DNA甲基化对LPS诱导牙髓细胞炎症反应的影响及调控机制

The major cause of pulpitis is the mixed infection mainly induced by gram-negative anaerobic bacteria. Lipopolysaccharide (LPS) is one of the key virulence factors of gram-negative bacteria and has been reported to facilitate the release of abundant inflammatory cytokines in human dental pulp cells (hDPCs). DNA methylation is a crucial epigenetic modification which plays critical roles in regulating the host inflammatory response; however, its role in dental pulp inflammation remains unknown. Our previous study demonstrated that DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidin (5-Aza-CdR) could up-regulate the expression level of several pro-inflammatory cytokines induced by LPS in hDPCs, and could activate the NF-κB and MAPK signal pathways. Meanwhile, the expression of DNA methyltransferases 1 (DNMT1) significantly declined, while DNMT3a and DNMT3b had no significant change in hDPCs induced by LPS. To clarify the contribution of DNA methylation to the inflammatory response of hDPCs, this project is designed to employ both ‘gain-of-function’ and ‘loss-of-function’ appoaches of DNMT1 for its target genes screening and validation. The research will also investigate the potential mechanism of DNMT1-dependent DNA methylation and its effect on the inflammatory response of hDPCs, which may repress candidate genes transcriptionally by impeding the binding of certain transcriptional factors to their promoters. To confirm this hypothesis, we test the interaction between the target promoters and corresponding transcriptional factors by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and dual-luciferase report gene system. This project may illuminate the regulatory effect of DNA methylation on the inflammatory response of hDPCs and may open new avenues for researches in dental pulp inflammation.

牙髓炎主要由革兰氏阴性厌氧菌为主的混合感染导致，LPS是革兰氏阴性菌最重要的毒力因子，可诱导人牙髓细胞（human dental pulp cells，hDPCs）释放大量炎症因子。DNA甲基化是重要的表观遗传修饰方式，在炎症免疫反应中发挥调控作用，但其对hDPCs炎症反应的影响尚未知。本课题组前期研究表明，DNA甲基转移酶抑制剂5-Aza-CdR可提高LPS诱导的hDPCs炎症反应中多种促炎因子的表达，活化NF-κB及MAPK信号通路；hDPCs经LPS刺激后DNA甲基转移酶DNMT1表达量显著降低。为阐明DNA甲基化对hDPCs炎症反应的影响及调控机制，本项目拟构建DNMT1过表达、沉默hDPCs模型，筛选下游靶基因并验证，探索DNA甲基化可否通过阻碍序列特异性转录因子与靶基因启动子区的结合抑制基因转录。本项目可望从表观遗传学角度阐明牙髓炎症的发生机制。

牙髓炎是口腔常见疾病，主要由厌氧菌为主的混合感染导致。LPS是G-菌最重要的毒力因子，可诱导人牙髓细胞（human dental pulp cells，hDPCs）释放大量炎症因子，LTA作为G+菌细胞壁重要的表面抗原，能引发牙髓第一道细胞防线成牙本质细胞的炎症反应。DNA甲基化/去甲基化是主要的表观遗传修饰方式，在炎症免疫反应中发挥重要调控作用，为阐明其对牙髓炎症反应的影响及调控机制，本项目研究了DNA甲基化抑制剂5-Aza-CdR及DNA甲基转移酶DNMT1在hDPCs及成牙本质样细胞（human odontoblast-like cells, hOBs）炎症反应中的作用，结果表明：5-Aza-CdR及DNMT1敲低可升高LPS诱导的hDPCs和LTA诱导的hOBs炎症反应中多种促炎因子的表达，激活炎症相关信号通路NF-κB和MAPK，引起牙髓炎症反应。DNA去甲基化酶TET2可通过NF-κB信号通路影响牙髓细胞炎性因子的表达，参与LPS诱导的牙髓细胞炎症反应。在分子机制方面，5-Aza-CdR通过降低LPS/TLR4信号通路关键转导分子TRAF6启动子区甲基化水平，激活NF-κB及MAPK信号通路，促进细胞产生炎性因子，DNMT1既可直接影响炎性因子IL-6启动子区甲基化水平，又可通过调控TRAF6启动子区甲基化水平，影响NF-κB及MAPK信号通路，参与牙髓细胞炎症反应。5-Aza-CdR或敲低DNMT1还能显著提高牙髓细胞miR-146a-5p的表达水平，间接调控LPS诱导的牙髓细胞炎性反应。5-Aza-CdR及DNMT1通过调控LPS/TLR4信号通路转导分子MyD88启动子区甲基化状态而改变其转录水平，影响下游NF-κB信号通路的激活，调控LTA诱导的hOBs炎症反应。本项目阐明了DNA甲基化/去甲基化对牙髓炎症反应的作用机制，扩展了牙髓炎症反应机制的研究领域。