整合素αvβ3抑制剂西仑吉肽在大鼠急性脑缺血中的作用机制

Efficiency of integrin αvβ3 inhibitor Cilengitide in acute cerebral ischemia in rats

目的探讨整合素αvβ3抑制剂西仑吉肽在大鼠急性脑缺血中对血脑屏障通透性、脑水肿、细胞凋亡的影响及其与血管内皮生长因子VEGF变化的关系。方法采用右侧大脑中动脉线栓法制作局灶性脑缺血再灌注模型MCAO，造模成功大鼠根据随机数字表法分为（1）治疗组A，尾静脉注射西仑吉肽100μg／kg（n=30）；（2）治疗组B，尾静脉注射西仑吉肽200μg／kg（n=28）；（3）假手术组，不置入线栓，尾静脉注射生理盐水（n=31）；（4）对照组，尾静脉注射生理盐水（n=27）。各组均在梗死后1h后治疗，2h再灌注及24h后处死。检测脑含水量、血脑屏障通透性、梗死面积、细胞凋亡、VEGF、P—Flk及Cleaved—Caspase-3等指标。结果治疗组A、治疗组B的脑含水量较对照组降低[（80．8±1．1）％比（84．8±1．4）％、（81．0±1．4）％比（84．8±1．4）％，P〈0．05]；治疗组A、治疗组B的EB含量较对照组降低[（9．2±1．1）μg／g比（12．2±0．8）μg/g、（8．6±0．6）μg/g比（12．2±0．8）μg／g，P〈0．05]；治疗组A、治疗组B的TUNEL阳性细胞数较对照组减少[（36±4）个比（69±6）个、（35±3）个比（69±6）个，P〈0．05]；治疗组A、治疗组B的梗死面积较对照组降低[（31．9±4．9）mm^3比（43．0±2．2）mm^3、（29．2±3．5）mm^3比（43．0±2．2）mm^3，P〈0．05]；与对照组比较，治疗组A、治疗组B的VEGF、P—Flk、Cleaved—Caspase-3等蛋白表达降低（P〈0．05）；治疗组A、治疗组B与假手术组比较，脑含水量、血脑屏障通透性、梗死面积及VEGF、P—Flk、Cleaved—Caspase-3蛋白表达及凋亡细胞数降低（P〈0．05）；治疗组A与治疗组B比较，脑含水量、血脑屏障通透性、梗死面积及VEGF、P-Flk、Cleaved-Caspase-3蛋白表达及凋亡细胞数差异无统计学意义（P〉0．05）。结论整合素αvβ3靶向抑制剂西仑吉肽可降低MCAO模型血脑屏障通透性，减轻脑水肿及抑制细胞凋亡，其作用机制可能与抑制VEGF介导的生物效应有关，梗死后1h后给予西仑吉肽100μg/kg及200μg/kg两个浓度治疗23h的效应未见剂量依赖关系。

Objective To explore the effect of the integrin αvβ3 inhibitor Cilengitide on the blood brain barrier （BBB） permeability, brain edema, neuronal cell apoptosis and the relation with the vascular endothelial growth factor （VEGF）expression in acute cerebral ischemia rats. Methods A rat focal cerebral ischemia/reperfusion model was established by middle cerebra] artery occlusion. Rats with middle cerebra] artery occlusion, in accordance with the random number table, were divided into four groups ： （ 1 ） the rats in Cilengitide group A （n = 30） were treated with Cilengitide at a dose of 100 μg/kg; （2） the rats in Cilengitide group B （ n = 28 ） were treated with Cilengitide at a dose of 200 μg/kg; （ 3 ） the rats in sham group （ n = 31 ） , without inserting thread into middle cerebral artery, were treated with normal saline ; （4） the rats in control group （n = 27 ） were treated with normal saline. All rats were treated with Cilengitide or saline 1 hour after infarction, given reperfusion 2 hours after infarction and were sacrificed 22 hours after reperfusion. The brain-water content was measured by dry/wet weight method. The permeability of BBB was measured by quantifying Evans Blue. The infarction volume was measured by 2,3,5-tripheyl tetrazolium Chloride （TTC） staining. Expression level of VEGF, P-Flk, Cleaved-Caspase-3 was measured by immunohistochemistry and Western blot, respectively. The neuronal cell apoptosis was evaluated by terminal deoxynucleotidyl transferased UTP nick end labeling （TUNEL）. Results Compared with Control group, treatment groups with cilengitide at the dose of 100 μg/kg and 200 μg/kg reduced brain-water content [ （ 80. 8 ± 1.1 ） % vs （ 84. 8±1.4） %, （ 81.0 ± 1.4） % vs （84. 8 ± 1.4） %, P 〈 0. 05 ], reduced exudation ofEvansblue[（9.2±1. 1） μg/gvs （12.2 ±0.8）μg/g,（8.6±0.6） μg/g vs （12.2 ±0.8） g/g,P〈 0.05 ], reduced infarction volume [ （ 31.9 ± 4. 9） mm^3 vs （43.0 ± 2. 2） mm^3, （29.2 ± 3.5 ） mm^3 vs （43.0 ± 2. 2） mm^3, P 〈 0.05 ] , reduced neuronal cell apoptosis [ （36 ± 4） vs （ 69 ± 6）, （ 35 ± 3 ） vs （ 69 ± 6 ）, P 〈 0. 05 ]. Compared with sham group, Cilengitide group A and Cilengitide group B had lower brain-water content, permeability of BBB, infarction volume, expression level of VEGF, P-Flk, Cleaved-Caspase-3 and neuronal cell apoptosis （P 〈 0.05 ）. When Cilengitide group A was compared with Cilengitide group B, there were no significant differences in brain-water content, permeability of BBB, infarction volume, expression level of VEGF, P-Flk, Cleaved-Caspase-3 and neuronal cell apoptosis （ P 〉 0. 05 ）. Conclusion The integrin αvβ3 inhibitor Cilengitide improves outcomes in the MCAO model by preserving the blood-brain barrier, attenuating brain edema and inhibiting neuronal cell apoptosis, which may occur in a VEGF-and VEGF-receptor-dependent manner, with the same efficacy between Cilengitide 100 μg/kg and 200 μg/kg after 23 hours treatment.