经典瞬时受体电位通道蛋白在β淀粉样蛋白诱导的阿尔茨海默病小鼠海马区的表达
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摘要
目的探讨经典瞬时受体电位(TRPC)通道蛋白在阿尔茨海默病(AD)小鼠海马区中的表达情况。方法将36只ICR小鼠随机分为AD组和对照组,每组18只。β淀粉样蛋白(Aβ)1-42侧脑室注射制作AD小鼠模型。Morris水迷宫试验测定小鼠学习记忆能力。逆转录聚合酶链反应(RT-PCR)检测海马区TRPC1~TRPC7 m RNA的表达。分别采用免疫荧光和Western blotting检测海马区的TRPC4蛋白表达。结果 AD组小鼠水迷宫测试的逃避潜伏期较对照组明显延长(P<0.01),在目标象限停留时间明显缩短(P<0.01),穿越平台次数明显减少(P<0.01)。RT-PCR结果显示,在AD组及对照组TRPC1~TRPC7 m RNA均有不同程度的表达,其中AD组TRPC4 m RNA表达较对照组明显增加(P<0.01)。免疫荧光显示TRPC4表达在海马区的神经细胞膜上,且与对照组相比AD组TRPC4表达增加。Western blotting结果显示AD组TRPC4蛋白表达较对照组增加(P<0.05)。结论侧脑室注射Aβ1-42寡聚体后,小鼠海马区TRPC4蛋白表达含量增加,提示TRPC4可能参与到钙稳态失调引起的AD的发病机制中。
Objective To investigate the protein expression of the canonical transient receptor potential(TRPC)channel in the hippocampus of amyloid β protein(Aβ)-induced Alzheimer's disease(AD)mice. Methods A total of 36 ICR mice were randomly divided into AD group and control group,with 18 rats in each group. AD mice models were established by Aβ 1-42 microinjection into the lateral intracerebroventricular.Learning and memory abilities of the mice were determined using Morris water maze. All TRPC1-TRPC7 m RNA levels in the hippocampus of the mice were detected using reverse transcriptase polymerase chain reaction(RT-PCR). Hippocampal TRPC4 protein expression was examined using immunofluorescence and Western blotting methods. Results Water maze test results showed that the escape latency of AD group were significantly longer than that of the control group(P < 0.01),and that the target quadrant occupancy of AD group was significantly shortened(P < 0.01)and the frequency of platform crossing of AD group was significantly reduced(P < 0.01). RT-PCR results showed that all TRPC(TRPC1-TRPC7)m RNA were expressed in the hippocampal of both AD group and control group. Among these channels,only TRPC4 m RNA levels of AD group was higher than that of the control group(P < 0.01). Immunofluorescence images showed that TRPC4 expressed on the membrane of neurons and the intensities of the immunofluorescence of TRPC4 in AD group were stronger than that of control group. Western blotting results showed that the TRPC4 protein expression of AD group was higher than that of control group(P < 0.05). Conclusion The increase of TRPC4 protein expression in the hippocampus of mice after intracerebroventricular injection of Aβ1-42 oligomers suggests that TRPC4 may be involved in the pathogenesis of AD induced by calcium homeostasis.
Objective To investigate the protein expression of the canonical transient receptor potential(TRPC)channel in the hippocampus of amyloid β protein(Aβ)-induced Alzheimer's disease(AD)mice. Methods A total of 36 ICR mice were randomly divided into AD group and control group,with 18 rats in each group. AD mice models were established by Aβ 1-42 microinjection into the lateral intracerebroventricular.Learning and memory abilities of the mice were determined using Morris water maze. All TRPC1-TRPC7 m RNA levels in the hippocampus of the mice were detected using reverse transcriptase polymerase chain reaction(RT-PCR). Hippocampal TRPC4 protein expression was examined using immunofluorescence and Western blotting methods. Results Water maze test results showed that the escape latency of AD group were significantly longer than that of the control group(P < 0.01),and that the target quadrant occupancy of AD group was significantly shortened(P < 0.01)and the frequency of platform crossing of AD group was significantly reduced(P < 0.01). RT-PCR results showed that all TRPC(TRPC1-TRPC7)m RNA were expressed in the hippocampal of both AD group and control group. Among these channels,only TRPC4 m RNA levels of AD group was higher than that of the control group(P < 0.01). Immunofluorescence images showed that TRPC4 expressed on the membrane of neurons and the intensities of the immunofluorescence of TRPC4 in AD group were stronger than that of control group. Western blotting results showed that the TRPC4 protein expression of AD group was higher than that of control group(P < 0.05). Conclusion The increase of TRPC4 protein expression in the hippocampus of mice after intracerebroventricular injection of Aβ1-42 oligomers suggests that TRPC4 may be involved in the pathogenesis of AD induced by calcium homeostasis.
引文
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[13]贺细菊,廖燕宏.瞬时受体电位通道在神经元中的研究进展[J].湖北医药学院学报,2014,33(1):81-84.DOI:10.13819/j.issn.1006-9674.2014.01.207.
[2]MAIA LF,KAESER SA,REICHWALD J,et al.Increased CSF Aβduring the very early phase of cerebral Aβdeposition in mouse models[J].EMBO Mol Med,2015,7(7):895-903.DOI:10.15252/emmm.201505026.
[3]WISNIEWSKI T,GONI F.Immunotherapeutic approaches for Alzheimer’s disease[J].Neuron,2015,85(6):1162-1176.DOI:10.1016/j.neuron.2014.12.064.
[4]SELVARAJ S,SUN Y,SINGH BB.TRPC channels and their implication in neurological diseases[J].CNS Neurol Disord Drug Targets,2010,9(1):94-104.DOI:10.2174/187152710790966650.
[5]YAMAMOTO S,WAJIMA T,HARA Y,et al.Transient receptor potential channels in Alzheimer’s disease[J].Biochim Biophys Acta,2007,1772(8):958-967.DOI:10.1016/j.bbadis.2007.03.006.
[6]苏秋香,张丽艳,汲坤,等.瞬时受体电位通道蛋白的跨膜片段分析[J].中国医科大学学报,2016,45(7):610-615.DOI:10.12007/j.issn.0258-4646.2016.07.008
[7]MORELLI MB,AMANTINI C,LIBERATI S,et al.TRP channels:new potential therapeutic approaches in CNS neuropathies[J].CNS Neurol Disord Drug Targets,2013,12(2):274-293.DOI:10.2174/18715273113129990056.
[8]高艳琴,高慧,周正怡,等.大鼠局灶性脑缺血再灌注损伤后纹状体和海马区瞬时受体电位通道蛋白4的表达增加[J].生理学报,2004,56(2):153-157.DOI:10.3321/j.issn.0371-0874.2004.02.005.
[9]OBUKHOV AG,NOWYCKY MC.TRPC4 can be activated by Gprotein-coupled receptors and provides sufficient Ca(2+)to trigger exocytosis in neuroendocrine cells[J].J Biol Chem,2002,277(18):16172-16178.DOI:10.1074/jbc.M111664200.
[10]MARAMBAUD P,DRESES-WERRINGLOVER U,VINDTDEUX V.Calcium signaling in neurodegeneration[J].Mol Neurodegener,2009,4:20.DOI:10.1186/1750-1326-4-20.
[11]CAHALAN MD.STIMulating store-operated Ca(2+)entry[J].Nat Cell Biol,2009,11(6):669-677.DOI:10.1038/ncb0609-669.
[12]PHELAN KD,MOCK MM,KRETZ O,et al.Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration[J].Mol Pharmacol,2012,81(3):384-392.DOI:10.1124/mol.111.075341.
[13]贺细菊,廖燕宏.瞬时受体电位通道在神经元中的研究进展[J].湖北医药学院学报,2014,33(1):81-84.DOI:10.13819/j.issn.1006-9674.2014.01.207.