褪黑素对胆红素脑病大鼠脑顶叶皮质AQP4的调控及其保护机制研究  

Melatonin down-regulates cerebral parietal cortex aquaporin 4 and induces neuroprotection through mTOR/PKC signaling in bilirubin encephalopathy rats

作　　者：张茗越 周龙洋[1] 蒲淞 崔爱洁 张丽荣 陈春燕 薛凯歌 樊萍[3] 甘胜伟[1,2] ZHANG Mingyue;ZHOU Longyang;PU Song;CUI Aijie;ZHANG Lirong;CHEN Chunyan;XUE Kaige;FAN Ping;GAN Shengwei(National Demonstration Center for Experimental Basic Medicine Education,Chongqing Medical University,Chongqing 400016,China;Institute of Neurosciences,School of Basic Medical Science,Chongqing Medical University,Chongqing 400016,China;Department of Gynecology and Obstetrics,The Fifth People's Hospital of Chongqing,Chongqing 400062,China)

机构地区：[1]重庆医科大学基础医学国家级实验教学示范中心,重庆400016 [2]重庆医科大学基础医学院神经科学研究中心,重庆400016 [3]重庆市第五人民医院妇产科,重庆400062

出　　处：《中国病理生理杂志》2023年第7期1199-1208,共10页Chinese Journal of Pathophysiology

基　　金：重庆市卫生计生委2015年科研计划项目(No.2015MSXM109);中华医学会医学教育分会立项课题(No.2020B-N13332);国家级大学生创新创业训练计划项目、重庆医科大学大学生科学研究与创新实验项目(No.SRIEP202004);重庆市2022年创新人才工程项目(No.CY220408)。

摘　　要：目的:研究脑顶叶皮质水通道蛋白4(aquaporin 4,AQP4)在褪黑素(melatonin,MT)治疗胆红素脑病(bilirubin encephalopathy,BE)中的表达变化,以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)/蛋白激酶C(protein kinase C,PKC)信号通路在MT对AQP4调控中的作用,以探讨MT对BE的治疗机制。方法:80只出生5~7 d的健康SD乳鼠,根据其处理方式,随机分为以下5组:假手术组、BE模型组、MT干预组、PKC抑制剂(PKC inhibitor,PKCi)干预组和mTOR抑制剂(mTOR inhibitor,mTORi)干预组。采用HE染色和尼氏染色检测各组鼠脑皮质病理改变;采用干-湿重法测定各组脑顶叶皮质含水量;应用免疫荧光染色法检测脑皮质AQP4的表达区域;运用TUNEL染色法检测脑顶叶皮质神经细胞的凋亡变化;使用Western blot法测定各组鼠脑顶叶皮质AQP4、caspase-3、cleaved caspase-3和PKCε的表达量。结果:HE和尼氏染色显示,MT可减轻胆红素所致神经细胞的损伤。干-湿重法结果显示,MT可降低BE乳鼠脑顶叶皮质脑含水量。免疫荧光染色显示,BE组AQP4荧光强度增加,主要表达于大脑顶叶皮质血管壁和星形胶质细胞膜;MT干预可阻止BE所致AQP4表达的增加。TUNEL染色显示,MT可减少BE所致细胞凋亡。Western blot显示,与假手术组相比,BE组脑顶叶皮质AQP4、cleaved caspase-3和caspase-3表达增加,PKCε表达降低;MT干预可降低BE鼠顶叶皮质AQP4、cleaved caspase-3和caspase-3表达,增加PKCε表达。PKCi和mTORi均可下调PKCε的表达,阻止MT通过mTOR/PKC信号通路降低AQP4的表达。结论:MT可通过减少BE大鼠脑顶叶皮质中AQP4的表达来缓解脑水肿,并减少caspase-3介导的神经细胞凋亡。MT下调AQP4表达的机制与mTOR/PKC信号通路的激活有关。AIM:To investigate the changes of cerebral parietal cortical aquaporin 4(AQP4)and neural apoptosis induced by melatonin(MT)in bilirubin encephalopathy(BE),and to determine the role of the mammalian target of the rapamycin(mTOR)/protein kinase C(PKC)signaling pathway in the therapeutic mechanism of MT for BE.METHODS:Eighty 7-day-old Sprague-Dawley(SD)rats were randomly divided into sham group,BE group,MT group,PKC inhibitor(PKCi)group,and mTOR inhibitor(mTORi)group.The BE model was established in neonatal SD rats by injecting bilirubin solution into the cisterna magna.In the mTORi group,the mTOR inhibitor solution was then injected intraperitoneally 1 h before modeling,and the MT solution was injected intraperitoneally 30 min before modeling.At 24 h after modeling,the brains of neonatal SD rats were removed,and hematoxylin-eosin(HE)and Nissl staining were used to detect cortical pathological changes in each group.The water content of the cerebral cortex was measured using the wet-dry weight method,and the expression level of AQP4 in cerebral cortex was detected by immunofluorescence staining.TUNEL staining was used to detect the neuronal apoptosis,and the expression levels of AQP4,caspase-3,cleaved caspase-3 and PKCεwere detected by Western blot.RESULTS:Compared with BE group,HE and Nissl staining showed that MT alleviated unconjugated bilirubin(UCB)-induced neural damage.The wet-dry weight method showed that the water content of the cerebral parietal cortical area was decreased after treatment with MT.Immunofluorescence staining showed that AQP4 was mainly expressed along the vessels on the end feet membrane of astrocytes,and MT prevented increased AQP4 levels induced by UCB.TUNEL staining showed that MT alleviated the cerebral parietal cortical neuronal apoptosis caused by BE.Western blot showed that in comparison with sham group,AQP4 and cleaved caspase-3 expression levels were increased,and PKC expression was decreased in the parietal cortex of the rats in BE group.MT intervention reduced the increased expres

关 键 词：胆红素脑病 褪黑素 水通道蛋白4 mTOR/PKC信号通路 

分 类 号：R363.13[医药卫生—病理学] R741[医药卫生—基础医学]