The effect of BMP9 on inflammation in the early stage of pulpitis

Abstract Bone morphogenetic protein 9 (BMP9) tends to be associated with various inflammatory responses of diseases, but its relationship with pulpitis remains unknown. Objective This study aimed to evaluate the effects and mechanisms of BMP9 in pulpitis. Methodology A rat model of pulpitis was used to evaluate the expression of BMP9, which was also analysed in Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-stimulated human dental pulp cells (hDPCs). The effects and mechanism of BMP9 on the regulation of inflammatory factors and matrix metalloproteinase-2 (MMP2) were evaluated using real-time quantitative PCR, western blotting, and immunocytofluorescence. Moreover, the migration ability of THP-1 monocyte-macrophages, treated with inflammatory supernate inhibited by BMP9, was previously tested by a transwell migration assay. Finally, a direct rat pulp capping model was used to evaluate in vivo the influence of the overexpression of BMP9 in pulpitis. Results The expression of BMP9 decreased after 24 h and increased after 3 and 7 d in rat pulpitis and inflammatory hDPCs. The overexpression of BMP9 inhibited the gene expression of inflammatory factors (IL-6, IL-8, and CCL2) and the secretion of IL-6 and MMP2 in Pg-LPS-stimulated hDPCs. The level of phosphorylated Smad1/5 was upregulated and the levels of phosphorylated ERK and JNK were downregulated. The inflammatory supernate of hDPCs inhibited by BMP9 reduced the migration of THP-1 cells. In rat pulp capping models, overexpressed BMP9 could partially restrain the development of dental pulp inflammation. Conclusion This is the first study to confirm that BMP9 is involved in the occurrence and development of pulpitis and can partially inhibit its severity in the early stage. These findings provided a theoretical reference for future studies on the mechanism of pulpitis and application of bioactive molecules in vital pulp therapy.摘要 骨形态发生蛋白9（Bone morphogenetic protein 9, BMP9）常与多种疾病的炎症反应相关，但其与牙髓炎的关联尚未明确。目的 本研究旨在探讨BMP9在牙髓炎中的作用及机制。方法 本研究通过构建大鼠牙髓炎模型，评估BMP9的表达水平；同时在牙龈卟啉单胞菌脂多糖（Porphyromonas gingivalis lipopolysaccharide, Pg-LPS）刺激的人牙髓细胞（human dental pulp cells, hDPCs）中开展相关分析。采用实时定量PCR（real-time quantitative PCR）、蛋白质印迹（western blotting）及免疫细胞荧光（immunocytofluorescence），以评估BMP9对炎症因子及基质金属蛋白酶2（matrix metalloproteinase-2, MMP2）的调控作用与机制。此外，本研究通过Transwell迁移实验，检测经BMP9抑制炎症作用的hDPCs炎症上清液处理的THP-1单核巨噬细胞的迁移能力。最后，采用大鼠直接盖髓模型，评估BMP9过表达对牙髓炎的体内干预效果。结果 在大鼠牙髓炎模型及Pg-LPS刺激的hDPCs中，BMP9的表达在24小时时下调，于3天和7天时上调。在Pg-LPS刺激的hDPCs中，BMP9过表达可抑制IL-6、IL-8及CCL2等炎症因子的基因表达，并降低IL-6与MMP2的分泌。同时，磷酸化Smad1/5的表达水平上调，而磷酸化ERK及JNK的表达水平下调。经BMP9抑制炎症作用的hDPCs炎症上清液可降低THP-1细胞的迁移能力。在大鼠直接盖髓模型中，BMP9过表达可部分抑制牙髓炎症的进展。结论 本研究首次证实BMP9参与牙髓炎的发生与发展，并可在早期阶段部分减轻其严重程度。上述研究结果为未来牙髓炎的机制研究及生物活性分子在活髓治疗中的应用提供了理论参考。