胡黄连苷Ⅱ通过增强自噬改善Aβ模型小鼠的学习和记忆功能

Picroside Ⅱ improves Aβ-induced learning and memory deficits in mice by enhancing autophagy

摘要:

目的 探究胡黄连苷（picroside Ⅱ，PicrⅡ）对体内外阿尔茨海默病（Alzheimer’s Disease，AD）模型的学习和记忆功能及自噬相关机制。方法 通过Western blot检测在PicrⅡ（12.5~400 μmol/L）对AD细胞模型中β淀粉样前体蛋白（β-amyloid precursor protein，APP）、β-位点淀粉样前体蛋白切割酶1（β-site APP-cleaving enzyme 1，BACE1）、早老素蛋白1（presenilin 1，PS1）和APP羧基末端片段（carboxyl terminal fragment of β-amyloid precursor protein，CTF）蛋白表达影响，以及PicrⅡ和氯喹（Chloroquine，CQ）（50 μmol/L）或巴佛洛霉素（bafilomycin A1，BafA1）（10 μmol/L）共处理后对APP、BACE1、PS1、C99、螯合体1（sequestosome 1，p62）、微管相关蛋白1轻链3（microtubule-associated protein light chain 3，LC3）、泛素化蛋白的表达影响；利用免疫荧光分析自噬通量；后期构建β-淀粉蛋白（amyloid β，Aβ）模型小鼠，分为WT+PBS组、WT+PicrⅡ组、Aβ+PBS组和Aβ+PicrⅡ组（20 mg/kg），通过Morris水迷宫实验验证小鼠学习和记忆功能，并检测小鼠海马蛋白中P62与LC3蛋白水平。结果 Western blot显示：与对照组相比，PicrⅡ（50 μmol/L）可显著降低APP（60.46±7.97，Ｐ=0.049）及相关代谢产物蛋白水平，并降低P62[（65.31±3.51）％，Ｐ=0.041]水平，增加LC3Ⅱ[（162.01±12.24）%，Ｐ<0.001]水平，并且在CQ作用下，PicrⅡ可降低P62[（147.24±10.69）%，Ｐ<0.001]及LC3水平[（826.23±39.18）%，Ｐ<0.001]；自噬通量分析显示：与对照组相比，PicrⅡ增加总荧光数（23.24±1.50，Ｐ<0.001）和红色荧光数（3.52±0.33，Ｐ<0.001）；行为学结果显示：与WT+Aβ组相比，Aβ+PicrⅡ组学习（F=17.25，Ｐ<0.001）与记忆能力（F=6.627，Ｐ<0.001）提高，且小鼠海马蛋白中Aβ+PicrⅡ组P62降低[（176.66±11.47）%，Ｐ=0.023]，LC3Ⅱ升高[（81.20±3.69）%，Ｐ<0.001]。结论 PicrⅡ可通过促进自噬体-溶酶体融合和提高自噬通量增强自噬，从而促进Aβ清除并改善其引起的学习和记忆障碍。

Abstract:

Objective To investigate the effect of picroside Ⅱ（PicrⅡ） on learning and memory functions in Alzheimer’s disease（AD） model in vivo and in vitro and autophagy-related mechanisms.Methods Western blot was used to observe the effect of PicrⅡ（12.5-400 μmol/L） on the expression of β-amyloid precursor protein（APP），β-site amyloid precursor protein cleaving enzyme 1（BACE1），presenilin 1（PS1），and carboxyl-terminal fragment of β-amyloid precursor protein（CTF） in the cell model of AD；after co-treatment with PicrⅡ and chloroquine（CQ）（50 μmol/L） or bafilomycin（BafA1）（10 μmol/L），its effect on the expression of APP，BACE1，PS1，C99，sequestosome 1（P62），microtubule-associated protein light chain 3（LC3），and ubiquitinated proteins in cells was observed；immunofluorescence assay was used to analyze autophagic flux. Then a mouse model of Aβ was established，and the mice were divided into WT+PBS group，WT+PicrⅡ group，Aβ+PBS group，and Aβ+PicrⅡ group（20 mg/kg）. The Morris water maze test was used to verify the learning and memory functions of Aβ model mice，and the protein expression levels of P62 and LC3 in the hippocampus of mice were measured.Results Western blot showed that compared with the control group，PicrⅡ（50 μmol/L） significantly reduced the protein expression levels of APP[（60.46±7.97）%，P=0.049] and related metabolites，and it also reduced the expression level of P62[（65.31±3.51）%，P=0.041] and increased the expression level of LC3Ⅱ[（162.01±12.24）%，P<0.001]；with the action of CQ，PicrⅡ could reduce the expression levels of P62[（147.24±10.69）%，P<0.001] and LC3Ⅱ[（826.23±39.18）%，P<0.001]. The analysis of autophagic flux showed that compared with the control group，PicrⅡ increased the total number of fluorescent dots（23.24±1.50，P<0.001） and the number of red dots（3.52±0.33，P<0.001）. The behavioral analysis showed that compared with the WT+Aβ group，the Aβ+PicrⅡ group had significant increases in learning ability（F=17.25，P<0.001） and memory ability（F=6.627，P<0.001），as well as a significant reduction in P62[（176.66±11.47）%，P=0.023] and a significant increase in LC3 Ⅱ[（81.20±3.69）%，P<0.001].Conclusion PicrⅡ can enhance autophagy by promoting autophagosome-lysosome fusion，thereby promoting the clearance of Aβ and improving Aβ-induced learning and memory deficits.