Abstract:

Objective To investigate the mechanism of the effect of melatonin (MT) on permeability of blood-brainbarrier (BBB) in chronic cerebral hypoperfusion (CCH) rats. Methods Therat 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 o fBBB 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 10mg/(kg·d) group (MT2 group) was better thanthat of the 5mg/(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.

Key words: Cerebral small vessel diseases, Melatonin, Blood-brain barrier, Disease models, animal

摘要：

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5mg/（kg·d）组和10mg/（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）、 Westernblotting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5mg/（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10mg/（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，10mg/（kg·d）组疗效优于5mg/（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。

关键词: 大脑小血管疾病, 褪黑激素, 血脑屏障, 疾病模型, 动物