中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (11): 2154-2161.doi: 10.4103/1673-5374.282271

• 原著:退行性病与再生 • 上一篇    下一篇

抑制TRPM2表达可改善氧糖剥夺PC12细胞损伤和NLRP3炎性体的激活

  

  • 出版日期:2020-11-15 发布日期:2020-08-23
  • 基金资助:
    国家自然科学基金(81671532,81771625);江苏省医学重点学科项目(ZDXKA2016013);江苏省医学青年人才项目(QNRC2016758);江苏省妇女儿童健康研究项目(F201750;苏州市公共卫生技术项目(SYS201765);苏州市儿科临床中心资助项目(szx201504)。

Knocking down TRPM2 expression reduces cell injury and NLRP3 inflammasome activation in PC12 cells subjected to oxygen-glucose deprivation

Tao Pan 1, Qiu-Jiao Zhu 2, Li-Xiao Xu 3 , Xin Ding 1 , Jian-Qin Li 4 , Bin Sun 1 , Jun Hua 2, Xing Feng 1   

  1. 1 Department of Neonatology, Children’s Hospital Affiliated to Suzhou University, Suzhou, Jiangsu Province, China
    2 Department of Critical Care Medicine, Children’s Hospital Affiliated to Suzhou University, Suzhou, Jiangsu Province, China
    3 Institute of Pediatrics, Children’s Hospital Affiliated to Suzhou University, Suzhou, Jiangsu Province, China
    4 Blood Section, Children’s Hospital Affiliated to Suzhou University, Suzhou, Jiangsu Province, China
  • Online:2020-11-15 Published:2020-08-23
  • Contact: Jun Hua, huajun_1970@126.com; Xing Feng, MD, feng_xing66@163.com.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, Nos. 81671532, 81771625 (to XF); the Jiangsu Provincial Key Medical Discipline of China, No. ZDXKA2016013 (to XF); the Jiangsu Provincial Medical Youth Talent of China, No. QNRC2016758 (to XF); the Jiangsu Province Women and Children Health Research Project of China, No. F201750 (to XF); the Public Health Technology Project of Suzhou City of China, No. SYS201765 (to XF); a grant from the Department of Pediatrics Clinical Center of Suzhou City of China, No. Szzx201504 (to XF).

摘要:

TRPM2是一种重要的阳离子通道,是缺血性脑损伤重要的调控靶点,然而下调TRPM2的表达能否干预缺氧复氧造成的神经元损伤及其机制尚不清楚。为此,实验以抑制TRPM2的表达对缺氧复氧诱导的PC12神经元细胞凋亡的影响及机制为研究目的。 将TRPM2干扰质粒转染至PC12细胞后,在无糖培养基,缺氧8 h,复氧24 h条件下培养建立氧糖剥夺细胞模型。(1)我们利用流式检测细胞凋亡、线粒体膜电位、ROS水平和钙离子浓度,利用荧光定量PCR和 Western blot 检测各组CXC 趋化因子12(CXCL2),炎症小体组分NLRP3和Caspase-1的表达中发现:与单纯氧糖剥夺的细胞相比,干扰 TRPM2 的氧糖剥夺模型细胞凋亡率、细胞线粒体膜电位、活性氧水平、Ca2+水平、CXCL2、NLRP3、Caspase-1 mRNA和蛋白表达均明显下降。(2)实验结果显示了,下调TRPM2的表达可减轻氧糖剥夺导致的神经元损伤,其机制可能和抑制细胞凋亡、氧化应激水平、细胞线粒体膜电位、内钙离子浓度和抑制NLRP3炎性体的激活有关。

orcid:0000-0001-6062-6919 (Xing Feng)
        0000-0001-9622-0558 (Jun Hua)

关键词: 细胞凋亡, 钙, 半胱氨酸天冬氨酸蛋白酶-1, NLRP3, 线粒体损伤, 氧化应激, 氧葡萄糖剥夺, PC12, shRNA, TrPM2

Abstract: Transient receptor potential melastatin 2 (TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mech- anism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma (PC12) cells injured by oxygen-glucose deprivation (OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reox- ygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2 (CXCL2), NACHT, LRR, and PYD domain– containing protein 3 (NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular cal- cium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.

Key words: apoptosis, calcium, caspase-1, NLRP3, mitochondrial impairment, oxidative stress, oxygen-glucose deprivation, PC12, shRNA,
TRPM2