摘要
目的 探讨泽泻醇A(alisol A, AA)通过抑制基质金属蛋白酶9(matrix metalloproteinase 9, MMP-9)改善皮层血脑屏障(blood brain barrier, BBB)障碍介导的脑缺血/再灌注损伤(cerebral ischemia-reperfusion injury, CIRI)。方法 构建小鼠全脑缺血/再灌注(global cerebral ischemia-reperfusion, GCI/R)体内动物模型,AA灌胃干预7 d(30 mg·kg^(-1)),改良神经功能缺损评分(modified neurological severity score, mNSS)、旷场和Y迷宫实验检测神经功能,磁共振波谱(magnetic resonance spectroscopy, MRS)检测皮层相关代谢物质水平,透射电镜(transmission electron microscope, TEM)观察皮层BBB超微结构,蛋白免疫印迹法和免疫组织化学检测皮层MMP-9蛋白表达,分子对接分析AA与MMP-9结合的可能性。结果 相较于假手术组(Sham), GCI/R组小鼠mNSS评分升高、旷场总路程和中心路程占总路程比值均减少、Y迷宫交替率降低(P<0.01),而AA干预组(GCI/R+AA)较GCI/R组小鼠mNSS评分降低、旷场总路程和中心路程占总路程比值增加、Y迷宫交替率增加(P<0.01)。MRS检测发现GCI/R组小鼠大脑皮层γ-氨基丁酸、N-乙酰天门冬氨酸表达降低,胆碱、肌醇和牛磺酸表达升高(P<0.01);GCI/R+AA组小鼠γ-氨基丁酸、N-乙酰天门冬氨酸表达升高,胆碱、肌醇和牛磺酸表达降低(P<0.01)。TEM发现GCI/R组小鼠脑微血管基膜塌陷、管腔变窄;内皮细胞被激活、质膜褶皱且间隙变大,紧密连接破坏;星形胶质细胞终足肿胀。GCI/R+AA组小鼠血管管腔充盈;内皮细胞质膜平整,紧密连接完好;星形胶质细胞终足相对正常。蛋白免疫印迹和免疫组织化学检测发现GCI/R组小鼠皮层MMP-9表达水平明显升高(P<0.01),GCI/R+AA组明显降低(P<0.05)。分子对接发现AA与MMP-9可通过TYR-50和ARG-106两个基团结合,结合能为-6.24 kcal·mol^(-1)。结论 AA治疗可改善GCI/R小鼠皮层CIRI,其机制可能是通过抑制MMP-9升高造成BBB损伤。
Aim To investigate whether alisol A(AA)could improve the blood brain barrier(BBB)mediated cortex cerebral ischemia-reperfusion injury(CIRI)by inhibiting matrix metalloproteinase 9(MMP-9).Methods The global cerebral ischemia-reperfusion(GCI/R)model in mice was established,and the AA was intragastric injected subsequently for seven days.The modified neurological severity scores(mNSS),open field test and Y-maze test were applied to detect neurological function.Magnetic resonance spectroscopy(MRS)was used to detect relevant neurosubstance metabolism in cortex of mice.Transmission electron microscope(TEM)was employed to observe the ultrastructure of BBB in cortex.Western blot and immunohistochemistry were used to detect the MMP-9 level in cortex.The binding possibility of AA and MMP-9 was determined by molecular docking.Results Compared with Sham group,mice in GCI/R group have an increased mNSS score but decreased at total distance and center distance to total distance ratio in open field test as well as alternation rate in Y-maze test(P<0.01).While mice in GCI/R+AA group have a decreased mNSS score but increased at total distance and center distance to total distance ratio in open field test as well as alternation rate in Y-maze test(P<0.01)compared with GCI/R group.MRS results found that in cortex of GCI/R group mice,the level of GABA and NAA significantly decreased while the Cho,mI and Tau level increased(P<0.01).Whereas in GCI/R+AA group mice,the GABA and NAA level increased and the Cho,mI and Tau decreased significantly(P<0.01).By TEM we observed that the basilemma of cerebral microvessels collapsed,the lumen narrowed,the endothelial cells were active and plasma membranes ruffled,gaps between cells were enlarged and tight junctions were damaged and the end feet of astrocytes were swollen in GCI/R group mice.While in GCI/R+AA group mice,the lumen was filled,plasma membranes of endothelial cells were smooth,tight junctions were complete and end feet of astrocytes were in normal condition.Western blot and immunohistochemistry both found that the MMP-9 level increased in GCI/R group mice(P<0.01)and decreased in GCI/R+AA group mice(P<0.05).Molecular docking proved the binding between aliso A and MMP9 through TYR-50 and ARG-106,and the binding energy was calculated as-6.24 kcal·mol^(-1).Conclusions The AA could improve CIRI in cortex of mice,and the potential mechanism might be inhibiting the BBB damage caused by increasing MMP-9 level.
作者
邓云飞
李惠红
魏伟
周阳杰
薛偕华
DENG Yun-fei;LI Hui-hong;WEI Wei;ZHOU Yang-jie;XUE Xie-hua(College of Rehabilitation Medicine.Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;the Affiliated Rehabilitation Hospital,Fujian University of Traditional Chinese Medicine,Fuzhou 350003,China;Fujian Key Laboratory of Rehabilitation Technology,Fuzhou 350122,China;Fujian Key Laboratory of Cognitive Rehabilitation,Fuzhou 350003,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2024年第1期83-90,共8页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 82274620)
福建省卫健委科技计划项目(No 2021zyyj69)
福建省自然科学基金资助项目(No 2021J01957)。
关键词
脑缺血/再灌注损伤
基质金属蛋白酶9
泽泻醇A
血脑屏障
神经保护
分子对接
cerebral ischemia reperfusion injury
matrix metalloproteinase 9
alisol A
blood brain barrier
neuroprotection
molecular docking
