Research question

Does the aggregation of M2 macrophages affect the expression of gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) in adenomyosis?

Design

Endometrial tissues were collected from patients with (n = 15) and without (n = 15) adenomyosis. Tissues were analysed for GRIM-19 and toll-like receptor 4 (TLR4) expression by immunohistochemistry and western blotting. Apoptosis was analysed by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. Human endometrial stromal cells (HESC) were transfected with GRIM-19 small interfering RNA (SiRNA) to knockdown GRIM-19 expression. The HESC were co-cultured with M2 macrophages to detect the influence of M2 macrophages in HESC cells. Analyses included GRIM-19, caspase-3 and TLR4 expression by western blotting, and GRIM-19 and TLR4 by quantitative real-time polymerase chain reaction. Apoptosis was measured by flow cytometry and TUNEL assay. Cell proliferation (Cell Counting Kit-8 assay) and migration assays were carried out.

Results

The expression of GRIM-19 was significantly lower in adenomyosis lesions compared with controls (P < 0.001). Deficiency of GRIM-19 induced by siRNA decreased apoptosis and increased proliferation and migration in HESC. A significant decrease in GRIM-19 expression occurred in HESC after co-culture with M2 macrophages (P = 0.018). After co-culture with M2 macrophage, apoptosis decreased and proliferation and cell invasion in HESC increased. Protein (P = 0.006) and mRNA (P = 0.013) expression of TLR4 in HESC also reduced after this co-culture. Up-regulation of GRIM-19 occurred in HESC treated with the activator TLR4 (P = 0.016). Up-regulation of GRIM-19 was significantly reversed in cells treated with the TLR4 inhibitor (P = 0.011).

Conclusion

M2 macrophages may be involved in regulating the expression of GRIM-19 partly through the TLR4 signalling axis in adenomyosis.

中文翻译：

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M2 巨噬细胞和子宫内膜细胞的相互作用诱导子宫腺肌病子宫内膜中 GRIM-19 的下调。

研究问题

M2 巨噬细胞的聚集是否会影响与维甲酸干扰素诱导的死亡率 19 (GRIM-19) 相关基因在子宫腺肌病中的表达？

设计

从有（n  = 15）和没有（n = 15) 子宫腺肌病。通过免疫组织化学和蛋白质印迹分析组织的 GRIM-19 和 toll 样受体 4 (TLR4) 表达。通过 TdT（末端脱氧核苷酸转移酶）介导的 dUDP 缺口末端标记（TUNEL）测定法分析细胞凋亡。用 GRIM-19 小干扰 RNA (SiRNA) 转染人子宫内膜基质细胞 (HESC) 以抑制 GRIM-19 表达。HESC 与 M2 巨噬细胞共培养以检测 M2 巨噬细胞对 HESC 细胞的影响。分析包括通过蛋白质印迹分析的 GRIM-19、caspase-3 和 TLR4 表达，以及通过定量实时聚合酶链反应分析的 GRIM-19 和 TLR4。通过流式细胞术和TUNEL测定法测量细胞凋亡。进行细胞增殖（Cell Counting Kit-8 测定）和迁移测定。

结果

与对照组相比，GRIM-19在子宫腺肌病病灶中的表达显着降低（P < 0.001）。siRNA 诱导的 GRIM-19 缺陷减少了 HESC 的细胞凋亡并增加了增殖和迁移。与 M2 巨噬细胞共培养后，HESC 中 GRIM-19 表达显着降低（P  = 0.018）。与M2巨噬细胞共培养后，HESC细胞凋亡减少，增殖和细胞侵袭增加。 在这种共培养后，HESC 中 TLR4 的蛋白质 ( P  = 0.006) 和 mRNA ( P = 0.013) 表达也降低。用激活剂 TLR4 处理的 HESC 中发生 GRIM-19 上调（P = 0.016）。在用 TLR4 抑制剂处理的细胞中，GRIM-19 的上调显着逆转（P  = 0.011）。

结论

M2巨噬细胞可能部分通过TLR4信号轴参与调节GRIM-19在子宫腺肌病中的表达。