Inhibitory effects of Fusobacterium nucleatum on osteogenic differentiation of osteoblasts and its molecular mechanism

Background: F.nucleatum is one of the important pathogens of periodontitis, and the main symptom of periodontitis is alveolar bone loss. The study of F.nucleatum in alveolar bone loss caused by periodontitis is still very insufficient. In this study, the biological behavior of osteoblasts stimulated by F.nucleatum was studied to elucidate the mechanism of bone loss in periodontitis. Methods:A primary rat calvarial osteoblast culture model was established and invaded repeatedly with F.nucleatum (ATCC 25586). Transcriptome analysis at multiple time points(days 1, 3, 7, 14, 21 and 28) was applied to identify gene expression changes in osteoblasts stimulated by F.nucleatum. Cell-counting and EDU-labeling assay were used to analyze the effects of F.nucleatum on the cell proliferation. Flow cytometry and enzyme linked immunosorbent assay (ELISA) was used to analysis the cell apoptosis, cell cycle and secreted inflammatory cytokines changes. ALP activity testing, Alizarin red mineralized nodules staining and calcium content assays, qPCR and western blot were carried on to evaluate the osteogenic differentiation and mineralization of osteoblasts. Results: Cytology experiments and RNA-seq analysis showed that F.nucleatum could inhibit cell proliferation, promote cell apoptosis, block cell cycle of osteoblasts and promote inflammatory cytokines production in a dose- and time-dependent manner. Besides, F. nucleatum could significantly inhibited osteoblasts differentiation and mineralization via a series inhibition of the differentiation regulatory genes and proteins. Whole-transcriptome analysis identified a total of 235 transcripts that were differentially expressed in all six time-points, most of which were inflammatory-related genes. The genes, Ccl2, Ccl20, Csf1, Cx3cl1, Cxcl1, Cxcl3, Il6, Birc3, Map3k8, Nos2, Nfkb2, Tnfrsf1b and Vcam1, played core roles in a PPI network and interacted closely with other ones in the infection. In addition, 133 osteogenesis-related DEGs were time-serially dynamically changed in a short time-series expression miner (STEM) analysis, which were enriched in multiple cancer-related pathways. The core dynamic DEGs (Mnda, Cyp1b1, Comp, Phex, Mmp3, Tnfrsf1b, Fbln5 and Nfkb2) had been reported to be closely related to the development and metastasis in tumor and cancer progress. This study is the first to evaluate the long-term interaction of F. nucleatum on osteoblasts, which might increase the risk of cell carcinogenesis of normal osteoblasts, and provides new insight into the pathogenesis of bacterial-induced bone destruction.背景：具核梭杆菌（F.nucleatum）是牙周炎的重要致病菌之一，牙周炎的主要症状为牙槽骨吸收。目前针对具核梭杆菌在牙周炎所致牙槽骨吸收中的研究仍较为匮乏。本研究通过探究具核梭杆菌刺激下成骨细胞的生物学行为，以阐明牙周炎骨丢失的发病机制。 方法：建立原代大鼠颅盖骨成骨细胞培养模型，并用具核梭杆菌（ATCC 25586）反复侵袭该模型。在第1、3、7、14、21及28天多个时间点开展转录组分析，以鉴定具核梭杆菌刺激后成骨细胞的基因表达变化。采用细胞计数法与5-乙炔基-2'-脱氧尿苷（EDU）标记实验分析具核梭杆菌对细胞增殖的影响；通过流式细胞术、酶联免疫吸附实验（ELISA）分别检测细胞凋亡、细胞周期变化以及分泌型炎性细胞因子的表达改变。此外，通过碱性磷酸酶（ALP）活性检测、茜素红矿化结节染色、钙含量测定、实时定量聚合酶链反应（qPCR）及蛋白质印迹法（western blot）实验，评估成骨细胞的成骨分化与矿化能力。 结果：细胞学实验与RNA测序（RNA-seq）分析结果显示，具核梭杆菌可通过剂量与时间依赖的方式抑制成骨细胞增殖、促进细胞凋亡、阻滞细胞周期，并诱导炎性细胞因子分泌。此外，具核梭杆菌可通过一系列分化调控基因与蛋白的表达抑制，显著削弱成骨细胞的分化与矿化能力。全转录组分析共鉴定出235个在全部6个检测时间点均呈差异表达的转录本，其中绝大多数为炎性相关基因。蛋白-蛋白相互作用（PPI）网络分析显示，Ccl2、Ccl20、Csf1、Cx3cl1、Cxcl1、Cxcl3、Il6、Birc3、Map3k8、Nos2、Nfkb2、Tnfrsf1b及Vcam1等基因处于核心调控地位，并在感染过程中与其他基因存在紧密相互作用。此外，通过短时间序列表达挖掘器（STEM）分析，共鉴定出133个成骨相关差异表达基因（DEGs）呈时序动态变化，这些基因富集于多条肿瘤相关通路。核心动态差异表达基因包括Mnda、Cyp1b1、Comp、Phex、Mmp3、Tnfrsf1b、Fbln5及Nfkb2，已有研究证实这些基因与肿瘤的发生发展及侵袭转移密切相关。本研究首次评估了具核梭杆菌与成骨细胞的长期相互作用，发现其可能增加正常成骨细胞的癌变风险，为细菌诱导的骨破坏发病机制提供了全新的研究视角。