烟碱通过N1受体促进热应激对RAW264.7细胞HSP70的诱导表达

中国临床药理学与治疗学 ›› 2017, Vol. 22 ›› Issue (2): 124-131.

烟碱通过N1受体促进热应激对RAW264.7细胞HSP70的诱导表达

谌崇峰^1,3，杨怀才²，涂自智³

¹暨南大学附属第一医院儿科，广州 510632，广东； ²清远市妇幼保健院外科，清远 511500，广东； ³中南大学湘雅医学院病理学与病理生理学教研室，长沙 410078，湖南

收稿日期:2016-09-06 修回日期:2017-01-10 出版日期:2017-02-26 发布日期:2017-03-02
作者简介:谌崇峰，女，博士，主治医师，研究方向：小儿重症医学及小儿神经。 Tel：020-38688838 E-mail:chenchfeng66@126.com

Nicotine enhanced the inducible expression of HSP70 under heat stress through N1 receptor in RAW264.7 macrophages

CHEN Chongfeng ^{1, 3}, YANG Huaicai ², TU Zizhi ³

¹Department of Pediatrics, the First Affiliated Hospital of Jinan University, Guangzhou 510632, Guangdong, China; ²Department of Surgery, Maternal and Child Health Hospital of Qingyuan City，Qingyuan 511500, Guangdong, China;³ Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha 410078, Hunan, China

Received:2016-09-06 Revised:2017-01-10 Online:2017-02-26 Published:2017-03-02

摘要/Abstract

摘要：

目的:本研究探讨在巨噬细胞中，低剂量烟碱联合温和热应激对热休克因子1（heat shock factor 1,HSF1）和热休克蛋白70（heat shock proteins70,HSP70）表达的影响，并揭示其受体机制。方法:用低剂量烟碱（浓度1 mmol/L）联合温和热应激（39 ℃）刺激RAW264.7巨噬细胞，采用Western-blot观察HSP70的表达，免疫荧光细胞化学观察HSF1的入核，凝胶滞留实验（EMSA）观察HSF1与热休克元件（heat shock element,HSE）的结合；接着用不同的阻断剂预处理RAW264.7巨噬细胞，再给予烟碱和热应激刺激，观察HSP70的表达及HSF1的入核。结果: 单纯低剂量烟碱（浓度1 mmol/L）刺激不能诱导HSP70的表达，但HSF1入核明显增多，HSF1与HSE的结合增强；在39 ℃时，再加入烟碱能增强HSP70的表达、HSF1的入核、HSF1与HSE的结合；六烃季铵预处理，HSF1的入核较单纯烟碱处理明显减少，且能明显阻断烟碱在39 ℃时诱导的HSP70表达的作用。结论:低剂量烟碱（浓度1 mmol/L）刺激可以促进HSF1入核并与HSE结合，但不能诱导HSP70的表达；烟碱通过N1受体诱导HSF1入核并促进温和热应激（39 ℃）对RAW264.7巨噬细胞中HSP70的诱导表达。

关键词: 烟碱, 热休克因子1, 热休克蛋白70, N1受体

Abstract:

AIM: To explore whether low doses of nicotine enhances the HSF1 nuclear translocation and expression of HSP70 in the RAW264.7 macrophages treated under lower heat stress (<40 ℃), and to uncover its receptor mechanism. METHODS:After stimulating with nicotine at doses of 1 mmol/L and its antagonists prior to nicotine treatment at 37 ℃ or 39 ℃, the expression of HSP70 in RAW264.7 macrophages was detected by Western-blot, translocation of HSF1 was detected by immunofluorescence and the combination of HSF1and heat shock element (HSE) was detected by EMSA, respectively. RESULTS:Western-blot showed that nicotine (1 mmol/L) alone could not induce HSPs expression, but was able to enhance the HSP70 expression at 39 ℃ in RAW246.7. Immunofluorescence showed nicotine (1 mmol/L) was able to induce translocation of HSF1 from cytoplasm to nucleus, which was blocked by the pre-treating with hexamethonium apparently; EMSA showed that nicotine (1 mmol/L) could promote the binding of HSF1 to HSE at 37℃. Furthermore, nicotine (1 mmol/L) induced HSF1 binding to HSE greatly at 39℃. CONCLUSION: Nicotine (1 mmol/L) alone can induce HSF1 nuclear translocation and its DNA-binding activity to HSE, but not the expression of HSP70; nicotine (1 mmol/L) can enhance the expression of HSP70 under mild heat stress(39℃) through N1-receptor in the RAW264.7 macrophages.

Key words: nicotine, HSF1, HSP70, N1 receptor

中图分类号:

R965.2

谌崇峰，杨怀才，涂自智. 烟碱通过N1受体促进热应激对RAW264.7细胞HSP70的诱导表达[J]. 中国临床药理学与治疗学, 2017, 22(2): 124-131.

CHEN Chongfeng, YANG Huaicai, TU Zizhi. Nicotine enhanced the inducible expression of HSP70 under heat stress through N1 receptor in RAW264.7 macrophages[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2017, 22(2): 124-131.

参考文献

［1］Xiao XZ, Benjamin IJ. Stress-response proteins in cardiovascular disease［J］. Am J Hum Genet, 1999, 64(3):685-690.
［2］Struzik J, Szulc-Dabrowska L, Niemiattowski M.Participation of heat shock proteins in modulation of NF-кB transcription factor activation during bacterial infections［J］.Postepy Hig Med Dosw,2015, 2(69):969-977.
［3］Singh IS, He JR, Calderwood S,et al.A high affinity HSF-1 binding site in the 5'-untranslated region of the murine tumor necrosis factor-alpha gene is a transcriptional repressor［J］.J Biol Chem, 2002, 277(7):4981-4988.
［4］Xie Y, Chen C, Stevenson MA,et al.Heat shock factor 1 represses transcription of the IL-1beta gene through physical interaction with the nuclear factor of interleukin 6［J］.J Biol Chem, 2002, 277(14):11802-11810.
［5］Takii R, Inouye S, Fujimoto M, et al.Heat shock transcription factor 1 inhibits expression of IL-6 through activating transcription factor 3［J］.J Immunol, 2010, 184(2):1041-1048.
［6］Bouchier-Hayes L, McBride S, Geelen CM,et al.Fas ligand gene expression is directly regulated by stress-inducible heat shock transcription factor-1［J］.Cell Death Differ,2010, 17(6):1034-1046.
［7］Zhang L, Yang M, Wang Q, et al.HSF1 regulates expression of G-CSF through the binding element for NF-IL6/CCAAT enhancer binding protein beta［J］.Mol Cell Biochem, 2011, 352(1-2):11-17.
［8］George M,Esther C, Elizabeh A.Mammalian Stress Proteins HSP70 and HSP28 Coinduced by Nicotine and either Ethanol or Heat［J］.Molecul and Cellular Biology,1991, 11(12):6034-6040.
［9］王宇萍, 蒋建东.热休克蛋白70的结构和功能［J］.中国细胞生物学学报,2010,32(2):305-313.
［10］MorimotoRI.Cells in stress: transcriptional activation of heat shock genes［J］. Science,1993,259(5100):1409-1410.
［11］Xia W, Voellmy R. Hyperphosphorylation of heat shock transcripttion factor 1 is correlated with transcriptional competence and slow dissociation of active factor trimers［J］. J Biol Chem, 1997, 272(7):4094-4102.
［12］Jurivich DA, Pachetti C,Qiu L,et al. Salicylate triggers heat shock factor differently than heat［J］. J Biol Chem, 1995, 270(41):24489-24495.
［13］Cotto JJ, Kline M, Morimoto RI. Activation of heat shock factor 1 DNA binding precedes stress-induced serine phosphorylation. Evidence for a multistep pathway of regulation［J］. J Biol Chem, 1996, 271(7):3355-3358.
［14］Toumy G, Frank B, William SL,et al. Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress［J］. BMC Biochemistry, 2005, 6:1-4.
［15］Nagy E, Balogi Z, Gombos I, et al. Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line［J］. Proc Nat Acad Sci USA,2007, 104(19):7945-7950.
［16］Bencherif M, Lippiello PM, Lucas R, et al.Alpha7 nicotinic receptors as novel therapeutic targets for inflammation-based diseases［J］.Cell Mol LifeSci, 2011, 68(6):931-949.
［17］杨义, 杨缙.加兰他敏减轻脓毒症炎症反应作用的研究［J］. 中国临床药理学与治疗学, 2015, 20(11):1217-1220.
［18］刘卫, 郑建全. 美加明和六烃季铵在交感神经元烟碱受体上作用位点的差异［J］. 生理学报, 2002, 54(6):497-500.
［19］Chen S, Zuo X, Yang M, et al. Severe multiple organ injury in HSF1 knockout mice induced by lipopolysaccharide is associated with an increase in neutrophil infiltration and surface expression of adhesion molecules［J］.J Leukoc Biol, 2012,92(4):851-857.
［20］Tong Z, Jiang B, Zhang L, et al. HSF-1 is involved in attenuating the release of inflammatory cytokines induced by LPS through regulating autophagy［J］.Shock, 2014 ,41(5):449-453.
［21］Naddafi F, Reza Haidari M, Azizi G, et al.Novel therapeutic approach by nicotine in experimental model of multiple sclerosis［J］.Innov Clin Neurosci, 2013, 10(4):20-25.
［22］Tracey KJ.Physiology and immunology of the cholinergic antiinflammatory pathway［J］.J Clin Invest, 2007, 117(2):289-296.
［23］Van Dijk JP, Madretsma GS, Keuskamp ZJ,et al.Nicotine inhibits cytokine synthesis by mouse colonic mucosa［J］.Eur J Pharmacol, 1995, 278(1):R11-R12.
［24］Yeboah MM, Xue X, Javdan M, et al.Nicotinic acetylcholine receptor expression and regulation in the rat kidney after ischemia-reperfusion injury［J］.Am J Physiol Renal Physiol, 2008, 295(3):F654-F661.
［25］Bao J, Liu Y, Yang J, et al.Nicotine inhibits LPS-induced cytokine production and leukocyte infiltration in rat placenta［J］.Placenta, 2016, 39:77-83.

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