Graduate School of Pharmaceutical Sciences, School of Pharmacy, Duquesne University, Pittsburgh, PA, United States of America.
PLoS One. 2020 Apr 14;15(4):e0231597. doi: 10.1371/journal.pone.0231597. eCollection 2020.
Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.
中枢神经系统的常驻小胶质细胞越来越被认为是神经病理性疼痛等疾病的关键参与者。神经病理性疼痛啮齿动物模型的生化和行为研究记录了令人信服的证据，表明 ATP 介导的 P2X4R-脑源性神经营养因子 (BDNF) 信号通路在痛觉过敏的起始和维持中起着关键作用，这一特征是驱动神经病理性疼痛相关行为的原因。本研究的目的是开发和表征一种激活的小胶质细胞体外细胞系模型，随后可用于筛选神经病理性疼痛治疗药物。在本研究中，我们使用生化技术组合对 SIM-A9 小胶质细胞系中 P2X4R-BDNF 信号轴的关键分子进行了表征，并开发了一种 ATP 激活的 SIM-A9 小胶质细胞模型。我们提出了三个新发现：首先，SIM-A9 细胞表达 P2X4R 和 BDNF 蛋白；其次，ATP 而不是 LPS 与 SIM-A9 细胞相容；第三，将细胞暴露于优化的 ATP 浓度下一定时间会增加 Iba1 和 BDNF 蛋白的细胞内表达。Iba1 水平的增加证实了小胶质细胞的激活，BDNF 表达的增加证实了 ATP 介导的 P2X4R 信号通路的刺激。我们提出，这种 ATP 激活的 SIM-A9 细胞系模型系统可用于筛选针对 BDNF 和/或 P2X4R 敲低的小分子和大分子神经病理性疼痛治疗药物。
