[Effect of electroacupuncture in regulating miRNA-21-5p/A20 to induce macrophages polarization in primary dysmenorrhea rats].

Abstract

<p>OBJECTIVE: To explore the mechanism of electroacupuncture on macrophages polarization by regulating microRNA-21-5p (miR-21-5p)/ubiquitin-editing enzyme A20 (A20) in primary dysmenorrhea (PDM) rats. METHODS: Twenty-four female SD rats of SPF grade in diestrus were randomly divided into a blank group, a model group, an electroacupuncture group and an ibuprofen group, 6 rats in each group. Except for the blank group, in the other 3 groups, PDM model was established using estradiol benzoate combined with oxytocin. In the electroacupuncture group, electroacupuncture was applied at "Guanyuan" (CV4) and bilateral "Sanyinjiao" (SP6), with continuous wave, in frequency of 50 Hz, 20 min each time, once a day for 10 days. In the ibuprofen group, ibuprofen solution of 125 mg/100 mL was given by gavage administration, 0.8 mL each time, once a day for 10 days. The writhing behavior was observed, the ultrastructure of M1 macrophages in the endometrium was observed by transmission electron, the numbers and distribution of M1/M2 macrophages in the uterus were analyzed by immunofluorescence double-labeling, the serum levels of prostaglandin F2α (PGF2α), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1) were measured by ELISA, the targeting relationship between miR-21-5p and A20 was validated by dual-luciferase reporter assay, the mRNA expression of miR-21-5p, A20, nuclear transcription factor-κB p65 subunit (NF-κB p65), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1β (IL-1β), cluster of differentiation 86 (CD86), iNOS, cluster of differentiation 206 (CD206) and Arg-1 in the uterus was detected by real-time PCR, and the protein expression of A20, CD86, iNOS, CD206, Arg-1 in the uterus was detected by Western blot. RESULTS: The bioinformatics prediction result indicated that A20 was a high-affinity target gene of miR-21-5p. Compared with the blank group, in the model group, the writhing behavior could be observed obviously; the M1 macrophages in the endometrium had severe edema, matrix dissolution and organelle damage; the density of CD86 positive cells was increased in the uterus (P&lt;0.01); the PGF2α, TNF-α and iNOS levels were increased (P&lt;0.05, P&lt;0.01), the Arg-1 level was decreased (P&lt;0.01) in the serum; the mRNA expression of miR-21-5p, NF-κB p65, NLRP3, IL-1β and CD86, as well as the protein expression of CD86 and iNOS were increased (P&lt;0.01), the mRNA and protein expression of A20 and CD206, as well as the protein expression of Arg-1 were decreased (P&lt;0.05, P&lt;0.01) in the uterus. Compared with the model group, in the electroacupuncture group and the ibuprofen group, the writhing numbers and writhing scores were decreased (P&lt;0.05), the writhing latency was prolonged (P&lt;0.01); the edema degree of the M1 macrophages was reduced and the swelling condition of organelles was improved of in the endometrium; the density of CD206 positive cells was increased in the uterus (P&lt;0.05, P&lt;0.01); the PGF2α, TNF-α and iNOS levels were decreased (P&lt;0.01, P&lt;0.05), the Arg-1 levels were increased (P&lt;0.01) in the serum; the mRNA expression of miR-21-5p, NF-κB p65, NLRP3, IL-1β and CD86, as well as the protein expression of CD86 and iNOS were decreased (P&lt;0.01), the mRNA expression of A20, CD206 and Arg-1, as well as the protein expression of A20 and Arg-1 were increased (P&lt;0.01, P&lt;0.05) in the uterus. Compared with the ibuprofen group, in the electroacupuncture group, the mRNA expression of IL-1β was decreased (P&lt;0.01), while that of CD86 was increased (P&lt;0.05) in the uterus. CONCLUSION: Electroacupuncture induces the polarization of M1 macrophages to M2 macrophages by down-regulating miR-21-5p and up-regulating A20, thus reducing the inflammatory reaction and relieving pain in PDM rats. 目的：探讨电针通过微小RNA-21-5p（miRNA-21-5p）/泛素编辑酶A20（A20）调控巨噬细胞极化对原发性痛经（PDM）模型大鼠的作用机制。 方法：将24只处于动情间期的SPF级雌性SD大鼠随机分为空白组、模型组、电针组和布洛芬组，每组6只。除空白组外，其余3组大鼠均采用苯甲酸雌二醇联合缩宫素制备PDM模型。电针组予电针“关元”和双侧“三阴交”干预，采用连续波，频率50 Hz，每次20 min，每天1次，连续10 d。布洛芬组予125 mg/100 mL布洛芬溶液灌胃，每次0.8 mL，每天1次，连续10 d。观察各组大鼠扭体反应，透射电镜观察大鼠子宫内膜M1型巨噬细胞超微结构，免疫荧光双标分析大鼠子宫组织M1型巨噬细胞和M2型巨噬细胞数量和分布，ELISA法检测大鼠血清前列腺素F2α（PGF2α）、肿瘤坏死因子-α（TNF-α）、诱导型一氧化氮合酶（iNOS）、精氨酸酶-1（Arg-1）含量，双荧光素酶报告实验（LUC）验证miR-21-5p与A20间的靶向调控关系，实时荧光定量PCR法检测大鼠子宫组织miR-21-5p、A20、核转录因子-κB p65亚基（NF-κB p65）、NOD样受体热蛋白结构域蛋白3（NLRP3）、白细胞介素-1β（IL-1β）、分化簇86（CD86）、iNOS、分化簇206（CD206）和Arg-1 mRNA表达，Western blot法检测大鼠子宫组织A20、CD86、iNOS、CD206和Arg-1蛋白表达。 结果：生物信息预测结果显示A20是miR-21-5p的高亲和力靶基因。与空白组比较，模型组大鼠出现明显扭体反应；M1型巨噬细胞水肿严重、基质溶解、细胞器受损；子宫组织CD86+细胞密度升高（P&lt;0.01）；血清PGF2α、TNF-α、iNOS含量升高（P&lt;0.05，P&lt;0.01），Arg-1含量降低（P&lt;0.01）；子宫组织miR-21-5p、NF-κB p65、NLRP3、IL-1β、CD86 mRNA及CD86、iNOS蛋白表达升高（P&lt;0.01），A20、CD206 mRNA、蛋白及Arg-1蛋白表达降低（P&lt;0.05，P&lt;0.01）。与模型组比较，电针组和布洛芬组大鼠扭体次数、扭体评分降低（P&lt;0.05），扭体潜伏期延长（P&lt;0.01）；M1型巨噬细胞水肿程度减轻、细胞器肿胀情况改善；子宫组织CD206+细胞密度升高（P&lt;0.05，P&lt;0.01）；血清PGF2α、TNF-α、iNOS含量降低（P&lt;0.01，P&lt;0.05），Arg-1含量升高（P&lt;0.01）；子宫组织miR-21-5p、NF-κB p65、NLRP3、IL-1β、CD86 mRNA及CD86、iNOS蛋白表达降低（P&lt;0.01），A20、CD206、Arg-1 mRNA及A20、Arg-1蛋白表达升高（P&lt;0.01，P&lt;0.05）。与布洛芬组比较，电针组大鼠子宫组织IL-1β mRNA表达降低（P&lt;0.01），CD86 mRNA表达升高（P&lt;0.05）。 结论：电针可通过下调miR-21-5p增加A20的表达水平，诱导巨噬细胞由M1型向M2型极化，减轻PDM大鼠炎症反应和疼痛程度。.</p>