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J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (5): 465-472    DOI: 10.3785/j.issn.1008-9292.2018.10.04
    
Neuroprotective effect of rapamycin against Parkinson's disease in mice
ZHU Feng(),FAN Miao,XU Ziwei,CAI Yiting,CHEN Yizhen,YU Shuang,ZENG Linghui*()
School of Medicine, Zhejiang University City College, Hangzhou 310015, China
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Abstract  

Objective: To investigate the effect of rapamycin on Parkinson's disease (PD) and its underlying mechanism in mice. Methods: Sixty SPF adult male C57BL/6 mice were randomly divided into control group, model group and treatment group. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP) was used to induce Parkinson's disease in model group and treatment group. All mice were trained to cross the runway and were subjected to computer-assisted CatWalk. The numbers of tyrosine hydroxylase positive (TH+) neurons in the substantia nigra (SN) were assessed by unbiased stereology using the optical fractionator method; protein expression was detected by Western blot analysis; and glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by spectrophotometry. Results: In the model group, a decrease in stride rate and an increase in variation of stance and swing were noted by CatWalk system (P < 0.05 or P < 0.01); the numbers of TH+ neurons decreased (P < 0.01); expression of p-Akt, p-S6K, p-S6 and p-ULK increased (all P < 0.01); LC3-Ⅱ/Ⅰ ratio decreased (P < 0.01); MDA level was elevated while the levels of SOD and GSH-PX were reduced (all P < 0.01). Compared with the model group, after treated with rapamycin, the abnormal behavior including the stride length, variation of stance and swing and step patterns induced by MPTP were all improved (P < 0.05 or P < 0.01); the numbers of TH+ neurons increased (P < 0.05); the expression of p-Akt, p-S6K, p-S6 and p-ULK was suppressed (all P < 0.01); the LC3-Ⅱ/Ⅰ ratio was upregulated (P < 0.05); MDA level decreased while the levels of GSH-Px and SOD increased (all P < 0.01). Conclusion: Rapamycin inhibits the activation of mTOR pathway, which contributes to protect against the loss of dopaminergic neurons and provide behavioral improvements in mice with Parkinson's disease. These results are partially related to the ability of rapamycin in inducing autophagy and reducing oxidative stress.



Key wordsParkinson disease/prevention & control      Sirolimus/pharmacology      Signal transduction      Autophagy      Oxidative stress     
Received: 15 September 2018      Published: 23 January 2019
CLC:  R742  
Corresponding Authors: ZENG Linghui     E-mail: zhuf@zucc.edu.cn;zenglh@zucc.edu.cn
Cite this article:

ZHU Feng,FAN Miao,XU Ziwei,CAI Yiting,CHEN Yizhen,YU Shuang,ZENG Linghui. Neuroprotective effect of rapamycin against Parkinson's disease in mice. J Zhejiang Univ (Med Sci), 2018, 47(5): 465-472.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2018.10.04     OR     http://www.zjujournals.com/med/Y2018/V47/I5/465


雷帕霉素对帕金森病小鼠的保护作用

目的: 探索雷帕霉素对帕金森病模型小鼠的保护作用及机制。方法: 60只SPF级成年健康雄性C57/B6小鼠随机分为对照组、模型组和治疗组。模型组和治疗组采用1-甲基4-苯基-1、2、3、6-四氢吡啶(MPTP)诱导建立帕金森病模型。治疗组在第7天MPTP注射后1 h开始腹腔注射雷帕霉素(3 mg/kg,1次/d,共7 d),模型组和对照组均予腹腔注射等体积的溶剂。CatWalk步态分析系统分析小鼠运动功能;免疫荧光法检测小鼠脑黑质部酪氨酸羟化酶(TH)阳性神经元数量;蛋白质印迹法检测mTOR信号通路相关蛋白及自噬相关蛋白的表达;试剂盒测定谷胱甘肽过氧化物酶(GSH-Px)、丙二醛和超氧化物歧化酶(SOD)等氧化应激产物的浓度。结果: 与对照组比较,模型组小鼠行走速度和步频变慢,速度变化率增加(P < 0.05或P < 0.01),被系统识别的落脚模式减少;小鼠脑黑质中TH阳性染色神经元数量减少,Akt、S6K、S6及UNC-51样激酶(ULK)磷酸化水平升高,LC3-Ⅱ/Ⅰ比值降低,氧化应激相关的SOD和GSH-Px含量减少而丙二醛含量增加(均P < 0.01)。与模型组比较,治疗组步态规律性恢复,落脚模式被系统识别的数量增加,行走速度和步频加快,速度变化率减小(P < 0.05或P < 0.01);小鼠脑黑质中TH阳性染色神经元数量增加,mTOR信号通路相关蛋白及ULK磷酸化水平降低,LC3-Ⅱ/Ⅰ比值升高,SOD、GSH-Px含量增加而丙二醛含量减少(P < 0.05或P < 0.01)。结论: 雷帕霉素可以抑制帕金森病小鼠mTOR信号通路活性,通过增强大脑黑质部自噬活性和降低氧化应激水平来减轻多巴胺能神经元损伤,改善帕金森病小鼠行为学异常。


关键词: 帕金森病/预防和控制,  西罗莫司/药理学,  信号传导,  自噬,  氧化性应激 
Fig 1 Representative illuminations of footprint view in three groups
Fig 2 Representative illuminations of footfall patterns in three groups
Fig 3 The run duration, cadence and walking speed variation in three groups (n=8)
Fig 4 Tyrosine hydroxylase(TH)-immunoreactive neurons in the compact part of substantia nigra in three groups
Fig 5 Phosphorylation levels of Akt, S6K and S6 in three groups(n=6)
Fig 6 Expression of ULK and LC3 in three groups (n=6)
($\bar x \pm s$)
组别 n SOD(U/mg) GSH-Px(U/mg) 丙二醛(nmol/mg)
与对照组比较,*P < 0.05,* *P < 0.01;与模型组比较,##P < 0.01.SOD:超氧化物歧化酶;GSH-Px:谷胱甘肽过氧化物酶.
对照组 6 107±22 94±16 3.0±1.7
模型组 6 58±7** 38±10** 7.5±1.3**
治疗组 6 89±5## 61±11*## 4.0±1.6*#
Tab 1 Levels of oxidative stress products in three groups
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