周期性张应力作用下Hippo-YAP信号通路调控人牙周膜细胞自噬

doi:10.7518/hxkq.2023.2022382

华西口腔医学杂志 ›› 2023, Vol. 41 ›› Issue (3): 260-268.doi: 10.7518/hxkq.2023.2022382

周期性张应力作用下Hippo-YAP信号通路调控人牙周膜细胞自噬

晚晓芳¹^,²(), 何海燕³, 吕佳岭⁴, 伍宇婕¹^,², 钟冠男¹^,², 徐晓梅¹^,²()

^1.西南医科大学附属口腔医院正畸科，泸州 646000
^2.西南医科大学口颌面修复重建和再生实验室，泸州 646000
^3.成都市温江区人民医院口腔科，成都 611130
^4.富顺县人民医院口腔科，自贡 643299

收稿日期:2022-09-26 修回日期:2023-02-02 出版日期:2023-06-01 发布日期:2023-06-02
通讯作者: 徐晓梅 E-mail:1226505065@qq.com;xuxiaomei@swmu.edu.cn
作者简介:晚晓芳，硕士，E-mail：1226505065@qq.com
基金资助:
泸州市人民政府与西南医科大学科技战略合作项目(20-);西南医科大学校级科研项目(2021LZMS019)

Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress

Wan Xiaofang¹^,²(), He Haiyan³, Jialing Lü⁴, Wu Yujie¹^,², Zhong Guannan¹^,², Xu Xiaomei¹^,²()

^1.Dept. of Orthodontics, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China
^2.Orofacial Reconstruction and Regeneration Laboratory, Southwest Medical University, Luzhou 646000, China
^3.Dept. of Stomatology, Chengdu Wenjiang District People’s Hospital, Chengdu 611130, China
^4.Dept. of Stomatology, Fushun County People’s Hospital, Zigong 643299, China.

Received:2022-09-26 Revised:2023-02-02 Online:2023-06-01 Published:2023-06-02
Contact: Xu Xiaomei E-mail:1226505065@qq.com;xuxiaomei@swmu.edu.cn
Supported by:
Science and Technology Strategic Cooperation Project between Luzhou Municipal Government and Southwest Medical University(2020LZXNYDZ06);School Scientific Research Project of Southwest Medical University(2021LZMS019)

摘要/Abstract

摘要：

目的探究周期性张应力（CTS）作用下人牙周膜细胞（hPDLCs）自噬激活的分子机制。方法分离培养正常牙周组织的hPDLCs，利用Forcel四点弯曲细胞加力仪对hPDLCs加载张应力模拟正畸牙移动过程中正畸力诱导的张力侧hPDLCs自噬，利用XMU-MP-1抑制Hippo信号通路探究Hippo-YAP信号通路在张应力激活hPDLCs自噬中的作用。采用实时荧光定量聚合酶链反应（RT-qPCR）检测hPDLCs自噬相关基因（Beclin-1、LC3、p62）的表达；采用蛋白免疫印迹（Western blot）检测hPDLCs自噬相关蛋白（Beclin-1、LC3-Ⅱ/LC3-Ⅰ、p62）及Hippo-YAP通路相关蛋白（active-YAP、p-YAP）的表达；采用免疫荧光染色定位hPDLCs自噬相关蛋白（LC3-Ⅱ、p62）及Hippo-YAP通路相关蛋白（active-YAP）。结果 CTS激活hPDLCs的自噬，自噬相关因子表达随加力时间的延长呈先升高后降低的趋势，30 min自噬开始，3 h达到高峰，随后出现下调（P<0.05）；CTS促进active-YAP蛋白表达增加，p-YAP蛋白表达降低（P<0.05）。XMU-MP-1抑制Hippo-YAP信号通路后（P<0.05），促进active-YAP蛋白进入细胞核，自噬表达增强（P<0.05）。结论 Hippo-YAP信号通路参与CTS作用下hPDLCs自噬激活的调控。

关键词: 自噬, Hippo-YAP信号通路, 张应力, 人牙周膜细胞

Abstract:

Objective This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs). Methods hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs. Results CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05). Conclusion The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Key words: autophagy, Hippo-YAP signaling pathway, tensile stress, human periodontal ligament cells

中图分类号:

R 783.5

晚晓芳, 何海燕, 吕佳岭, 伍宇婕, 钟冠男, 徐晓梅. 周期性张应力作用下Hippo-YAP信号通路调控人牙周膜细胞自噬[J]. 华西口腔医学杂志, 2023, 41(3): 260-268.

Wan Xiaofang, He Haiyan, Jialing Lü, Wu Yujie, Zhong Guannan, Xu Xiaomei. Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress[J]. West China Journal of Stomatology, 2023, 41(3): 260-268.

图/表 5

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基因名称	引物序列（5'-3'）
GAPDH	F：CAATGACCCCTTCATTGACC R：GACAAGCTTCCCGTTCTCAG
Beclin-1	F：CGTGTCACCATCCAGGAACT R：ATCTCCAAACAGCGTCTGGCT
LC3	F：GAGAAGCAGCTTCCTGTTCTGG R：GTGTCCGTTCACCAACAGGAAG
p62	F：GGACCCGTCTACAGGTGAAC R：GAGAGGGACTCAATCAGCCG

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周期性张应力作用下Hippo-YAP信号通路调控人牙周膜细胞自噬

Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress

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