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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (2): 162-170    DOI: 10.3724/zdxbyxb-2021-0099
专题报道     
白介素-17 诱导自噬促进破骨前体细胞分化的机制
沈烨琦(),王中秀,谭静怡,钟佳慧,陈莉丽()
浙江大学医学院附属第二医院牙周病专科，浙江 杭州 310009
TRAF6/ERK/p38 pathway is involved in interleukin-17-mediated autophagy to promote osteoclast precursor cell differentiation
SHEN Yeqi(),WANG Zhongxiu,TAN Jingyi,ZHONG Jiahui,CHEN Lili()
Department of Periodontology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
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摘要：

目的：探讨白介素-17（IL-17）诱导的自噬及其相关蛋白在促进牙槽骨破坏过程中的具体机制。方法：对照组以含30?ng/mL巨噬细胞集落刺激因子、50?ng/mL核因子κB 受体活化因子配体（RANKL）的培养基诱导小鼠骨髓来源巨噬细胞（BMM），IL-17组分别以 0.01、0.1、1.0、10 ng/mL IL-17 处理，采用抗酒石酸酸性磷酸酶（TRAP）染色观察 TRAP 阳性多核破骨细胞；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情况。另设对照组使用含50?ng/mL RANKL 的培养基诱导 RAW264.7 细胞，IL-17组分别以 0.01、0.1、1.0、10?ng/mL的 IL-17 处理，采用蛋白质印迹法检测不同浓度 IL-17 处理后自噬相关蛋白 Beclin-1、微管相关蛋白 1 轻链3（LC3）及破骨细胞相关蛋白 c-fos、活化 T 细胞核因子 1（NFATc1）的表达；检测1.0?ng/mL IL-17 处理及加入自噬抑制剂 3-MA 后 RAW264.7 细胞 LC3、NFATc1、TRAF6/ERK/p38 信号通路相关蛋白的表达。结果：0.01、0.1、1.0?ng/mL IL-17 处理 BMM 后，TRAP 阳性多核破骨细胞数量、肌动蛋白环周长以及骨吸收陷窝面积较对照组均增加，且1.0?ng/mL IL-17 处理 RAW264.7 细胞后，c-fos、NFATc1、Beclin-1、LC3、TRAF6、磷酸化 ERK、磷酸化 p38 蛋白表达均上调（均P<0.05）；而采用自噬抑制剂 3-MA 处理后，LC3、NFATc1、TRAF6、磷酸化 ERK、磷酸化 p38 的表达水平均下降（均P<0.05）。结论：IL-17 可促进 Beclin-1、LC3 等自噬关键蛋白表达，增强破骨前体细胞的分化能力，TRAF6/ERK/p38 信号通路参与该过程。
关键词： 破骨细胞;  细胞分化;  白介素-17;  自噬;  TRAF6/ERK/p38 通路    
Abstract:

Objective: 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. Methods: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 50?ng/mL 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, 10?ng/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 1.0 ?ng/mL IL-17 and autophagy inhibitor 3-MA group. Results: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, 1.0?ng/mL) 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 P <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 P <0.05). Conclusion: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.
Key words: Osteoclast    Cell differentiation    Interleukin-17    Autophagy    TRAF6/ERK/p38 pathway
收稿日期: 2021-01-14 出版日期: 2021-06-18
CLC:  R781.4  
基金资助: 国家自然科学基金（81771072，81800972）
通讯作者: 陈莉丽     E-mail: 21918660@zju.edu.cn;chenlili_1030@zju.edu.cn
作者简介: 沈烨琦，硕士研究生，主要从事牙周炎发生发展机制研究；E-mail：21918660@zju.edu.cn；https：//orcid.org/0000-0003-3265-6787
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沈烨琦
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引用本文:

沈烨琦,王中秀,谭静怡,钟佳慧,陈莉丽. 白介素-17 诱导自噬促进破骨前体细胞分化的机制[J]. 浙江大学学报（医学版）, 2021, 50(2): 162-170.

SHEN Yeqi,WANG Zhongxiu,TAN Jingyi,ZHONG Jiahui,CHEN Lili. TRAF6/ERK/p38 pathway is involved in interleukin-17-mediated autophagy to promote osteoclast precursor cell differentiation. J Zhejiang Univ (Med Sci), 2021, 50(2): 162-170.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0099    或    http://www.zjujournals.com/med/CN/Y2021/V50/I2/162

图 1  3-MA 及不同浓度IL-17 处理后 RAW264.7 细胞增殖活性变化
图 2  不同浓度 IL-17 处理小鼠骨髓来源巨噬细胞后 TRAP 染色结果
图 3  不同浓度 IL-17 处理小鼠骨髓来源巨噬细胞后肌动蛋白环染色结果
图 4  不同浓度IL-17处理小鼠骨髓来源巨噬细胞后骨吸收陷窝的变化
图 5  不同浓度 IL-17 处理破骨细胞相关蛋白表达变化A：c-fos 蛋白表达电泳图；B：NFATc1 蛋白表达电泳图.IL：白介素；NFATc：活化 T 细胞核因子；GAPDH： 甘油醛-3-磷酸脱氢酶.

组 别

n

c-fos

NFATc1

Beclin-1

LC3

对照组

3

1.000±0.026

1.000±0.028

1.000±0.012

1.000±0.020

IL-7 0.01 ng/mL

3

1.068±0.100*

1.142±0.039**

1.070±0.059*

1.053±0.043*

0.1 ng/mL

3

1.127±0.136*

1.121±0.085*

1.050±0.031*

1.053±0.051*

1.0 ng/mL

3

1.162±0.101*

1.146±0.034**

1.046±0.032*

1.168±0.089*

10 ng/mL

3

0.800±0.116*

0.900±0.048*

0.789±0.153*

0.827±0.095*
表 1  不同浓度 IL-17 处理后破骨细胞分化和自噬相关蛋白表达量比较
图 6  IL-17 对自噬相关蛋白表达的影响
图 7  自噬抑制剂对破骨细胞 LC3、NFATc1 及 TRAF6/ERK/p38 通路相关蛋白表达的影响“－”：无；“＋”：有.LC3：微管相关蛋白 1 轻链 3；NFATc：活化 T 细胞核因子；TRAF：肿瘤坏死因子受体相关因子；ERK：胞外信号调节激酶；GAPDH： 甘油醛-3-磷酸脱氢酶； IL：白介素.

组 别

n

LC3

NFATc1

TRAF6

磷酸化 ERK

磷酸化 p38

对照组

3

1.000±0.027

1.000±0.038

1.000±0.017

1.000±0.025

1.000±0.023

IL-17 组

3

1.337±0.099**

1.106±0.061*

1.397±0.084**

1.090±0.042*

1.211±0.115*

3-MA 组

3

0.600±0.051**

0.673±0.021**

0.705±0.016**

0.800±0.023**

0.536±0.090**

IL-17+3-MA 组

3

0.612±0.089**

0.757±0.038**

0.754±0.039**

0.873±0.037**

0.719±0.041**
表 2  自噬抑制剂处理后破骨细胞 LC3、NFATc1、TRAF6、磷酸化 ERK 及磷酸化 p38 蛋白表达量比较
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