组蛋白去甲基化酶JMJD3通过调控巨噬细胞极化抑制实验性牙周炎牙槽骨破坏的研究  

Histonedemethylase JMJD3 inhibits alveolar bone loss by regulating macrophage polarization in periodontitis

作　　者：王瑞玲 鲁嘉韦 罗礼君 Wang Ruiing;Lu Jiawei;Luo Lijun(Department of Periodontology,Stomatological Hospital and Dental School,Tongji University&Shanghai Engineering Research Center of Tooth Restoration and Regeneration&Tongji Research Institute of Stomatology,Shanghai 200072,China)

机构地区：[1]同济大学附属口腔医院牙周病科、同济大学口腔医学院、上海牙组织修复与再生工程技术研究中心、同济大学口腔医学研究所,上海200072

出　　处：《中华口腔医学杂志》2024年第8期823-832,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82071123)。

摘　　要：目的探究组蛋白去甲基化酶——含Jumonji结构域蛋白3(JMJD3)在炎症牙周组织中的表达及其对牙周炎调控的潜在机制。方法分析2022年发表在基因表达综合数据库(GEO)中牙周组织单细胞测序的结果,并收集2021年6至12月同济大学附属口腔医院牙周病科和口腔颌面外科牙周手术和拔牙术中获取的健康及牙周炎患者的牙龈样本各9例,进行免疫组织化学染色、实时荧光定量PCR(RT-qPCR)。构建小鼠牙周炎模型,实验分组为:健康对照+生理盐水组、丝线结扎+生理盐水组、丝线结扎+GSK-J4(JMJD3抑制剂)组。体外使用牙龈卟啉单胞菌(Pg)来源的脂多糖(LPS)(Pg-LPS)模拟牙周炎症微环境,并使用靶向Jmjd3的小干扰RNA(siRNA)、GSK-J4分别处理巨噬细胞。siRNA转染实验分组为:阴性对照序列转染(NC)组、siRNA-Jmjd3组、NC+LPS组、siRNA-Jmjd3+LPS组。抑制剂实验分组为:二甲基亚砜(DMSO)组、GSK-J4组、DMSO+LPS组、GSK-J4+LPS组。使用蛋白质免疫印迹法、免疫荧光染色等方法探索JMJD3在体内、体外环境下对巨噬细胞极化及牙周炎症的影响。结果牙周炎患者牙龈组织的JMJD3表达(1.97±0.91)显著高于健康牙龈组织(1.00±0.33)(t=2.45,P=0.048)。体外实验RT-qPCR结果显示,siRNA敲低JMJD3或使用GSK-J4抑制JMJD3均可促进炎症环境下巨噬细胞的M1极化,抑制M2极化:NC+LPS组精氨酸酶Ⅰ(Arg1)的表达(0.90±0.06)显著高于siRNA-Jmjd3+LPS组(0.61±0.11)(P<0.01);NC+LPS组白细胞介素(Il)-6、Il-1β和肿瘤坏死因子α(Tnf-α)的表达(分别为8.50±0.16、5.56±0.20、3.44±0.16)均显著低于siRNA-Jmjd3+LPS组(分别为14.63±0.48、8.55±0.10、11.72±0.58)(均P<0.01)。DMSO+LPS组Arg1、类几丁质酶3样分子(Ym1)、Il-10的表达(分别为0.82±0.01、0.35±0.16、1.47±0.11)均显著高于GSK-J4+LPS组(分别为0.55±0.03、0.22±0.21、0.51±0.11)(均P<0.01);DMSO+LPS组Il-6、Il-1β、Tnf-α的表达(分别为2.03±0.13、3.63±0.14和4.06±0.03)均显著低于GSObjective To investigate the expression of histone demethylase,Jumonji domain-containing protein 3(JMJD3),in inflammatory periodontal tissues and its potential mechanism for the regulation of periodontitis.Methods The results of single-cell sequencing of periodontal tissues published in the Gene Expression Omnibus(GEO)database in 2022 were analyzed.Nine gingival samples each from healthy and inflamed periodontal patients were collected during periodontal surgery or tooth extractions for immunohistochemical staining and real-time fluorescence quantitative PCR(RT-qPCR).Mice periodontitis models were constructed,and the experimental groups were:healthy control+saline group,silk ligation+saline group,silk ligation+GSK-J4(inhibitor of JMJD3)group.Lipopolysaccharide(LPS)derived from Porphyromonas gingivalis(Pg)(Pg-LPS)was used to mimic the periodontal inflammatory microenvironment.The macrophages were treated with small interfering RNA(siRNA)targeting Jmjd3 and the JMJD3 inhibitor GSK-J4.siRNA transfection experiments were grouped into the following:the NC group(negative control sequence transfection group),the siRNA-Jmjd3 group,the NC+LPS group,siRNA-Jmjd3+LPS group.Inhibitor experiments were grouped as dimethyl sulfoxide(DMSO)group,GSK-J4 group,DMSO+LPS group,GSK-J4+LPS group.Western blotting and immunofluorescence staining were used to explore the effects of JMJD3 on macrophage polarization and periodontal inflammation in the in vivo and in vitro settings.Results RT-qPCR results showed that JMJD3 expression in gingival tissues of periodontitis patients(1.97±0.91)was significantly higher than that in healthy gingival tissues(1.00±0.33)(t=2.45,P=0.048).RT-qPCR results of in vitro experiments showed that either siRNA knockdown of JMJD3 or inhibition of JMJD3 using GSK-J4 promoted M1 polarization and inhibited M2 polarization in macrophages under inflammatory environment:the expression of arginaseⅠ(Arg 1)in the NC+LPS group(0.90±0.06)was significantly higher than that in the siRNA-Jmjd3+LPS group(0.61±0.11)(P<0.01

关 键 词：牙周炎 牙槽骨质丢失 单细胞测序 含Jumonji结构域蛋白3 巨噬细胞 巨噬细胞极化 

分 类 号：R781.42[医药卫生—口腔医学]