[1]潘燕,杨越,丛丽娜,等.烟碱型乙酰胆碱受体α7亚基在帕金森病细胞模型中调控多巴胺神经元的作用机制[J].卒中与神经疾病杂志,2024,31(05):457-464.[doi:10.3969/j.issn.1007-0478.2024.05.007]
　Pan Yan,Yang Yue,Cong Lina,et al.The mechanism of nicotinic acetylcholine receptor α7 subunit regulating dopamine neurons in Parkinson's cell model[J].Stroke and Nervous Diseases,2024,31(05):457-464.[doi:10.3969/j.issn.1007-0478.2024.05.007]
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烟碱型乙酰胆碱受体α7亚基在帕金森病细胞模型中调控多巴胺神经元的作用机制()
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《卒中与神经疾病》杂志[ISSN:1007-0478/CN:42-1402/R]

卷:
第31卷
期数:
2024年05期
页码:
457-464
栏目:
论著
出版日期:
2024-10-20

文章信息/Info

Title:
The mechanism of nicotinic acetylcholine receptor α7 subunit regulating dopamine neurons in Parkinson's cell model
文章编号:
1007-0478(2024)05-0457-08
作者:
潘燕; 杨越; 丛丽娜; 童书杰
830011 乌鲁木齐,新疆医科大学第五附属医院神经内科
Author(s):
Pan Yan;  Yang Yue;  Cong Lina;  et al.
Department of Neurology, Fifth Affiliated Hospital of Xinjiang Medical University, Urumuqi 830011
关键词:
烟碱型乙酰胆碱α7亚基 人神经母细胞瘤细胞 帕金森病
Keywords:
Nicotinic acetylcholine receptors(nAChRs)α7 Human neuroblastoma cells Parkinson's disease
分类号:
R742.5
DOI:
10.3969/j.issn.1007-0478.2024.05.007
文献标志码:
A
摘要:
目的 分析烟碱型乙酰胆碱受体(Nicotinic acetylcholine receptors,nAChRs)α7对人神经母细胞瘤细胞(Human neuroblastoma cells,SH-SY5Y)增殖及凋亡的影响,阐明α7nAChR可以通过正向调控多巴胺(Dopamine,DA)神经元的表达来发挥其生物学功能,从而治疗帕金森病。方法 本实验通过选取 SH-SY5Y细胞系进行细胞培养、复苏、传代、制作帕金森病细胞模型以及细胞转染; 实验分组为对照组、模型组、α7nAChR过表达组、α7nAChR过表达空载组; 分别进行细胞增殖实验(Cell counting kit-8,CCK8)检测细胞活性; 流式细胞术检测细胞凋亡; 酶联免疫吸附测定(Enzyme-linked immuno sorbent assay,ELISA)检测多巴胺(DA)的表达水平; Western blot检测α7nAChR、磷酸化钙调素依赖性蛋白激酶2(Phosphorylated calmodulin kinaseⅡ,p-CAMKⅡ)、磷酸化细胞外信号调节激酶(Phosphorylated extracellular signal regulated kinase,p-ERK)、磷酸化Kirsten大鼠肉瘤病毒癌基因同源物(Phosphorylated Kirsten rats arcomaviral oncogene homolog,p-Ras)的蛋白表达水平; 免疫荧光检测酪氨酸羟化酶(Tyrosine hydroxylase,TH)、α7nAChR、α-突触核蛋白(Alpha synuclein,αSYN)的表达水平。结果 Model组α7nAChR的蛋白表达水平较Control组显著降低(P<0.05),而α7nAChR-OE组的蛋白表达水平较Model组显著升高(P<0.05)。CCK8表明,Model组细胞的增殖活性较Control组显著降低(P<0.05); α7nAChR-OE组细胞的增殖活性较Model组显著升高(P<0.05)。流式细胞术表明,Model组细胞的凋亡率较Control组显著升高(P<0.05); α7nAChR-OE组细胞的凋亡率较Model组显著降低(P<0.05)。Model组的αSYN水平较Control组显著升高(P<0.05); α7nAChR-OE组的αSYN水平较Model组显著降低(P<0.05)。Model组的TH,α7nAChR,DA水平较Control组显著降低(P<0.05); α7nAChR-OE组的TH,α7nAChR,DA水平较Model组显著升高(P<0.05)。Model组的Kirsten大鼠肉瘤病毒癌基因同源物(Kirsten rats arcomaviral oncogene homolog,KRAS),CAMK II,ERK的蛋白磷酸化水平较Control组显著升高(P<0.05); α7nAChR-OE组的KRAS,CAMK II,ERK的蛋白磷酸化水平较Model组显著降低(P<0.05)。结论 α7nAChR的高表达能促进帕金森病模型细胞的增殖、抑制凋亡,且α7nAChR 可以通过抑制Ca2+/CAMK/ERK通路活性来发挥神经保护作用,提高DA神经元表达,改善帕金森病的症状。
Abstract:
ObjectiveTo investigate the effects of nicotinic acetylcholine receptors(nAChRs)α7 on the proliferation and apoptosis of human neuroblastoma cells(SH-SY5Y), and to clarify that α7nAChR can exert its biological function by positively regulating the expression of dopamine(DA)neurons, so as to treat Parkinson's disease.Methods In this experiment, SH-SY5Y cell line was selected for cell culture, resuscitation, passage, production of Parkinson's cell model and cell transfection. The experimental subjects were divided into control group, model group, α 7nAChR overexpression group, and α 7nAChR overexpression no-load group. CCK8 was performed to detect cell activity. Flow cytometry was used to detect apoptosis. The expression of dopamine(DA)was detected by ELISA. The protein expressions of α7nAChR, p-CAMKⅡ, p-ERK and p-Ras were detected by Western Blot Analysis. The expressions of TH, α7nAChR and αSYN were detected by immunofluorescence.Results The protein expression level of α7nAChR in Model group was significantly lower than that in Control group(P<0.05). The protein expression level in α7nAChR-OE group was significantly higher than that in Model group(P<0.05). The results of CCK8 showed that the cell proliferation activity in Model group was significantly decreased compared with that in control group(P<0.05). The cell proliferation activity in α7nAChR-OE group was significantly higher than that in Model group, P<0.05. Flow cytometry showed that the apoptosis rate of cells in Model group was significantly higher than that in Control group(P<0.05). The apoptosis rate of α7nAChR-OE group was significantly decreased compared with that in Model group(P<0.05). The αSYN in Model group was significantly increased compared with that in Control group(P<0.05). The αSYN in α7nAChR-OE group was significantly decreased compared with that in Model group(P<0.05). The TH, α7nAChR and DA in Model group were significantly decreased compared with those in Control group(P<0.05). The TH, α7nAChR and DA in α7Nachr-OE group were significantly increased compared with those in Model group(P<0.05). The protein phosphorylation levels of KRAS, CAMK II and ERK in Model group were significantly increased compared with those in Control group(P<0.05). The protein phosphorylation levels of KRAS, CAMK II and ERK in α7nAChR-OE group were significantly decreased compared with those in Model group(P<0.05).Conclusion The high expression of α7nAChR can promote the proliferation and inhibition of the apoptosis in Parkinson's model cells. Besides, α7nAChR can play a neuroprotective role by inhibiting the activity of Ca2+/CAMK/ERK pathway, increasing the expression of DA neurons, and improving Parkinson's disease.

参考文献/References:

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备注/Memo

备注/Memo:
基金项目:新疆维吾尔自治区自然科学基金项目(编号为2022D01C570)
更新日期/Last Update: 2024-10-20