羟基红花黄色素A对糖氧剥夺后bEnd.3细胞自噬作用的影响  

Effects of hydroxysafflor yellow A on autophagy in bEnd.3 cells after oxygen-glucose deprivation

作　　者：戴瑶瑶 舒梦琦 魏汝恒 苗珠月 丁智斌 马东[3] 黄建军[3] 宋丽娟 马存根 DAI Yao-yao;SHU Meng-qi;WEI Ru-heng;MIAO Zhu-yue;DING Zhi-bin;MA Dong;HUANG Jian-jun;SONG Li-juan;MA Cun-gen(The Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine,Research Center of Neurobiology,Shanxi University of Chinese Medicine,Jinzhong 030619,Shanxi Province,China;Department of Neurology,Shanxi Bethune Hospital,Taiyuan 030032,Shanxi Province,China3;.Department of Neurosurgery,General Hospital of Sinopharm TongliangGroup,Datong 037003,Shanxi Province,China;Ministry of Education Key Laboratory of Cell Physiology,Shanxi Medical University,Taiyuan 03000l,Shanxi Province,China)

机构地区：[1]山西中医药大学神经生物学研究中心,国家中医药管理局多发性硬化益气活血重点研究室,山西晋中030619 [2]山西白求恩医院神经内科,山西太原030032 [3]国药同煤集团总医院神经外科,山西大同037003 [4]山西医科大学细胞生理学省部共建教育部重点实验室,山西太原030001

出　　处：《中国临床药理学杂志》2024年第12期1734-1738,共5页The Chinese Journal of Clinical Pharmacology

基　　金：国家自然科学基金资助项目(82004028,81473577);中国博士后科学基金面上资助项目(2020M680912);山西省科技创新人才青年团队基金资助项目(202204051001028);山西省卫健委医学科技领军团队基金资助项目(2020TD05);山西省研究生科研创新基金资助项目(2023KY677);山西中医药大学2022年度科技创新团队基金资助项目(2022TD2010);山西中医药大学基地平台开放课题资助项目(2022JD-KF-19);山西中医药大学学科建设经费(2023XKJS—02);山西中医药大学研究生教育创新计划基金资助项目(2023CX024)。

摘　　要：目的探究羟基红花黄色素A(HSYA)对糖氧剥夺(OGD)后bEnd.3细胞自噬作用的影响及机制。方法将bEnd.3细胞分为正常组(常规培养)、模型组(OGD模型)、HSYA组(OGD模型+75μmol·L^(-1)HSYA)、3-甲基腺嘌呤(3-methyladenine,3MA)抑制药组(5 mmol·L^(-1)3MA+OGD模型)、3MA+HSYA组(5 mmol·L^(-1)3MA+OGD模型+75μmol·L^(-1)HSYA)。用TUNEL荧光染色法测定细胞凋亡水平;用蛋白质印迹法测定自噬、血脑屏障(BBB)相关蛋白的表达水平;用实时荧光定量聚合酶链反应法测定沉默信息调节因子1(SIRT1)、人叉头蛋白O3A(FOXO3A)mRNA的表达水平。结果正常组、模型组、HSYA组、3MA抑制药组和3MA+HSYA组中TUNEL染色阳性细胞分别为5.00±1.00、28.00±2.00、21.00±3.00、35.33±2.51和29.67±2.52;微管相关蛋白1轻链3-Ⅱ/-Ⅰ(LC3-Ⅱ/-Ⅰ)表达水平分别为0.90±0.20、1.34±0.10、1.95±0.14、0.76±0.15和1.14±0.09;泛素结合蛋白P62(P62)表达水平分别为0.99±0.02、0.60±0.02、0.38±0.01、0.67±0.04和0.54±0.01;闭合蛋白(Occludin)表达水平分别为1.39±0.17、0.62±0.15、1.00±0.09、0.40±0.13和0.80±0.15;紧密连接蛋白1(ZO-1)表达水平分别为1.63±0.20、0.64±0.06、0.98±0.14、0.37±0.14和0.87±0.04;SIRT1 mRNA表达为1.00±0.00、0.75±0.07、1.69±0.09、0.31±0.02和0.56±0.01;FOXO3A mRNA表达为1.00±0.00、0.80±0.05、1.47±0.09、0.40±0.01和0.62±0.09。模型组与正常组比较,HSYA组与模型组比较,3MA+HSYA组与3MA抑制药组比较,差异均有统计学意义(P<0.05,P<0.01,P<0.001)。结论HSYA可能通过SIRT1/FOXO3A通路增强OGD后bEnd.3细胞自噬水平,抑制细胞凋亡减轻BBB损伤。Objective To explore the effect and mechanism of hydroxysafflor yellow A(HSYA)on autophagy in bEnd.3 cells after oxygen-glucose deprivation(OGD).Methods The bEnd.3 cells were divided into normal group(conventional culture),model group(OGD model),HSYA group(OGD model+75μmol·L^(-1)HSYA),3-methyladenine(3MA)group(5 mmol·L^(-1)3MA+OGD model)and 3MA+HSYA group(5 mmol·L^(-1)3MA+OGD model+75μmol·L^(-1)HSYA).The level of apoptosis was determined by TUNEL fluorescence staining;Western blot was used to detect the expression of autophagy,blood brain barrier(BBB)related proteins;real time fluorescence quantitative polymerase chain reaction method for determining the expression of sirtuin-1(SIRT1)and forkhead box protein 03 a(FOXO3A)mRNA.Results In the normal group,model group,HSYA group,3MA group and 3MA+HSYA group,the positive cells selected for TUNEL staining were 5.00±1.00,28.00±2.00,21.00±3.00,35.33±2.51 and 29.67±2.52;the expression levels of microtubule-associated protein 1 light chain 3-Ⅱ/-Ⅰ(LC3-Ⅱ/-Ⅰ)were 0.90±0.20,1.34±0.10,1.95±0.14,0.76±0.15 and 1.14±0.09;sequestosome 1(P62 were 0.99±0.02,0.60±0.02,0.38±0.01,0.67±0.04 and 0.54±0.01;occludin were 1.39±0.17,0.62±0.15,1.00±0.09,0.40±0.13 and 0.80±0.15;zonula occludens-1(ZO-1)were 1.63±0.20,0.64±0.06,0.98±0.14,0.37±0.14 and 0.87±0.04;SIRT1 mRNA were 1.00±0.00,0.75±0.07,1.69±0.09,0.31±0.02 and 0.56±0.01;FOXO3A mRNA were 1.00±0.00,0.80±0.05,1.47±0.09,0.40±0.01 and 0.62±0.09,respectively.Significant differences were found between model group and normal group,HSYA group and model group,3MA+HSYA group and 3MA group(P<0.05,P<0.01,P<0.001).Conclusion HSYA may enhance autophagy levels in bEnd.3 cells after OGD through the SIRT1/FOXO3A pathway,inhibit cell apoptosis and alleviate BBB damage.

关 键 词：羟基红花黄色素A 糖氧剥夺 自噬 bEnd.3细胞 沉默信息调节因子1/人叉头蛋白O3A通路 

分 类 号：R28[医药卫生—中药学]