远隔缺血后处理调节脑源性神经营养因子甲基化修饰对脑缺血-再灌注大鼠运动功能的改善作用研究

Remote ischemic postconditioning regulates brain-derived neurotrophic factor methylation modification to improve motor function in rats with cerebral ischemia-reperfusion

目的探索远隔缺血后处理(RIP)对脑缺血-再灌注大鼠运动功能障碍的改善作用和对运动皮质脑源性神经营养因子(BDNF)表达的调控及机制。方法选用成年雄性Sprague Dawley大鼠制作大脑中动脉闭塞(MCAO)模型,将大鼠完全随机分为假手术组(Sham组)、MCAO组和RIP组(MCAO+RIP组),MCAO造模成功后24 h对MCAO+RIP组大鼠进行连续21 d RIP干预。采用旷场实验和转棒实验检测3组大鼠运动功能(包括运动速度、运动总距离及停留时间),蛋白免疫印迹(Western blot)和免疫荧光染色检测大鼠皮质BDNF、酪氨酸激酶受体B(TrkB)的蛋白表达以及神经元核抗原(NeuN)和含半胱氨酸的半胱氨酸蛋白水解酶3(Caspase3)蛋白的表达水平,实时荧光定量逆转录聚合酶链反应(RT-qPCR)检测皮质BDNF信使核糖核酸(mRNA)水平。点杂交实验和甲基化特性聚合酶链反应(PCR)分别检测大鼠皮质总体的5甲基胞嘧啶(5mC)水平和BDNF的5mC甲基化修饰水平,以及Western blot检测了DNA甲基化相关酶的蛋白表达水平,包括DNA甲基转移酶1(DNMT1)、DNA甲基转移酶3a(DNMT3a)、DNA甲基转移酶3b(DNMT3b)和5甲基胞嘧啶羟化酶(TET1),并进行3组间的比较。结果与Sham组比较,术后第21天旷场实验结果显示,MCAO组大鼠的运动总距离和运动速度减少[(12±6)m比(25±6)m,(0.30±0.12)m/s比(0.50±0.06)m/s,均P<0.01],术后第22天转棒实验停留时间减少[(11±7)s比(34±12)s,P<0.01]。与MCAO组大鼠比较,MCAO+RIP组大鼠的旷场实验运动总距离[(20±4)m]和运动速度[(0.44±0.05)m/s]增加(均P<0.05),转棒实验停留时间[(24±8)s]增加(P<0.05)。免疫荧光染色结果显示,与Sham组大鼠比较,MCAO组大鼠运动皮质神经元数量显著减少[阳性细胞数:(19±3)个/视野比(92±6)个/视野,P<0.01],但MCAO+RIP组[阳性细胞数:(55±8)个/视野]较MCAO组运动皮质神经元数量增多(P<0.01)。Western blot和RT-qPCR结果显示,MCAO组大鼠皮质的BDNF mRNA和蛋白相对表达水平均较Sham组降低(均P<0.05),而与MCAO组比较,MCAO+RIP组BDNF mRNA和蛋白相对表达水平均升高,差异均有统计学意义(均P<0.05)。与Sham组比较,点杂交实验结果显示MCAO组大鼠运动皮质总体5mC修饰水平较高,Western blot结果表明MCAO组DNMT1和DNMT3b蛋白的相对表达水平升高,甲基化特异性PCR结果表明MCAO组BDNF甲基化水平升高,差异均有统计学意义(均P<0.05);与MCAO组比较,MCAO+RIP组皮质总体5mC水平和DNMT1和DNMT3b的蛋白相对表达水平以及相对BDNF甲基化水平均明显降低,差异均有统计学意义(均P<0.05)。结论RIP可能通过调控BDNF的5mC甲基化修饰,使运动皮质BDNF的表达水平升高,从而改善MCAO大鼠的运动功能障碍。

Objective To investigate the effect of remote ischemic postconditioning(RIP)on motor dysfunction and the regulation of brain-derived neurotrophic factor(BDNF)expression in motor cortex after cerebral ischemia-reperfusion in rats.Methods Adult male Sprague Dawley rats were selected to establish the middle cerebral artery occlusion(MCAO).Rats were randomly divided into sham operation group(Sham group),MCAO group and remote ischemic postconditioning group(MCAO+RIP group),and the rats in the MCAO+RIP group were subjected to RIP for 21 consecutive days 24 hours after successful MCAO modeling.Open field test and the rotarod test were carried out to examine the motor function of rats.Western blot and immunofluorescence staining detected the protein expression of BDNF and Tyrosine Kinase receptor B(TrkB),cysteinyl aspartate specific proteinase(Caspase3),the expression of neuronal core antigen(NeuN).And the level of BDNF messenger ribonucleic acid(mRNA)was detected by quantitative real-time polymerase chain reaction(RT-qPCR).Dot blot and methylation-specific polymerase chain reaction(PCR)showed the 5-methylcytosine(5mC)level and BDNF 5mC methylation modification level.In addition,Western blot detected the expression levels of DNA methylation-related enzymes respectively,including DNA methyltransferase 1(DNMT1),DNA methyltransferase 3a(DNMT3a),DNA methyltransferase 3b(DNMT3b)and ten-eleven translocation 1(TET1).The above observation indicators were compared among the Sham group,MCAO group and MCAO+RIP group.Results Compared with the Sham group,rats in MCAO group showed a significant reduction in movement distance and speed in open filed test 21 days after operation and dwell time in the rotarod test 22 days after operation([12±6]m vs.[25±6]m,[0.30±0.12]m/s vs.[0.50±0.06]m/s,[11±7]s vs.[34±12]s,all P<0.01),while the movement distance([20±4]m)and speed([0.44±0.05]m/s)and the dwell time([24±8]s)of the MCAO+RIP group rats increased(all P<0.05).The results of immunofluorescence staining showed that compared with Sham group,the number of motor cortex neurons in MCAO group was significantly reduced(number of positive cells:[19±3]cells/field vs.[92±6]cells/field,P<0.01),but MCAO+RIP group(number of positive cells:[55±8]cells/field)were more than those in MCAO group(P<0.01).The results of Western blot and RT-qPCR showed that the expression levels of BDNF mRNA and protein in the cortex of MCAO group were lower than those of Sham group(both P<0.05).Compared with MCAO group,the expression levels of BDNF mRNA and protein in MCAO+RIP group were increased,and the differences were statistically significant(all P<0.05).Compared with Sham group,point hybridization experiment results showed that the overall level of 5mC modification in the cortex of rats in MCAO group was higher;the Western blot results showed that the expression levels of DNMT1 and DNMT3b in MCAO group were increased;methylation-specific PCR results showed that the methylation level of BDNF was increased in MCAO group rats;all differences were statistically significant(all P<0.05).The Methylation-specific PCR results showed that the methylation level of BDNF increased,with statistical significance(all P<0.05).However,compared with MCAO group,the overall level of 5mC,the expression levels of DNMT1 and DNMT3b,and the methylation level of BDNF in the cortex of MCAO+RIP group were significantly decreased(all P<0.05).Conclusion RIP regulates the 5mC methylation modification of BDNF and increases the expression level of BDNF in motor cortex,thereby improving the motor dysfunction of MCAO rats.