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

The mechanism of nicotinic acetylcholine receptorα7 subunit regulating dopamine neurons in Parkinson's cell model

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Ca^(2+)/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。

Objective To investigate the effects of nicotinic acetylcholine receptors(nAChRs)α7 on the proliferation and apoptosis of human neuroblastoma cells(SH-SY5Y),and to clarify thatα7nAChR 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,α7nAChR overexpression group,andα7nAChR 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 ofα7nAChR,p-CAMKⅡ,p-ERK and p-Ras were detected by Western Blot Analysis.The expressions of TH,α7nAChR andαSYN were detected by immunofluorescence.Results The protein expression level ofα7nAChR in Model group was significantly lower than that in Control group(P<0.05).The protein expression level inα7nAChR-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 inα7nAChR-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 ofα7nAChR-OE group was significantly decreased compared with that in Model group(P<0.05).TheαSYN in Model group was significantly increased compared with that in Control group(P<0.05).TheαSYN inα7nAChR-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 inα7Nachr-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 inα7nAChR-OE group were significantly decreased compared with those in Model group(P<0.05).Conclusion The high expression ofα7nAChR 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 Ca^(2+)/CAMK/ERK pathway,increasing the expression of DA neurons,and improving Parkinson's disease.