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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (1): 106-112    DOI: 10.3724/zdxbyxb-2021-0044
原著     
瞬时受体电位 M2抑制剂 A10对缺糖缺氧后复糖复氧细胞的保护作用
黄卓群1(),余夏飞2,刘星宇2,马康3,黄敏华2,李芳芳2,杨巍2,*(),牛建国1,3
1. 宁夏医科大学宁夏颅脑疾病重点实验室，宁夏 银川 750004
2. 浙江大学基础医学院生物物理学系，浙江 杭州 310058
3. 宁夏医科大学基础医学院人体解剖与组织胚胎学系，宁夏 银川 750004
Protective effect of transient receptor potential melastatin 2 inhibitor A10 on oxygen glucose deprivation/reperfusion model
HUANG Zhuoqun1(),YU Xiafei2,LIU Xingyu2,MA Kang3,HUANG Minghua2,LI Fangfang2,YANG Wei2,*(),NIU Jianguo1,3
1. Laboratory of Brain，Ningxia Medical University，Yinchuan 750004，China;
2. Department of Biophysics，Zhejiang University School of Medicine，Hangzhou 310058，China;
3. Department of Human Anatomy，Histology and Embryology，School of Basic Medicine，Ningxia Medical University，Yinchuan 750004，China
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摘要：

目的:探讨瞬时受体电位M2（TRPM2）抑制剂A10对缺糖缺氧后复糖复氧（OGD/R）细胞模型的保护作用。 方法:采用SH-SY5Y细胞系制备OGD/R损伤模型。将细胞随机分为空白对照组、模型对照组和A10组。细胞计数试剂盒8检测细胞存活率；活性氧检测试剂盒检测细胞活性氧水平；四甲基罗丹明甲酯法检测线粒体膜电位；一步法TUNEL细胞凋亡检测试剂盒检测凋亡细胞数量；蛋白质印迹法测定cleaved caspase 3 蛋白表达。 结果:相对于3、20、30、50和 100?μmol/L， 10?μmol/L浓度的A10具有较低的细胞毒性及较好的通道活性抑制作用。与模型对照组比较，A10组活性氧水平降低（ P<0.05），线粒体膜电位降低程度改善（ P<0.05），凋亡细胞数减少（ P<0.05），凋亡相关蛋白cleaved caspase 3表达减少（ P<0.05）。 结论:A10可以通过抑制TRPM2通道功能、减少细胞外钙离子内流、降低细胞活性氧水平、稳定线粒体膜电位水平和减少细胞凋亡缓解OGD/R后细胞的损伤。

关键词： 瞬时受体电位M2通道;  缺糖缺氧/复糖复氧;  活性氧;  线粒体膜电位;  细胞凋亡;  Cleaved caspase 3;  SH-SY5Y细胞    
Abstract:

Objective:To investigate the effect of transient receptor potential melastatin 2 （TRPM2） inhibitor A10 on oxygen glucose deprivation/reperfusion （OGD/R） injury in SH-SY5Y cells． Methods:Human neuroblastoma SH-SY5Y cells were subject to OGD/R injury，and then were divided into blank control group，model control group and A10 group randomly. The cell survival rate was detected by cell counting kit 8 （CCK-8）; the level of cellular reactive oxygen species （ROS） was detected by reactive oxygen detection kit; the mitochondrial membrane potential was detected by tetramethylrhodamine （TMRM） method; the number of apoptotic cells was detected by TUNEL apoptosis assay kit; the protein expression level of cleaved caspase 3 was detected by Western blot． Results:Compared with 3，20，30，50， 100?μmol/L， 10?μmol/L?A10 has lower cytotoxicity and better inhibition effect on channel activity. Compared with the model control group，ROS level was reduced，the mitochondrial membrane potential was improved，the number of apoptosis cells was reduced ，and the expression of cleaved caspase 3 was significantly reduced in the A10 group（all P<0.05）. Conclusion: A10 can alleviate cell damage after OGD/R by inhibiting TRPM2 channel function，reducing extracellular calcium influx，reducing cell ROS levels，stabilizing mitochondrial membrane potential levels，and reducing apoptosis.

Key words: Transient receptor potential melastatin 2 channel    Oxygen glucose deprivation/reperfusion    Reactive oxygen species    Mitochondrial membrane potential    Apoptosis    Cleaved caspase 3    SH-SY5Y cells
收稿日期: 2020-11-28 出版日期: 2021-05-14
CLC:  R743.31  
基金资助: 宁夏回族自治区重点研发（东西部合作）项目（2019BFH02003）
通讯作者: 杨巍     E-mail: huangzhuoqun1985@126.com;yangwei@zju.edu.cn
作者简介: 黄卓群，主治医师，主要从事脑缺血与神经退行性疾病研究；E-mail：huangzhuoqun1985@126.com；https:// orcid.org/0000-0002-2642-9425
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引用本文:

黄卓群,余夏飞,刘星宇,马康,黄敏华,李芳芳,杨巍,牛建国. 瞬时受体电位 M2抑制剂 A10对缺糖缺氧后复糖复氧细胞的保护作用[J]. 浙江大学学报（医学版）, 2021, 50(1): 106-112.

HUANG Zhuoqun,YU Xiafei,LIU Xingyu,MA Kang,HUANG Minghua,LI Fangfang,YANG Wei,NIU Jianguo. Protective effect of transient receptor potential melastatin 2 inhibitor A10 on oxygen glucose deprivation/reperfusion model. J Zhejiang Univ (Med Sci), 2021, 50(1): 106-112.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0044    或    http://www.zjujournals.com/med/CN/Y2021/V50/I1/106

图 1  不同浓度瞬时受体电位M2抑制剂A10对SH-SY5Y细胞的药物毒性 与浓度比较，<0.01.

组别

n

细胞存活率

活性氧水平

线粒体膜电位

相对细胞凋亡水平

cleaved caspase 3蛋白相对表达量

空白对照组

3

101.5±5.5

100.0±4.5

100.0±5.0

100.0±20.8

100.0

模型对照组

3

60.1±6.2 *

187.6±4.9 *

45.0±15.0 *

452.0±130.7 *

125.6±12.0 *

A10组

3

74.5±8.2 *#

125.1±11.5 *#

70.0±7.3 *#

166.2±51.9 #

93.4±12.8 #

F值

—

64.94

104.10

30.92

15.59

14.11

P值

—

<0.05

<0.05

<0.05

<0.05

<0.05
表 1  缺糖缺氧复糖复氧处理后TRPM2抑制剂A10对细胞存活率、活性氧水平、线粒体膜电位、细胞凋亡数以及cleaved caspase 3蛋白表达水平的影响
图 2  缺糖缺氧复糖复氧处理后A10对细胞活性氧水平的影响 A：空白对照组细胞的绿色荧光强度弱；B：模型对照组细胞的绿色荧光强度强，活性氧水平升高；C：A10组细胞的绿色荧光强度较模型对照组减弱. 标尺
图 3  缺糖缺氧复糖复氧处理后A10对细胞线粒体膜电位变化的影响 A：空白对照组细胞红色荧光强度强；B：模型对照组细胞红色荧光强度弱，线粒体膜电位降低；C：A10组细胞红色荧光强度较模型对照组增强. 标尺
图 4  缺糖缺氧复糖复氧处理后A10对细胞凋亡的影响 A：空白对照组，TUNEL阳性细胞数较少；B：模型对照组，TUNEL阳性细胞数较空白对照组增多；C：A10组TUNEL阳性细胞数较模型对照组减少. 标尺
图 5  缺糖缺氧复糖复氧处理后A10对凋亡相关蛋白cleaved caspase 3表达的影响
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