Arginase1敲低通过增强Aβ降解和减轻神经炎症改善APP/PS1小鼠认知功能

Arginase1 Knockdown Improves Cognitive Function by Enhancing AβDegradation and Reducing Neuroinflammation in APP/PS1 Mouse

【目的】探讨表达 M2型小胶质细胞标志物精氨酸酶 1(Arginase1,Arg1)敲低对阿尔茨海默病模型小鼠 (APP/PS1)认知功能的影响。【方法】采用 Arginase1基因工程小鼠,分为 C57BL/6对照组(WT)、Arginase1低表达组 (Arg1^(+/-))、阿尔茨海默病小鼠模型组(APP/PS1)、Arginase1、APP/PS1转基因小鼠 Arginase1低表达组(Arg1^(+/-);APP/ PS1),旷场实验(OFT)、Morris水迷宫(MWM)实验分别测试小鼠自主活动和适应能力、学习记忆能力,硫磺素 S染色 法和免疫荧光染色法检测小鼠脑内 β-淀粉样蛋白(Aβ)沉积;小鼠海马区域 Arginase1,小胶质细胞特征性标志物离 子钙接头蛋白分子 1(Iba1)、跨膜蛋白 119(Tmem119)及溶酶体蛋白(CD68)表达量;ELISA法检测海马和皮质组织 Aβ_(1-40)、Aβ_(1-42)表达量,蛋白免疫印迹(Western blot)法检测淀粉样蛋白前体蛋白(APP)、APP-β位点剪切酶(BACE)、 脑啡肽酶(NEP)、白细胞介素-1β(IL-1β)表达量。【结果】OFT结果显示,与 APP/PS1组比较 Arg1^(+/-);APP/PS1组在中 央格区域的持续时间和运动频率明显增高(P<0.001);MWM实验结果显示,与 APP/PS1组比较 Arg1^(+/-);APP/PS1组小 鼠逃避潜伏期明显减少(P<0.001)、目标象限停留时间和距离及穿越平台次数均明显增多(均 P<0.05);硫磺素 S和 免疫荧光染色法测定,与 APP/PS1组比较 Arg1^(+/-);APP/PS1组海马区域 Aβ水平显著降低(P<0.001),与 APP/PS1组比 较 Arg1^(+/-);APP/PS1组海马区域小胶质细胞表达 CD68增多(P=0.039 2,P=0.000 3);ELISA与 Western blot结果显示, 与 APP/PS1组比较,Arg1^(+/-);APP/PS1组 Aβ_(1-40)、Aβ_(1-42)、APP、IL-1β水平下降,NEP蛋白表达量增加(P<0.000 1),BACE 蛋白表达量无明显差异(P=0.497 7)。【结论】在 APP/PS1模型小鼠中,下调 Arginase1可能促进脑内小胶质细胞激 活,上调 Aβ降解酶 NEP水平,减少脑内 Aβ沉积和神经炎症,从而提高 AD模型小鼠空间学习能力。

【Objective】To investigate the effect of the M2 microglia marker Arginase1(Arg1)knockdown on cognitive function in APP/PS1 transgenic mouse model of Alzheimer's disease(AD).【Methods】Arginase1 genetically engineered mice were used and divided into C57BL/6 control group(WT),Arginase1 low expression group(Arg^(1+/-)),Alzheimer's disease mouse model group(APP/PS1),Arginase1,APP/PS1 transgenic mice Arginase1 low expression group(Arg^(1+/-);APP/PS1).We used open field test(OFT)to assess the autonomous activity and adaptive capacity of mice;morris water maze experiment(MWM)to evaluate learning and memory function of mice;thioflavine-S staining to observe the brain amyloidβ-protein(Aβ)deposition in mice;immunofluorescence staining to detect the expression of microglia markers ionized calcium-binding adaptor molecule 1(Iba1),transmembrane protein 119(Tmem119)and lysosomal protein(CD68);Elisa to determine Aβ_(1-40)and Aβ_(1-42)expression in hippocampal and cortical tissues;and protein immunoblot(Western blot)to measure the expression of amyloid precursor protein(APP),beta-site APP-cleaving enzyme(BACE),enkephalinase(NEP),and interleukin-1β(IL-1β).【Results】Compared with APP/PS1 group,Arg^(1+/-);APP/PS1 group showed significantly increased duration and frequency in central area(P<0.001);significantly reduced escape latency(P<0.001),significantly increased dwell time,path length in the target quadrant and times of crossing the platform(all P<0.05);significant decrease of Aβdeposition in the hippocampus(P<0.0001)and significant increased expression of CD68 by microglia in the hippocampal region(P=0.0392,P=0.0003);decreased levels of Aβ_(1-40),Aβ_(1-42),APP,IL-1βand increased expression of NEP protein(P<0.0001).No significant difference in BACE protein expression(P=0.4977)was found.【Conclusion】Down-regulation of Arg1 may promote microglia activation in the brain,up-regulate the level of Aβ-degrading enzyme NEP,reduce Aβdeposition and neuroinflammation in the brain,thus improve the spatial learning capacity of APP/PS1 mouse model of AD.