Background

3, 3’-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition.

Purpose

This study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition.

Methods

Cytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo.

Results

DIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67⁺ F4/80⁺ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain.

Conclusion

DIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via “AhR-Nrf2/Arg-1” signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.

中文翻译：

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3, 3'-二吲哚基甲烷通过 AhR/Nrf2/Arg-1 介导的精氨酸代谢途径增强巨噬细胞胞吐作用并随后缓解内脏疼痛

背景

3, 3'-二吲哚基甲烷 (DIM) 是一种经典的芳烃受体 (AhR) 激动剂，已被证明可以缓解神经性疼痛，但很少有研究报道 DIM 在结肠炎情况下对内脏痛的疗效。

目的

本研究旨在探讨DIM对结肠炎状态下内脏痛的影响及机制。

方法

使用 MTT 测定法进行细胞毒性。应用 RT-qPCR 和 ELISA 测定来确定致痛物质 P (SP)、神经生长因子 (NGF) 和脑源性神经营养因子 (BDNF) 的表达和释放。流式细胞术用于检查细胞凋亡和胞吐作用。使用蛋白质印迹法检测 Arg-1-精氨酸代谢相关酶的表达。ChIP 测定用于检查 Nrf2 与 Arg-1 的结合。建立了葡聚糖硫酸钠 (DSS) 的小鼠模型来说明 DIM 的作用并验证体内机制。

结果

DIM 不直接影响肠神经胶质细胞 (EGC) 中产藻 SP、NGF 和 BDNF 的表达和释放。然而，当与 DIM 预处理的 RAW264.7 细胞共培养时，脂多糖刺激的 EGC 中 SP 和 NGF 的释放减少。此外，DIM 在体外增加EGCs 和 RAW264.7 细胞共培养系统中PKH67 ⁺ F4/80 ⁺细胞的数量，并通过调节 SP 和 NGF 的水平以及肌电图值​​减轻结肠炎条件下的内脏疼痛（ EMG)、腹部退缩反射 (AWR) 和甩尾潜伏期 (TFL)在体内，这被细胞增多症抑制剂显着抑制。随后，发现 DIM 可下调细胞内精氨酸的水平，上调鸟氨酸、腐胺和 Arg-1 的水平，但不会上调细胞外精氨酸或其他代谢酶的水平，并且多胺清除剂逆转了 DIM 对胞吐作用和 SP 和神经生长因子。展望未来，DIM 增强了 Nrf2 转录和 Nrf2 与 Arg-1-0.7 kb 的结合，AhR 拮抗剂 CH223191 消除了 DIM 对 Arg-1 和胞吐作用的促进作用。最后，nor-NOHA 验证了 Arg-1 依赖性精氨酸代谢在减轻 DIM 内脏痛中的重要性。

结论

DIM通过“AhR-Nrf2/Arg-1”信号以精氨酸代谢依赖性方式增强巨噬细胞胞吐作用，并抑制 SP 和 NGF 的释放，从而缓解结肠炎情况下的内脏疼痛。这些发现为治疗结肠炎患者的内脏痛提供了一种潜在的治疗策略。