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北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (3): 438-443. doi: 10.19723/j.issn.1671-167X.2020.03.007

• 论著 • 上一篇    下一篇

苯并[a]芘对脑内多巴胺能神经元和α-突触核蛋白的影响及其机制

祁宇泽,权会会,徐卫星,李清如,周辉()   

  1. 北京大学公共卫生学院劳动卫生与环境卫生学系,北京 100191
  • 收稿日期:2020-02-18 出版日期:2020-06-18 发布日期:2020-06-30
  • 通讯作者: 周辉 E-mail:zhouhui@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(21577004);北京市自然科学基金(7162104)

Effect of benzo(a)pyrene on dopaminergic neurons and α-synuclein in brain and its mechanism involved

Yu-ze QI,Hui-hui QUAN,Wei-xing XU,Qing-ru LI,Hui ZHOU()   

  1. Department of Occupational and Environmental Health, Peking University School of Public Health, Beijing 100191, China
  • Received:2020-02-18 Online:2020-06-18 Published:2020-06-30
  • Contact: Hui ZHOU E-mail:zhouhui@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(21577004);Natural Science Foundation of Beijing(7162104)

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摘要：

目的 分析苯并[a]芘[benzo(a)pyrene,BaP]暴露对帕金森病病理特征多巴胺能神经元和α-突触核蛋白表达的影响,并探讨可能的机制。方法 8月龄人源SNCA转基因小鼠随机分为BaP染毒组和对照组,分别腹腔注射1.0 mg/kg体质量的BaP和玉米油溶剂,连续注射60 d。通过转轮实验观察小鼠运动功能障碍状况,通过免疫组织化学与免疫蛋白印迹实验观察BaP对多巴胺能神经元和α-突触核蛋白的影响,采用实时荧光定量PCR法检测相关mRNA的表达。研究中涉及的基因主要与神经递质转运蛋白、神经递质受体、细胞自噬和α-突触核蛋白聚集与降解相关。结果 BaP染毒后,转轮实验中小鼠运动时间显著降低(P<0.05),小鼠黑质多巴胺能神经元明显减少,为对照组的62%(P<0.05),中脑α-突触核蛋白表达增多,为对照组的1.36倍(P<0.05)。BaP染毒后,小鼠中脑14种mRNA表达显著下调(P均<0.05),主要涉及α-突触核蛋白降解与细胞自噬、神经元转运体、神经递质受体等功能;而Synphilin-1表达显著上调(P<0.01),与α-突触核蛋白合成有关。结论 BaP暴露抑制神经递质受体、多巴胺转运体蛋白功能和细胞自噬作用,阻碍α-突触核蛋白降解,从而诱导黑质多巴胺能神经元变性死亡和α-syn聚集体形成,增加帕金森病发病风险。

关键词: 苯并[a]芘, 多巴胺能神经元, α-突触核蛋白, 帕金森病

Abstract:

Objective: To analyze the effect of benzopyrene on the decrease of dopaminergic neurons, and the increase and aggregation of α-synuclein, which are the pathological features of Parkinson’s disease, and to explore its possible mechanisms.Methods: Eight-month-old transgenic mice with human SNCA gene were randomly divided into a BaP-exposed group and a control group. BaP and solvent corn oil were injected intraperitoneally to BaP-exposed group and control group respectively, once a day for 60 days. The motor dysfunction of mice was tested by rotarod test. The effects of BaP on the decrease of dopaminergic neurons and increase and aggregation of α-synuclein were observed by immunohistochemistry and Western blot experiments respectively, and the expression of related mRNA was detected by quantitative real-time PCR (qRT-PCR). Twenty genes were tested in the study, mainly related to neurotransmitter transporter (2 genes), neurotransmitter receptor function (10 genes), cellular autophagy (5 genes), and α-synuclein aggregation and degradation (3 genes).Results: After BaP exposure, the movement time of the mice in the rotarod test was significantly reduced (P<0.05). The substantia nigra dopami-nergic neurons in the mice were significantly reduced, which was 62% of the control group (P<0.05), and the expression of α-synuclein in the midbrain increased, which was 1.36 times that of the control group (P<0.05). After BaP exposure, mRNA expressions of 14 genes in the midbrain of the mice were significantly down-regulated (P<0.05). Alpha-synuclein degradation and cell autophagy (5 genes), neuron transporters (2 genes), and neurotransmitter receptor functions (5 genes) were involved. The expression of one gene, Synphilin-1, was significantly up-regulated (P<0.01), which was related to α-synuclein aggregation.Conclusion: BaP exposure not only inhibited function of neurotransmitter receptor and dopamine transporter, but also interfered cell autophagy, thereby hindering the degradation of α-synuclein, which could lead to decrease of dopaminergic neurons in substantia nigra and increase and aggregation of α-synuclein in midbrain, as the significant pathology of Parkinson’s disease. Therefore, BaP exposure may increase the risk of Parkinson’s disease.

Key words: Benzo(a)pyrene, Dopaminergic neurons, Alpha-synuclein, Parkinson’s disease

中图分类号: 

  • R12

表1

目的基因引物序列"

Genes Forward primer Reverse primer
Alphy-syn degradation and cell autophagy
Beclin1 ATGGAGGGGTCTAAGGCGTC TGGGCTGTGGTAAGTAATGGA
LC3-Ⅱ GACCGCTGTAAGGAGGTGC CTTGACCAACTCGCTCATGTTA
Hspa1a TGGTGCAGTCCGACATGAAG GCTGAGAGTCGTTGAAGTAGGC
Hsc70 TCTCGGCACCACCTACTCC CCCGATCAGACGTTTGGCA
Hsp90 CCAGCAAACAGGACCGAAC GCAATGGTGCCAAGGTTATTGAT
Neurotransmitter transporter
DAT GATGCACATAGCAGCAACTCT GCACACCACGCTCAAAATACTC
Vmat2 ATGCTGCTCACCGTCGTAG GGCAGTCTGGATTTCCGTAGT
Alphy-syn aggregate formation
Synphilin-1 CTTCCCTCTAATGCGCTGGTT CGTTGCGTTTCTTTTGATGTCA
Septin4 ACGGAATCGCAACAAACTGAC TCTTTCTCCCGGATTAGCTTCTC
GAK GGATGGAGCAAAACTTCGGAT TGGAAGACTGTGTAACGAGTGT
Neurotransmitter receptor
Htr1a GACAGGCGGCAACGATACT CCAAGGAGCCGATGAGATAGTT
Htr1d CACCCGCACCTGGAACTTT AGTGGAGGGATGGAGATACAAA
Htr5b CTGGTGAGCGAGTTGTCCG GCGTGATAGTCCAGTAGCGA
Adora2a TTCCACTCCGGTACAATGGC CGATGGCGAATGACAGCAC
Adra1b ACATTGGGGTGCGATACTCTC TTGGGCGCAGGTTCTTTCC
Drd1 GACATACGCCATTTCATCCTCC ATGCGCCGGATTTGCTTCT
Drd2 CAAGCGCCGAGTTACTGTCAT ATGGAGGAGTAGACCACGAAG
Gabra5 TTGGACGGACTCTTGGATGG TGTTCGCACCTGCGTGATT
Hrh3 CTCCGCACCCAGAACAACTT GCACGATGTTGAAGACTGAGG
Lepr GTCTTCGGGGATGTGAATGTC ACCTAAGGGTGGATCGGGTTT

图1

转轮实验落棒时间变化"

图2

黑质部多巴胺能神经元染色"

图3

中脑α-syn表达量变化"

表2

BaP暴露对基因表达的影响"

Genes Multiple of change P value
Alphy-syn degradation and cell autophagy
Beclin1 0.34 0.026
LC3-Ⅱ 0.43 0.023
Hsc70 0.29 0.026
Hspa1a 0.22 0.023
Hsp90 0.33 0.030
Neurotransmitter transporter
DAT 0.24 0.018
Vmat2 0.12 0.032
Alphy-syn aggregate formation
Synphilin-1 3.52 0.016
Septin4 0.18 0.006
GAK 0.50 0.140
Neurotransmitter receptor
Htr1a 0.26 0.007
Htr5b 0.20 0.005
Gabra5 0.26 0.007
Hrh3 0.26 0.008
Lepr 0.31 0.030
Htr1d 0.45 0.140
Adora2a 0.80 0.757
Adra1b 0.40 0.107
Drd1 0.78 0.582
Drd2 0.53 0.155
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