MiR-381通过靶向基质细胞衍生因子-1对多发性肌炎组织浸润的巨噬细胞的作用机制

doi:10.16098/j.issn.0529-1356.2022.02.007

摘要/Abstract

摘要：

目的探讨miR-381通过靶向基质细胞衍生因子-1（SDF-1）对多发性肌炎（PM）组织浸润的巨噬细胞的作用机制。方法通过兔肌球蛋白（1.5 mg）、结核分枝杆菌（5 mg）和百日咳毒素（500 ng）构建PM小鼠模型。将30只PM模型小鼠随机分为PM组和PM+miR-381组（每组15只），另取同期15只健康小鼠作为对照组。PM+miR-381组小鼠腹腔内注射miR-381 agomir（300 μg）2周。检测和比较各组小鼠血清中血清肌酸激酶（s-CK）、白细胞介素（IL）-1β 和IL-6水平及肌肉组织病理学变化。通过免疫组织化学染色检测巨噬细胞标志蛋白F4/80，评估组织中浸润的巨噬细胞情况，检测各组肌肉组织中miR-381、SDF-1 mRNA和蛋白表达水平。通过双荧光素酶报告基因实验验证miR-381和SDF-1的靶向关系。将小鼠巨噬细胞分为miR-381 NC组和miR-381 mimics组，通过转染miR-381 mimics过表达miR-381，通过Real-time PCR和Western blotting检测各组SDF-1 mRNA和蛋白水平，通过Transwell实验检测细胞迁移水平评估侵袭能力。结果动物实验上述指标3组间差异显著（P<0.05）。PM组肌肉组织中miR-381水平显著低于对照组，s-CK、IL-1β、IL-6、组织学评分、组织中浸润的巨噬细胞情况、SDF-1 mRNA和蛋白表达水平显著高于对照组（P<0.05）。PM+miR-381组肌肉组织中miR-381水平显著高于PM组，s-CK、 IL-1β、IL-6、组织学评分、组织中浸润的巨噬细胞情况、SDF-1 mRNA和蛋白表达水平显著低于PM组（P<0.05）。双荧光素酶报告基因实验结果表明，miR-381可以与SDF-1靶向结合。细胞实验结果表明，miR-381 mimics组巨噬细胞中的SDF-1 mRNA和蛋白表达水平显著低于miR-381 NC组（P<0.05）。MiR-381 mimics组的迁移细胞数目显著低于miR-381 NC组（P<0.05）。结论提高miR-381水平可通过靶向抑制SDF-1的表达而抑制巨噬细胞的炎性浸润能力，从而缓解PM。

关键词: 多发性肌炎, 基质细胞衍生因子-1, 巨噬细胞浸润, 免疫组织化学, 双荧光素酶报告基因实验, 小鼠

Abstract:

Objective To explore the mechanism of miR-381 on the infiltration of polymyositis (PM) macrophages by targeting stromal cell derived factor-1 (SDF-1). Methods PM model mouse was constructed by rabbit myosin (1.5 mg), mycobacterium tuberculosis (5 mg) and pertussis toxin (500 ng). The 30 PM model mice were divided into control group and PM+miR-381 group (n=15/group). During the same period, 15 healthy mice were used as a control group. Mice in the PM+miR-381 group were injected with miR-381 agomir (300 μg) intraperitoneally for 2 weeks. Serum creatine kinase (s-CK), interleukin (IL)-1β and IL-6 levels in serum of each group of mice, and the pathological changes of muscle tissue were detected and compared. The macrophage marker protein F4/80 was detected by immunohistochemical staining to assess the infiltration of macrophages. The expression levels of miR-381 and SDF-1 mRNA and protein in muscle tissues of each group were detected. The target relationship between miR-381 and SDF-1 was verified by dual luciferase report. Mouse macrophages were divided into miR-381 NC group and miR-381 mimic group. The SDF-1 mRNA and protein levels in each group were detected by Real-time PCR and Western blotting. Transwell was used to detect the level of cell migration to evaluate the infiltration capacity. Results The above indicators of the three groups were significantly different (P<0.05). The level of miR-381 in the muscle tissue of the PM group was significantly lower than that of the control group, s-CK, IL-1β, IL-6, histological score, macrophage infiltration, and SDF-1 mRNA and protein expression levels were significantly higher than those of the control group (P<0.05). The level of miR-381 in the muscle tissue of the PM+miR-381 group was significantly higher than that of the PM group, s-CK, IL-1β, IL-6, histological score, macrophage infiltration, and SDF-1 mRNA and protein expression levels were significantly lower than those in the PM group (P<0.05). The dual luciferase report result indicated that miR-381 could target binding to SDF-1. The expression levels of SDF-1 mRNA and protein in macrophages in the miR-381 mimic group were significantly lower than those in the miR-381 NC group (P<0.05). The number of migrating cells in the miR-381 mimic group was significantly lower than that in the miR-381 NC group (P<0.05). Conclusion Increasing the level of miR-381 can inhibit the inflammatory infiltration ability of macrophages by targeting the expression of SDF-1, thereby alleviating PM.

Key words: Polymyositis, Stromal cell derived factor-1, Macrophage infiltration, Immunohistochemistry, Dual luciferase report, Mouse

中图分类号:

R632.9

陈昭英洪文轲. MiR-381通过靶向基质细胞衍生因子-1对多发性肌炎组织浸润的巨噬细胞的作用机制[J]. 解剖学报, 2022, 53(2): 183-189.

CHEN Zhao-ying HONG Wen-ke. Mechanism of miR-381 on macrophage infiltration in polymyositis by targeting stromal cell derived actor-1[J]. Acta Anatomica Sinica, 2022, 53(2): 183-189.

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