Figure 2.

Metabolite distribution in periapical lesions and healthy pulp tissues. (A) Venn diagram representing the number of unique and shared metabolites in periapical abscess, radicular cyst, periapical granuloma, and healthy control. The color code is the same as that of Table 3 which lists the most important metabolites in full. The unique metabolites in each group were 14, 12, 11, and 8 metabolites in radicular cysts, healthy control, periapical abscesses, and periapical granulomas, respectively. The shared metabolites were as follows: 24 metabolites in periapical abscesses and granulomas; 14 metabolites in healthy control and radicular cysts; 9 metabolites in all groups; 3 metabolites in all periapical lesion groups; 2 metabolites in healthy control, periapical abscesses, and radicular cysts; and 2 metabolites in healthy control, periapical abscesses, and periapical granulomas. Only one metabolite was found to be shared between periapical abscesses and radicular cysts; between radicular cysts and periapical granulomas; between healthy control and periapical granulomas; and between healthy control, radicular cysts, and periapical granulomas. (B) Unsupervised hierarchical clustering and heatmap analysis of the identified metabolites in the periapical lesions and healthy control. The values of metabolites represented the weighted average of area under the peak in each group of healthy control, periapical abscess, radicular cyst, and periapical granuloma. Euclidean distance measure and Ward linkage analysis were used to carry out unsupervised hierarchical clustering using the metabolomics data. Heatmap analysis showed that the metabolites clustered into two separate groups, a group representing the healthy control and radicular cyst and the other group representing the periapical abscess and granuloma. In the healthy control group, carboxylic acid metabolites in cluster C are included in dental pellet composition, while cluster D metabolites are responsible for normal signal transduction and antimicrobial activity. Cluster E metabolites enhance NK cells with some antifungal activities. Oleic acid in cluster F has anti-inflammatory and antimicrobial activities, in addition to dendritic cell activation. Cluster G metabolites are known to activate NK cells and dendritic cells and induce apoptotic cellular response. In the periapical abscess, cluster H metabolites are correlated to M2 polarization and IL-10 expression. Cluster I metabolites activate NK cell; increase MMP-9, IL-6, and IL-10 release; and downregulate M1 macrophage polarization. Cluster J metabolites have a pro-inflammatory activity and activate the production of IL-8, CYP4F3, and VEGF. In the radicular cyst, cluster K metabolites are generally related to cellular membrane flexibility and radicular cyst expansion. Cluster L metabolites are highly hydrophobic lipid metabolites released during lipophagy. Cluster M metabolites are significantly correlated to induce apoptosis. In the periapical granuloma, cluster N metabolites induce anti-inflammatory M2 polarization. Cluster O metabolites maintain the chronic inflammation and induce M2 macrophage polarization. Cluster P contains L-(+)-lactic acid and ethylene glycol that appear to be involved in granuloma formation, polarize more M2 macrophage, and reduce the cytotoxic effect of NK cells.