FTY720改善颅脑损伤大鼠神经功能的机制研究

doi:10.3877/cma.j.issn.1674-6880.2016.04.006

目的

探讨FTY720对颅脑损伤大鼠神经功能的影响及其相关机制研究。

方法

60只大鼠分成假手术组、模型组和治疗组，每组20只，采用改进的Feeney自由落体损伤装置建立大鼠颅脑损伤模型。治疗组大鼠按1 mg/kg剂量予FTY720腹腔注射，假手术组及模型组大鼠予以腹腔注射1 ml生理盐水。术后24 h，每组各取10只大鼠断头处死，分离海马组织，采用Western-blotting检测核因子κB表达水平，采用免疫组织化学染色法检测大鼠小胶质细胞OX-42表达水平。同时采用前肢放置试验评分、平衡实验评分及改良神经功能缺损程度评分进行神经功能评估。

结果

三组大鼠海马组织核因子κB蛋白表达水平（F=95.962，P<0.001）及小胶质细胞（F=108.853，P<0.001）比较，差异均有统计学意义。且模型组核因子κB蛋白及小胶质细胞OX-42表达水平较假手术组及治疗组明显增加（P均< 0.05）。同时，模型组大鼠术后7、14、21、28 d前肢放置试验评分[（7.0 ± 0.7）分vs.（6.3 ± 0.5）分，（5.9 ± 0.7）分vs.（5.0 ± 0.8）分，（4.9 ± 1.0）分vs.（3.8 ± 0.8）分，（3.7 ± 1.1）分vs.（2.3 ± 0.7）分；t=2.689、2.586、2.741、3.525，P=0.015、0.019、0.013、0.002]、平衡实验评分[（4.3 ± 0.7）分vs.（3.6 ± 0.7）分，（3.5 ± 1.1）分vs.（2.6 ± 0.7）分，（2.9 ± 0.9）分vs.（1.9 ± 0.7）分，（2.5 ± 0.7）分vs.（1.8 ± 0.6）分；t=2.278、2.212、2.762、2.333，P=0.035、0.040、0.013、0.031]及改良神经功能缺损程度评分[（10.1 ± 1.0）分vs.（8.7 ± 1.6）分，（8.8 ± 0.8）分vs.（7.5 ± 1.5）分，（7.3 ± 1.0）分vs.（5.6 ± 1.3）分，（5.7 ± 1.3）分vs.（4.1 ± 1.4）分；t=2.385、2.414、3.400、2.726，P=0.028、0.027、0.003、0.014]均明显高于治疗组。

结论

FTY720可显著改善颅脑损伤大鼠神经功能，其作用机制可能与FTY720的中枢炎症抑制作用有关。

关键词: 颅脑损伤, 大鼠, FTY720, 神经功能

Objective

To investigate the effect of FTY720 on neurological function in rats with traumatic brain injury and the related mechanism.

Methods

A total of 60 rats were randomly divided to the sham-operation group, model group and treatment group, 20 rats in each group. Modified Feeney free fall injury device was used to build rat craniocerebral injury model. Rats in the treatment group were administrated intraperitoneally with 1 mg/kg FTY720. Rats in the sham-operation group and model group were given 1 ml normal saline by intraperitoneal injection. After 24 hours, 10 rats in each group were sacrifice and their hippocampus tissues were used to determine the expression of nuclear factor-kappa B and microglial OX-42 by Western blotting and immunohistochemistry respectively. Additionally, the forelimb-placement test, balance test and modified neurological severity scores were used to assess the neurological function in each group.

Results

The expression of nuclear factor-kappa B (F=95.962, P<0.001) and microglial OX-42 (F=108.853, P<0.001) were all showed significant differences among the three groups, and were higher in the model group than those in the sham-operation group and treatment group (all P<0.05). Meanwhile, the forelimb-placement test [(7.0 ± 0.7) vs. (6.3 ± 0.5), (5.9 ± 0.7) vs. (5.0 ± 0.8), (4.9 ± 1.0) vs. (3.8 ± 0.8), (3.7 ± 1.1) vs. (2.3 ± 0.7); t=2.689, 2.586, 2.741, 3.525, P=0.015, 0.019, 0.013, 0.002], balance test [(4.3 ± 0.7) vs. (3.6 ± 0.7), (3.5 ± 1.1) vs. (2.6 ± 0.7), (2.9 ± 0.9) vs. (1.9 ± 0.7), (2.5 ± 0.7) vs. (1.8 ± 0.6); t=2.278, 2.212, 2.762, 2.333, P=0.035, 0.040, 0.013, 0.031] and modified neurological severity scores [(10.1 ± 1.0) vs. (8.7 ± 1.6), (8.8 ± 0.8) vs. (7.5 ± 1.5), (7.3 ± 1.0) vs. (5.6 ± 1.3), (5.7 ± 1.3) vs. (4.1 ± 1.4); t=2.385, 2.414, 3.400, 2.726, P=0.028, 0.027, 0.003, 0.014] on 7, 14, 21, 28 d after operation in the model group were higher as compared with the treatment group.

Conclusion

FTY720 could markedly improve the neurological function of rats with trauma brain injury and its mechanism might be related to the inhibition of FTY720 on central nervous system inflammation.

Key words: Craniocerebral trauma, Rat, FTY720, Neurological function

图1 FTY720对三组大鼠核因子κB蛋白表达的影响图。注：a图为各组大鼠核因子κB蛋白表达相对值的比较，与假手术组比较，^*P<0.05，与模型组比较，^#P<0.05；b图为Western-blotting检测下各组大鼠κB蛋白表达电泳图（n=10）

图2 FTY720对三组颅脑损伤大鼠海马组织小胶质细胞增生的影响图。注：a~c分别为假手术组、模型组及治疗组大鼠小胶质细胞OX-42表达情况（免疫组织化学染色× 20）

图3 FTY720对三组颅脑损伤大鼠海马组织OX-42染色阳性小胶质细胞的影响。注：与假手术组比较，^*P<0.05，与模型组比较，^#P<0.05（n=10）

表1 两组颅脑损伤大鼠不同时间点前肢放置实验评分的比较（分，±s）

组别	例数	1 d	3 d	7 d	14 d	21 d	28 d
模型组	10	8.5 ± 0.9	7.6 ± 0.8	7.0 ± 0.7	5.9 ± 0.7	4.9 ± 1.0	3.7 ± 1.1
治疗组	10	8.3 ± 0.7	7.3 ± 0.7	6.3 ± 0.5	5.0 ± 0.8	3.8 ± 0.8	2.3 ± 0.7
t值	?	0.583	0.878	2.689	2.586	2.741	3.525
P值	?	0.567	0.391	0.015	0.019	0.013	0.002

表1 两组颅脑损伤大鼠不同时间点前肢放置实验评分的比较（分，±s）

组别	例数	1 d	3 d	7 d	14 d	21 d	28 d
模型组	10	8.5 ± 0.9	7.6 ± 0.8	7.0 ± 0.7	5.9 ± 0.7	4.9 ± 1.0	3.7 ± 1.1
治疗组	10	8.3 ± 0.7	7.3 ± 0.7	6.3 ± 0.5	5.0 ± 0.8	3.8 ± 0.8	2.3 ± 0.7
t值	?	0.583	0.878	2.689	2.586	2.741	3.525
P值	?	0.567	0.391	0.015	0.019	0.013	0.002

表2 两组颅脑损伤大鼠不同时间点平衡实验评分的比较（分，±s）

组别	例数	1d	3d	7 d	14 d	21 d	28 d
模型组	10	5.4 ± 0.5	4.8 ± 0.6	4.3 ± 0.7	3.5 ± 1.1	2.9 ± 0.9	2.5 ± 0.7
治疗组	10	5.6 ± 0.7	4.3 ± 0.7	3.6 ± 0.7	2.6 ± 0.7	1.9 ± 0.7	1.8 ± 0.6
t值	?	0.728	1.709	2.278	2.212	2.762	2.333
P值	?	0.476	0.105	0.035	0.040	0.013	0.031

表2 两组颅脑损伤大鼠不同时间点平衡实验评分的比较（分，±s）

组别	例数	1d	3d	7 d	14 d	21 d	28 d
模型组	10	5.4 ± 0.5	4.8 ± 0.6	4.3 ± 0.7	3.5 ± 1.1	2.9 ± 0.9	2.5 ± 0.7
治疗组	10	5.6 ± 0.7	4.3 ± 0.7	3.6 ± 0.7	2.6 ± 0.7	1.9 ± 0.7	1.8 ± 0.6
t值	?	0.728	1.709	2.278	2.212	2.762	2.333
P值	?	0.476	0.105	0.035	0.040	0.013	0.031

表3 两组颅脑损伤大鼠不同时间点改良神经功能缺损程度评分的比较（分，±s）

组别	例数	1d	3d	7 d	14 d	21 d	28 d
模型组	10	13.8 ± 1.7	11.4 ± 1.8	10.1 ± 1.0	8.8 ± 0.8	7.3 ± 1.0	5.7 ± 1.3
治疗组	10	13.6 ± 1.7	10.9 ± 2.0	8.7 ± 1.6	7.5 ± 1.5	5.6 ± 1.3	4.1 ± 1.4
t值	?	0.263	0.578	2.385	2.414	3.400	2.726
P值	?	0.795	0.570	0.028	0.027	0.003	0.014

表3 两组颅脑损伤大鼠不同时间点改良神经功能缺损程度评分的比较（分，±s）

组别	例数	1d	3d	7 d	14 d	21 d	28 d
模型组	10	13.8 ± 1.7	11.4 ± 1.8	10.1 ± 1.0	8.8 ± 0.8	7.3 ± 1.0	5.7 ± 1.3
治疗组	10	13.6 ± 1.7	10.9 ± 2.0	8.7 ± 1.6	7.5 ± 1.5	5.6 ± 1.3	4.1 ± 1.4
t值	?	0.263	0.578	2.385	2.414	3.400	2.726
P值	?	0.795	0.570	0.028	0.027	0.003	0.014

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