褪黑素对慢性脑低灌注大鼠血-脑屏障的保护作用

Protective effect of melatonin on blood-brain barrier dysfunction in rats with chronic cerebral hypoperfusion

目的探讨褪黑素对慢性脑低灌注模型大鼠血-脑屏障通透性的影响及其作用机制。方法健康雄性Sprague-Dawley(SD)大鼠通过改良双侧颈总动脉结扎法制备慢性脑低灌注模型,随机分为假手术组、模型组、褪黑素5 mg/(kg·d)组和10 mg/(kg·d)组,以伊文思蓝和异硫氰酸荧光素标记的右旋糖酐双重染色检测血-脑屏障通透性,聚合酶链反应测定基质金属蛋白酶-2和9(MMP-2和MMP-9)、Western blotting法测定Occludin蛋白和Claudin-5蛋白表达变化。结果与假手术组相比,模型组大鼠基底节荧光强度增强(P=0.000),MMP-2(P=0.000)和MMP-9(P=0.000)水平升高,Occludin蛋白(P=0.000)和Claudin-5蛋白(P=0.000)水平降低;经褪黑素5 mg/(kg·d)治疗后,大鼠基底节荧光强度减弱(P=0.021)、MMP-2水平降低(P=0.000)、Claudin-5蛋白水平升高(P=0.000),与假手术组差异仍有统计学意义(P=0.000,0.006,0.000);而褪黑素10 mg/(kg·d)治疗后,大鼠基底节荧光强度进一步减弱(P=0.000)、MMP-2(P=0.000)和MMP-9(P=0.000)水平持续降低且接近假手术组水平(均P>0.05),而Occludin蛋白(P=0.000)和Claudin-5蛋白(P=0.000)水平升高但仍低于假手术组(P=0.003,0.000);褪黑素两剂量组比较,10 mg/(kg·d)组疗效优于5 mg/(kg·d)组(均P<0.05)。结论褪黑素通过抑制基质金属蛋白酶的表达而使Occludin和Claudin-5蛋白降解减少,进而保护血-脑屏障的完整性。

Objective To investigate the mechanism of the effect of melatonin(MT)on permeability of blood-brain barrier(BBB)in chronic cerebral hypoperfusion(CCH)rats.Methods The rat model of CCH was established by bilateral common carotid artery occlusion(BCCAO).A total of 72 male Sprague-Dawley rats were randomly divided into 4 groups:sham group(N=18),BCCAO group(N=18),the melatonin[5 mg/(kg·d)]treatment model group(MT1 group,N=18),and the melatonin[10 mg/(kg·d)]treatment model group(MT2 group,N=18).Evans blue staining and fluorescein-labeled glucoside(FITC-Dextran)staining marked by fluorescein isothiocyanate(FITC)were used to assess the permeability of BBB in rats.Expression of matrix metalloproteinase-2(MMP-2)and MMP-9 mRNA levels in basal ganglia of the brain were measured by quantitative real-time polymerase chain reaction(qRT-PCR).Expression of Occludin and Claudin-5 protein in rat basal ganglia were measured by Western blotting method.Results Compared with the sham group,the fluorescence density in basal ganglia was increased(P=0.000),expression of MMP-2 and MMP-9 were increased(all P=0.000),and expression of Occludin and Claudin-5 were decreased(all P=0.000)of BCCAO group.Compared with the BCCAO group,the fluorescence density in basal ganglia was decreased(P=0.021),the expression of MMP-2 was decreased(P=0.000)and the expression of Claudin-5 was increased(P=0.000)in MT1 group,and the differences with sham group were statistically significant(P=0.000,0.006,0.000).Compared with the BCCAO group,in MT2 group,the fluorescence degree in basal ganglia was further decreased(P=0.000),the expression of MMP-2 and MMP-9 were decreased(all P=0.000),and were similar to the sham group(all P>0.05),while expression of Occludin and Claudin-5 were increased(all P=0.000)but lower than those of sham group(P=0.003,0.000).Compared with the two melatonin treatment groups,the efficacy of the 10 mg/(kg·d)group(MT2 group)was better than that of the 5 mg/(kg·d)group(MT1 group,all P<0.05).Conclusions Melatonin may protect the integrity of BBB by inhibiting the degradation of Occludin and Claudin-5 protein through inhibiting the expression of matrix metalloproteinases.