Guan Xin-Ying, Yuan Bao-Tong, Li Meng-Na, Wang Tian, Zhu Lin-Peng, Song Si-Yuan, Ji Lu-Lu, Gao Yong-Jing, Ma Ling-Jie
Department of Neurology, The Affiliated Hospital of Kangda College of Nanjing Medical University, The First People's Hospital of Lianyungang, Jiangsu, 222001, China.
Institute of Pain Medicine and Special Environmental Medicine, Co-innovation Center of Neuroregeneration, Nantong University, Jiangsu, 226019, China.
J Headache Pain. 2025 Jul 15;26(1):160. doi: 10.1186/s10194-025-02093-1.
Chronic neuropathic pain involves complex molecular adaptations, and emerging evidence indicates that cerebellins (CBLNs) play a role in sensory processing. This study investigates the role of CBLN2 in trigeminal neuropathic pain (TNP) and examines its regulation through epigenetic mechanisms.
Bioinformatics analyses of CBLNs were performed using publicly available expression data from the trigeminal ganglion (TG). A mouse model of TNP was established through partial infraorbital nerve transection (pIONT). Facial allodynia in mice was assessed using the von Frey test. Quantitative real-time PCR (qRT-PCR), Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the expression of CBLN2. Immunofluorescence was used to determine CBLN2's cellular localization. DNA methylation of Cbln2's promoter region was examined using methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). Neuronal excitability was assessed through whole-cell patch-clamp recordings.
Integration of cross-species single-nucleus RNA sequencing (snRNA-seq) datasets identified dominant CBLN2 expression in Aδ, Aβ and c-fiber low-threshold mechanoreceptors (LTMRs), with significant upregulation observed in a murine model of inflammatory migraine. Consistently, CBLN2 expression was upregulated in the TG of pIONT-induced TNP mice and localized to neurons with myelinated axons, peptidergic and nonpeptidergic nociceptors. siRNA-mediated Cbln2 knockdown attenuated mechanical allodynia, confirming its role in pain initiation and maintenance. Notably, Cbln2 expression was partially dependent on promoter demethylation. MSP and BSP analyses revealed significantly reduced methylation of the Cbln2 promoter in pIONT mice compared to sham controls. Furthermore, pIONT induced persistent upregulation of ten-eleven translocation 3 (TET3) in the TG, while Tet3 knockdown alleviated neuropathic pain and downregulated both Tet3 and Cbln2 expression. Additionally, exogenous CBLN2 potentiated neuronal excitability and activated extracellular signal-regulated kinase (ERK) signaling. Inhibition of the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) pathway abolished CBLN2-induced hypersensitivity and suppressed the expression of proinflammatory cytokines, including Cxcl1, Cxcr2, Cxcl9, Cxcl10, and Il-6.
TET3-mediated demethylation of the Cbln2 promoter drives ERK-dependent neuronal hyperexcitability and neuroinflammation following pIONT. The dual regulatory effects on neuroinflammatory cascades establish CBLN2 as a novel therapeutic target for the treatment of TNP.
慢性神经性疼痛涉及复杂的分子适应性变化，并且新出现的证据表明小脑素（CBLNs）在感觉处理中发挥作用。本研究调查了CBLN2在三叉神经病理性疼痛（TNP）中的作用，并通过表观遗传机制研究其调控。
使用来自三叉神经节（TG）的公开可用表达数据对CBLNs进行生物信息学分析。通过部分眶下神经横断（pIONT）建立TNP小鼠模型。使用von Frey试验评估小鼠的面部异常性疼痛。进行定量实时PCR（qRT-PCR）、蛋白质免疫印迹和酶联免疫吸附测定（ELISA）以评估CBLN2的表达。使用免疫荧光确定CBLN2的细胞定位。使用甲基化特异性PCR（MSP）和亚硫酸氢盐测序PCR（BSP）检测Cbln2启动子区域的DNA甲基化。通过全细胞膜片钳记录评估神经元兴奋性。
跨物种单核RNA测序（snRNA-seq）数据集的整合确定了CBLN2在Aδ、Aβ和c纤维低阈值机械感受器（LTMRs）中占主导地位的表达，在炎症性偏头痛小鼠模型中观察到其显著上调。同样，在pIONT诱导的TNP小鼠的TG中，CBLN2表达上调，并定位于具有有髓轴突的神经元、肽能和非肽能伤害感受器。siRNA介导的Cbln2敲低减轻了机械性异常性疼痛，证实了其在疼痛起始和维持中的作用。值得注意的是，Cbln2表达部分依赖于启动子去甲基化。MSP和BSP分析显示，与假手术对照组相比，pIONT小鼠中Cbln2启动子的甲基化显著降低。此外，pIONT诱导TG中十一-十一易位3（TET3）持续上调，而Tet3敲低减轻了神经性疼痛，并下调了Tet3和Cbln2的表达。此外，外源性CBLN2增强了神经元兴奋性并激活了细胞外信号调节激酶（ERK）信号传导。丝裂原活化蛋白激酶（MAPK）/ERK激酶（MEK）途径的抑制消除了CBLN2诱导的超敏反应，并抑制了促炎细胞因子的表达，包括Cxcl1、Cxcr2、Cxcl9、Cxcl10和Il-6。
TET3介导的Cbln2启动子去甲基化驱动pIONT后ERK依赖性神经元兴奋性过高和神经炎症。对神经炎症级联反应的双重调节作用使CBLN2成为治疗TNP的新治疗靶点。
