中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (1): 170-177.doi: 10.4103/1673-5374.314314

• 原著:神经损伤修复保护与再生 • 上一篇    下一篇

Ghrelin可减轻6-OHDA诱导SH-SY5Y细胞的神经毒性

  

  • 出版日期:2022-01-05 发布日期:2021-09-22

Ghrelin alleviates 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells

Xin He1, 2, Wei Yuan3, Chun-Qing Yang4, Lu Zhu1, Fei Liu2, Juan Feng2, Yi-Xue Xue1, *   

  1. 1Department of Neurobiology, School of Life Sciences, China Medical University, Shenyang, Liaoning Province, China; 2Department of Neurology, Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China; 3Department of Orthopedics, First Hospital of China Medical University, Shenyang, Liaoning Province, China; 4Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning Province, China
  • Online:2022-01-05 Published:2021-09-22
  • Contact: Yi-Xue Xue, PhD, xyxcmu@hotmail.com or xueyixue888@163.com.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, No. 81901417 (to XH); the Natural Science Foundation Doctoral Research Initiation Plan of Liaoning Province of China, No. 2019-BS-287 (to XH); the China Postdoctoral Science Foundation, No. 2019M661173 (to XH).

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摘要：

有研究证实，具有多样生理功能的神经多肽Ghrelin在多种神经系统疾病模型中可发挥神经保护作用，但其对帕金森病是否也有保护作用及机制尚不明确。实验旨在明确ghrelin在6-OHDA诱导帕金森病模型中的作用及潜在机制。①实验采用6-OHDA诱导帕金森病SH-SY5Y细胞模型，通过CCK-8及Annexin V/PI凋亡检测证实不同剂量ghrelin(1 nM，10 nM，100 nM)预处理30 min后并与6-OHDA共处理24 h可以有效减轻6-OHDA的神经毒性；②通过qRT-PCR及Western blot检测发现，6-OHDA处理可以上调α-突触核蛋白及lincRNA-p21表达水平，并下调转录因子TGIF1(α-突触核蛋白编码基因SNCA的潜在转录调控因子)的水平，而ghrelin可显著逆转上述效应；③Annexin V/PI凋亡检测实验证实，应用小干扰RNA抑制α-突触核蛋白及lincRNA-p21表达，均可显著降低6-OHDA所致细胞凋亡水平，而抑制lincRNA-p21水平可部分上调TGIF1表达；④通过检索生物信息学数据库、应用双荧光素酶实验及RNA免疫沉淀实验证实，lincRNA-p21与TGIF1可以形成Stau1蛋白结合位点，继而启动Stau1蛋白介导mRNA降解途径，而TGIF1可以与α-突触核蛋白编码基因SNCA启动子区结合发挥转录调控作用；⑤在上述实验结果基础上，应用Annexin V/PI凋亡检测证实，TGIF1小干扰RNA或lincRNA-p21过表达载体均可显著削弱ghrelin在6-OHDA诱导帕金森病细胞模型中的神经保护作用。⑥数据证实ghrelin减轻6-OHDA诱导的SH-SY5Y细胞神经毒性，lincRNA-p21/TGIF1/α-突触核蛋白途径介导了ghrelin在帕金森病模型中的神经保护作用。

https://orcid.org/0000-0002-0452-9572 (Yi-Xue Xue)

关键词: font-family:宋体, ">凋亡, font-family:Arial, sans-serif, ">α-font-family:宋体, ">突触核蛋白, font-family:Arial, sans-serif, ">ghrelinfont-family:宋体, ">, font-family:Arial, sans-serif, ">lincRNA-p21font-family:宋体, ">, 神经多肽, 神经毒性, 帕金森病, font-family:Arial, sans-serif, ">Stau1font-family:宋体, ">介导font-family:Arial, sans-serif, ">mRNAfont-family:宋体, ">降解, font-family:Arial, sans-serif, ">TGIF1font-family:宋体, ">, font-family:Arial, sans-serif, ">6-OHDA

Abstract: Ghrelin is a neuropeptide that has various physiological functions and has been demonstrated to be neuroprotective in a number of neurological disease models. However, the underlying mechanisms of ghrelin in Parkinson’s disease remain largely unexplored. The current study aimed to study the effects of ghrelin in a 6-hydroxydopamine (6-OHDA)-induced Parkinson’s disease model and evaluate the potential underlying mechanisms. In the present study, we treated an SH-SY5Y cell model with 6-OHDA, and observed that pretreatment with different concentrations of ghrelin (1, 10, and 100 nM) for 30 minutes relieved the neurotoxic effects of 6-OHDA, as revealed by Cell Counting Kit-8 and Annexin V/propidium iodide (PI) apoptosis assays. Reverse transcription quantitative polymerase chain reaction and western blot assay results demonstrated that 6-OHDA treatment upregulated α-synuclein and lincRNA-p21 and downregulated TG-interacting factor 1 (TGIF1), which was predicted as a potential transcription regulator of the gene encoding α-synuclein (SNCA). Ghrelin pretreatment was able to reverse the trends caused by 6-OHDA. The Annexin V/PI apoptosis assay results revealed that inhibiting either α-synuclein or lincRNA-p21 expression with small interfering RNA (siRNA) relieved 6-OHDA-induced cell apoptosis. Furthermore, inhibiting lincRNA-p21 also partially upregulated TGIF1. By retrieving information from a bioinformatics database and performing both double luciferase and RNA immunoprecipitation assays, we found that lincRNA-p21 and TGIF1 were able to form a double-stranded RNA-binding protein Staufen homolog 1 (STAU1) binding site and further activate the STAU1-mediated mRNA decay pathway. In addition, TGIF1 was able to transcriptionally regulate α-synuclein expression by binding to the promoter of SNCA. The Annexin V/PI apoptosis assay results showed that either knockdown of TGIF1 or overexpression of lincRNA-p21 notably abolished the neuroprotective effects of ghrelin against 6-OHDA-induced neurotoxicity. Collectively, these findings suggest that ghrelin exerts neuroprotective effects against 6-OHDA-induced neurotoxicity via the lincRNA-p21/TGIF1/α-synuclein pathway. 

Key words: 6-hydroxydopamine, apoptosis, ghrelin, lincRNA-p21, neuropeptide, neurotoxicity, Parkinson’s disease, STAU1-mediated mRNA decay, TGIF1, α-synuclein