NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

doi:10.12047/j.cjap.5601.2017.123

中国应用生理学杂志 ›› 2017, Vol. 33 ›› Issue (6): 519-523.doi: 10.12047/j.cjap.5601.2017.123

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

马洁¹, 郭康², 吕林亚³, 李新娟¹, 王璐¹, 魏林郁¹, 赵红岗¹, 李东亮¹

1. 新乡医学院生理学与神经生物学教研室, 河南新乡 453003;
2. 新乡医学院第三附属医院肿瘤内科, 河南新乡 453003;
3. 新乡医学院第三附属医院神经外科, 河南新乡 453003

收稿日期:2017-01-16 修回日期:2017-07-20 出版日期:2017-11-28 发布日期:2018-06-19
基金资助:
国家自然科学基金资助项目（81271376，81371346）；河南省高等学校重点科研项目计划（16A310011，16A310013）

Effect of NaHS on ATP-induced P2X receptor expression in rat microglia

MA Jie¹, GUO Kang², LV Lin-ya³, LI Xin-juan¹, WANG Lu¹, WEI Lin-yu¹, ZHAO Hong-gang¹, LI Dong-liang¹

1. Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang 453003, China;
2. Department of Internal Medicine-Oncology of the Third Affiliated Hospital, Xinxiang Medical University, Xinxiang 453003, China;
3. Department of Neurosurgery of the Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, China

Received:2017-01-16 Revised:2017-07-20 Online:2017-11-28 Published:2018-06-19
Contact: 李东亮,Tel:0373-3029104;E-mail:xyldl8@126.com E-mail:xyldl8@126.com
Supported by:
国家自然科学基金资助项目（81271376，81371346）；河南省高等学校重点科研项目计划（16A310011，16A310013）

摘要/Abstract

摘要： 目的：观察硫化氢（H₂S）供体硫氢化钠（NaHS）对ATP致伤的大鼠小胶质细胞细胞活力、细胞膜通透性及P2X7受体表达的影响。方法：实验取对数期形态结构及生长分化良好的大鼠小胶质细胞，随机分4组，每组设3个复孔。①正常对照组：常规培养，不进行ATP处理。②ATP组：接种细胞24 h后ATP处理。③NaHS+ATP组：NaHS预先孵育30 min后再用ATP处理，并且NaHS始终存在于反应体系中。④KN-62（P2X₇受体阻断剂）+ATP组：KN-62预先孵育30 min，其余同NaHS+ATP组。MTT检测各组细胞活力，荧光染料YO-PRO-1检测各组相对荧光单位（RFU）反映膜的通透性，Western blot检测各组P2X₇受体表达水平。结果：①与对照组相比，不同浓度的ATP （1、3、5、10 mmol/L）作用3 h均可明显降低大鼠小胶质细胞活力，NaHS （200 μmol/L）干预后大鼠小胶质细胞活力较ATP组明显增加（P<0.01），但NaHS达400 μmol/L浓度时，其保护作用未进一步增加。②随着ATP浓度的增加，大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加，NaHS预处理可明显减少细胞对YO-PRO-1的摄取（P<0.01）。③ATP （3 mmol/L）能上调P2X₇受体蛋白表达水平，而NaHS （200 μmol/L）预孵育则可明显抑制ATP引起的P2X₇受体蛋白表达的上调（P<0.01）。结论：NaHS可减少ATP致伤的大鼠小胶质细胞的P2X₇受体表达、降低通透性、增加细胞活力，提示调控P2X₇受体的表达和功能可能是H₂S神经保护作用的重要环节。

关键词: 三磷酸腺苷, 嘌呤能P2X7受体, 大鼠小胶质细胞, NaHS

Abstract: Objective: To observed the effect of sodium hydrosulphide (NaHS), a donor of H₂S on the cell viability,the membrane permeability and the expression of P2X₇ receptor induced by adenosine triphosphate(ATP) in rat microglia.Methods: Rat microglia in logarithmic growth phase was randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 hours. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, the others were as the same as NaHS+ATP group. The cell viability was detected by MTT. Fluorescent dyes YO-PRO-1 was used to observe the membrane permeability. The expression of P2X7 receptor was examined by immunofluorescence staining.Results: ① Compared with control group, the cell viability dropped after treatment with ATP (1、3、5、10 mmol/L) for 3 hours. When pre-incubation with NaHS(200 μmol/L), the cell viability was apparently higher than that of ATP alone group(P<0.01), while 400 μmol/L had no further beneficial.②The YO-PRO-1 fluorescence intensity was obviously elevated by ATP in rat microglia, but this effect was counteracted by NaHS pretreatment (P<0.01). ③ The expression of P2X7 receptor protein was significantly increased after ATP(3 mmol/L) for 3 h. While the expression upregulation of P2X7 receptor protein induced by ATP was significantly counteracted by pretreating with NaHS(200 μmol/L) (P<0.01).Conclusion: NaHS could reduce the expression of P2X7 receptor, decrease membrane permeability, and increase the cell viability in rat microglia injured by ATP. So the cytoprotection of hydrogen sulfide may be related to the expression and function of P2X7 receptor.

Key words: adenosine triphosphate, purinergic P2X7 receptor, rat microglia, hydrogen sulfide

马洁, 郭康, 吕林亚, 李新娟, 王璐, 魏林郁, 赵红岗, 李东亮. NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响[J]. 中国应用生理学杂志, 2017, 33(6): 519-523.

MA Jie, GUO Kang, LV Lin-ya, LI Xin-juan, WANG Lu, WEI Lin-yu, ZHAO Hong-gang, LI Dong-liang. Effect of NaHS on ATP-induced P2X receptor expression in rat microglia[J]. CJAP, 2017, 33(6): 519-523.

参考文献

[1] Monif M, Reid CA, Powell KL, et al. The P2X7 receptor drives microgl ial acti vation and proliferation:a trophic role for P2X7R pore[J]. J Neurosci, 2009, 29(12):3781-3791.
[2] 魏林郁, 卢娜, 孟莉, 等. 人P2X7真核表达载体的构建及稳定转染细胞株的建立[J]. 中国应用生理学杂志, 2016, 32(5):471-475.
[3] Shiratori M, Tozaki-Saitoh H, Yoshitake M, et al. P2X7 receptor a ctivation induces CXCL2 production in microglia through NFAT and PKC/MAPK pathways[J]. J Neurochem, 2010, 114(3):810-819.
[4] Franceschini A, Capece M, Chiozzi P, et al. The P2X7 receptor direc tly inter acts with the NLRP3 inflammasome scaffold protein[J]. FASEB J, 2015, 29(6):2450-2461.
[5] Zarjou A, Agarwal A. ATP as a death factor:purinergic signaling in renal epithelial-fibroblast cross talk[J]. Am J Physiol, 2011, 300(1):60-61.
[6] Le Feuvre R, Brough D, Rothwell N. Extracellular ATP and P2X7 receptors in neurodegeneration[J]. Eur J Pharmacol, 2002, 7(447):261-269.
[7] Bernardino L, Balosso S, Ravizza T, et al. Inflammatory events in hippocampal slice cultures prime neuronal susceptibility to excitotoxic injury:A crucial role of P2X7 receptor mediated IL-1beta release[J]. J Neurochem, 2008, 106(1):271-280.
[8] Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator[J]. J Neurosci, 1996, 16(3):1066-1071.
[9] Caseley EA, Muench SP, Baldwin SA, et al. Docking of competi tive inhibitors to the P2X7 receptor family reveals key differences responsible for changes in response between rat and human[J]. Bioorg Med Chem Lett, 2015, 25(16):3164-3167.
[10] 杨锐, 贾强, 刘小粉, 等. 硫化氢对大鼠糖尿病心肌病氧化应激及内质网应激的影响[J]. 中国应用生理学杂志, 2016, 32(1):8-12.
[11] 马洁, 沈慧, 王璐, 等. 硫化氢保护被ATP损伤的PC12细胞[J]. 中国病理生理杂志, 2015, 31(7):1231-1236.
[12] 马洁, 王娇娇, 王璐, 等. H₂S抑制ATP诱导的大鼠小胶质细胞活化及IL-1β的释放[J]. 中国病理生理杂志, 2016, 32(8):1408-1412.
[13] Costa-Junior HM, Sarmento Vieira F, Coutinho-Silva R. C terminus of the P2X7 receptor:treasure hunting[J]. Purinergic Signal, 2011, 7(1):7-19.
[14] Volonté C, Apolloni S, Skaper SD, et al. P2X7 receptors:c hannels, pores and more[J]. CNS Neurol Disord Drug Targets, 2012, 11(6):705-721.

NaHS对ATP诱导大鼠小胶质细胞P2X受体表达的影响

Effect of NaHS on ATP-induced P2X receptor expression in rat microglia

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