炎症微环境中TOM40对牙髓成纤维细胞线粒体呼吸功能的影响  

The effect of TOM40 on mitochondrial respiration of dental pulp fibroblasts in the inflammatory microenvironment

作　　者：李兆扬 刘肖晨 张旗[1] LI Zhaoyang;LIU Xiaochen;ZHANG Qi(Department of Endodontics,Stomatological Hospital and Dental School of Tongji University,Shanghai Engineering Research Center of Tooth Restoration and Regeneration,Shanghai 200072,China)

机构地区：[1]同济大学口腔医学院·同济大学附属口腔医院牙体牙髓科,上海牙组织修复与再生工程技术研究中心,上海200072

出　　处：《口腔生物医学》2024年第3期123-130,共8页Oral Biomedicine

基　　金：国家自然科学基金(82170945,82370950);临床“五新”创新研发项目(SHDC2020CR83058B)。

摘　　要：目的:探究炎症微环境中线粒体外膜转位酶(TOM)复合体亚基TOM40对牙髓成纤维细胞线粒体呼吸功能的影响。方法:开髓暴露法构建大鼠磨牙牙髓炎模型,苏木精-伊红染色观察牙髓坏死面积;免疫组化染色观察牙髓组织促炎因子白介素(IL)-1β、抗肿瘤坏死因子(TNF)-α及DNA氧化损伤标志物8-OHDG的表达情况;TUNEL法检测牙髓组织细胞凋亡情况。体外培养人牙髓成纤维细胞(hDPFs),分别以1、5、10μg/mL脂多糖(LPS)炎性刺激,实时荧光定量PCR及Western blot检测细胞促炎因子IL-1β、TNF-α及促凋亡因子Bax、Bak的表达情况;通过TMRE、DCFH-DA探针及腺苷三磷酸(ATP)含量检测试剂盒检测hDPFs线粒体呼吸功能变化;实时荧光定量PCR筛选TOM复合体中表达下调亚基,并通过免疫荧光染色加以验证;基于筛选结果构建TOM40过表达hDPFs细胞株,并加入LPS刺激,检测线粒体呼吸功能变化。结果:大鼠磨牙牙髓炎模型中,随着暴露时间延长,牙髓坏死面积增大,IL-1β、TNF-α及8-OHDG表达升高,细胞凋亡增多(P<0.05)。炎症hDPFs模型中,LPS浓度增加,IL-1β、TNF-α、Bax及Bak蛋白和基因表达升高,线粒体呼吸功能受损,TOM40表达特异性下调(P<0.05);而过表达TOM40可有效改善LPS刺激导致的hDPFs线粒体膜电位下降、活性氧积累与ATP含量下降(P<0.05)。结论:牙髓炎的发展过程伴随牙髓组织氧化应激与hDPFs线粒体呼吸功能障碍,过表达TOM40可挽救hDPFs受损的线粒体呼吸功能。Objective:To explore the effect of TOM40 on mitochondria respiration of dental pulp fibroblasts in the inflammatory microenvironment.Methods:SD rats mandibular first molar were used to construct the pulpitis model.Hematoxylin-eosin was used to observe the inflammatory infiltration of pulpitis.Immunohistochemical staining was used to visualize the expression of pro-inflammatory factor IL-1β,TNF-αand 8-OHDG in dental pulp tissue.The apoptosis of dental pulp tissue was detected by TUNEL.Human dental pulp fibroblasts(hDPFs)were cultured in vitro and stimulated with LPS at concentrations of 1,5 and 10μg/mL,respectively.The expressions of pro-inflammatory cytokines IL-1β,TNF-αand pro-apoptotic factors Bax and Bak were detected by qRT-PCR and Western blot,and the changes of mitochondrial respiratory function of hDPFs were detected by TMRE,DCFH-DA probe and ATP content detection kit.The expression of down-regulated subunits in TOM complex was screened by qRT-PCR and verified by immunofluorescence staining.TOM40 overexpression hDPFs cell line was constructed based on the screening results,and LPS stimulation was added to detect the changes of mitochondrial respiratory function.Results:In rats molar pulpitis model,with the prolongation of exposure time,the necrotic area of dental pulp,the expression of IL-1β,TNF-α,8-OHDG and apoptosis were increased(P<0.05).In the inflammatory hDPFs model,the expression of IL-1β,TNF-α,Bax and Bak were increased,the respiratory function of mitochondria was impaired,and the expression of TOM40 was specifically down-regulated(P<0.05).Overexpression of TOM40 could effectively improve the decrease of mitochondrial membrane potential,ROS accumulation and ATP content of hDPFs induced by LPS stimulation(P<0.05).Conclusions:The development of pulpitis is accompanied by oxidative stress and mitochondrial respiratory dysfunction of hDPFs,mitochondrial impaired respiratory function could be saved by overexpress TOM40.

关 键 词：TOM40 牙髓炎 牙髓成纤维细胞 线粒体 细胞呼吸 

分 类 号：R781.31[医药卫生—口腔医学]