Department of Periodontology.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2021 Apr 25;50(2):162-170. doi: 10.3724/zdxbyxb-2021-0099.
To investigate the effects of interleukin (IL)-17-mediated autophagy on the TNF receptor associated factor (TRAF6)/extracellular signal-regulated kinase (ERK)/p38 pathway and osteoclast differentiation. Mouse bone marrow-derived macrophages (BMM) were cultured with a medium containing 30 ng/mL macrophage colony stimulating factor and 50 ng/mL receptor activator of nuclear factor-kappa B ligard (RANKL), and IL-17 (0.01, 0.1, 1.0, 10 ng/mL) was added for intervention (IL-17 group). Tartrate-resistant acid phosphatase (TRAP) staining was used to observe TRAP positive multinucleated cells; phalloidin fluorescent staining was used to detect actin ring circumference; toluidine blue staining was used to analyze bone resorption lacuna formation. To further examine the mechanism of the effect of IL-17-mediated autophagy on the differentiation of osteoclasts, the control group used RANKL medium to culture mouse macrophage RAW264.7 cells, while the IL-17 group was treated with IL-17 (0.01, 0.1, 1.0, /mL). Western blot was used to detect the expression of autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) and osteoclast-related proteins c-fos and nuclear factor of activated T cell 1 (NFATc1) after treatment with different concentrations of IL-17. The expression of LC3, NFATc1, TRAF6/ERK/p38 signaling pathway related proteins were detected in IL-17 and autophagy inhibitor 3-MA group. The number of TRAP positive multinucleated cells, the circumference of the actin ring and the area of bone resorption lacuna in IL-17 group treated with IL-17 (0.01, 0.1, were significantly higher than those in the control group. In IL-17 treated RAW264.7 cells, the expression of c-fos, NFATc1, Beclin-1, LC3, TRAF6, p-ERK, and p-p38 was all significantly up-regulated (all 0.05). After treatment with the autophagy inhibitor 3-MA, the expression levels of LC3, NFATc1, TRAF6, p-ERK, and p-p38 all decreased significantly (all 0.05). IL-17 can promote the expression of autophagy proteins and enhance the differentiation ability of osteoclast precursor cells, and the TRAF6/ERK/p38 signaling pathway may be involved in this process.
探讨白细胞介素（IL）-17 介导致Autophagy 对肿瘤坏死因子受体相关因子（TRAF6）/细胞外信号调节激酶（ERK）/p38 通路和破骨细胞分化的影响。培养小鼠骨髓来源的巨噬细胞（BMM），培养基中含 30ng/ml 巨噬细胞集落刺激因子和 50ng/ml 核因子κB 配体受体激活剂（RANKL），加入 IL-17（0.01、0.1、1.0、10ng/ml）进行干预（IL-17 组）。采用抗酒石酸酸性磷酸酶（TRAP）染色观察 TRAP 阳性多核细胞；鬼笔环肽荧光染色检测肌动蛋白环周长；甲苯胺蓝染色分析骨吸收陷窝形成。为进一步研究 IL-17 介导的 Autophagy 对破骨细胞分化的影响机制，对照组用 RANKL 培养基培养小鼠巨噬细胞 RAW264.7 细胞，IL-17 组用 IL-17（0.01、0.1、1.0/ml）处理。采用 Western blot 检测不同浓度 IL-17 处理后自噬相关蛋白 Beclin-1、微管相关蛋白 1 轻链 3（LC3）和破骨细胞相关蛋白 c-fos 和活化 T 细胞核因子 1（NFATc1）的表达。检测 IL-17 和自噬抑制剂 3-MA 组 LC3、NFATc1、TRAF6/ERK/p38 信号通路相关蛋白的表达。IL-17 组 IL-17（0.01、0.1、1.0/ml）处理后 TRAP 阳性多核细胞数、肌动蛋白环周长和骨吸收陷窝面积明显高于对照组。IL-17 处理 RAW264.7 细胞后，c-fos、NFATc1、Beclin-1、LC3、TRAF6、p-ERK、p-p38 的表达均明显上调（均 P<0.05）。用自噬抑制剂 3-MA 处理后，LC3、NFATc1、TRAF6、p-ERK、p-p38 的表达水平均明显下降（均 P<0.05）。IL-17 可促进自噬蛋白表达，增强破骨细胞前体细胞的分化能力，TRAF6/ERK/p38 信号通路可能参与这一过程。
