基于线粒体MCU通道探究甲基莲心碱促血管再生抗脑缺血的作用机制  

Exploring Mechanism of Neferine in Promoting Vascular Regeneration Against Cerebral Ischemia Based on Mitochondrial MCU Channel

作　　者：张琪曼 高艳华 吴文洁 杨维[1] 刘陈 李淑婷 蔡冰洁 杨家霖 张滢 马菁 李韶菁[1] ZHANG Qiman;GAO Yanhua;WU Wenjie;YANG Wei;LIU Chen;LI Shuting;CAI Bingjie;YANG Jialin;ZHANG Ying;MA Jing;LI Shaojing(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Anhui University of Chinese Medicine,Hefei 230012,China;Beijing City University,Beijing 100191,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]安徽中医药大学,合肥230012 [3]北京城市学院,北京100191

出　　处：《中国实验方剂学杂志》2024年第24期103-113,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家“重大新药创制”科技重大专项(2019ZX09201-004);中国中医科学院科技创新工程项目(CI2021A02001);中国中医科学院新入职青年科研人员培养专项(ZZ17-XRZ-088)。

摘　　要：目的:探究甲基莲心碱(Nef)通过调控线粒体钙单向转运体(MCU)离子通道促进血管再生,进而抗脑缺血的作用机制。方法:以微血管缺失斑马鱼肠下血管面积为指标,对Nef进行血管再生功效评价,并计算半数有效浓度(EC50)。将大鼠随机分为假手术组、模型组、阳性药组(丁苯酞,6 mg·kg^(-1))、Nef低、中、高剂量组(0.125、0.625、3.125μg·kg^(-1)),除假手术组外,其余各组建立大鼠大脑中动脉栓塞(MCAO)模型,造模完成后各组按相应剂量灌胃给药,假手术组、模型组灌胃给予等体积生理盐水,1次/d,连续7 d。对各组大鼠进行神经行为学评分,并通过2,3,5-氯化三苯基四氮唑(TTC)染色计算缺血侧脑组织梗死率;散斑血流成像系统测定各组大鼠局部脑血流量(rCBF);免疫荧光及蛋白免疫印迹法(Western blot)检测各组大鼠血管内皮生长因子(VEGF)、血小板内皮细胞黏附分子-1(CD31)、缺氧诱导因子-1α(HIF-1α)蛋白表达。将人脐静脉内皮细胞(HUVECs)分为正常组、模型组、阳性药(黄芪甲苷,10μmol·L^(-1))组、Nef组(32 nmol·L^(-1));在验证Nef线粒体保护作用及促血管再生机制时,增设精胺组(MCU激动剂)、Nef+精胺组,除正常组外,其余各组建立糖氧剥夺(OGD)的HUVECs模型,通过噻唑蓝(MTT)法检测细胞活力,划痕实验与管成实验评估细胞迁移能力,应用Rhod-2 AM、Fluo-3 AM、JC-1、Calcein AM荧光探针,以及细胞能量代谢分析仪分析Nef的线粒体保护作用。通过分子对接预测Nef与MCU、HIF-1α的结合能力,采用Western blot检测Nef对OGD模型HUVECs中MCU、B细胞淋巴瘤-2相关X蛋白(Bax)、胱天蛋白酶-3(Caspase-3)和HIF-1α蛋白表达的影响。结果:对微血管缺失斑马鱼的血管生成结果显示,与正常组比较,模型组的肠下血管面积显著下降(P<0.01);与模型组比较,不同浓度Nef组肠下血管面积显著提高(P<0.01),最大耐受浓度为10.24μmol·L^(-1),EC50为0.23μmol·L^(-1)。对MCAO大Objective:To investigate the mechanism of neferine(Nef)in promoting vascular regeneration against cerebral ischemia through modulation of mitochondrial calcium uniporter(MCU)ion channel.Method:Taking the area of subintestinal vessels in microvascular deficiency zebrafish as an index,the vascular regenerative efficacy of Nef was evaluated,and the median effective concentration(EC50)was calculated.Rats were randomly divided into a sham operation group,a model group,a positive drug group(butylphthalide,6 mg·kg^(-1)),and Nef low,medium,and high dose groups(0.125,0.625,3.125μg·kg^(-1)).Except for the sham operation group,the middle cerebral artery occlusion(MCAO)model was established in other groups.After modeling,the groups were administered the corresponding dose of drugs by gavage,while the sham operation and model groups received equal volumes of saline,once a day for 7 consecutive days.Neurobehavioral scores were assessed for each group of rats,and the infarct rate of ischemic brain tissue was calculated by 2,3,5-triphenyltetrazolium chloride(TTC)staining.The regional cerebral blood flow(rCBF)of each group was measured using a speckle contrast imaging.Immunofluorescence and Western blot were conducted to detect the expression of vascular endothelial growth factor(VEGF),platelet endothelial cell adhesion molecule-1(CD31),and hypoxia-inducible factor-1α(HIF-1α)proteins in each group.Human umbilical vein endothelial cells(HUVECs)were divided into the normal group,model group,positive drug group(astragalosideⅣ,10μmol·L^(-1)),and Nef group(32 nmol·L^(-1)).In the verification of mitochondrial protection of Nef and its mechanism in promoting vascular regeneration,the spermine(MCU agonist)and Nef+spermine group were added.HUVECs model of oxygen-glucose deprivation(OGD)was established in all groups except the normal group,the cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,and cell migration ability was evaluated through scratch and tube formation assays.F

关 键 词：脑缺血 甲基莲心碱 斑马鱼 血管再生 线粒体钙单向转运体(MCU) 缺氧诱导因子-1 线粒体 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R743R654.3