24-乙酰泽泻醇A通过miR-98-5p/TRPM2改善脑微血管内皮细胞缺血/再灌注损伤  

Alisol A 24-acetate ameliorates cerebral ischemia reperfusion injury in brain microvascular endothelial cells via miR-98-5p/TRPM2

作　　者：魏伟 李惠红 徐沛韬 陶大梅 邓云飞 詹增土 WEI Wei;LI Hui-hong;XU Pei-tao;TAO Da-mei;DENG Yun-fei;ZHAN Zeng-tu(The Affiliated Rehabilitation of Fujian University of Traditional Chinese Medicine,Fuzhou 350003,China;Key Laboratory of Cognitive Rehabilitation of Fujian Province,Fuzhou 350003,China;College of Rehabilitation Medical,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)

机构地区：[1]福建中医药大学附属康复医院,福建福州350003 [2]福建省认知功能康复重点实验室,福建福州350003 [3]福建中医药大学康复医学院,福建福州350122

出　　处：《中国药理学通报》2025年第4期695-702,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No.82004436);福建省卫生健康科研项目医学创新课题(No.2021CXA041);福建省教育厅中青年教师教育科研项目(No.JAT90269);福建中医药大学2023年福建省认知功能康复重点实验室开放课题(No.XKF2023005);国家中医药管理局高水平中医药重点学科建设项目中医康复学(No.zyyzdxk-2023102)。

摘　　要：目的探讨24-乙酰泽泻醇A(Alisol A 24-acetate,24A)改善脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)缺血/再灌注损伤分子机制及其与miR-98-5p/瞬时受体电位离子通道-2(TRPM2)的相关性。方法bEnd.3细胞OGD 8 h/R 16 h构建离体BMECs缺血/再灌注损伤模型,miR-98-5p mimics转染后18.77μmol·L-124A干预24 h,分为对照、OGD/R、OGD/R+24A、OGD/R+24A+miR-98-5p mimics及OGD/R+miR-98-5p mimics组;qPCR检测miR-98-5p和TRPM2 mRNA水平;ELISA检测IL-1β、TNF-α水平;Western blot检测TRPM2、p-AKT、p-GSK3β、AKT、GSK3β、Bcl-2、Bax、ZO-1、Occludin、Claudin-5蛋白表达水平;流式细胞术检测凋亡及活性氧(ROS)水平;双荧光素酶验证miR-98-5p与TRPM2靶向关系。结果OGD/R组比对照组凋亡明显、Bcl-2/Bax降低,ZO-1、Occludin、Claudin-5减少,IL-1β、TNF-α和ROS增加,miR-98-5p、p-AKT/AKT、p-GSK3β/GSK3β降低但TRPM2增加;但与OGD/R组相比,除对照组外其余三组在以上方面表现出了相反趋势;与OGD/R+24A组相比,OGD/R+24A+miR-98-5p mimics组凋亡减轻,ZO-1、Occludin、Claudin-5降解减少,炎症和ROS减轻,miR-98-5p、p-AKT/AKT、p-GSK3β/GSK3β增加且TRPM2降低;但与OGD/R+24A+miR-98-5p mimics组相比,OGD/R+miR-98-5p mimics组则逆转了这种趋势。双荧光素酶证实miR-98-5p靶向调控TRPM2。结论24A通过miR-98-5p抑制BMECs内TRPM2表达,调控AKT/GSK3β信号通路,减少OGD/R炎症及氧化应激介导的细胞凋亡,阻止ZO-1、Occludin及Claudin-5降解,改善血脑屏障(blood brain barrier,BBB)渗透性。Aim To explore the underlying molecular mechanism of Alisol A 24-acetate(24A)in improving oxygen-glucose deprivation/reoxygenation(OGD/R)injury in brain microvascular endothelial cells(BMECs)and its correlation with miR-98-5p/transient receptor potential melastatin-2(TRPM2).Methods The ischemia-reperfusion injury in brain microvascular endothelial cells(BMECs)was established using bEnd.3 cells subjected to 8 h of oxygen-glucose deprivation followed by 16 h of re-oxygenation.The cells were intervened by miR-98-5p mimics and/or 18.77μmol·L-124A for 24 h and divided into the control group,OGD/R group,OGD/R+24A group,OGD/R+24A+miR-98-5p mimics group and OGD/R+miR-98-5p mimics group.The mRNA levels of miR-98-5p and TRPM2 were detected by qPCR.IL-1βand TNF-αlevels were detected by ELISA.The expression levels of TRPM2,p-AKT,p-GSK3β,AKT,GSK3β,Bcl-2,Bax,ZO-1,Occludin,Claudin-5 were detected by Western blot.Apoptosis and reactive oxygen species(ROS)levels were detected by flow cytometry.The targeting relationship between miR-98-5p and TRPM2 was verified using dual luciferase assay.Results Compared with the control group,the apoptosis of OGD/R group was obvious,Bcl-2/Bax decreased,ZO-1,Occludin,Claudin-5 decreased,IL-1β,TNF-αand ROS increased,miR-98-5p,p-AKT/AKT,p-GSK3β/GSK3βdecreased but TRPM2 increased.But compared with the OGD/R group,except the control group,the other three groups showed the opposite trend in the above aspects;compared with the OGD/R+24A group,OGD/R+24A+miR-98-5p mimics group showed decreased apoptosis,decreased degradation of ZO-1,Occludin and Claudin-5,and decreased inflammation and ROS.miR-98-5p,p-AKT/AKT,p-GSK3β/GSK3βincreased and TRPM2 decreased.However,compared with the OGD/R+24A+miR-98-5p mimics group,the OGD/R+miR-98-5p mimics group reversed this trend.Dual luciferase confirmed that miR-98-5p targeted regulation of TRPM2.Conclusion 24A inhibits the expression of TRPM2 in BMECs through miR-98-5p,regulates AKT/GSK3βsignal pathway,reduces OGD/R inflammation and oxidative stress-mediate

关 键 词：24-乙酰泽泻醇A 脑缺血/再灌注损伤 脑微血管内皮细胞 miR-98-5p TRPM2 

分 类 号：R-332[医药卫生] R284.1R322.81R329.24R342.2R743.31