自噬对脑外伤引起的神经细胞死亡及神经功能障碍的影响
发布时间：2019-04-04 12:51
【摘要】： 目的:研究自噬/溶酶体途径对脑外伤后神经细胞死亡和神经功能障碍的影响并探讨其相关机制。 方法:建立小鼠定量脑外伤(traumatic brain injury, TBI)动物模型,运用影响自噬体成熟的工具药3-甲基腺嘌呤(3-methyladenine,3-MA)、布雷菲德菌素(Brefeldin A,BFA)侧脑室(lateral cerebral ventricle)注射给药,对3-MA组、BFA组及生理盐水对照组用PI标记和体视学方法观测损伤区及其周边区(皮层与海马)神经细胞死亡情况,通过连续切片苏木精染色和体视学软件测量脑缺损体积(lesion volume, LV),并通过行为学试验方法(Motor test和Morris water maze)检测TBI引起的神经功能障碍,研究自噬对TBI引起的神经细胞死亡和神经功能障碍的影响;同时应用免疫组织化学和免疫印记法检测损伤侧的脑皮质与海马中自噬/溶酶体途径及凋亡信号通路相关蛋白Cathepsin-B,LC3,Beclin-1,Caspase-3,Bcl-2,Bax的表达情况,研究自噬/溶酶体途径影响TBI引起的神经细胞死亡和神经功能障碍的可能机制。 结果:(1)PI阳性细胞计数:TBI后1 h和6 h,3-MA组与生理盐水组PI阳性细胞数均开始逐渐增加,12 h后明显增加,但两组之间无统计学差异,24 h阳性细胞数达高峰,且两组的PI阳性细胞数有统计学差异(P0.05),TBI后48 h组仍有大量的PI阳性细胞,但3-MA组与生理盐水组比较无统计学差异;同样,BFA组与生理盐水组比较,TBI后24 h,两组的PI阳性细胞数有统计学差异(P0.05)。(2)LV检测:与生理盐水组比较,3-MA组、BFA组脑外伤后LV显著减小(P0.05);(3)行为学检测:与生理盐水组比较,3-MA组和BFA组在TBI后24 h均能改善运动功能(P0.05),48 h以后差异不显著;3-MA组在TBI后7 d到10 d能改善学习记忆能力(P0.05);(4)蛋白表达检测:TBI后24 h和48 h,3-MA组相对与生理盐水组Cathepsin-B, Caspase-3表达减少(P0.05),Beclin-1/ Bcl-2的比值减小(P0.05),Bcl-2 /Bax的比值增大(P0.05);与生理盐水对照组比较,TBI后24 h和48 h BFA组LC3Ⅱ/LC3Ⅰ的比值减小(P0.05)。 结论: 1.自噬/溶酶体途径参与了TBI后的病理生理过程。阻滞自噬体形成可以减少TBI引起的神经细胞死亡和脑组织缺损体积,并改善运动和学习记忆功能。 2.自噬/溶酶体途径通过影响细胞凋亡信号通路来调节TBI引起的神经细胞死亡和神经功能障碍。
[Abstract]:Aim: to study the effects of autophagy / lysosome pathway on neuronal death and neurological dysfunction after traumatic brain injury. Methods: the animal model of (traumatic brain injury, TBI) was established in mice. The tool drugs, 3-methyladenine (3-methyladenine, 3-methyladenine), (Brefeldin A, which affect the maturation of autophages, were used to establish the animal model of brain injury in mice. (lateral cerebral ventricle) was injected into the lateral ventricle of BFA. The death of nerve cells in 3-MA group, BFA group and normal saline control group was observed by PI labeling and stereology in the injured area and its peripheral area (cortex and hippocampus). The volume (lesion volume, LV), of brain defect was measured by serial section hematoxylin staining and stereology software, and the neurological dysfunction caused by TBI was detected by behavioral test methods (Motor test and Morris water maze). To study the effect of autophagy on nerve cell death and neurological dysfunction induced by TBI. At the same time, the expression of autophagy / lysosome pathway and apoptosis signal pathway-related protein Cathepsin-B,LC3,Beclin-1,Caspase-3,Bcl-2,Bax in cortex and hippocampus of injured side were detected by immunohistochemistry and immuno-blotting. To investigate the possible mechanism of autophagy / lysosome pathway affecting neuronal death and neurological dysfunction induced by TBI. Results: (1) PI positive cell count: 1 h and 6 h after TBI, the number of PI positive cells in 3MA group and saline group began to increase gradually and increased significantly after 12 h, but there was no statistical difference between the two groups, and the number of PI positive cells reached the peak at 24 h. There was significant difference in the number of PI positive cells between the two groups (P0.05) 48 hours after), TBI there were still a large number of PI positive cells, but there was no statistical difference between the 3-MA group and the saline group. Similarly, the number of PI positive cells in BFA group was significantly different from that in saline group 24 hours after TBI (P0.05). (2) LV test: compared with saline group, 3-MA group, The LV of BFA group was significantly decreased after brain injury (P0.05). (3) Behavioral test: compared with saline group, both 3-MA group and BFA group could improve motor function at 24 h after TBI (P0.05), but there was no significant difference after 48 h. The ability of learning and memory was improved in 3-MA group from 7 days to 10 days after TBI (P0.05). (4) protein expression: 24 h and 48 h after TBI, the expression of Cathepsin-B, Caspase-3 and the ratio of Beclin-1/ Bcl-2 in 3MA group were significantly lower than those in normal saline group (P0.05). The ratio of Bcl-2 / Bax increased (P0.05); Compared with the saline control group, the ratio of LC3 鈪，
本文编号：2453831
[Abstract]:Aim: to study the effects of autophagy / lysosome pathway on neuronal death and neurological dysfunction after traumatic brain injury. Methods: the animal model of (traumatic brain injury, TBI) was established in mice. The tool drugs, 3-methyladenine (3-methyladenine, 3-methyladenine), (Brefeldin A, which affect the maturation of autophages, were used to establish the animal model of brain injury in mice. (lateral cerebral ventricle) was injected into the lateral ventricle of BFA. The death of nerve cells in 3-MA group, BFA group and normal saline control group was observed by PI labeling and stereology in the injured area and its peripheral area (cortex and hippocampus). The volume (lesion volume, LV), of brain defect was measured by serial section hematoxylin staining and stereology software, and the neurological dysfunction caused by TBI was detected by behavioral test methods (Motor test and Morris water maze). To study the effect of autophagy on nerve cell death and neurological dysfunction induced by TBI. At the same time, the expression of autophagy / lysosome pathway and apoptosis signal pathway-related protein Cathepsin-B,LC3,Beclin-1,Caspase-3,Bcl-2,Bax in cortex and hippocampus of injured side were detected by immunohistochemistry and immuno-blotting. To investigate the possible mechanism of autophagy / lysosome pathway affecting neuronal death and neurological dysfunction induced by TBI. Results: (1) PI positive cell count: 1 h and 6 h after TBI, the number of PI positive cells in 3MA group and saline group began to increase gradually and increased significantly after 12 h, but there was no statistical difference between the two groups, and the number of PI positive cells reached the peak at 24 h. There was significant difference in the number of PI positive cells between the two groups (P0.05) 48 hours after), TBI there were still a large number of PI positive cells, but there was no statistical difference between the 3-MA group and the saline group. Similarly, the number of PI positive cells in BFA group was significantly different from that in saline group 24 hours after TBI (P0.05). (2) LV test: compared with saline group, 3-MA group, The LV of BFA group was significantly decreased after brain injury (P0.05). (3) Behavioral test: compared with saline group, both 3-MA group and BFA group could improve motor function at 24 h after TBI (P0.05), but there was no significant difference after 48 h. The ability of learning and memory was improved in 3-MA group from 7 days to 10 days after TBI (P0.05). (4) protein expression: 24 h and 48 h after TBI, the expression of Cathepsin-B, Caspase-3 and the ratio of Beclin-1/ Bcl-2 in 3MA group were significantly lower than those in normal saline group (P0.05). The ratio of Bcl-2 / Bax increased (P0.05); Compared with the saline control group, the ratio of LC3 鈪，
本文编号：2453831
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