Identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid

The inflammatory disease periodontitis results in tooth loss and can even lead to diseases of the whole body if not treated. Gingival crevicular fluid (GCF) reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamed sites. In this study, we aimed to discover potential protein biomarkers for periodontitis in GCF proteome using LC-MS/MS. We identified 305 proteins from GCF of healthy individuals and periodontitis patients collected using a sterile gel loading tip by ESI-MS/MS coupled to nano-LC. Among these proteins, about 45 proteins were differentially expressed in the GCF proteome of moderate periodontitis patients when compared to the healthy individuals. We first identified azurocidin in the GCF, but not the saliva, as an upregulated protein in the periodontitis patients and verified its increased expression during periodontitis by ELISA using the GCF of the classified periodontitis patients compared to the healthy individuals. In addition, we found that azurocidin inhibited the differentiation of bone marrow-derived macrophages to osteoclasts. Our results show that GCF collection using a gel loading tip and subsequent LC-MS/MS analysis following 1D-PAGE proteomic separation are effective for the analysis of the GCF proteome. Our current results also suggest that azurocidin could be a potential biomarker candidate for the early detection of inflammatory periodontal destruction by gingivitis and some chronic periodontitis. Our data also suggest that azurocidin may have an inhibitory role in osteoclast differentiation and, thus, a protective role in alveolar bone loss during the early stages of periodontitis.