目的 探讨六味地黄丸对β淀粉样蛋白 1-40(Aβ1-40)损伤的小鼠脑微血管内皮细胞(bEnd.3)的保护作用及其机制.方法 采用CCK8 法检测Aβ1-40和六味地黄丸含药血清(MSLDP)对细胞活性的影响,筛选合适的作用浓度.将bEnd.3 细胞分为对照组、Aβ1-40组、MSLDP+Aβ1-40组和MSLDP组,采用Western blot检测低密度脂蛋白相关蛋白 1(LRP1)、晚期糖基化终末产物受体(RAGE)、基质金属蛋白酶 2(MMP-2)、MMP-9、闭锁小带蛋白-1(ZO-1)、脑源性神经营养因子(BDNF)蛋白表达,免疫荧光检测LRP1、RAGE、ZO-1 表达;再将bEnd.3 细胞分为对照组、Aβ1-40组、FPS-ZM1(RAGE抑制剂)+Aβ1-40组和FPS-ZM1+Aβ1-40+MSLDP组,Western blot检测RAGE、MMP-9、MMP-2、ZO-1蛋白表达.结果 Aβ1-40呈剂量依赖性降低bEnd.3 细胞活性(P<0.01),MSLDP对Aβ1-40损伤的细胞活性具有保护作用(P<0.05,P<0.01),因此选择 10 μmol/L Aβ1-40 和 10%MSLDP 进行后续实验.与对照组比较,Aβ1-40组RAGE、MMP-2、MMP-9蛋白表达升高(P<0.01),LRP1、ZO-1、BDNF蛋白表达降低(P<0.05,P<0.01),并且LRP1、ZO-1荧光强度降低(P<0.01),RAGE荧光增强(P<0.01);与Aβ1-40组比较,MSLDP组RAGE、MMP-2、MMP-9蛋白表达和RAGE荧光强度降低(P<0.05,P<0.01),而LRP1、ZO-1、BDNF蛋白表达和LRP1、ZO-1 荧光强度升高(P<0.05,P<0.01).与Aβ1-40 组比较,Aβ1-40+FPS-ZM1 组MMP-2、MMP9、RAGE蛋白表达降低(P<0.05,P<0.01),ZO-1蛋白表达升高(P<0.05);Aβ1-40+FPS-ZM1+MSLDP 组MMP-2、MMP9、RAGE蛋白表达降低(P<0.01),ZO-1蛋白表达升高(P<0.01),FPS-ZM1 和MSLDP 联合使用的效果更佳.结论 六味地黄丸能够保护Aβ1-40损伤的脑微血管内皮的细胞紧密连接,减轻血脑屏障障碍,保护神经血管单元防治阿尔茨海默病,可能通过调节RAGE途径抑制MMP-2/MMP-9途径实现.
AIM To investigate the protective effects and the mechanism of the Liuwei Dihuang Pills on mouse brain microvascular endothelial(bEnd.3)cells damaged by β-Amyloid protein1-40(Aβ1-40).METHODS CCK8 method was used to detect the effects of Aβ1-40 and medicated serum of Liuwei Dihuang Pills(MSLDP)on cell activity,and to screen the appropriate concentration.bEnd.3 cells of the control group,the Aβ1-40 group,the MSLDP+Aβ1-40 group and the MSLDP group had their low density lipoprotein-associated protein 1(LRP1),receptor for advanced glycation end products(RAGE),matrix metalloproteinase-2(MMP-2),MMP-9,scaffold protein zonule protein-1(ZO-1)detected by Western blot.bEnd.3 cells assigned into the control group,the Aβ1-40 group,the FPS-ZM1(RAGE inhibitor)+Aβ1-40 group and the FPS-ZM1+Aβ1-40+MSLDP group had their expressions of RAGE,MMP-9,MMP-2 and ZO-1 detected by Western blot as well.RESULTS The cell activity of bEnd.3,was dose-dependently decreased by Aβ1-40(P<0.01),but was protected by MSLDP(P<0.05,P<0.01).And 10 μmol/L Aβ1-40 and 10%MSLDP were selected for subsequent experiments.Compared with the control group,the Aβ1-40 group displayed increased protein expressions of RAGE,MMP-2 and MMP-9(P<0.01),decreased protein expressions of LRP1,ZO-1 and BDNF(P<0.05,P<0.01),and decreased fluorescence intensities of LRP1 and ZO-1(P<0.01).Compared with the Aβ1-40 group,the MSLDP group shared decreased expressions of RAGE,MMP-2,MMP-9 proteins and RAGE fluorescence intensity(P<0.05,P<0.01),and increased expressions of LRP1,ZO-1 and BDNF proteins,and the fluorescence intensity of LRP1 and ZO-1(P<0.05,P<0.01);the Aβ1-40+FPS-ZM1 group displayed decreased protein expressions of MMP-2,MMP9 and RAGE(P<0.05,P<0.01),and increased ZO-1 protein expression(P<0.05);and the Aβ1-40+FPS-ZM1+ MSLDP group displayed an even more decreased protein expressions of MMP-2,MMP9 and RAGE(P<0.01),increased ZO-1 protein expression(P<0.01)due to the the combination use of FPS-ZM1 and MSLDP.CONCLUSION Liuwei Dihuang Pills can protect the tight junction of bEnd.3 injured by Aβ1-40 and neurovascular units from Alzheimer's disease by alleviating the dysfunction of the blood-brain barrier via RAGE-mediated MMP-2/MMP-9 pathway inhibition.