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J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (5): 487-492    DOI: 10.3785/j.issn.1008-9292.2018.10.07
    
G protein-coupled receptor 17 is involved in CoCl2-induced hypoxic injury in RGC-5 cells
LIN Kana1(),LIN Meili2,GU Yingfen1,ZHANG Shunguo1,HUANG Shiying1,*()
1. Department of Pharmacy, Shanghai Children's Medical Center Affiliated to School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China
2. Department of Pharmacy, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
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Abstract  

Objective: To investigate the effect of G protein-coupled receptor 17 (GPR17) on hypoxia injury in retinal ganglion cells in vitro. Methods: CoCl2 (400 μmol/L) was used to induce hypoxic injury in RGC-5 cells. The expression of GPR17 and the effect of GPR17 ligands were investigated, and the role of GPR17 in hypoxia injury was further studied by transfection of RGC-5 cells with GPR17 small interfering RNA (siRNA). The cell viability was determined by MTT and the cell apoptosis rate was detected by flow cytometry analysis. The expression of GPR17 mRNA was determined with RT-PCR. Results: mRNA expressions of GPR17 in RGC-5 cells with and without CoCl2 treatment were 0.36±0.05 and 0.26±0.08(P < 0.01). Compared with hypoxia without any treatment, pretreatment with GPR17 agonists (LTD4, UDP, UDP-G) significantly reduced cell viability (the survival rates of cells decreased by 29.6%, 31.8% and 33.9%, all P < 0.01), while the effect of GPR17 antagonist (cangrelor) was the opposite (the survival rates of cells increased by 33.2%, P < 0.01). Transfection with GPR17 SiRNA inhibited hypoxia-induced up-expression of GPR17 mRNA (P < 0.01)and reduced cell apoptosis[rates of cell apoptosis were(39.73±2.06)%, (42.50±3.64)% and (24.98±2.16)% for blank control, NC siRNA and GPR17 siRNA groups, P < 0.01]. Conclusion: GPR17 may mediate hypoxia injury in RGC-5 cells, while the knockdown of GPR17 can reduce the hypoxia injury.



Key wordsReceptors, G-protein-coupled/antagonists & inhibitors      Cobalt/pharmacology      Retinal ganglion cells/drug effects      Cell line      Anoxia/drug therapy      Apoptosis      Cells, cultured     
Received: 25 June 2018      Published: 23 January 2019
CLC:  R774  
Corresponding Authors: HUANG Shiying     E-mail: linkana@163.com;huangshiying@scmc.com.cn
Cite this article:

LIN Kana,LIN Meili,GU Yingfen,ZHANG Shunguo,HUANG Shiying. G protein-coupled receptor 17 is involved in CoCl2-induced hypoxic injury in RGC-5 cells. J Zhejiang Univ (Med Sci), 2018, 47(5): 487-492.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2018.10.07     OR     http://www.zjujournals.com/med/Y2018/V47/I5/487


G蛋白偶联受体17在视网膜神经节细胞缺氧损伤中的作用

目的: 探讨G蛋白偶联受体17(GPR17)在视网膜神经节细胞缺氧损伤中的作用。方法: 采用氯化钴(400 μmol/L)诱导RGC-5细胞化学缺氧损伤,观察GPR17的表达及GPR17配体的作用,并通过小干扰RNA(siRNA)降低GPR17的表达深入研究GPR17在细胞缺氧损伤中的作用。MTT法检测细胞存活率;流式细胞术检测细胞凋亡率;RT-PCR法检测GPR17 mRNA相对表达量。结果: 氯化钴处理后,细胞中GPR17 mRNA相对表达量由0.26±0.08上调至0.36±0.05(P < 0.01)。与未经GPR17配体预处理的缺氧细胞比较,GPR17激动剂尿苷5'-二磷酸二钠盐、尿苷5'-二磷酸葡萄糖二钠盐、LTD4预处理细胞的存活率分别降低了29.6%、31.8%、33.9%(均P < 0.01);而其拮抗剂坎格雷洛预处理细胞的存活率升高了33.2%(P < 0.01)。GPR17 siRNA处理可以抑制缺氧导致的GPR17 mRNA表达上调(P < 0.01),减少缺氧导致的细胞凋亡[未加siRNA、NC siRNA和GPR17 siRNA组细胞的凋亡率分别为(39.73±2.06)%,(42.50±3.64)%和(24.98±2.16)%,P < 0.01]。结论: GPR17介导了RGC-5细胞的缺氧损伤,抑制GPR17表达可减轻缺氧损伤。


关键词: 受体, G-蛋白偶联/拮抗剂和抑制剂,  钴/药理学,  视网膜神经节细胞/药物作用,  细胞系,  缺氧/药物疗法,  细胞凋亡,  细胞, 培养的 
引物名称 序列(5'→3')
GPR17 上游:AGACTGTGCAGACCAACCAC
下游:CCGCGGTAGTGAAGCACATA
β-actin 上游:TGACGTTGACATCCGTAAAG
下游:AAAGGGTGTAAAACGCAGCTC
Tab 1 RT-PCR primer sequences
Fig 1 Effect of hypoxia on the expression of GPR17 mRNA
Fig 2 Effect of GPR17 ligands on CoCl2-induced hypoxia in RGC-5 cells(n=6)
Fig 3 The effect of GPR17 siRNA on the expression of GPR17 mRNA under hypoxic conditions(n=4)
Fig 4 Effect of GPR17 siRNA on cell apoptosis under hypoxic conditions(n=4)
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