中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (10): 1560-1566.doi: 10.12307/2023.302

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

高迁移率族蛋白B1及ERK1/2通路对张应力下人牙周膜细胞自噬的影响

张  闵1，2，白书林3，李胜鸿1，2，范智博1，2，谢乙加1，2，徐晓梅1，2   

  1. 1西南医科大学附属口腔医院正畸科，四川省泸州市   646000；2西南医科大学口颌面修复重建和再生实验室，四川省泸州市   646000；3内江市第一人民医院正畸科，四川省内江市   641000
  • 收稿日期:2022-04-16 接受日期:2022-06-02 出版日期:2023-04-08 发布日期:2022-09-08
  • 通讯作者: 徐晓梅，博士，副教授，硕士生导师，西南医科大学附属口腔医院正畸科，四川省泸州市 646000；西南医科大学口颌面修复重建和再生实验室，四川省泸州市 646000
  • 作者简介:张闵，女，1996年生，四川省达州市人，汉族，西南医科大学在读硕士，医师，主要从事口腔正畸学方面的研究。
  • 基金资助:
    四川省科学技术厅科研项目(2021YJ0151)，项目负责人：徐晓梅；西南医科大学科研项目(2021LZMS019)，项目负责人：徐晓梅

Effects of high mobility group box 1 and ERK1/2 pathway on autophagy of human periodontal ligament cells under tensile stress

Zhang Min1, 2, Bai Shulin3, Li Shenghong1, 2, Fan Zhibo1, 2, Xie Yijia1, 2, Xu Xiaomei1, 2   

  1. 1Department of Orthodontics, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 3Department of Orthodontics, First People’s Hospital of Neijiang, Neijiang 641000, Sichuan Province, China
  • Received:2022-04-16 Accepted:2022-06-02 Online:2023-04-08 Published:2022-09-08
  • Contact: Xu Xiaomei, MD, Associate professor, Master’s supervisor, Department of Orthodontics, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Zhang Min, Master candidate, Physician, Department of Orthodontics, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Oral & Maxillofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    Scientific Research Project of Sichuan Provincial Department of Science and Technology, No. 2021YJ0151 (to XXM); Scientific Research Project of Southwest Medical University, No. 2021LZMS019 (to XXM)

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摘要：

文题释义：
高迁移率族蛋白B1(HMGB1)：正畸牙移动机械力刺激信号转导途径及其调控机制是正畸生物力学和生物学研究的热点，正畸牙移动过程中张力侧人牙周膜细胞对机械刺激反应的复杂分子信号网络机制目前尚不太明确。HMGB1是一种高度保守的核蛋白，已证实可以响应多种刺激，在炎症、凋亡、自噬等生物过程中发挥作用。
ERK1/2通路：是胞内的一种蛋白激酶，ERK1/2的活化将各类信号从细胞膜表面受体转导至胞内，促进某些基因的转录与表达，与细胞的增殖、分化及自噬密切相关。

背景：正畸牙移动骨重塑的机械力信号转导通路及其调控机制是正畸生物力学和生物学研究的热点。目前正畸牙移动过程中张力侧牙周膜细胞对机械刺激反应的复杂分子信号网络机制尚不太明确。
目的：探究正畸牙移动过程中张应力作用下高迁移率族蛋白B1(high mobility group protein 1，HMGB1)和ERK1/2通路对人牙周膜细胞自噬的影响。
方法：选取第3-5代生长状态良好的人牙周膜细胞，施加周期性张应力，利用免疫荧光观察HMGB1在细胞中的分布情况，RT-qPCR及Western blot检测自噬相关蛋白LC3-Ⅱ、Beclin-1和HMGB1的表达水平，完成张应力加载后分别提取胞核蛋白和胞浆蛋白，Western blot分别检测胞核及胞浆中HMGB1蛋白水平。为探究HMGB1通过何种机制参与调控细胞自噬，使用丙酮酸乙酯(HMGB1胞浆抑制剂)进行细胞处理，通过免疫荧光、RT-qPCR和Western blot检测细胞自噬水平。最后，选用PD98059抑制ERK1/2通路，通过RT-qPCR以及Western blot检测自噬水平变化，探讨HMGB1通过ERK1/2通路调控自噬的机制。
结果与结论：①通过体外张应力加载，诱导了人牙周膜细胞内自噬水平升高，HMGB1的表达增加，同时HMGB1发生了由胞核到胞质的转位；②人牙周膜细胞通过HMGB1核质转位机制参与张应力作用下自噬的调控；③使用ERK1/2通路抑制剂PD98059显著抑制了张应力作用下 HMGB1引起的细胞自噬；④结果表明，张应力作用下HMGB1从细胞核转移到细胞质，并通过ERK1/2途径调节自噬，维持人牙周膜细胞的稳态和牙周动态平衡。

https://orcid.org/0000-0003-2211-655X (徐晓梅) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 张应力, 牙周膜细胞, 正畸牙移动, HMGB1, 自噬, ERK1/2, 稳态

Abstract: BACKGROUND: The mechanical force signal transduction pathway and its regulation mechanism of bone remodeling after orthodontic tooth movement are hot topics in orthodontic biomechanics and biology. In the present study, the mechanism of complex molecular signal network in the response of periodontal ligament cells to mechanical stimulation during orthodontic tooth movement is not clear.  
OBJECTIVE: To investigate the effect of high mobility group box 1 (HMGB1) and ERK1/2 pathway on autophagy in human periodontal ligament cells under tensile stress during orthodontic tooth movement.
METHODS:  The passages 3-5 human periodontal ligament cells were selected and the HMGB1 distribution in the cells was observed by immunofluorescence under cyclic tensile stress. The expression levels of autophagy-associated protein LC3-II, Beclin-1 and HMGB1 were detected by real-time fluorescence quantitative PCR (RT-qPCR) and western blot assay. After tensile stress loading, nuclear and cytoplasmic proteins were extracted respectively. Western blot assay was used to detect HMGB1 protein level in nucleus and cytoplasm. The mechanisms by which HMGBI participated in regulating autophagy were investigated. Cellular autophagy levels were measured by immunofluorescence, RT-qPCR and western blot assay using ethyl pyruvate, a cytosolic inhibitor of HMGB1. Finally, PD98059 was used to inhibit ERK1/2 pathway, and the autophagy level of HMGB1 was detected by RT-qPCR and western blot assay. The mechanism of HMGB1 regulating autophagy through ERK1/2 pathway was explored.  
RESULTS AND CONCLUSION: (1) By external tensile stress loading, the autophagy level and HMGB1 expression of human periodontal ligament cells were increased and HMGB1 translocated from nucleus to cytoplasm. (2) Human periodontal ligament cells participated in the regulation of autophagy induced by tensile stress through HMGB1 nuclear-cytoplasmic translocation. (3) PD98059, an inhibitor of the ERK1/2 pathway, significantly inhibited the autophagy induced by HMGB1 under tensile stress. (4) These findings suggested that under tensile stress, HMGB1 transferred from the nucleus to the cytoplasm, and regulated autophagy by ERK12 pathway to maintain the homeostasis of human periodontal ligament cells and periodontal dynamic equilibrium.

Key words: tensile stress, periodontal ligament cell, orthodontic tooth movement, HMGB1, autophagy, ERK1/2, homeostasis

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