重复经颅磁刺激对AD小鼠脑组织中Aβ1~42水平的影响及机制

Effect and mechanism of repetitive transcranial magnetic stimulation on Aβ1-42 level in brain tissues of AD mice

目的探讨重复经颅磁刺激(rTMS)对阿尔茨海默病(AD)小鼠脑组织中β样淀粉蛋白1~42(Aβ1~42)水平的影响及机制。方法将小鼠随机分为假手术组、AD组、低频rTMS组、高频rTMS组,每组8只。AD组、低频rTMS组、高频rTMS组颅内注射1μg/μL的Aβ1~42溶液3μL建立AD模型,假手术组注射无菌生理盐水3μL。造模成功后,低频rTMS组、高频rTMS组分别使用1、10 Hz rTMS治疗2周。末次治疗后1 d处死小鼠,取其脑组织。用ELISA法检测脑组织中Aβ1~42水平,用实时荧光定量PCR法检测脑组织中PTEN诱导激酶1(PINK1)、Parkin mRNA表达,用Western blotting法检测脑组织中PINK1、Parkin蛋白及自噬标志蛋白(LC3Ⅰ、Ⅱ及p62、Beclin-1)表达。结果与假手术组比较,AD组脑组织中Aβ1~42水平高(P<0.05);与AD组比较,低、高频rTMS组脑组织中Aβ1~42水平低(P均<0.05);与低频rTMS组比较,高频rTMS组脑组织中Aβ1~42水平低,但差异无统计学意义(P>0.05)。与假手术组比较,AD组PINK1、Parkin mRNA、蛋白相对表达量低(P均<0.05);与AD组比较,低、高频rTMS组PINK1、Parkin mRNA、蛋白相对表达量高(P均<0.05),且高频rTMS组较低频rTMS组变化更明显(P均<0.05)。与假手术组比较,AD组p62蛋白相对表达量高,Beclin-1蛋白相对表达量、LC3Ⅱ/Ⅰ低(P均<0.05);与AD组比较,低、高频rTMS组p62蛋白相对表达量低,Beclin-1蛋白相对表达量、LC3Ⅱ/Ⅰ高(P均<0.05),且高频rTMS组较低频rTMS组变化更明显(P均<0.05)。结论rTMS可清除AD小鼠脑组织中沉积的Aβ1~42,其作用机制可能为通过激活PINK1/Parkin通路促进线粒体自噬。

Objective To investigate the effect and mechanism of repetitive transcranial magnetic stimulation(rTMS)on the level of Aβ1-42 in the brain of Alzheimer's disease(AD)mice.Methods The mice were randomly divided into four groups:the sham operation group,AD group,low-frequency rTMS group,and high-frequency rTMS group,with 8 mice in each.In the AD group,low-frequency rTMS group and high-frequency rTMS group,the AD models were established by intracranial injection of 1μg/μL Aβ1-423μL,meanwhile,3μL saline was injected into mice in the sham operation group.The mice in the low-frequency rTMS group and high-frequency rTMS group were treated with 1 and 10 HzrTMS for 2 weeks after successful modeling.The mice were killed 1 d after the last treatment and their brain tissues were taken.The contents of Aβ1-42 in the brain tissues were detected by ELISA,the expression levels of PINK1,Parkin mRNA and protein were detected by real-time PCR,the protein expression levels of PINK1,Parkin and autophagy marker protein of LC3Ⅰ/Ⅱ,p62,and Beclin-1 were detected by Western blotting.Results Compared with the sham operation group,the content of Aβ1-42 in the brain tissues of AD group increased(P<0.05).Compared with the AD group,the content of Aβ1-42 in the brain tissues of mice in the low-frequency and high-frequency rTMS groups decreased(all P<0.05).Compared with the low-frequency rTMS group,the content of Aβ1-42 in the brain tissues of mice in the high-frequency rTMS group decreased,but showed no significant different(P>0.05).Compare with the sham operation group,the expression of PINK1,Parkin mRNA and protein in the brain tissues of mice in AD group decreased(all P<0.05).Compare with the AD group,the expression of PINK1,Parkin mRNA and protein in the low-frequency rTMS group and high-frequency rTMS group increased(all P<0.05),and the change of the high-frequency rTMS group was more obvious than that of the low-frequency rTMS group(P<0.05).Compare with the sham operation group,the protein relative expression of p62 in the AD group increased,and the protein relative expression of Beclin-1 and LC3Ⅱ/Ⅰin the AD group decreased(all P<0.05).Compare with the AD group,the protein relative expression of p62 in the low-frequency rTMS group and high-frequency rTMS group decreased,the protein relative expression of Beclin-1 and LC3Ⅱ/Ⅰin the low-frequency rTMS group and high-frequency rTMS group increased(all P<0.05),and the change of the high-frequency rTMS group was more obvious than that of the low-frequency rTMS group(P<0.05).Conclusion rTMS can scavenge Aβ1-42 in the brain of AD mice,and the mechanism may be that it promotes mitochondrial autophagy by activating PINK1/Parkin pathway.