苓桂术甘汤对阿尔茨海默病血脑屏障损伤的影响

Mechanism of Linggui Zhugantang in Repairing Blood-brain Barrier Injury of Alzheimer’s Disease

目的:通过观察苓桂术甘汤(LG)对体外阿尔茨海默病(AD)病理状态下血脑屏障模型的影响,探讨其对AD血脑屏障损伤后修复的具体机制。方法:将50只雄性SPF级大鼠,随机分为5组,分别灌胃7 d,2次/d,LG高、中、低剂量组以4.8,2.4,1.2 g·kg^(-1),西药组以盐酸多奈哌齐0.5 mg·kg^(-1),正常组以等体积生理盐水灌胃,末次灌胃1 h后经腹主动脉取含药血清;以细胞共培养法构建AD体外血脑屏障损伤模型,实验分为正常组,模型组,LG高、中、低剂量组和盐酸多奈哌齐组,模型组加入β淀粉样蛋白1-42(Aβ;)100μL,终浓度为5μmol·L^(-1),LG高、中、低剂量组以及盐酸多奈哌齐组在模型组基础上分别加入10%含药血清。通过噻唑蓝(MTT)比色法检测各组细胞存活率;采用蛋白免疫印迹法(Western blot)测定各组血脑屏障相关骨架蛋白跨膜密蛋白-5(Claudin-5),外周支架蛋白-1(ZO-1),闭合蛋白(Occludin),基质金属蛋白酶-2(MMP-2),基质金属蛋白酶-9(MMP-9)的表达;采用酶联免疫吸附测定法(ELISA)检测炎症因子白细胞介素-1β(IL-1β),IL-6,肿瘤坏死因子-α(TNF-α)的含量变化;采用实时荧光定量聚合酶链式反应(Real-time PCR)和Western blot检测LG高、中、低剂量组血脑屏障不同时间点Aβ转运蛋白低密度脂蛋白受体相关蛋白1(LRP-1)和晚期糖基化终产物受体(RAGE)表达。结果:MTT结果分析显示,与正常组比较,模型组细胞存活率明显下降(P<0.05);与模型组比较,盐酸多奈哌齐组和LG高剂量组细胞存活率明显增强(P<0.05);Western blot结果分析显示,与正常组比较,模型组骨架蛋白显著下调,MMP-2,MMP-9蛋白明显上调(P<0.05);与模型组比较,盐酸多奈哌齐组和LG高剂量骨架蛋白含量明显上调(P<0.05),MMP-2,9水平明显下降(P<0.05);与模型组比较,仅LG中剂量组Claudin-5蛋白水平明显上调(P<0.05),MMP-2水平明显下调(P<0.05);ELISA结果分析显示,与正常组比较,模型组IL-1β,IL-6,TNF-α含量明显上调(P<0.05);与模型组比较,盐酸多奈哌齐组和LG高、中剂量组IL-1β,IL-6,TNF-α表达明显下调(P<0.05);PCR结果分析显示,与0 h比较,3 h时LRP-1表达明显上调,RAGE表达明显下调(P<0.05),6,12,24,36 h后LRP-1,RAGE表达逐渐和缓;Western blot结果分析显示,3 h时,与正常组比较,模型组LRP-1含量明显下调,RAGE含量明显上调(P<0.05);与模型组比较,盐酸多奈哌齐组和LG高剂量组LRP-1含量显著上调,RAGE含量明显下调(P<0.05)。结论:LG对AD体外血脑屏障损伤具有修复作用,其机制可能与抑制炎症因子和MMP-2,MMP-9表达、促进骨架蛋白表达并调节转运蛋白平衡相关。

Objective: To observe the effect of Linggui Zhugantang(LG)on the blood-brain barrier(BBB)model of Alzheimer’s disease(AD)in vitro and to explore the mechanism of LG in repairing the BBB injury in AD. Method:A total of 50 male SPF rats were randomized into five groups:high-dose(4.8 g·kg^(-1)),medium-dose(2.4 g·kg^(-1)),and low-dose(1.2 g·kg^(-1))LG groups,western medicine(0.5 g·kg^(-1)donepezil hydrochloride) group, and normal group(normal saline of equivalent volume). They received(ig)corresponding drugs twice a day for 7 d. Drug-containing serum was respectively collected from the abdominal aorta 1 h after the last administration. The BBB injury of AD in vitro was induced with the cell co-culture method,and 6 groups were designed:normal group,model group,high-,medium-,and low-dose LG groups,and western medicine group. The model group was added with 100 μL amyloid β1-42(Aβ;),final concentration:5 μmol·L^(-1)),and high-dose,medium-dose,and low-dose LG groups and the western medicine group were added with corresponding 10% drug-containing serum in addition to the 100 μL Aβ;(final concentration:5 μmol·L;). Cell survival rate was detected by methyl thiazolyl tetrazolium(MTT)assay,expression of BBBrelated skeleton proteins(claudin-5,ZO-1,occludin),matrix metalloproteinase-2(MMP-2),and matrix metalloproteinase-9(MMP-9)by Western blot,and content of inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)by enzyme-linked immunosorbent assay(ELISA).BBB Aβ transporter low-density lipoprotein receptor-related protein 1(LRP-1)and advanced glycation end product receptor(RAGE)at different time points in high-dose,medium-dose,and low-dose LG groups were determined by Real-time PCR and Western blot. Result: Cell survival rate of the model group was lower than that of the normal group(P<0.05)and the survival rates of the western medicine group and high-dose LG group was higher than that in the model group(P<0.05). The skeleton proteins were down-regulated and MMP-2 and MMP-9 were up-regulated in the model group compared with those in the normal group(P<0.05). The expression of skeleton proteins was higher(P<0.05)and that of MMP-2 and MMP-9 was lower(P<0.05)in the western medicine group and high-dose LG group than in the model group. Compared with the model group,only the medium-dose LG group showed the up-regulation(P<0.05)of claudin-5(P<0.05)and the decrease(P<0.05)of MMP-2. IL-1β,IL-6,and TNF-α in the model group were up-regulated(P<0.05)compared with those in the normal group,and those inflammatory factors in the western medicine group and high-dose and mediumdose LG groups were lower(P<0.05)than those in the model group. LRP-1 expression was up-regulated and RAGE expression was down-regulated at 3 h compared with those at 0 h(P<0.05),while the expression of the two became stable at 6,12,24,36 h. At 3 h,LRP-1 expression was down-regulated and RAGE expression was up-regulated in model group compared with those in the normal group at 3 h(P<0.05). Moreover,the LRP-1 content was higher and RAGE content was lower in the western medicine group and high-dose LG group than in the model group. Conclusion: LG can repair the BBB injury in vitro by inhibiting the expression of inflammatory factors and MMP-2,MMP-9,promoting the expression of skeletal proteins,and regulating the balance of transporters.