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

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.