Zheng Bingjie, Li Shengwen, Xiang Yufeng, Zong Wentian, Ma Qingliang, Wang Shiyu, Wu Haihao, Song Haixin, Ren Hong, Chen Jian, Liu Junhui, Zhao Fengdong
Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310027, China.
Key Laboratory of Musculoskeletal System Degeneration and Regeneration Translational Research of Zhejiang Province, Hangzhou, Zhejiang, 310027, China.
J Orthop Translat. 2022 Dec 23;39:21-33. doi: 10.1016/j.jot.2022.11.006. eCollection 2023 Mar.
Discogenic low back pain (LBP) is associated with nociceptive nerve fibers that grow into degenerated intervertebral discs (IVD) but the etiopathogenesis of disease is not fully understood. The purpose of this study was to clarify the role of Netrin-1 in causing discogenic LBP.
The level of nociceptive nerve innervation was examined in disc degenerative patients and rat needle-punctured models by immunohistochemistry. Nucleus pulposus (NP) cells were isolated from IVD tissues of rats and induced degeneration by interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα). The candidate genes related to neuron outgrowth and migration were selected by Next-generation sequencing (NGS). CRISPR/Cas9 was used to knockdown Netrin-1 in NP cells. The impact of Netrin-1 on nerve innervation were evaluated with P2X2、NF200 staining and microfluidics assay. Meanwhile the CD31 staining and transwell assay were used to evaluate the impact of Netrin-1 in angiogenesis. The proteins and RNA extracted from NP cells related to catabolism and anabolism were examined by western blot assay and RT-qPCR experiment. ChIP and luciferase experiments were used to assess the intracellular transcriptional regulation of Netrin-1. Further, a needle-punctured rat model followed by histomorphometry and immunofluorescence histochemistry was used to explore the potential effect of Netrin-1 on LBP .
The level of nerve innervation was increased in severe disc degenerative patients while the expression of Netrin-1 was upregulated. The supernatants of NP cells stimulated with IL-1β or TNFα containing more Netrin-1 could promote axon growth and vascular endothelial cells migration. Knocking down Netrin-1 or overexpressing transcription factor TCF3 as a negative regulator of Netrin-1 attenuated this effect. The needle-punctured rat model brought significant spinal hypersensitivity, nerve innervation and angiogenesis, nevertheless knocking down Netrin-1 effectively prevented disc degeneration-induced adverse impacts.
Discogenic LBP was induced by Netrin-1, which mediated nerve innervation and angiogenesis in disc degeneration. Knocking down Netrin-1 by CRISPR/Cas9 or negatively regulating Netrin1 by transcription factor TCF3 could alleviate spinal hypersensitivity.
This study on Netrin-1 could provide a new target and theoretical basis for the prevention and treatment for discogenic back pain.
椎间盘源性下腰痛（LBP）与长入退变椎间盘（IVD）的伤害性神经纤维相关，但该病的发病机制尚未完全阐明。本研究旨在明确Netrin-1在椎间盘源性LBP发病中的作用。
采用免疫组织化学方法检测椎间盘退变患者及大鼠针刺模型中伤害性神经支配水平。从大鼠IVD组织中分离出髓核（NP）细胞，并用白细胞介素-1β（IL-1β）或肿瘤坏死因子α（TNFα）诱导其退变。通过下一代测序（NGS）筛选与神经元生长和迁移相关的候选基因。利用CRISPR/Cas9敲低NP细胞中的Netrin-1。通过P2X2、NF200染色及微流控分析评估Netrin-1对神经支配的影响。同时，采用CD31染色及Transwell分析评估Netrin-1对血管生成的影响。通过蛋白质印迹分析和RT-qPCR实验检测NP细胞中与分解代谢和合成代谢相关的蛋白质和RNA。采用染色质免疫沉淀和荧光素酶实验评估Netrin-1的细胞内转录调控。此外，采用针刺大鼠模型，随后进行组织形态计量学和免疫荧光组织化学分析，以探讨Netrin-1对LBP的潜在影响。
严重椎间盘退变患者的神经支配水平升高，而Netrin-1的表达上调。用IL-1β或TNFα刺激的NP细胞上清液中含有更多的Netrin-1，可促进轴突生长和血管内皮细胞迁移。敲低Netrin-1或过表达作为Netrin-1负调控因子的转录因子TCF3可减弱这种作用。针刺大鼠模型导致明显的脊髓超敏反应、神经支配和血管生成，然而敲低Netrin-1可有效预防椎间盘退变引起的不良影响。
Netrin-1诱导椎间盘源性LBP，其介导椎间盘退变中的神经支配和血管生成。通过CRISPR/Cas9敲低Netrin-1或通过转录因子TCF3负调控Netrin-1可减轻脊髓超敏反应。
本研究对Netrin-1的研究可为椎间盘源性背痛的防治提供新的靶点和理论依据。
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