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Title: Effect of electroacupuncture on inflammatory response and immune cells in mice with chronic inflammatory pain.
Author: Yang JL, Shi JJ, Ma NQ, Jiang MJ, Peng R, Chen LY, Yu ML, Lu SF.
Journal: Zhen Ci Yan Jiu; 2024 Jan 25; 49(1):30-36. PubMed ID: 38239136.
Abstract:
OBJECTIVES: To observe the effects of electroacupuncture(EA) on local inflammatory mediators and macrophage polarization, and immune cells in the spleen of mice with chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in the hind paw, so as to investigate the immunoinflammatory regulatory mechanisms of EA in relieving pain and swelling in mice with chronic inflammatory pain. METHODS: Thirty C57BL/6 mice were randomly divided into control, model, and EA groups, with 10 mice in each group. Chronic inflammatory pain model were established by subcutaneous injection of 20 μL CFA solution in the left hind paw for 7 consecutive days. After modeling, mice in the EA group received EA at bilateral "Zusanli"(ST36) for 20 min (2 Hz/100 Hz, 1 mA) once a day for 18 consecutive days. Mechanical pain threshold, heat pain thresholds, and paw thickness were measured before and after mode-ling, and after interventions. Western blot was used to detect the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and NOD-like receptor protein 3 (NLRP3) in the paw tissue. Immunohistochemistry was used to detect the positive expression of M1-type macrophage marker inducible nitric oride synthase (iNOS) and M2-type marker CD206 in the paw, and flow cytometry was used to detect the proportion of F4/80⁺ CD11b⁺ macrophages, Ly6G⁺ CD11b⁺ neutrophils, and CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen. RESULTS: Compared with the control group, mechanical pain and heat pain thresholds were significantly reduced(P<0.000 1), while paw thickness, expressions of IL-1β, TNF-α, and NLRP3 in the paw, and positive expression of M1 macrophage marker iNOS in the paw, the proportions of macrophages and neutrophils in the spleen were significantly increased (P<0.000 1, P<0.001) in the model group. Compared with the model group, mechanical pain threshold and heat pain thresholds, CD206 positive expression in the paw, and Treg cell proportion in spleen were significantly increased (P<0.01), while paw thickness, the expressions of IL-1β, TNF-α and NLRP3 in the paw, as well as the positive expression of M1 macrophage marker iNOS in the paw, the proportions of macrophages and neutrophils in the spleen were significantly reduced (P<0.001, P<0.01, P<0.05)in mice of the EA group after intervention. CONCLUSIONS: EA may alleviate pain and swelling in mice with chronic inflammatory pain by regulating the numbers of macrophages, neutrophils, and Treg cells, as well as promoting M2 polarization of local macrophages and inhibiting the release of pro-inflammatory cytokines. 目的: 观察电针对完全弗氏佐剂（CFA）诱导的慢性炎性痛小鼠足掌局部炎性介质、巨噬细胞极化及脾脏中相关免疫细胞的影响，探究电针缓解慢性炎性痛小鼠疼痛及肿胀的免疫炎性调控机制。方法: C57BL/6小鼠随机分为对照组、模型组、电针组，每组10只。连续7 d左后足掌皮下注射20 μL CFA溶液建立慢性炎性痛小鼠模型。造模后电针组给予双侧“足三里”电针治疗20 min（2 Hz/100 Hz，1 mA），每天1次，连续18 d。于造模前后、干预后分别测量各组小鼠机械痛阈值、热痛阈值和足掌厚度，Western blot法检测足掌组织肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、NOD样受体热蛋白结构域相关蛋白3（NLRP3）表达，免疫组织化学法检测足掌局部巨噬细胞M1型标记物iNOS、M2型标记物CD206的阳性表达，流式细胞术检测脾脏F4/80⁺ CD11b⁺巨噬细胞、Ly6G⁺ CD11b⁺中性粒细胞、CD25⁺ Foxp3⁺调节性T细胞（Treg）占比。结果: 与对照组相比，模型组小鼠机械痛阈值、热痛阈值明显降低（P<0.000 1），左后足掌厚度，足掌局部IL-1β、TNF-α、NLRP3表达量，足掌局部iNOS阳性表达，脾脏中巨噬细胞、中性粒细胞占比明显升高（P<0.000 1，P<0.001）。与模型组相比，电针组小鼠干预后机械痛阈值、热痛阈值，足掌CD206阳性表达，脾脏Treg占比显著升高（P<0.01），足掌厚度，足掌局部IL-1β、TNF-α、NLRP3蛋白表达量，足掌局部iNOS阳性表达，脾脏中巨噬细胞、中性粒细胞占比显著降低（P<0.001，P<0.01，P<0.05）。结论: 电针可能通过调控脾脏巨噬细胞、中性粒细胞、Treg占比，并促进局部巨噬细胞向M2型极化，抑制促炎因子释放，从而缓解慢性炎性痛小鼠的疼痛及肿胀。.

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