OBJECTIVE Alzheimer disease(AD) is the most common type of dementia and is featured by the accumulation of β-amyloid peptide(Aβ) in the brain. The Alpha 7 nicotinic acetylcholine receptor family(α7 nAChR) was widel...OBJECTIVE Alzheimer disease(AD) is the most common type of dementia and is featured by the accumulation of β-amyloid peptide(Aβ) in the brain. The Alpha 7 nicotinic acetylcholine receptor family(α7 nAChR) was widely considered to interact with that Aβ, mediate neuroprotection and improve cognitive performance. However, the mechanisms underlying these interactions remain elusive. The present study aimed to determine how this interaction contribute to AD pathology. METHODS In vitro model of AD(primary culture of mice hippocampus treated with Aβ) and in vivo, a mouse model of AD(APPswe/PSEN1 d E9 double transgenic mice, APP/PS1_DT mice) were used to study to the possible inter-action of α7 nAChR and Aβ in the pathogenesis of AD. In vitro experiments, the primary hippocampal neurons cell was exposed to Aβ1-42 peptides in combination with PNU. In vivo experiments, different drugs/operations was applied to APP/PS1_DT mice for setting up of the following groups: WP group, wild-type C57 mice treated with PNU(α7 nAChR specific agonist);AP group, APP/PS1_DT mice treated with PNU;APP/PS1 group, the APP/PS1_DT mice injected intraperitoneally with the same amount of normal saline for 5 d;Control group, wild-type C57 mice injected intraperitoneally with the same amount of normal saline for 5 d. A transmission electron microscope was used to observed the synaptic morphological changes of hippocampal neurons. Reverse transcription quantitative PCR(RT-q PCR) and Western blot analysis were used to detect the expression levels of synaptic-associated proteins(SYN, SNAP25 etc). The learning and memory abilities of mice were detected by Morris water maze. RESULTS In vitro, it was found that α7 nAChR acts as an anti-Aβ-induced synaptic injury to nerve cell by increased the expression of synaptic-associated proteins and attenuated apoptosis induced by Aβ oligomers. In vivo, α7 nAChR attenuated synaptic loss induced by Aβ1-42, reduced the deposition of Aβ1-42 in the hippocampus and maintained the integrity of synaptic structures in the hippocampus. Furthermore, in the Morris water maze test, α7 nAChR improved the learning and memory ability of the APP/PS1_DT mice. CONCLUSION Theα7 nAChR attenuate the toxic effect of Aβ in vivo and in vitro, eg reduced the deposition of Aβ in the hippocampus,prevented the synaptic loss, partially restored the expression levels of synaptic-associated proteins, and improved the learning and memory abilities of APP/PS1_DT mice. Our results also suggested that the α7 nAChR interacted with Aβby mediated by Ca M-Ca MKⅡ-CREB signalling pathway, which was imbalanced by deposition of Aβ.展开更多
Acetylcholine(ACh)regulates inflammation viaα7 nicotinic acetylcholine receptor(α7 nAChR).Acetylcholinesterase(AChE),an enzyme hydrolyzing ACh,is expressed in immune cells suggesting non-classical function in inflam...Acetylcholine(ACh)regulates inflammation viaα7 nicotinic acetylcholine receptor(α7 nAChR).Acetylcholinesterase(AChE),an enzyme hydrolyzing ACh,is expressed in immune cells suggesting non-classical function in inflammatory responses.Here,the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages.In lipopolysaccharide-induced inflammatory processes,AChE was upregulated by the binding of NF-κB onto the ACHE promotor.Conversely,the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration,which was in contrast to that of applied AChE inhibitors.AChEmt,a DNA construct without enzymatic activity,was adopted to identify the protein role of AChE in immune system.Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration.The co-localization ofα7 nAChR and AChE was found in macrophases,suggesting the potential interaction ofα7 nAChR and AChE.Besides,immunoprecipitation showed a close association ofα7 nAChR and AChE protein in cell membrane.Hence,the novel function of AChE in macrophage by interacting withα7 nAChR was determined.Together with hydrolysis of ACh,AChE plays a direct role in the regulation of inflammatory response.As such,AChE could serve as a novel target to treat age-related diseases by antiinflammatory responses.展开更多
【目的】观察青藤碱(sinomenine,SIN)对环氧化酶2(COX2)、α7烟碱型乙酰胆能受体(α7nAChR)和腺苷受体(A2A)表达变化的影响,探讨青藤碱对A549细胞增殖的抑制作用及机制。【方法】采用四甲基偶氮唑盐微量酶反应比色(MTT)法检测青藤碱和4...【目的】观察青藤碱(sinomenine,SIN)对环氧化酶2(COX2)、α7烟碱型乙酰胆能受体(α7nAChR)和腺苷受体(A2A)表达变化的影响,探讨青藤碱对A549细胞增殖的抑制作用及机制。【方法】采用四甲基偶氮唑盐微量酶反应比色(MTT)法检测青藤碱和4-甲基亚硝胺基-1-3-吡啶基-1-丁酮(NNK)对A549细胞增殖的影响;细胞划痕实验观察青藤碱和NNK对A549细胞迁移的影响;Western blot法观察青藤碱和NNK对A549细胞COX2蛋白表达的影响;RT-PCR和Western blot法观察青藤碱和NNK对A549细胞α7n ACh R、A2A表达的影响。【结果】NNK能促进增殖和迁移,青藤碱能抑制A549细胞增殖和迁移;NNK组COX2蛋白质水平增加,青藤碱组COX2蛋白质水平下降;NNK组α7n ACh R、A2A的表达增加,青藤碱组α7n ACh R、A2A表达下降。【结论】青藤碱能通过抑制COX2蛋白表达发挥抗A549细胞增殖和迁移作用;青藤碱对α7n ACh R和A2A受体的表达均有抑制作用。展开更多
文摘OBJECTIVE Alzheimer disease(AD) is the most common type of dementia and is featured by the accumulation of β-amyloid peptide(Aβ) in the brain. The Alpha 7 nicotinic acetylcholine receptor family(α7 nAChR) was widely considered to interact with that Aβ, mediate neuroprotection and improve cognitive performance. However, the mechanisms underlying these interactions remain elusive. The present study aimed to determine how this interaction contribute to AD pathology. METHODS In vitro model of AD(primary culture of mice hippocampus treated with Aβ) and in vivo, a mouse model of AD(APPswe/PSEN1 d E9 double transgenic mice, APP/PS1_DT mice) were used to study to the possible inter-action of α7 nAChR and Aβ in the pathogenesis of AD. In vitro experiments, the primary hippocampal neurons cell was exposed to Aβ1-42 peptides in combination with PNU. In vivo experiments, different drugs/operations was applied to APP/PS1_DT mice for setting up of the following groups: WP group, wild-type C57 mice treated with PNU(α7 nAChR specific agonist);AP group, APP/PS1_DT mice treated with PNU;APP/PS1 group, the APP/PS1_DT mice injected intraperitoneally with the same amount of normal saline for 5 d;Control group, wild-type C57 mice injected intraperitoneally with the same amount of normal saline for 5 d. A transmission electron microscope was used to observed the synaptic morphological changes of hippocampal neurons. Reverse transcription quantitative PCR(RT-q PCR) and Western blot analysis were used to detect the expression levels of synaptic-associated proteins(SYN, SNAP25 etc). The learning and memory abilities of mice were detected by Morris water maze. RESULTS In vitro, it was found that α7 nAChR acts as an anti-Aβ-induced synaptic injury to nerve cell by increased the expression of synaptic-associated proteins and attenuated apoptosis induced by Aβ oligomers. In vivo, α7 nAChR attenuated synaptic loss induced by Aβ1-42, reduced the deposition of Aβ1-42 in the hippocampus and maintained the integrity of synaptic structures in the hippocampus. Furthermore, in the Morris water maze test, α7 nAChR improved the learning and memory ability of the APP/PS1_DT mice. CONCLUSION Theα7 nAChR attenuate the toxic effect of Aβ in vivo and in vitro, eg reduced the deposition of Aβ in the hippocampus,prevented the synaptic loss, partially restored the expression levels of synaptic-associated proteins, and improved the learning and memory abilities of APP/PS1_DT mice. Our results also suggested that the α7 nAChR interacted with Aβby mediated by Ca M-Ca MKⅡ-CREB signalling pathway, which was imbalanced by deposition of Aβ.
基金supported by Shenzhen Science and Technology Committee Research Grant(JCYJ20170413173747440,ZDSYS 201707281432317,JCYJ20180306174903174,CKFW2016082916015476,China)China Post-doctoral Science Foundation(2019M653087)+3 种基金Zhongshan Municipal Bureau of Science and Technology(ZSST20SC03,China)Guangzhou Science and Technology Committee Research Grant(GZSTI16SC02 and GZSTI17SC02,China)Hong Kong RGC Theme-based Research Scheme(T13-607/12R,China)Hong Kong Innovation Technology Fund(UIM/340,UIM/385,ITS/500/18FP,TCPD/17e9,PD18SC01 and HMRF18SC06,China)
文摘Acetylcholine(ACh)regulates inflammation viaα7 nicotinic acetylcholine receptor(α7 nAChR).Acetylcholinesterase(AChE),an enzyme hydrolyzing ACh,is expressed in immune cells suggesting non-classical function in inflammatory responses.Here,the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages.In lipopolysaccharide-induced inflammatory processes,AChE was upregulated by the binding of NF-κB onto the ACHE promotor.Conversely,the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration,which was in contrast to that of applied AChE inhibitors.AChEmt,a DNA construct without enzymatic activity,was adopted to identify the protein role of AChE in immune system.Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration.The co-localization ofα7 nAChR and AChE was found in macrophases,suggesting the potential interaction ofα7 nAChR and AChE.Besides,immunoprecipitation showed a close association ofα7 nAChR and AChE protein in cell membrane.Hence,the novel function of AChE in macrophage by interacting withα7 nAChR was determined.Together with hydrolysis of ACh,AChE plays a direct role in the regulation of inflammatory response.As such,AChE could serve as a novel target to treat age-related diseases by antiinflammatory responses.
文摘【目的】观察青藤碱(sinomenine,SIN)对环氧化酶2(COX2)、α7烟碱型乙酰胆能受体(α7nAChR)和腺苷受体(A2A)表达变化的影响,探讨青藤碱对A549细胞增殖的抑制作用及机制。【方法】采用四甲基偶氮唑盐微量酶反应比色(MTT)法检测青藤碱和4-甲基亚硝胺基-1-3-吡啶基-1-丁酮(NNK)对A549细胞增殖的影响;细胞划痕实验观察青藤碱和NNK对A549细胞迁移的影响;Western blot法观察青藤碱和NNK对A549细胞COX2蛋白表达的影响;RT-PCR和Western blot法观察青藤碱和NNK对A549细胞α7n ACh R、A2A表达的影响。【结果】NNK能促进增殖和迁移,青藤碱能抑制A549细胞增殖和迁移;NNK组COX2蛋白质水平增加,青藤碱组COX2蛋白质水平下降;NNK组α7n ACh R、A2A的表达增加,青藤碱组α7n ACh R、A2A表达下降。【结论】青藤碱能通过抑制COX2蛋白表达发挥抗A549细胞增殖和迁移作用;青藤碱对α7n ACh R和A2A受体的表达均有抑制作用。
