Xue Song, Cao Zhen-Xin, Wang Jun-Nan, Zhao Qing-Xiang, Han Jie, Yang Wen-Jie, Sun Tao
Department of Pain Management, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.
Departments of Pain Management, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.
Front Mol Neurosci. 2022 Apr 19;15:861312. doi: 10.3389/fnmol.2022.861312. eCollection 2022.
Neuroinflammation is critical in developing and maintaining neuropathic pain after spinal cord injury (SCI). The receptor-interacting protein kinase 3 (RIPK3) has been shown to promote inflammatory response by exerting its non-necroptotic functions. In this study, we explored the involvement of RIPK3 in neuropathic pain after SCI.
Thoracic (T10) SCI rat model was conducted, and the mechanical threshold in rats was measured. The expressions of RIPK3, nod-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, and nuclear factor-κB (NF-κB) were measured with western blotting analysis or quantitative real-time polymerase chain reaction (qRT-PCR). Double immunofluorescence staining was used to explore the colabeled NLRP3 with NeuN, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (IBA1). In addition, enzyme-linked immunosorbent assay (ELISA) was applied to analyze the levels of proinflammatory factors interleukin 1 beta (IL-1β), interleukin 18 (IL-18), and tumor necrosis factor alpha (TNF-α).
The expression of RIPK3 was elevated from postoperative days 7-21, which was consistent with the development of mechanical allodynia. Intrathecal administration of RIPK3 inhibitor GSK872 could alleviate the mechanical allodynia in SCI rats and reduce the expression levels of RIPK3. The activation of NLRP3 inflammasome and NF-κB was attenuated by GSK872 treatment. Furthermore, immunofluorescence suggested that NLRP3 had colocalization with glial cells and neurons in the L4-L6 spinal dorsal horns. In addition, GSK872 treatment reduced the production of inflammatory cytokines.
Our findings indicated that RIPK3 was an important facilitated factor for SCI-induced mechanical allodynia. RIPK3 inhibition might relieve mechanical allodynia by inhibiting NLRP3 inflammasome, NF-κB, and the associated inflammation.
神经炎症在脊髓损伤（SCI）后神经性疼痛的发生和维持中起关键作用。受体相互作用蛋白激酶3（RIPK3）已被证明可通过发挥其非坏死性功能促进炎症反应。在本研究中，我们探讨了RIPK3在SCI后神经性疼痛中的作用。
建立胸段（T10）SCI大鼠模型，并测量大鼠的机械阈值。采用蛋白质印迹分析或定量实时聚合酶链反应（qRT-PCR）检测RIPK3、含NOD样受体家族吡咯结构域蛋白3（NLRP3）、半胱天冬酶-1和核因子-κB（NF-κB）的表达。采用双重免疫荧光染色法探讨NLRP3与神经元特异性核蛋白（NeuN）、胶质纤维酸性蛋白（GFAP）和离子钙结合衔接分子1（IBA1）的共定位情况。此外，应用酶联免疫吸附测定（ELISA）分析促炎因子白细胞介素1β（IL-1β）、白细胞介素18（IL-18）和肿瘤坏死因子α（TNF-α）的水平。
RIPK3的表达在术后7至21天升高，这与机械性异常性疼痛的发展一致。鞘内注射RIPK3抑制剂GSK872可减轻SCI大鼠的机械性异常性疼痛，并降低RIPK3的表达水平。GSK872处理可减弱NLRP3炎性小体和NF-κB的激活。此外，免疫荧光显示NLRP3在L4-L6脊髓背角与胶质细胞和神经元共定位。此外，GSK872处理可减少炎性细胞因子的产生。
我们的研究结果表明，RIPK3是SCI诱导的机械性异常性疼痛的重要促进因子。抑制RIPK3可能通过抑制NLRP3炎性小体、NF-κB及相关炎症来减轻机械性异常性疼痛。
