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J Zhejiang Univ (Med Sci)  2021, Vol. 50 Issue (1): 106-112    DOI: 10.3724/zdxbyxb-2021-0044
    
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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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 wordsTransient receptor potential melastatin 2 channel      Oxygen glucose deprivation/reperfusion      Reactive oxygen species      Mitochondrial membrane potential      Apoptosis      Cleaved caspase 3      SH-SY5Y cells     
Received: 28 November 2020      Published: 14 May 2021
CLC:  R743.31  
  R743.31  
  A  
Corresponding Authors: YANG Wei     E-mail: huangzhuoqun1985@126.com;yangwei@zju.edu.cn
Cite this article:

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.

URL:

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


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

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 
Figure 1 Toxic effect of TRPM2 inhibitor A10 at different concentrations on SH-SY5Y cells

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

64.94

104.10

30.92

15.59

14.11

P

<0.05

<0.05

<0.05

<0.05

<0.05

Table 1 Effects of TRPM2 inhibitor A10 on the survival rate,reactive oxygen species level,mitochondrial membrane potential,cell apoptosis number,and cleaved caspase 3 protein level of cells after OGD/R treatment
Figure 2 Effect of A10 on reactive oxygen species levels of cells after OGD/R treatment
Figure 3 Effect of A10 on the mitochondrial membrane potential of cells after OGD/R treatment
Figure 4 Effect of A10 on the apoptosis of cells after OGD/R treatment
Figure 5 Effects of A10 on the expression of cleaved caspase 3 in cells after OGD/R treatment
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