CRMP2可通过改善神经细胞凋亡减轻缺血/再灌注大鼠神经功能缺损

CRMP2 alleviates neurological deficit by reducing neuron apoptosis in rats after cerebral ischemia/reperfusion injury

目的探讨CRMP2(collapsin response mediator protein2)对大鼠脑缺血/再灌注损伤后神经细胞凋亡的影响及其可能的机制。方法 192只♂成年SD大鼠分成4组:假手术组(sham)、脑缺血/再灌注组(MCAO)、脑缺血+质粒对照组(MCAO+GFP)、脑缺血+CRMP2真核质粒干预组(MCAO+CRMP2/GFP)。大鼠MCA阻塞手术前1 d将真核质粒注射入脑内,缺血/再灌注后48 h、1周,采用RT-PCR检测各组大鼠脑组织CRMP2、BCL2、p53、Caspase-3和Caspase-8的mRNA的表达;Western blot检测脑组织CRMP2蛋白的表达;TUNEL染色检测凋亡细胞;免疫组化检测脑源性神经营养因子(brain derived neurotrophic factor,BDNF)的表达;TTC染色检测脑梗死体积并进行神经功能缺损评分。结果脑缺血/再灌注48 h及1周,与sham组比较,MCAO组及MCAO+GFP组CRMP2和BCL2的表达水平明显降低(P<0.01),而Caspase-3、Caspase-8及p53的mRNA表达升高(P<0.01),TUNEL阳性细胞数量明显升高(P<0.01)。MCAO+CRMP2/GFP组CRMP2和BCL2较MCAO及MCAO+GFP组明显增高(P<0.01),同时该组p53、Caspase-3及Caspase-8表达明显降低(P<0.01)。过表达CRMP2使TUNEL阳性细胞数明显减少(P<0.01)。脑缺血/再灌注后BDNF表达升高(P<0.01),而脑内过表达CRMP2使BDNF的水平上调更明显(P<0.01)。TTC染色显示,MCAO+CRMP2/GFP组脑梗死体积较MCAO组及MCAO+GFP组明显减小(P<0.01),且神经功能缺损明显减轻(P<0.01)。结论过表达CRMP2可能通过对线粒体凋亡通路的调控减少了脑缺血/再灌注损伤后的神经细胞凋亡、减少脑梗死体积而起到神经保护作用。

Aim To investigate the influence of the overexpression of CRMP2 on neural cell apoptosis after ischemia reperfusion injury in rats and its possible mechanism. Methods A total of 192 male adult SD rats were divided into four groups: sham group,cerebral ischemia / reperfusion group( MCAO group),cerebral ischemia with blank plasmid control group( MCAO+ GFP group),cerebral ischemia with CRMP2 eukaryotic plasmid group( MCAO + CRMP2 / GFP group). One day after injecting eukaryotic plasmid,the rats were operated for 120-min ischemia through MCA occlusion and reperfused. At 48 h and 1 wk,the expression of CRMP2,p53,Caspase-3,Caspase-8 and BCL2 in brain tissue was tested by RT-PCR and Western blot. Apoptotic cells were observed by TUNEL test. TTC staining was use to detect cerebral infarction volume. The neural function of the rats were also evaluated. Results Compared with the sham group,the expression levels of CRMP2 and BCL2 in MCAO group and MCAO + GFP group were significantly decreased( P < 0. 01),while p53,Caspase-3,Caspase-8 and TUNEL positive cells were elevated( P < 0. 01). Intervention of CRMP2 eukaryotic plasmid resulted in the increased expression of CRMP2 and BCL2( P < 0. 01)and the decreased p53,Caspase-3 and Caspase-8 expression. In TUNEL test,overexpression of CRMP2 obviously decreased the number of TUNEL positive cells( P < 0. 01). The expression of BDNF was upregulated after cerebral ischemic injury( P < 0. 01),while overexpression of CRMP2 increased BDNF more significantly( P < 0. 01). TTC staining showed cerebral infarction volumn of MCAO + CRMP2 / GFP group was obviously decreased( P < 0. 01),and neurologic deficits were significantly improved( P < 0. 01). Conclusion The overexpression of CRMP2 reduces nerve cell apoptosis possibly by regulating the mitochondrial apoptosis pathway after cerebral ischemia / reperfusion injury to protect nervous system.