自噬對腦外傷引起的神經(jīng)細(xì)胞死亡及神經(jīng)功能障礙的影響
發(fā)布時間：2019-04-04 12:51
【摘要】： 目的:研究自噬/溶酶體途徑對腦外傷后神經(jīng)細(xì)胞死亡和神經(jīng)功能障礙的影響并探討其相關(guān)機(jī)制。 方法:建立小鼠定量腦外傷(traumatic brain injury, TBI)動物模型,運用影響自噬體成熟的工具藥3-甲基腺嘌呤(3-methyladenine,3-MA)、布雷菲德菌素(Brefeldin A,BFA)側(cè)腦室(lateral cerebral ventricle)注射給藥,對3-MA組、BFA組及生理鹽水對照組用PI標(biāo)記和體視學(xué)方法觀測損傷區(qū)及其周邊區(qū)(皮層與海馬)神經(jīng)細(xì)胞死亡情況,通過連續(xù)切片蘇木精染色和體視學(xué)軟件測量腦缺損體積(lesion volume, LV),并通過行為學(xué)試驗方法(Motor test和Morris water maze)檢測TBI引起的神經(jīng)功能障礙,研究自噬對TBI引起的神經(jīng)細(xì)胞死亡和神經(jīng)功能障礙的影響;同時應(yīng)用免疫組織化學(xué)和免疫印記法檢測損傷側(cè)的腦皮質(zhì)與海馬中自噬/溶酶體途徑及凋亡信號通路相關(guān)蛋白Cathepsin-B,LC3,Beclin-1,Caspase-3,Bcl-2,Bax的表達(dá)情況,研究自噬/溶酶體途徑影響TBI引起的神經(jīng)細(xì)胞死亡和神經(jīng)功能障礙的可能機(jī)制。 結(jié)果:(1)PI陽性細(xì)胞計數(shù):TBI后1 h和6 h,3-MA組與生理鹽水組PI陽性細(xì)胞數(shù)均開始逐漸增加,12 h后明顯增加,但兩組之間無統(tǒng)計學(xué)差異,24 h陽性細(xì)胞數(shù)達(dá)高峰,且兩組的PI陽性細(xì)胞數(shù)有統(tǒng)計學(xué)差異(P0.05),TBI后48 h組仍有大量的PI陽性細(xì)胞,但3-MA組與生理鹽水組比較無統(tǒng)計學(xué)差異;同樣,BFA組與生理鹽水組比較,TBI后24 h,兩組的PI陽性細(xì)胞數(shù)有統(tǒng)計學(xué)差異(P0.05)。(2)LV檢測:與生理鹽水組比較,3-MA組、BFA組腦外傷后LV顯著減小(P0.05);(3)行為學(xué)檢測:與生理鹽水組比較,3-MA組和BFA組在TBI后24 h均能改善運動功能(P0.05),48 h以后差異不顯著;3-MA組在TBI后7 d到10 d能改善學(xué)習(xí)記憶能力(P0.05);(4)蛋白表達(dá)檢測:TBI后24 h和48 h,3-MA組相對與生理鹽水組Cathepsin-B, Caspase-3表達(dá)減少(P0.05),Beclin-1/ Bcl-2的比值減小(P0.05),Bcl-2 /Bax的比值增大(P0.05);與生理鹽水對照組比較,TBI后24 h和48 h BFA組LC3Ⅱ/LC3Ⅰ的比值減小(P0.05)。 結(jié)論: 1.自噬/溶酶體途徑參與了TBI后的病理生理過程。阻滯自噬體形成可以減少TBI引起的神經(jīng)細(xì)胞死亡和腦組織缺損體積,并改善運動和學(xué)習(xí)記憶功能。 2.自噬/溶酶體途徑通過影響細(xì)胞凋亡信號通路來調(diào)節(jié)TBI引起的神經(jīng)細(xì)胞死亡和神經(jīng)功能障礙。
[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，
本文編號：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，
本文編號：2453831
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