中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (10): 1842-1850.doi: 10.4103/1673-5374.238621

• 原著:退行性病与再生 • 上一篇    下一篇

MAPK磷酸酶1保护PC12细胞免受Aβ诱导的神经毒性

  

  • 收稿日期:2018-07-18 出版日期:2018-10-15 发布日期:2018-10-15

Mitogen-activated protein kinase phosphatase 1 protects PC12 cells from amyloid beta-induced neurotoxicity

Yue Gu1, Lian-Jun Ma2, Xiao-Xue Bai3, Jing Jie1, Xiu-Fang Zhang1, Dong Chen1, Xiao-Ping Li4   

  1. 1 Department of Respiratory Medicine, the First Hospital of Jilin University, Changchun, Jilin Province, China
    2 Endoscopy Center, the China-Japan Hospital of Jilin University, Changchun, Jilin Province, China
    3 Cadre’s Wards, the First Hospital of Jilin University, Changchun, Jilin Province, China
    4 Department of Pediatrics, the First Hospital of Jilin University, Changchun, Jilin Province, China
  • Received:2018-07-18 Online:2018-10-15 Published:2018-10-15
  • Contact: Xiao-Ping Li,xiaopingli@jlu.edu.cn.

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摘要：

MAPK信号通路在调控细胞生长、增殖、分化、转化和死亡中起重要作用，而MAPK磷酸酶1对p38MAPK和JNK有显著抑制作用，但其是否在Aβ诱导的氧化应激和神经元炎症中起作用尚无公认。实验以MKP1 shRNA、MKP1慢病毒颗粒或对照慢病毒感染PC12细胞12h，然后以0.1，1，10，100μM Aβ42干预细胞。以CCK8分析检测细胞存活率，以定量实时PCR分析MAPK磷酸酶1、肿瘤坏死因子α和白细胞介素1β基因表达水平，以Western blot分析MAPK磷酸酶1以及磷酸化JNK表达水平，以DCFH-DA检测活性氧水平，以流式细胞仪检测线粒体膜电位，以比色法检测超氧化物歧化酶活性和丙二醛水平，以酶标仪检测乳酸脱氢酶活性，以ELISA检测Caspase 3的表达水平，以TUNEL染色检测细胞凋亡。这些层面的数据结果综合显示，MAPK磷酸酶1过表达可抑制Aβ诱导的PC12细胞中JNK磷酸化，活性氧升高，肿瘤坏死因子α和白细胞介素1β高表达以及细胞凋亡。而利用RNA干扰敲除MAPK磷酸酶1基因，则加剧Aβ诱导的PC12细胞中的氧化应激、炎症和细胞损伤。进一步的研究使用JNK特异性抑制剂SP600125抑制MAPK磷酸酶1，也使得Aβ诱导的神经毒性作用加重。表明MAPK磷酸酶1可通过抑制JNK信号通路来减轻Aβ诱导的细胞凋亡、氧化应激和神经炎症而发挥了神经保护作用。

orcid:0000-0002-1782-8010(Xiao-Ping Li)
        0000-0002-1505-0014(Yue Gu)

关键词: MAPK磷酸酶1, JNK信号通路, 阿尔茨海默病, 神经元, 痴呆, 细胞凋亡, RNA干扰, 慢病毒, 炎症, 氧化应激, 神经再生

Abstract:

The mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation,transformation and death. Mitogen-activated protein kinase phosphatase 1 (MKP1) has an inhibitory effect on the p38MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 shRNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42 (Aβ42). The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) mRNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase (JNK) expression levels were assessed using western blot assay. Reactive oxygen species (ROS) levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.

Key words: nerve regeneration, mitogen-activated protein kinase phosphatase 1, c-Jun N-terminal kinase signaling pathway, Alzheimer’s disease, neurons, dementia, apoptosis, RNA interference, lentivirus, inflammation, oxidative stress, neural regeneration