摘要
目的探讨基质金属蛋白酶(MMP)抑制剂伊洛马司他(GM6001)对癫痫模型大鼠学习记忆功能及海马髓鞘相关蛋白的影响。方法30只SPF级健康8周龄雄性SD大鼠采用随机数字表法分为对照组、干预对照组、短时模型组、长时模型组、长时干预组,每组6只。采用氯化锂-匹罗卡品注射法诱导大鼠癫痫持续状态(SE)发作,短时模型组、长时模型组与长时干预组分别于SE发作15 min、30 min、30 min予以腹腔注射地西泮(10 mg/kg)终止发作,干预对照组及长时干预组均予以尾静脉注射GM6001(20μg/kg)。Morris水迷宫实验评估各组大鼠学习记忆能力,伊文思蓝(EB)染料检测血脑屏障通透性,ELISA及Western blot法检测血清MMP、海马髓鞘碱性蛋白(MBP)及2',3'-环核苷酸3'-磷酸二酯酶(CNPase)蛋白表达水平。采用SPSS 21.0进行统计分析,水迷宫数据多组间比较采用重复测量方差分析,其他数据多组间比较采用单因素方差分析,进一步两两比较采用LSD检验。结果(1)水迷宫实验中5组大鼠到达平台潜伏期的时间和组别交互作用不显著(F_(交互)=0.430,P_(交互)=0.970),时间主效应及组别主效应均显著(F_(时间)=144.521,F_(组别)=12.819,均P<0.001),第2~5天长时模型组大鼠到达平台潜伏期均高于对照组及长时干预组(均P<0.05)。各组大鼠首次到达原平台时间、目标象限停留时间、穿越平台次数、距原平台中心距离均差异有统计学意义(F=7.008,7.306,15.704,11.559,均P<0.05)。长时干预组首次到达原平台时间[(13.71±4.41)s]及距原平台中心距离[(60.37±8.59)cm]均低于长时模型组[(23.52±5.34)s,(73.06±12.01)cm](均P<0.05),目标象限停留时间[(10.93±2.99)s]及穿越平台次数[2.00(1.75,2.75)次]均高于长时模型组[(6.04±4.33)s,1.00(0,1.00)次](均P<0.05)。(2)5组大鼠海马区域EB荧光强度及血清MMP-2、MMP-9水平均差异有统计学意义(F=15.249,7.951,33.299,均P<0.001)。长时模型组EB荧光强度[(0.18±0.03)/100μm^(2)]高于对照组[(0.09±0.02)/100μm^(2)]及长时干预组[(0.12±0.04)/100μm^(2)](均P<0.05)。长时模型组MMP-2及MMP-9水平[(10.91±1.36)ng/mL,(10.96±1.28)ng/mL]均高于对照组[(7.89±1.04)ng/mL,(4.20±1.48)ng/mL]及长时干预组[(6.49±1.12)ng/mL,(8.06±0.91)ng/mL](均P<0.05)。(3)5组大鼠海马组织MBP、CNPase蛋白表达水平均差异有统计学意义(F=8.757,4.116,均P<0.05)。长时模型组MBP、CNPase蛋白表达水平[(1.00±0.19),(0.93±0.21)]均低于对照组[(1.57±0.27),(1.36±0.22)]及长时干预组[(1.45±0.19),(1.23±0.26)](均P<0.05)。结论MMP抑制剂GM6001能有效改善癫痫模型大鼠学习记忆功能障碍,这可能与改善血脑屏障渗漏、减轻海马区域髓鞘损伤有关。
Objective To investigate the effects of a matrix metalloproteinase(MMP)inhibitor(ilomastat,GM6001)on learning and memory function and hippocampal myelin-related proteins in epileptic model rats.Methods Thirty 8-week-old male SPF Sprague-Dawley rats were randomly divided into control group,intervention control group,short-term model group,long-term model group and long-term intervention group,with 6 rats in each group.The lithium-pilocarpine method was used to induce status epilepticus(SE).SE was terminated respectively by intraperitoneal injection of diazepam(10 mg/kg)at 15 min in short-term model group and at 30 min both in the long-term model group and long-term intervention group.Rats in intervention control group and long-term intervention group were treated with GM6001(20μg/kg)via tail vein injection.Morris water maze test was used to assess the learning and memory function and the blood-brain barrier permeability was evaluated by Evans blue(EB)staining.The levels of serum MMP,the hippocampal myelin basic protein(MBP)and 2',3'-cyclic nucleotide 3'-phosphodiesterase(CNPase)were measured by ELISA and Western blot.Statistical analysis was performed by SPSS 21.0 software,with repeated measures ANOVA for Morris water maze data and one-way ANOVA with LSD post hoc test for other comparisons.Results(1)In the water maze test,the interaction between the time and group on the latencies reached to the platform showed no significance among the 5 groups of rats(F=0.430,P=0.970).The main effects of time and group on latencies were both significant among the 5 groups(F=144.521,12.819,both P<0.001).From the second day to the fifth day,the latencies reached to the platform of rats in the long-term model group were significantly higher than those in the control group and in the long-term intervention group(both P<0.05).There were statistically significant differences in the latency of the first time reached the platform,the duration in the target quadrant,the frequency of crossing the platform and the distance to the platform among the 5 groups(F=7.008,7.306,15.704,11.559,all P<0.05).In the long-term intervention group,the latency of the first time reached the platform((13.71±4.41)s)and the distance to the platform((60.37±8.59)cm)were significantly lower than those in the long-term model group((23.52±5.34)s,(73.06±12.01)cm)(both P<0.05),and the duration in the target quadrant((10.93±2.99)s)and the frequency of crossing the platform(2.00(1.75,2.75)times)were significantly higher than those of the long-term model group((6.04±4.33)s,1.00(0,1.00)times)(both P<0.05).(2)The hippocampal EB fluorescence intensity and the serum levels of MMP-2 and MMP-9 were significantly different among the 5 groups(F=15.249,7.951,33.299,all P<0.001).The EB fluorescence intensity of the long-term model group((0.18±0.03)/100μm^(2))was significantly higher than that in control group((0.09±0.02)/100μm^(2))and the long-term intervention group((0.12±0.04)/100μm^(2))(both P<0.05).The serum MMP-2 and MMP-9 levels in the long-term model group((10.91±1.36)ng/mL,(10.96±1.28)ng/mL)were significantly higher than those of the control group((7.89±1.04)ng/mL,(4.20±1.48)ng/mL)and the long-term intervention group((6.49±1.12)ng/mL,(8.06±0.91)ng/mL)(all P<0.05).(3)The expression levels of hippocampal MBP and CNPase were significantly different among the 5 groups(F=8.757,4.116,both P<0.05).The expression levels of MBP and CNPase in long-term model group((1.00±0.19),(0.93±0.21))were significantly lower than those in the control group((1.57±0.27),(1.36±0.22))and the long-term intervention group((1.45±0.19),(1.23±0.26))(all P<0.05).Conclusion The MMP inhibitor GM6001 can ameliorate learning and memory deficits in epileptic model rats,potentially by reducing blood-brain barrier permeability and alleviating hippocampal myelin damage.
作者
肖扬
赵亚楠
Xiao Yang;Zhao Yanan(Department of Neurology,Zhongshan Hospital Affiliated to Fudan University,Shanghai 200032,China;Department of Neurology,Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University,Shanghai 201700,China)
出处
《中华行为医学与脑科学杂志》
北大核心
2025年第6期481-487,共7页
Chinese Journal of Behavioral Medicine and Brain Science
基金
上海市青浦区卫生健康委员会科研课题(QWJ2022-12)
复旦大学附属中山医院青浦分院科研课题(QY2021-01)。
关键词
癫痫
认知功能
髓鞘
血脑屏障
基质金属蛋白酶
大鼠
Epilepsy
Cognitive function
Myelin sheath
Blood-brain barrier
Matrix metalloproteinase
Rat
