Annexin A1 (ANXA1) exerts anti-nociceptive effect through ANXA1 receptor formyl peptide receptor 2 (FPR2/ALX (receptor for lipoxin A4), FPR2) at the dorsal root ganglia (DRG) level. However, the mechanisms remain elucidated. By using radiant heat, hot/cold plate, tail flick, von Frey, and Randall-Selitto tests to detect nociception in intact and chemical (capsaicin, menthol, mustard oil, formalin or CFA) injected AnxA1 conditional knockout (AnxA1−/−) mice, applying calcium imaging and patch clamp recordings in cultured DRG neurons to measure neuronal excitability, conducting immunofluorescence and western blotting to detect the protein levels of TRPV1, FPR2 and its downstream molecules, and performing double immunofluorescence and co-immunoprecipitation to investigate the interaction between Calmodulin (CaM) and TRPV1; we aim to uncover the molecular and cellular mechanisms of ANXA1’s role in antinociception. AnxA1−/− mice exhibited significant sensitivity to noxious heat (mean ± SD, 6.2 ± 1.0 s vs. 9.9 ± 1.6 s in Hargreaves test; 13.6 ± 1.5 s vs. 19.0 ± 1.9 s in hot plate test; n = 8; P < 0.001), capsaicin (101.0 ± 15.3 vs. 76.2 ± 10.9; n = 8; P < 0.01), formalin (early phase: 169.5 ± 32.8 s vs. 76.0 ± 21.9 s; n = 8; P < 0.05; late phase: 444.6 ± 40.1 s vs. 320.4 ± 33.6 s; n = 8; P < 0.01) and CFA (3.5 ± 0.8 s vs. 5.9 ± 1.4 s; n = 8; P < 0.01). In addition, we found significantly increased capsaicin induced Ca2+ response, TRPV1 currents and neuronal firing in AnxA1 deficient DRG neurons. Furthermore, ANXA1 mimic peptide Ac2-26 robustly increased intracellular Ca2+, inhibited TRPV1 current, activated PLCβ and promoted CaM-TRPV1 interaction. And these effects of Ac2-26 could be attenuated by FPR2 antagonist Boc2. Selective deletion of AnxA1 in DRG neurons enhances TRPV1 sensitivity and deteriorates noxious heat or capsaicin induced nociception, while ANXA1 mimic peptide Ac2-26 desensitizes TRPV1 via FPR2 and the downstream PLCβ-Ca2+-CaM signal. This study may provide possible target for developing new analgesic drugs in inflammatory pain.

中文翻译：

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Annexin A1调节背根神经节神经元TRPV1和伤害感受的机制

膜联蛋白 A1 (ANXA1) 通过背根神经节 (DRG) 水平的 ANXA1 受体甲酰肽受体 2（FPR2/ALX（脂氧素 A4 受体）、FPR2）发挥抗伤害性作用。然而，机制仍有待阐明。通过使用辐射热、热/冷板、甩尾、von Frey 和 Randall-Selitto 测试来检测完整和化学（辣椒素、薄荷醇、芥子油、福尔马林或 CFA）注射的 AnxA1 条件性敲除 (AnxA1-/-) 中的伤害感受小鼠，在培养的 DRG 神经元中应用钙成像和膜片钳记录来测量神经元兴奋性，进行免疫荧光和蛋白质印迹以检测 TRPV1、FPR2 及其下游分子的蛋白质水平，并进行双重免疫荧光和共免疫沉淀以研究两者之间的相互作用钙调蛋白 (CaM) 和 TRPV1; 我们的目标是揭示 ANXA1 在镇痛作用中的分子和细胞机制。AnxA1-/- 小鼠对有害热量表现出显着的敏感性（平均值 ± SD，6.2 ± 1.0 s 与 Hargreaves 试验中的 9.9 ± 1.6 s；热板试验中 13.6 ± 1.5 s 与 19.0 ± 1.9 s；n = 8；P < 0.001)、辣椒素（101.0 ± 15.3 与 76.2 ± 10.9；n = 8；P < 0.01）、福尔马林（早期：169.5 ± 32.8 s 与 76.0 ± 21.9 s；n = 8；P < 晚期） ：444.6 ± 40.1 s 与 320.4 ± 33.6 s；n = 8；P < 0.01）和 CFA（3.5 ± 0.8 s 与 5.9 ± 1.4 s；n = 8；P < 0.01）。此外，我们发现 AnxA1 缺陷的 DRG 神经元中辣椒素诱导的 Ca2+ 反应、TRPV1 电流和神经元放电显着增加。此外，ANXA1 模拟肽 Ac2-26 显着增加细胞内 Ca2+，抑制 TRPV1 电流，激活 PLCβ 并促进 CaM-TRPV1 相互作用。FPR2拮抗剂Boc2可以减弱Ac2-26的这些作用。选择性删除 DRG 神经元中的 AnxA1 可增强 TRPV1 敏感性并降低有害热或辣椒素诱导的伤害感受，而 ANXA1 模拟肽 Ac2-26 通过 FPR2 和下游 PLCβ-Ca2+-CaM 信号使 TRPV1 脱敏。该研究可能为开发新的炎症性疼痛镇痛药物提供可能的靶点。