瞬时受体电位通道1在大鼠脑缺血后钙超载致神经元损伤中的作用机制

Mechanism of transient receptor potential channel 1 in neuronal injury induced by calcium overload after cerebral ischemia in rats

目的研究大鼠脑缺血后瞬时受体电位通道1(transient receptor potential channel 1,TRPC1)在神经元钙超载中的作用,探讨TRPC1参与脑缺血后神经元凋亡及突触功能障碍的机制。方法120只6周龄SD大鼠随机分为正常对照组、全脑缺血模型组、SKF96365干预模型组、DMSO干预对照组。四血管阻塞法建立大鼠全脑缺血模型,全脑缺血前立体定向注射SKF96365建立干预模型组,注射DMSO建立干预对照模型组。在缺血后的1,3,5,7,10 d收集大鼠双侧海马标本,进行形态学染色,用电镜观察细胞超微结构,Fura-2AM检测胞内Ca 2+浓度,Western blot检测TRPC1蛋白的动态表达,TUNEL检测海马神经元凋亡情况,用Morris水迷宫评估大鼠海马记忆功能,通过体视学分析计算海马CA1区中存活神经元的数量。结果形态学研究发现,全脑缺血后神经元肿胀,髓鞘结构破坏,片层结构消失。与正常组相比,全脑缺血模型组海马TRPC1蛋白表达持续升高,在3 d达到高峰,同时海马神经元胞内Ca 2+浓度及TUNEL阳性细胞数达到峰值(P<0.05)。给予TRPC1通道阻断剂SKF96365后,与全脑缺血模型组相比,干预模型组海马TRPC1的蛋白表达被抑制,海马神经元钙内流减低,TUNEL阳性细胞数显著降低,大鼠神经行为学及记忆功能评分改善(P<0.05)。结论TRPC1与脑缺血后钙超载导致的迟发性神经元凋亡密切相关,抑制TRPC1产生的钙内流可缓解海马神经元细胞凋亡,改善海马的空间记忆功能。

Objective To investigate the role of canonical transient receptor potential channel 1(TRPC1)in neuronal calcium overload,and to explore the mechanism of TRPC1 involved in neuronal apoptosis and synaptic dysfunction after cerebral ischemia.Methods A total of 120 SD rats of 6 weeks old were randomly divided into normal control group,whole cerebral ischemia model group,SKF96365 intervention model group and DMSO intervention control group.The rat model of global cerebral ischemia was established by four-vessel occlusion method.The intervention model was established by stereotactic injection of SKF96365 before cerebral ischemia.The intervention control model was established by injection of DMSO.Rat hippocampal specimens were collected at 1 d,3 d,5 d,7 d and 10 d after ischemia for morphological staining.Ultrastructure was observed by electron microscopy,intracellular Ca 2+concentration was detected by Fura-2AM,and TRPC1 protein was detected by Western blot.The TUNEL was used to detect the apoptosis of hippo-campal neurons.The memory function of hippocampus was assessed by Morris water maze.The number of surviving neurons in hippo-campal CA1 region was calculated by stereological analysis.Results After global cerebral ischemia,the neurons were swollen,the myelin structure was destroyed,and the lamellar structure disappeared.Compared with normal group,the expression of TRPC1 protein in hippocampus increased in whole cerebral ischemia model group and reached the peak at 3 d,meanwhile the intracellular Ca 2+concentration and TUNEL positive cells of hippocampal neurons reached the peak(P<0.05).Compared with global cerebral ischemia model group,the protein expression of hippocampal TRPC1 was inhibited in intervention model group,the calcium influx of hippocampal neurons was reduced,the number of TUNEL positive cells was significantly decreased,and the memory function score was improved(all P<0.05).Conclusion TRPC1 is closely related to the delayed neuronal apoptosis induced by calcium overload after cerebral ischemia.Inhibition of calcium influx produced by TRPC1 can alleviate hippocampal neuronal apoptosis and improve spatial memory function of hippocampus.