CX3CR1在慢性偏头痛小鼠三叉神经脊束核尾部的表达及作用

THE EXPRESSION OF CX3CR1 IN TRIGEMINAL NUCLEUS CAUDALIS OF MICE WITH CHRONIC MIGRAINE

摘要:

-目的：观察趋化因子C-X3-C基序受体1 (C-X3-C Chemokine Receptor 1, CX3CR1) 在慢性偏头痛 (chronic migraine, CM) 小鼠三叉神经脊束核尾部 (trigeminal nucleus caudalis, TNC) 中的表达变化，并探讨其在CM中的作用。 -方法：通过腹腔隔日重复注射硝酸甘油 (nitroglycerin, NTG) 建立CM模型小鼠。使用von Frey测定小鼠眶周和足底机械痛阈值。采用Western blot、免疫荧光检测TNC脑区降钙素基因相关肽 (calcitonin gene-related peptide, CGRP) 表达水平。Western blot及实时荧光定量聚合酶链反应(Quantitative Real-time Polymerase Chain Reaction, qPCR)检测TNC脑区CX3CR1蛋白及mRNA表达，免疫荧光检测CX3CR1和离子钙结合蛋白1 (ionized calcium binding adapter molecule 1, Iba1) 在TNC的定位情况。酶联免疫吸附实验 (enzyme-linked immunosorbent assay, ELISA) 检测血浆肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、白细胞介素-6 (interleukin-6, IL-6)、白细胞介素-1β (interleukin-1β, IL-1β) 及白细胞介素-10 (interleukin-10, IL-10) 含量。 -结果：与对照组相比，模型组小鼠眶周及足底机械痛阈值显著降低 (P < 0.01)，TNC中CX3CR1、CGRP的表达水平明显增高 (P < 0.01)。荧光染色显示TNC脑区CX3CR1主要表达于小胶质细胞。给予CX3CR1拮抗剂AZD8797后模型组小鼠眶周及足底痛阈改善 (P < 0.05)，ELISA结果示AZD8797组血浆TNF-α、IL-1β及IL-6等促炎因子水平减低 (P < 0.01)，抗炎因子IL-10水平升高 (P < 0.01)。结论：CX3CR1可能通过激活小胶质细胞从而参与偏头痛中枢敏化。 -关键词 慢性偏头痛；CX3CR1；三叉神经脊束核尾部；小胶质细胞

Abstract:

-Objective: To observe the expression changes of C-X3-C Chemokine Receptor 1 (CX3CR1) in the trigeminal nucleus caudalis (TNC) of mice with chronic migraine (CM) and investigate its role in CM. -Methods: Nitroglycerin (NTG) was repeatedly intraperitoneally injected (i.p.) in mice to establish CM model. Mechanical pain thresholds of periorbital region and hind paw were detected by von Frey. Western blotting and immunofluorescence were performed to detect the protein expression levels of calcitonin gene-related peptide (CGRP) in TNC. Western blotting and Quantitative Real-time Polymerase Chain Reaction(qPCR) were performed to detect CX3CR1 protein and mRNA expression in the TNC. Immunofluorescence staining was used to determine the localization of CX3CR1 and Iba-1 in the TNC. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the plasma levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10). -Results: After repeated NTG injections, the pain threshold in CM group in periorbital regions and hind paw regions was remarkably decreased (P < 0.01), while the expression of CX3CR1 and CGRP was increased (P < 0.01). Immunofluorescence staining revealed that CX3CR1 was expressed in microglia in TNC. Administration of the CX3CR1 antagonist AZD8797 improved the pain threshold of CM model mice (P < 0.05).ELISA results showed that the plasma levels of TNF-α, IL-1β and IL-6 were reduced in the AZD8797-treated group (P < 0.01), while the expression of IL-10 increased (P < 0.01). Conclusion: CX3CR1 could be involved in migraine central sensitization by activating microglia. -Keywords chronic migraine; CX3CR1; trigeminal nucleus caudalis; microglia