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

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GSK-J4+LPS组(分别为2.69±0.16、15.04±1.15、4.36±0.10)(均P<0.01)。体内实验结果发现,抑制JMJD3加剧了小鼠实验性牙周炎的骨丧失,增加了小鼠炎症牙周组织中巨噬细胞的M1极化,降低了M2极化。丝线结扎+生理盐水组的颊侧釉质牙骨质界至牙槽嵴顶的距离(CEJ-ABC)、腭侧CEJ-ABC及M1/M2型巨噬细胞比值[分别为(0.26±0.03)、(0.24±0.01)mm和0.35±0.10]均显著低于丝线结扎+GSK-J4组[分别为(0.34±0.04)、(0.30±0.05)mm和2.50±0.58](t=3.65,P=0.006;t=2.67,P=0.049;t=7.31,P=0.004)。结论单细胞测序及体内外实验验证JMJD3在牙周炎牙周组织中表达上调,JMJD3可能通过调控巨噬细胞极化抑制牙周炎的牙槽骨破坏,从而在牙周炎症过程中发挥保护作用。

Objective 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);the expression of interleukin(Il)-6,Il-1β,and tumor necrosis factor alpha(Tnf-α)in the NC+LPS group(8.50±0.16,5.56±0.20,3.44±0.16)were significantly lower than those in the siRNA-Jmjd3+LPS group(14.63±0.48,8.55±0.10,11.72±0.58)(P<0.01).The expression of Arg1,chitinase-like 3(Ym1),Il-10 in the DMSO+LPS group(0.82±0.01,0.35±0.16,1.47±0.11)were significantly higher(P<0.01)than the GSK-J4+LPS group(0.55±0.03,0.22±0.21,0.51±0.11);the expression of Il-6,Il-1β,and Tnf-αin the DMSO+LPS group(2.03±0.13,3.63±0.14,4.06±0.03)were significantly lower than the GSK-J4+LPS group(2.69±0.16,15.04±1.15,4.36±0.10)(P<0.01).The results of the in vivo experiments revealed that inhibition of JMJD3 exacerbated bone loss in experimental periodontitis mice,increased macrophage M1 polarization,and decreased M2 polarization in inflamed periodontal tissues.The buccal cemento-enamel junction(CEJ)-alveolar bone crest(ABC),palatal CEJ-ABC,as well as the ratio of M1/M2 type macrophages were significantly lower in the silk ligation+saline group[(0.26±0.03),(0.24±0.01)mm,0.35±0.10]than in the silk ligation+GSK-J4 group[(0.34±0.04),(0.30±0.05)mm,2.50±0.58](t=3.65,P=0.006;t=2.67,P=0.049;t=7.31,P=0.004;respectively).Conclusions Single-cell sequencing as well as the in vitro and in vivo experiments verified that JMJD3 expression was upregulated in periodontitis periodontal tissues.JMJD3 may exert a protective role in periodontitis by regulating macrophage polarization,thereby inhibiting alveolar bone destruction associated with the periodontitis.