灯盏花素通过Wnt/β-catenin通路对阿尔茨海默病大鼠模型Aβ跨血脑屏障的转运清除机制研究

Breviscapine promotes the transportof Aβ across the blood-brain barrier via Wnt/β-catenin pathway in a rat model of Alzheimer’s disease

目的 探讨灯盏花素(Breviscapine, BRE)对阿尔茨海默病(Alzheimer disease, AD)大鼠模型淀粉样蛋白β(Amyloidβ,Aβ)跨血脑屏障(blood brain barrier, BBB)的转运清除的影响及其可能作用机制。方法 随机选择15只Wistar大鼠作为空白组,剩余大鼠于侧脑室内注射淀粉样蛋白β25-35(Aβ25-35)以诱导AD大鼠模型;后随机分为模型组、BRE低(50 mg·kg^(-1)·d^(-1))、高(100 mg·kg^(-1)·d^(-1))剂量组及多奈哌齐组(1 mg·kg^(-1)·d^(-1)),每组各15只,连续灌服给药21 d;Morris水迷宫行为测试检测大鼠空间学习记忆能力;海马组织行尼氏、刚果红染色;酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)法检测海马组织中Aβ42、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素1β(Interleukin 1β,IL-1β)、丙二醛(Malondialdehyde, MDA)、超氧化物歧化酶(Superoxide dismutase, SOD)水平;Western blot法检测海马Wnt3a/β-连环蛋白(β-catenin)通路因子及Aβ相关转运体蛋白表达水平。结果 与空白组比较,模型组大鼠逃避潜伏期延长,穿台次数减少,海马CA3区存活神经元数减少,Aβ斑块状严重沉积,Aβ42,TNF-α,IL-1β,MDA水平升高,SOD水平、Wnt3a,β-catenin、低密度脂蛋白受体相关蛋白1(Low density lipoprotein receptor-associated protein 1,LRP-1)及P糖蛋白(P glycoprotein, P-gp)相对表达水平降低,磷酸化糖原合酶激酶3β(Phosphorylated GSK3β,p-GSK3β)、轴抑制蛋白2(Axis inhibition protein 2,Axin2)相对表达水平升高(P<0.05);与模型组比较,BRE低、高剂量组及多奈哌齐组大鼠逃避潜伏期缩短,穿台次数增加,海马CA3区存活神经元数增加,Aβ斑块状沉积减轻,Aβ42,TNF-α,IL-1β,MDA水平降低,SOD水平、Wnt3a,β-catenin, LRP-1及P-gp相对表达水平升高,p-GSK3β,Axin2相对表达水平降低(P<0.05);低、高剂量BRE及多奈哌齐作用效果逐渐增强(P<0.05)。结论 BRE可促进AD大鼠模型Aβ跨BBB转运清除,其作用机制可能与激活Wnt/β-catenin通路有关。

Objective To investigate the effect of breviscapine(BRE) on Amyloid-β(Aβ)transport and clearance through the blood-brain barrier(BBB) in a rat model of Alzheimer’s disease(AD), and to illustrate the molecular mechanism.Methods Fifteen Wistar rats were randomly selected as the blank group. The remaining rats were injected with Aβ25-35 into the lateral ventricle to induce AD rat model. The rats were randomly divided into the model group, BRE low dose(50 mg·kg^(-1)·d^(-1)), BRE high dose(100 mg·kg^(-1)·d^(-1)) group, and Donepezil group(1 mg·kg^(-1)·d^(-1)), with 15 rats in each group. Morris water maze test was used to detect the spatial learning and memory ability of the rats. The hippocampal sections were stained with Nessie’s and Congo red. The levels of Aβ42, tumor necrosis factor-α(TNF-α), interleukin 1β(IL-1β), malondialdehyde(MDA), and superoxide dismutase(SOD) in the hippocampus were determined by ELISA. Western blot was used to detect the expression of Wnt3 a/β-catenin pathway and Aβ-associated transporter protein in the hippocampus.Results Compared with the blank group, the rats in the model group had longer escape latency time, reduced number of stage penetration, reduced number of surviving neurons in the CA3 region of the hippocampus, more severe Aβ plaque-like deposition, increased levels of Aβ42, TNF-α, IL-1β, MDA, and decreased levels of SOD, Wnt3 a, β-catenin, low density lipoprotein receptor-associated protein 1(LRP-1), P glycoprotein(P-gp), phosphorylated glycogen synthase kinase 3β(phosphorylated GSK3 β(p-GSK3β), and axis inhibition protein 2(Axin 2)(P<0.05). Compared with the model group, rats in the low and high dose BRE and donepezil groups had shorter escape latency, increased number of stage penetration, increased number of surviving neurons in the hippocampal CA3 area, reduced Aβ plaque-like deposition, decreased expression level of Aβ42, TNF-α, IL-1β, MDA levels, SOD levels, Wnt3 a, β-catenin, LRP-1, P-gp protein, p-GSK3β and Axin2(P<0.05).The effects of BRE and donepezil are dose-dependent(P<0.05).Conclusion BRE could promote the trans-BBB transport and clearance of Aβ in AD rats, and its mechanism may be related to the activation of the Wnt/β-catenin pathway.