖宗宇,潘琪,等.改良线栓法大鼠脑缺血再灌注损伤模型的建立[J].中国医学创新,2015,12(3):24-27.
[9]陈卫伟,杨留才,潘施文,等.线栓法制备SD大鼠局灶性脑缺血再灌注损伤模型[J].中国组织工程研究与临床康复,2011,15(50):9377-9380.
[10]Henninger N,Bouley J,Bratane BT,et al.Laser Doppler flowmetry predicts occlusion but not t PA-mediated reperfusion success after rat embolic stroke[J].Exp Neurol,2009,215(2):290-297.
[11]Hu Q,Chen C,Yan J,et al.Therapeutic application of gene silencing MMP-9 in a middle cerebral artery occlusion-induced focal ischemia rat model[J].Exp Neurol,2009,216(1):35-46.
[12]Grabowski J,Vazquez DE,Costantini T,et al.Tumor necrosis factor expression is ameliorated after exposure to an acidic environment[J].J Surg Res,2012,173(1):127-134.
[13]Rosko AE,McColl KS,Zhong F,et a1.Acidosis sensing receptor GPR65 correlates with anti-apoptotic Bcl-2 family member expression in CLL cells:potential implications for the CLL microenvironment[J].J Leuk(Los Angel),2014,2(5).pii:160.
[14]马晓冬,邵东华,杭黎华,等.TDAG8与氧糖缺失-复糖复氧诱发大鼠神经元凋亡时内源性保护机制的关系[J].中华麻醉学杂志,2016,36(9):1080-1084.
[15]Osuka K,Watanabe Y,Usuda N,et al.Modification of endothelial NO synthase through protein phosphorylation after forebrain cerebral ischemia/reperfusion[J].Stroke,2004,35(11):2582-2586.