AQP4在右美托咪定降低机械通气小鼠血脑屏障通透性中的作用:与PKC的关系

Role of AQP4 in dexmedetomidine-induced reduction of blood-brain barrier permeability in mechanically ventilated mice: relationship with PKC

目的评价水通道蛋白4(AQP4)在右美托咪定降低机械通气小鼠血脑屏障通透性中的作用及其与蛋白激酶C(PKC)的关系。方法清洁级健康雄性C57BL6小鼠150只,体质量20~25 g,8~12周龄,采用随机数字表法分为5组(n=30):对照组(C组)、机械通气组(V组)、LY317615组(L组)、右美托咪定组(D组)和右美托咪定+PMA组(DP组)。C组自主呼吸空气6 h;V组机械通气6 h;L组在机械通气前30 min时腹腔注射PKC抑制剂LY31761510μg/kg;D组和DP组机械通气前30 min时腹腔注射右美托咪定50μg/kg;DP组机械通气前60 min时腹腔注射PKC激活剂PMA 15μg/kg。机械通气后1 d时麻醉小鼠,处死取海马组织,光镜下观察CA1和CA3区组织病理学结果;取小鼠脑组织,测定脑组织含水量,检测脑组织伊文氏蓝(EB)含量;取小鼠脑组织,采用Western blot法检测PKC和AQP4的表达。机械通气后3 d,行新物体识别实验评估小鼠认知功能。结果与C组比较,V组和DP组机械通气后脑组织含水量和EB含量升高,脑组织PKC和AQP4表达上调,新物体探索百分比和辨别指数降低(P<0.05),海马CA1区和CA3区组织病理学损伤加重;与V组比较,D组和L组机械通气后脑组织含水量和EB含量降低,脑组织PKC和AQP4表达下调,新物体探索百分比和辨别指数增高(P<0.05),海马CA1区和CA3区组织病理学损伤减轻;与D组比较,DP组机械通气后脑组织含水量和EB含量升高,脑组织PKC和AQP4表达上调,新物体探索百分比和辨别指数降低(P<0.05),海马CA1区和CA3区组织病理学损伤加重。结论AQP4参与了右美托咪定降低机械通气小鼠血脑屏障通透性的过程,其机制与抑制PKC激活有关。

Objective To evaluate the role of aquaporin 4(AQP4)in dexmedetomidine-induced reduction of blood-brain barrier permeability in mechanically ventilated mice and the relationship with protein kinase C(PKC).Methods One hundred and fifty clean-grade healthy male C57BL6 mice,weighing 20-25 g,aged 8-12 weeks,were divided into 5 groups(n=30 each)using a random number table method:control group(group C),mechanical ventilation group(group V),LY317615 group(group L),dexmedetomidine group(group D),and dexmedetomidine+PMA group(group DP).Group C spontaneously breathed air for 6 h.The animals were mechanically ventilated for 6 h in group V.PKC inhibitor LY317615μg/kg was intraperitoneally injected at 30 min before mechanical ventilation in group L.Dexmedetomidine 50μg/kg was intraperitoneally injected at 30 min before mechanical ventilation in D and DP groups.PKC activator PMA 15μg/kg was intraperitoneally injected at 60 min before mechanical ventilation in group DP.Mice were anesthetized at 1 day after mechanical ventilation,then sacrificed and hippocampal tissues were taken for microscopic examination of pathological changes in the hippocampal CA1 and CA3 areas(with a light microscope).Brain tissues were also taken to measure the water content and content of Evans blue(EB)and to detect the expression of PKC and AQP4(by Western blot).The cognitive function was evaluated using a novel object recognition task at 3 days after mechanical ventilation.Results Compared with group C,the water content and EB content of brain tissues were significantly increased after mechanical ventilation,the expression of PKC and AQP4 in brain tissues was up-regulated,the percentage of novel object exploration and discrimination index were decreased(P<0.05),and the histopathological damage in the hippocampal CA1 and CA3 areas was aggravated in group V and group DP.Compared with group V,the water content and EB content of brain tissues were significantly decreased after mechanical ventilation,the expression of PKC and AQP4 in brain tissues was down-regulated,the percentage of novel object exploration and discrimination index were increased(P<0.05),and the histopathological damage in the hippocampal CA1 and CA3 areas was significantly attenuated in group D and group L.Compared with group D,the water content and EB content of brain tissues were significantly increased after mechanical ventilation,the expression of PKC and AQP4 in brain tissues was up-regulated,the percentage of novel object exploration and discrimination index were decreased(P<0.05),and the histopathological damage in the hippocampal CA1 and CA3 areas was aggravated in group DP.Conclusions AQP4 is involved in dexmedetomidine-induced reduction of blood-brain barrier permeability in mechanically ventilated mice,and the mechanism is related to inhibiting activation of PKC.