Table 4.

The relation between the unique metabolites, enrichment pathways, and the related functional genes in periapical lesions and healthy controls.

Group	Unique metabolites	Enrichment pathway	Associated genes to the enriched metabolic pathway and tissue condition
Periapical abscess	•17-Octadecynoic acid	•R-HSA-2142816: synthesis of (16–20)-hydroxyeicosatetraenoic acids (HETE) •GO:0007005: mitochondrion organization	•The amount of total lipid increased during abscess development (40). 20-HETE is a lipid eicosanoid metabolite derived from the metabolism of arachidonic acid (41). Human PMNs express CYP4F3 (42), which produces 20-HETE (43). HETE potentiates angiogenesis, inflammation, and apoptosis (44, 45). HETE potentiates VEGF expression (46) and MMP-9 (47) but compromises the survival and function of endothelial cells for collateral vessel formation (48), thus interfering with blood vessel formation. HETE increases reactive oxygen species production and NF-κB activity. This results in endothelial activation characterized by the increase in the expression of IL-8 (49). Bacteroides surface protein A (BspA) is identified in oral bacteria like Tannerella forsythia (50), the major oral pathogens in abscess formation (51). Binding of BspA to TLR2 causes the release of chemokine IL-8 from the human gingival epithelial cells (52). •Abscess in general is an osmotically active environment (53) and mitochondrial remodeling occurs during hyperosmotic stress (54). •In conclusion, upregulation of CYP4F3 in periapical abscess enhances the production of 20-HETE, which in turn upregulated VEGF and MMP-9 and activated NF-κB and hence increased the expression of IL-8. Furthermore, upregulation of TLR2 has a stimulatory effect on the upregulation of IL-8.
Radicular cyst	•Beta-sitosterol •Ethanimidic acid	•R-HSA-9613354: lipophagy •GO:0005977: glycogen metabolic process •GO:1901983: regulation of protein acetylation	•Lipophagy is used to describe the autophagic degradation of lipid droplets (55). Nutrient depletion promotes lipophagy (56, 57). Nutritional deficiency is the most proposed theory for the formation of radicular cyst (58). Lipophagy was reported in lipid-laden macrophages (foam macrophages) (59, 60) and in memory CD8 T cells (61) in odontogenic cyst (62–64). IL-12 is important in CD8 T-cell clonal expansion in addition to the generation of memory CD8 T cells (65). IL-12 released from macrophages inhibits VEGF and MMP-9 (66). IL-17A released from Th-17 enhances either directly bone resorption in periapical lesions (67) or indirectly through stimulating autophagy in macrophages and osteoclast differentiation (68). •High cellular metabolism in radicular cyst is due to its inflammatory origin (69) and glycogenolysis is necessary for inflammatory macrophage survival (70). •Histone acetyltransferase p300 showed significant higher expression in periapical cyst in comparison with healthy tissue (71). •In conclusion, IL-12A was the most prominent upregulated gene, which is highly correlated to CD8 effector and memory CD8 T cells, which were reported in our study to be the highest lymphocytes present in radicular cyst (Figure 7 and Table S7) with enhanced lipophagy. Additionally, IL-12 has an inhibitory effect on the expression of VEGF and MMP-9. IL-17, which is related to bone resorption in periapical lesions, also stimulates the autophagy in macrophages and osteoclasts.
Periapical granuloma	•L-(+)-Lactic acid •Octanoic acid •Decanoic acid •Petroselinic acid	•R-HSA-383280: nuclear receptor transcription pathway M162: PID RXR VDR PATHWAY •GO:0060149: negative regulation of posttranscriptional gene silencing •R-HSA-1989781: PPARA activates gene expression	•Retinoid X receptor (RXR) has sequence similarities to ROR subfamily of NRs (72). Retinoic acid-related orphan receptor gamma t (RORγt) is a nuclear receptor, which is selectively expressed by various lymphocytes. RORγt is critical for the development of secondary and tertiary lymphoid organs and for the thymic development of T-cell lineage (73). RORγt has been extensively studied as the master transcription factor of IL-17 expression and Th17 cells, which are strongly associated with various inflammatory and autoimmune conditions (73). Positive correlations between RORγt and IL-17 protein levels were observed in periapical granulomas (74). Vitamin D receptor (VDR) is an endocrine member of the nuclear receptor superfamily (75). The 1α,25-(OH)2 D3/VDR complex functions to regulate gene transcription through heterodimerization with any of three retinoid X receptor (RXR) isoforms and binds to cognate vitamin D responsive elements (VDREs) in the promoter region of target genes (76). This explains the massive local production of 1,25-(OH)2D by disease-associated macrophages that is seen in patients with granulomatous diseases (77, 78) and even granulation tissue formation in normal wound healing (79). Abnormality in VDR gene related to apical periodontitis (80). Activation of VDR can downregulate MMP-9 in vascular cells (81). •Methylation is related to gene silencing (82). Hypomethylation of TLR2 promoter exacerbates periapical inflammatory and angiogenic responses in association with symptomatic apical periodontitis (83). •PPAR-α is activated under conditions of energy deprivation and prolonged starvation (84, 85). This condition can be observed with a high level of lactic acid (86). MMP-9 protein was not affected by PPARα activators (87) but negatively regulated by VDR pathway (81). PPARα negatively regulates TLR4 activity and therefore exerts anti-inflammatory actions (88). PPARα agonists are found to inhibit endothelial VEGFR2 expression (89). •In conclusion, IL-17A was significantly upregulated due to the activation of RORγt in Th17. TLR4 was significantly downregulated in granuloma compared with healthy (P  < 0.05), which was most probably due to the negative regulation of PPARα. Due to the inhibitory effect of PPARα and VDR pathways, VEGFR and MMP-9 were also downregulated, respectively.
Healthy control	•Oleic acid •L-Glutamic acid •L-Aspartic acid •Pentadecanoic acid •2-Butenedioic acid •Itaconic acid •Glycolic acid	•ko04724: glutamatergic synapse •GO:0035249: synaptic transmission, glutamatergic •ko05033: nicotine addiction •ko05030: cocaine addiction •GO:0006820: anion transport •R-HSA-451306: ionotropic activity of kainate receptors •GO:0070997: neuron death	•Vesicular glutamate transporters (VGLUTs) are involved in the transport of transmitter glutamate into synaptic vesicles and are used as markers for glutamatergic neurons. VGLUT1 is involved mainly in the glutamate-mediated signaling of pain, primarily at the level of healthy peripheral dental pulp (90). •Some patients are smokers which explain the inclusion of nicotine-enriched pathway. •Lidocaine used as local anesthetic solution is one of cocaine derivatives (91). •The organic anion transporter family is known to play an important role in the elimination of a variety of endogenous and exogenous harmful substances from the body (92). Tooth enamel formation or amelogenesis is roughly divided into two consecutive stages: the secretory stage and the maturation stage. In the secretory stage, tall columnar ameloblasts synthesize and secrete enamel matrix proteins. Once the full thickness of the enamel is laid down, the ameloblasts become typical transporting cells and regulate calcium influx and matrix removal in and out of the enamel throughout the process of enamel maturation (93, 94). For the highly mineralized enamel to form, extensive degradation and reabsorption of the organic matrix are essential (93). •Kainate receptors are ionotropic glutamate receptors that mediate fast excitatory neurotransmission and are localized to the presynaptic and postsynaptic sides of excitatory synapses (95). Glutamate receptor ionotropic kainate 1 (GRIK1) has been implicated in tooth development and root formation (96). •In neural death, apoptosis is a part of normal pulp homeostasis (97). Most apoptotic cells in normal pulp can be found at the periphery and are usually associated with the subodontoblastic region rather than with the odontoblastic layer (98). •In conclusion, the healthy control is characterized by a pulp with densely innervated and highly vascularized soft tissues, and hence, VGLUT1 is necessary for the continuous homeostasis of healthy dental pulp. Healthy dental pulps were obtained from completely impacted wisdom tooth expecting the final stages of enamel mineralization in which the organic matrix should be replaced by the inorganic one through organic anion transporter mechanism.