Does Zn-mediated regulation of the kynurenine pathway provide the link between periodontal disease and diabetes?

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Ebru Afşar, Kadirhan Doğan, Erdem Arslan, Işıl Eranil, Neşe Oral, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6641065/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract It has long been known that there is a relationship between periodontal diseases and diabetes. The present study aimed to assess the effect of pancreatic zinc (Zn) levels on Kynurenin pathways (KP) and glucose homeostasis and the impact of Thymoquinone (TQ) in the periodontal disease animal model. Methods : 10 µl Porphyromonas gingivalis-Lipopolysaccharide (P. gingivalis-Lps) (1mg/ml) was injected 6 times at 48-hour intervals into the palatal gingiva of rats. TQ was given by oral gavage (10 mg/kg per day) for 2 weeks. Glucose homeostasis was assessed using the Homeostatic Model Assessment (HOMA-IR), and β-cell function (HOMA-β Levels). Kynurenine (KYN), Tryptophan (TRP), kynurenic acid (KYNA), quinolinic acid (QA), KYN 3-monooxygenase (KMO), kynureninase, interferon-γ (IFN-γ), insulin, ZIP10, and caspase-3 levels measured by the enzyme-linked immunosorbent assay (ELISA). Zinc levels in the pancreas tissue and plasma samples were measured using a colorimetric method. Morphological changes in the pancreas were identified by hematoxylin and eosin staining, and X-ray radiography determined bone resorption in the maxillary bone. Results : In the LPS group, pancreas ZIP10 and Zn levels increased, the KP pathway was changed to favor KYNA, and impaired glucose homeostasis. TQ administration decreased pancreatic Zn levels, changed KP to favor QA, and improved morphological changes in the pancreas. Conclusion : During the periodontal diseases, KP may be altered by Zn levels through ZIP10 in the pancreas, and impair pancreatic function. Regulation of Zn levels may be key to shared pathways between periodontal diseases and diabetes. Kynurenine Porphyromonas gingivalis Diabetes Thymoquinone Zinc Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Microbes highly colonize the gingival tissues in the oral cavity surrounding each tooth. Correlations have been found between oral health and cardiovascular disease ( 1 ), pregnancy outcomes ( 2 ), Rheumatoid Arthritis ( 3 ), and type II diabetes (T2DM) ( 4 ). Porphyromonas gingivalis (P. gingivalis) is a gram-negative anaerobic bacterium found in the mouth. This species plays a crucial role as a keystone pathogen in the progression of periodontal disease (PD) and contributes to various systemic diseases ( 5 ). P. gingivalis or virulence factors such as lipopolysaccharide (LPS) can spread systemically through active periodontal lesions or by invading cells directly ( 6 – 8 ). Therefore, inflammatory conditions caused by LPS of P. gingivalis may be the cause of the molecular link between periodontal health and diabetes ( 6 ). Previous studies showed the relationship between P. gingivalis and diabetes ( 5 , 9 ). However, many unknown points exist about the molecular connections between oral health impaired by P. gingivalis and the pathogenesis of T2DM. The disruption of zinc may lead to insulin resistance and the development of diabetic complications ( 10 ). In addition, recent studies have revealed that there may be a causal relationship between periodontitis pathology and Zn ( 11 ). Therefore, Zn disregulation may provide molecular connections between PD and T2DM. Previous studies showed that PD and T2DM mellitus share many pathological pathways, including the kynurenine pathway (KP) ( 12 – 15 ). Tryptophan (Trp) is an essential amino acid metabolized by the serotonin or the KP to various bioactive molecules. Pro-inflammatory cytokines, such as interferon-γ (IFN-γ), upregulate the enzyme indoleamine 2,3-dioxygenase (IDO), causing decreases in L-Trp and increasing the metabolite kynurenine (KYN) levels ( 15 ). Then, KYN is converted to kynurenic acid (KYNA) by kynurenine aminotransferase (KAT) or quinolinic acid (QA) by two different pathways: 1) KYN can be converted to anthranilic acid by kynureninase and then converted to QA, or 2) KYN can be converted to 3-hydroxyKYN (3-HK) by KYN 3-monooxygenase (KMO) and then converted to QA by kynureninase-mediated pathway. It is evident from the previous study that increasing concentrations of Zn 2+ inhibit the kynureninase, whereas they activate the KAT ( 16 ). However, it is unknown how the change in Zn levels caused by P. gingivalis alters the KP pathway and whether it contributes to the link between PD and diabetes. Thymoquinone (TQ) is one of the main active components of the essential oil obtained from black cumin (Nigella sativa) seeds. Many studies have demonstrated TQ's antibacterial, hypoglycemic, anti-inflammatory, and anti-oxidative activities ( 17 , 18 ). Thus, this study investigated the effect of Zn on KP metabolism in the pancreas on glucose regulation in impaired oral health rat models created by P. gingivalis-LPS injection and whether TQ affects this change. Our study is the first to examine the molecular link between P. gingivalis-specific periodontal health deterioration and diabetes in pancreatic tissue. It may contribute to understanding the pathogen-specific molecular link between periodontal diseases and diabetes. 2. Methods 2.1 Animals All experimental protocols conducted on male rats were performed by the standards established by the Institutional Animal Care and Use Committee at Aksaray University (2024/9–56). The rats were obtained from the Aksaray University Animal Care Unit. Male albino Wistar rats aged 3 months, weighing 200–300 g, were used throughout all experiments. Standard rat, cow, and tap water were given ad libitum, and the animals were housed in stainless steel cages at standard conditions (23 ± 1°C and humidity 50 ± 5%) with 12:12 h light-dark cycles at all times. 2.2 Establishment of animal model and treatment P. gingivalis is one of the pathogens that mediates PD; for this reason, we injected P. gingivalis-LPS into the palatal gingiva to create a model of impaired periodontal health according to previous studies ( 19 – 22 ). TQ was supplied from Sigma Aldrich (Saint Louis, MO, USA), with purity ≥ 98%. TQ administration was performed using the dose previously reported in the literature. The water solubility of TQ is reported to be > 0,5 mg/mL, which is enough to exert pharmacologic effects ( 23 ). Animals were randomly divided into four groups ( n = 8 for each group). Oral injections were done 6 times at 48-hour intervals into the palatal gingiva between the right and left side first and second upper molars with 10 µl P. gingivalis-LPS or saline; tap water or TQ solution (10 mg/kg per day) was given by oral gavage for 2 weeks. TQ administration was started on the day of injection and continued for 14 days. The Control group received saline injections and tap water; the LPS group (LPS) received P. gingivalis-LPS injections and tap water; the TQ group (TQ) received saline injections and TQ; and the LPS + TQ group (LPS + TQ) received P. gingivalis-LPS injections and TQ. At the end of the experimental period, the rats were examined orally, and their clinical features of periodontitis, such as swelling, redness, and gingival bleeding, were evaluated [2]. After rats were anesthetized intraperitoneally with a mixture of ketamine (80 mg/kg) and xylazine hydrochloride (5 mg/kg), the animals were sacrificed. 2.3 Tissue Collection and Preparation The maxillary jawbone was separated for X-ray analysis, and the other was separated for biochemical analysis. Some pancreatic tissues were stored at -80°C for biochemical analysis, and some were separated for hematoxylin and eosin staining. The excised pancreas and gingival tissues were homogenized in cold PBS and centrifuged at 10,000×g for 5 min for biochemical analysis. The plasma samples were separated by centrifugation at 1,500×g for 15 min. 2.4 Biochemical Analysis KYN, TRP, KYNA, QA, IFN-γ, caspase-3 levels, KMO, and ZIP10 levels were measured in the pancreas tissue, and insulin levels were also measured in plasma samples using the enzyme-linked immunosorbent assay technique, employing commercial kits from Bioassay Technology Laboratory (BT Lab) according to the manufacturer's instructions. Zinc levels in the pancreas tissue and plasma samples were measured using a colorimetric method (Fully Automatic Clinical Biochemistry Analyzer, Mindray BS400). Fasting glucose levels assessment The evening before the measurement day, 8–10 hours before the measurement, animals were restricted from food access, and the tail region was bled and measured by glucometer (plusMED Blood GlucoseMeter, Accuro, pM1-300, Bionime Corporation, Taiwan). Homeostatic model (HOMA) assessment Insulin resistance was assessed using the Homeostatic Model Assessment (HOMA-IR) (insulin (mIU/L) multiplied by fasting blood glucose (mM) divided by 22.5) ( 24 ). β cell function was assessed using the analysis of HOMA-β levels, calculated by the Eq. (360 x fasting plasma insulin)/(fasting plasma glucose − 63) (%) ( 25 ). Determination of Kyn/Trp ratio (KTR) The Kyn/Trp ratio change reflects the degradation rate of Trp. Therefore, the Trp/Kynurenine ratio determined IDO activity ( 26 ). Protein measurements Protein concentrations of tissue samples were measured at 595 nm by a modified Bradford assay using Coomassie Plus reagent with bovine serum albumin as a standard (Pierce Chemical Company, Rockford, IL) ( 27 ). 2.5 Histopathologic examinations of pancreatic tissues The pancreas tissues were fixed in a 10% paraformaldehyde/phosphate-buffered saline solution and then subjected to a standard paraffin-embedding process, obtaining 5–6 µm sections. Finally, the sections were stained with Hematoxylin-eosin (H&E) and evaluated under light microscopy (Leica DFC450, Almanya) ( 28 , 29 ). 2.6 X-ray films of the periodontal tissue The dissected maxillary bone was examined with a periapical X-ray device (KaVo Focus (KaVo Dental, Bieberich, Germany) at 10x magnification to determine the alveolar bone level. The distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) between the alveolar bone was measured medially and distally. The CEJ was connected with a line drawn in silico and indicated on the tooth's mesial and distal surface projections. Then, perpendicular lines were drawn to the alveolar bone crest along the CEJ and the distance was automatically measured by the computer-aided system (CLINIVIEW™ software, Instrumentarium Dental, Tuusula, Finland) ( 30 , 31 ). 2.7 Statistical analysis The statistical analyses of the obtained data were performed using SPSS 23.0 (SPSS, Chicago, IL, USA) software for Windows. Biochemical parameters were analyzed by a one-way analysis of variance (ANOVA) followed by Tukey’s Post Hoc Test for normally distributed variables, and Kruskal-Wallis followed by the Mann-Whitney U test for non-normally distributed variables. Results are expressed as the mean ± SEM. A p-value < 0.05 was considered significant. 3. Results 3.1 Oral examination and radiography results of the experimental groups We observed that the rats' gum tissues became red and swollen, displaying a dark red color and experiencing spontaneous bleeding after two weeks of LPS injection. These preliminary findings indicated the successful induction of impaired oral health. Oral findings of rats in the TQ group were similar to those of the healthy groups. In addition, the gingival tissues of the LPS+TQ group appeared pink, while their gingival margins became red and swollen. No bleeding was detected. X-ray films revealed slightly alveolar bone resorption in the second maxillary molar in the rats injected with LPS, in contrast to the control and TQ-administered rats. Also, alveolar bone resorption was alleviated in the LPS+TQ group (Figure 1). 3.2 Analysis of Zn-related parameters As shown in Table 1, ZIP10 levels in the pancreatic tissue of the control groups were significantly higher than in the TQ and LPS+TQ groups (p=0.001, 0.000), and significantly lower than in the LPS+TQ group (p=0.042). Also, TQ administration significantly decreased ZIP10 levels in the pancreas of the LPS+TQ group compared to the LPS group. The control group's plasma Zn levels were significantly higher than those of the TQ and LPS groups (p=0.017, p=0.000) and significantly lower than those of the LPS+TQ group (p=0.000). Also, the LPS+TQ group's plasma Zn levels significantly increased compared to the LPS group (p=0.000). Pancreas Zn levels of the control group were significantly higher than those of the TQ and LPS+TG groups (p=0.000, p=0.000), and significantly lower than those of the LPS group (p=0.003). Also, pancreas Zn levels of the LPS+TQ group significantly decreased compared to the LPS group (p=0.000). 3.3 Analysis of Cytokine and Caspase-3 Levels in Pancreas Tissue Pro-inflammatory cytokine levels in various groups are shown in Table 1. IFN-γ levels in pancreas samples of the control group were lower than those of the TQ group (p=0.055), the LPS group (p=0.025), and the LPS+TQ group (p=0.037). Also, IFN-γ levels in pancreas samples of the LPS+TQ group were higher than those of the LPS group. However, it is not significant. Caspase-3 levels in pancreas samples of the LPS group significantly decreased compared to the control group (p=0.026) and markedly reduced compared to the LPS+TQ group (p=0.055). 3.3 Analysis Of Glucose Homeostasis Firstly, we investigated the fasting glucose and insulin levels in various groups. As seen in Table 2, the fasting glucose levels of the control group were significantly lower than those of the LPS group and the LPS+TQ group (p=0.000, p=0.000), and the TQ group compared to the LPS group and the LPS+TQ group (p=0.000, p=0.000). Plasma insülin levels of the TQ group were significantly lower than those of the control, LPS, and LPS+TQ groups (p=0.030, p=0.000, p=0.000). TQ administration markedly increased plasma insülin levels of the LPS+TQ group compared to LPS; however, this trend did not reach significant levels. Then, we determined HOMA-IR and HOMA-β levels. The HOMA-IR levels of the control groups were lower than those of the LPS and LPS+TQ groups (p=0.000, p=0.000), and those of the TQ group were lower than those of the LPS and LPS+TQ groups (p=0.000, p=0.000).TQ administration slightly decreased HOMA-IR levels of the TQ group compared to the control group and increased somewhat LPS+TQ group compared to the LPS group; however, these trends did not reach significant levels. Additionally, HOMA-β levels were higher in the control groups compared to TQ, LPS, and LPS+TQ groups (p=0.002, p=0.001, p=0.006). HOMA-β Levels of LPS+TQ groups markedly increased compared to the LPS group. However, this increasing trend did not reach significant levels 3.4 Analysis of Change in KYN Pathways 3.4.1 Analysis of Trp, KYN, KYNA, and QA levels in the pancreas tissue and plasma samples We investigated differences in the various groups' Trp and KYN levels of the pancreas tissue. There was no difference in the Trp and KYN levels of pancreas tissue (p =0.567; p = 0.071). TRP levels determined for the control group 99.727 ± 47.40 ng/g protein, the LPS group 43.495 ± 8.16 ng/g protein, the LPS+TQ group 9.369 ± 31.75 ng/g protein, and KYN levels determined for the control group 0.510±0.132 ng/mg protein, the LPS group 0.871 ± 0.151 ng/mg protein, TQ group 2.156 ± 0.770 ng/mg protein, LPS+TQ group 2.737 ± 0.868 ng/mg protein (Figure 2. A and Figure 2.C). Pancreatic KYNA levels of the LPS group (27.328 ± 3.553 ng/mg protein) were higher than those of the control group (18.601 ± 3.319 ng/mg protein); however, with this increasing trend, there was no significant difference (p=0.078).KYNA levels of the LPS+TQ group (5.679 ± 0.643 ng/mg protein) were decreased compared to the control group, the TQ group (38.023 ± 9.697 ng/mg protein), and the LPS group (p=0.016, p=0.004, p=0.004) (Figure 2B). QA levels of the LPS group (1,029±0,207 ng/mg protein) were lower than those of the control group (2,585±0,317 ng/mg protein) and LPS+TQ group (11,109±2,400 ng/mg protein) (P=0.004, p=0.004) (Figure 2. D ). 3.4.2. Analysis of Kyn/Trp ratio , KMO, and Kynureninase levels in the pancreas tissue Significant differences in the Kyn/Trp ratio of various groups in the pancreas tissue. The pancreas Kyn/Trp ratio is lower in the control group (0.0110 ± 0.0013 µg/ng) compared to the LPS group (0,0213±0,0024 µg/ng) and the LPS+TQ group (0.0301±0.0026 µg/ng) (respectively p=0.010, p=0.004). TQ administration increased the Kyn/Trp ratio in the pancreas of LPS+TQ group rats compared to the LPS group. However, this increasing trend does not reach significant levels (p=0.078) (Figure 3.A). The pancreatic KMO levels of the control group (144.022 ± 25.075 ng/g protein) were higher than those of the LPS group (54.046 ± 11.874 ng/g protein) and the LPS+TQ group (48.342 ± 8.306 ng/g protein) (p=0.048, p=0.033). Also, KMO levels of the TQ group (156.276 ± 34.649 ng/g protein) were higher than those of the LPS group (p=0.021, p=0.015) (Figure 3.B). It was observed that the pancreatic kynureninase levels of the LPS group (1.399 ± 0.059 ng/mg protein) decreased compared to the control group (3.136 ± 0.712 ng/mg protein) and compared to the LPS+TQ group (6.401 ± 2,848 ng/mg protein) (p=0.025, p=0.004). TQ administration increased kynureninase levels of the TQ group (7.328 ± 3.171 ng/mg protein). However, with this increasing trend, there was no significant (Figure 3.C). 3.5 Histological analysis. The results of HE of the pancreatic tissues were evaluated regarding edema, inflammatory cell infiltration, acinar cell degeneration, and hemorrhage parameters (Figure 4). The severity of each criterion was graded from 0 to 3: 0: absent or rare, 1: mild, 2: moderate, 3: severe (28, 29). As seen in Table 3, the damage score scores of the LPS group were statistically higher than those of the control group (p=0.05). The TQ group scores were similar to those of the control group. The damage score of the LPS+TQ group decreased statistically significantly compared to the LPS group but increased significantly compared to the control and TQ groups (p=0.05). 4. Discussion Periodontal health and diabetes have long been known to have reciprocal relationships ( 32 ); however, how these connections are achieved remains unclear. In this study, we investigated the effect of oral pathogen P. gingivalis-LPS on glucose homeostasis by changes in Zn-mediated KYN metabolism and whether TQ affects these conditions. Our results showed that P. gingivalis-LPS injection impaired glucose homeostasis by causing peripheral insulin resistance and deterioration of beta cell functions. Insulin resistance and defects in pancreatic beta cells are the two significant pathophysiologic abnormalities that underlie type 2 diabetes ( 33 ), resulting in hyperglycemia ( 34 ). Additionally, reduced insulin signalling and/or insulin resistance and the associated diminution in glucose transport promote a compensatory increase in pancreatic insulin production, resulting in hyperinsulinaemia. However, even before a diagnosis of type 2 diabetes mellitus (T2DM), excess insulin levels can indicate who is likely to develop the disease ( 35 ). Therephore, increased insulin synthesis, which was observed in the present study, may be an adaptation mechanism against LPS-treatment. Abdelmageed et al. showed that T2D rats exhibited significant increases in fasting glucose and insulin levels, higher HOMA-IR, and lower HOMA-β than the control group ( 36 ). Therefore, our findings are consistent with other studies that show the relationship between PD and DM ( 37 ). Additionally, Zn levels decreased in the plasma of the LPS group, which is consistent with reduced plasma Zn levels in the T2DM and ( 38 ) periodontitis patient ( 10 ). Also, ZIP10 and Zn levels were increased in the pancreas tissue of the LPS group. Zn is an essential component for the normal function of the pancreas, and deficiency and overload of Zn are linked to diverse disorders, including diabetes and obesity ( 38 ). Therephore, the physiological and cellular Zn concentrations are regulated by Zn transporters (ZnTs), and Zn importers (ZIPs, Zrt- and Irt-like proteins) ( 39 ). ZIP10 is part of the ZIP (SLC39) class of transporters that enhance Zn concentration in the cytoplasm by facilitating its influx from the extracellular space or promoting its efflux from intracellular vesicles. Research has shown that ZIP10 transcripts are expressed in pancreatic alpha ( 40 ) and beta cells ( 41 ), suggesting that ZIP10 may regulate glucose homeostasis ( 42 ). This view supports the idea that in isolated breast cancer cells treated with glucose concentrations equivalent to hyperglycemia in humans, the expression of ZIP10 is reported to be up-regulated with a concomitant increase in cellular Zn concentrations ( 43 ). Moreover, Zn is crucial in insulin synthesis, crystallization, storage, secretion, and signaling in the pancreatic β-cells ( 38 ). Our results suggest that increased ZIP10 may lead to transport of Zn into the β cell and increased insülin syntesis. Thus, our results also indicate that changing Zn levels in the pancreas may affect the KP pathway. A study supporting this view demonstrated that Zn triggered the degradation of tryptophan by IDO and the production of kynurenine by dendritic cells (DCs) while also significantly suppressing the pro-inflammatory response to stimulation by Toll-like receptor (TLR) ligands ( 44 ). Similarly, inflammatory cytokine IFN-γ levels and IDO activity in the pancreas tissue of the LPS group were increased in our study. These results may depend on the pathogen type because multiple pathogens are involved in PD pathogenesis ( 45 ). Furthermore, our findings revealed that the effect of P. gingivalis-LPS activates the KP pathway and changes metabolite levels in the pancreas. This may be related to increased pancreatic Zn levels. It is demonstrated that Zn2 + inhibits the kynureninase, which is responsible for converting KYN to QA, whereas it activates the KAT, which converts KYN to KYNA ( 16 ). Consistent with these findings in our study, kynureninase levels decreased in the plasma of the LPS group compared to the control group. Also, our results show that caspase-3 levels decreased in the pancreas of the LPS group, suggesting increased apoptosis. Caspase-3 is a key zymogen in cell apoptosis. It is not activated until it cleaves by initiator caspases during apoptotic flux ( 46 ). Therephore, decreasing zymogen form caspase-3 levels may increase cleaved caspase-3, active caspase form. Previous in vivo and in vitro studies show that QA has apoptotic properties ( 37 ) while KYNA has anti-apoptotic properties ( 47 ). However, Arya et al. showed that the combination dose of quercetin (QE) and QA (50 mg/kg) exhibited maximum inhibition of the pro-apoptotic protein Bax expression and enhanced the anti-apoptotic protein Bcl-2 expression, suggesting a protective role in the kidneys of diabetic rats ( 48 ). Therephore, our results indicate that decreased QA levels in the pancreas of the LPS group may be related to the decrease in HOMA β levels and pancreatic beta cell structure. İmportantly, increased ZIP10-mediated Zn levels may be responsible for these changes. Supporting this view, TQ administration decreased ZIP10 and Zn levels in the pancreas tissue of the LPS-TQ group. Also, IDO activity increased more in the LPS + TQ group than in the LPS group, while QA levels increased and KYNA levels decreased, contrary to the LPS group. Decreased Zn levels and increased IDO activity may be related to Zn's dose-dependent effects. Moreover, our results suggest that QA levels in the pancreas tissue of the LPS + TQ group, due to the suppressive effect on kynureninase, may be eliminated by decreasing the Zn level, as mentioned earlier ( 16 ). Also, results showed that insulin levels in the plasma and zymogen form of caspase-3 levels in the pancreas were increased, and pancreatic morphology was improved in the LPS + TQ group. QA is an agonist of neuronal N-methyl-D-aspartate receptors (NMDARs) [45], and KYNA is a competitive NMDAR antagonist ( 49 ). Lockridge et al. showed that D-serine can have acute antidiabetic effects in mice and potentiates insulin secretion through excitatory β-cell NMDAR co-agonism ( 50 ). Thus, our results suggest that changes in QA and KYNA levels may be responsible for decreasing β-cell number and function in the LPS group's pancreas. TQ administration may improve this by regulating Zn levels through ZIP10. Our findings were consistent with previous studies, which showed that TQ protects against STZ-induced diabetes by repressing apoptosis of β-cells, ameliorating β-cell ultrastructure, and leading to insülin secretion ( 51 , 52 ); KYNA, which has potentiated hyperglycemic effects by inhibiting proinsulin synthesis and insulin secretion in rat pancreatic islet cells ( 13 ) and an apoptotic effect on cancer cells [55, 56]. Additionally, TQ administration decreased plasma and pancreatic Zn levels and ZIP10 levels in the pancreas tissue of the TQ group. Our result showed decreased HOMA-IR and plasma insülin levels in the TQ group. Zn is crucial in insulin synthesis, crystallization, storage, secretion, and signaling in the pancreatic β-cells. In the process of ınsulın synthesis, insülin is transformed into an insoluble crystalline hexamer, including two Zn ions (Zn²⁺) and one calcium ion (Ca²⁺) in granules. As β-cells secrete hexameric insulin into the extracellular space, the hexamers quickly dissociate into active monomers within a few seconds [20]. Therephore, decreased plasma insulin levels may be related to suppressed β-cell function and insülin secterion due to the effect of TQ on insulin sensitivity ( 53 ). Also, this may be due to the increased KYNA levels in the pancreas of rats administered only TQ because KYNA and QA continue to act oppositely in inflammation and other functions ( 54 ). Our results suggest that Zn-mediated alteration of the pancreatic KP pathway may provide the molecular link between PD and T2DM. Furthermore, ZIP10's regulation of Zn levels in the pancreas may mediate TQ's anti-diabetic effect. However, the increasing impact of TQ on insulin in the LPS group may be a risk of developing diabetes in the long term. This issue needs to be investigated further. Interestingly, our results showed that the TQ administration increased the inflammatory cytokines in the pancreas of LPS + TQ groups. Cytokines are central mediators of immune responses, and T helper (Th) cells are professional cytokine-producing cells. Once activated, CD4 + T helper cells further differentiate into Th1 cells, which specialize in producing IL-2, IL-6, IL-12, and IFNγ. Therefore, agents that can influence the Th cells' differentiation have the potential to alter the adaptive immune response in various diseases and medical conditions ( 55 ). Th1 cells are crucial for host defense against intracellular pathogens, including viruses, protozoa, and bacteria. One of their primary functions is to activate macrophages by producing IFN-γ ( 56 ). Additionally, our findings were consistent with the observation that purified protein extracts of N. sativa seeds significantly enhanced the production of TNF-α from unstimulated and PWM-activated lymphocytes [52] and serum IFN-γ levels in CMV-infected mice ( 57 ). Also, our results are consistent with the study's findings, which show that TQ reduces bone resorption in an experimental periodontitis model ( 58 ). Therephore, our results may indicate that activation of the immune response by TQ has a beneficial effect on impaired glucose regulation and bone resorption by P. gingivalis-LPS. When the results obtained from our research are evaluated together, it can be thought that the increase in pancreatic Zn concentration via ZIP10 due to LPS treatment induces the KP pathway and increased insulin synthesis as an adaptation mechanism against disturbance of pancreatic function. TQ treatment reversed the effect of LPS on KP pathways, improved the pancreas morphological structure, and increased insülin syntesis. 5. Conclusion In conclusion, our results suggest that oral application of P. gingivalis-LPS causes an increase in pancreatic Zn levels by ZIP10, and causes a change in KP metabolites, favoring KYNA production and resulting in apoptosis of pancreatic cells. TQ administration reverses these changes induced by P. gingivalis, alleviating the impairment of β-cell function. Our study examined the relationship between periodontal diseases and diabetes in the early period when oral health deterioration and bone resorption begin through the Zn-mediated changes in the KP pathway, specifically for P. gingivalis. According to the results of our study, the P. gingivalis pathogen may mediate the formation of diabetogenic conditions via the KP pathway in the deterioration of oral health. The results obtained from our research are expected to contribute to understanding this link. Abbreviations LPS Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) injected group TQ Thymoquinone administered group LPS + TQ P. gingivalis-LPS injected and TQ administered group. Declarations Statements & Declarations Funding This work was supported by KÜN.2024-BAGP-012 Grant numbers and Author E.A. has received research support from the Cappodoccia University Research Foundation, Turkey Competing Interests The authors declare no conflict of interest. Author contribution statement EA (Ebru Afşar), EA (Erdem Arslan), and MO did a P.gingivalis injection and oral gavage application. EA (Erdem Arslan) and MO also monitored laboratory animals' living conditions and nutrition. KD and TC sacrificed experimental animals and collected samples. They were also responsible for transferring samples from the experimental animal unit to Cappadocia University, where the analyses would be conducted. NO performed X-ray analyses and evaluated these analyses. IE performed hematoxylin and eosin staining analyses and evaluation of these analyses. SS performed oral examinations of experimental animals before and after the injection procedure. EA (Ebru Afşar) and SS conducted the literature review and designed the study. EA (Ebru Afşar) designed the study, performed biochemical and statistical analyses, and interpreted the analysis results. Data Availability Statement The data supporting this study's findings are available from the corresponding author upon reasonable request. Ethics approval Ethics approval provided by the Institutional Animal Care and Use Committee at Aksaray University (2024/9-56). 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Supplementary Files TABLE1.docx TABLE2.docx TABLE3.docx Graphicalabstract.png Graphical Abstract A: The effect of P.gingivalis-LPS injection on pancreatic KP and glucose homeostasis B: The effect of TQ administration on changes of P.gingivalis-LPS-Induced pancreatic KP and glucose homeostasis Abbreviations: P.gingivalis-LPS, Porphyromonas gingivalis-lipopolysaccharide; Trp, Tryptophan; KYN, Kynurenine. KP, Kynurenine pathway; KYNA, kynurenic acid; QA, quinolinic acid; KMO, Kynurenine 3-monooxygenase, IDO, indoleamine 2,3-dioxygenase TDO, tryptophan-2,3-dioxygenase. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6641065","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456313984,"identity":"3038021f-1047-46d6-b8c4-762e40d974a0","order_by":0,"name":"Ebru Afşar","email":"data:image/png;base64,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","orcid":"","institution":"Cappadocia University","correspondingAuthor":true,"prefix":"","firstName":"Ebru","middleName":"","lastName":"Afşar","suffix":""},{"id":456313985,"identity":"8391ceeb-7c83-4e8a-baa5-09d0b62eb5e3","order_by":1,"name":"Kadirhan Doğan","email":"","orcid":"","institution":"Cappadocia University","correspondingAuthor":false,"prefix":"","firstName":"Kadirhan","middleName":"","lastName":"Doğan","suffix":""},{"id":456313986,"identity":"9dca7067-7c34-4d8e-85de-974188430d37","order_by":2,"name":"Erdem Arslan","email":"","orcid":"","institution":"Aksaray University","correspondingAuthor":false,"prefix":"","firstName":"Erdem","middleName":"","lastName":"Arslan","suffix":""},{"id":456313987,"identity":"14fec6a3-87bb-4094-b276-cacdcfab9200","order_by":3,"name":"Işıl Eranil","email":"","orcid":"","institution":"Cappadocia University","correspondingAuthor":false,"prefix":"","firstName":"Işıl","middleName":"","lastName":"Eranil","suffix":""},{"id":456313988,"identity":"7a5784a3-1fc3-4edc-a2b3-6ca2847d41bc","order_by":4,"name":"Neşe Oral","email":"","orcid":"","institution":"Cappadocia University","correspondingAuthor":false,"prefix":"","firstName":"Neşe","middleName":"","lastName":"Oral","suffix":""},{"id":456313989,"identity":"98fd0a28-c3e4-4287-84ca-864d6341d87e","order_by":5,"name":"Bahadır Kadir Kuzzu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bahadır","middleName":"Kadir","lastName":"Kuzzu","suffix":""},{"id":456313990,"identity":"688b3ea6-7091-4ab0-90b7-39697a45adc7","order_by":6,"name":"Tayfun Ceylan","email":"","orcid":"","institution":"Cappadocia University","correspondingAuthor":false,"prefix":"","firstName":"Tayfun","middleName":"","lastName":"Ceylan","suffix":""},{"id":456313991,"identity":"2d7452ed-ef90-4995-b443-d53e200a00e7","order_by":7,"name":"Mehmet Öz","email":"","orcid":"","institution":"Aksaray University","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"Öz","suffix":""}],"badges":[],"createdAt":"2025-05-11 18:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6641065/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6641065/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82922116,"identity":"1d71f86e-ac28-4c64-a0e4-782ead73b0ab","added_by":"auto","created_at":"2025-05-16 17:57:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eX-Ray Films of the Periodontal Tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: LPS, \u003c/strong\u003ePorphyromonas gingivalis-lipopolysaccharide\u003cstrong\u003e \u003c/strong\u003e(P. gingivalis-LPS) injected group; \u003cstrong\u003eTQ,\u003c/strong\u003eThymoquinone administered group; \u003cstrong\u003eLPS+TQ,\u003c/strong\u003e P. gingivalis-LPS injected and TQ administered group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/19ddf4b772340010b3c7143a.png"},{"id":82922118,"identity":"508c8771-f4b2-4e92-9de1-02889f5702c6","added_by":"auto","created_at":"2025-05-16 17:57:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Trp, KYN, KYNA, and QA levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eThe Trp levels of pancreas tissue; \u003cstrong\u003eB:\u003c/strong\u003e KYNA levels of pancreas tissue; \u003cstrong\u003eC:\u003c/strong\u003e The KYN levels of pancreas tissue; \u003cstrong\u003eD:\u003c/strong\u003e The QA levels of pancreas tissue.\u003c/p\u003e\n\u003cp\u003eStatistical analyses were done using Kruskal-Wallis's One Way Analysis of Variance on Ranks. All pairwise multiple comparison procedures were done using the Mann-Whitney U test. All values are mean ± SEM and n=6 for each group. *, p\u0026lt;0,05 vs control; **, p\u0026lt;0,01 vs control; ***, p\u0026lt;0,001 vs control; \u003csup\u003e#\u003c/sup\u003e, p\u0026lt;0,05 vs TQ; \u003csup\u003e##\u003c/sup\u003e, p\u0026lt;0,01 vs TQ; \u003csup\u003e###\u003c/sup\u003e, p\u0026lt;0,001 vs TQ; \u003csup\u003eƐ\u003c/sup\u003e, p\u0026lt;0,05 \u003csup\u003eƐƐ\u003c/sup\u003e, p\u0026lt;0,01 vs LPS \u003csup\u003eƐƐƐ\u003c/sup\u003e, p\u0026lt;0,001 vs LPS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: Trp, \u003c/strong\u003eTryptophan; \u003cstrong\u003eKYN,\u003c/strong\u003e Kynurenine; \u003cstrong\u003eKYNA, \u003c/strong\u003eKynurenic acid;\u003cstrong\u003e QA.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/e90994e6de32fcad1e94c8fd.png"},{"id":82922123,"identity":"78f2b052-6e61-4c71-aa47-3b0441220ff3","added_by":"auto","created_at":"2025-05-16 17:57:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Kyn/Trp ratio\u003c/strong\u003e, \u003cstrong\u003eKMO, and Kynureninase levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: Kyn/Trp \u003c/strong\u003elevels of pancreas tissue; \u003cstrong\u003eB:\u003c/strong\u003e KMO levels of pancreas tissue; \u003cstrong\u003eC:\u003c/strong\u003e Kynureninase levels. Statistical analysis of KMO levels of pancreas tissue was done using a one-way analysis of variance (ANOVA) followed by Tukey’s Post Hoc. Statistical analysis of \u003cstrong\u003eKyn/Trp\u003c/strong\u003e and Kynureninase levels in pancreas tissue was done using Kruskal-Wallis's One Way Analysis of Variance on Ranks. All pairwise multiple comparison procedures were done using the Mann-Whitney U test. All values are mean ± SEM and n=6 for each group.*, p\u0026lt;0,05 vs control; **, p\u0026lt;0,01 vs control; ***, p\u0026lt;0,001 vs control; \u003csup\u003e#\u003c/sup\u003e, p\u0026lt;0,05 vs TQ; \u003csup\u003e##\u003c/sup\u003e, p\u0026lt;0,01 vs TQ; \u003csup\u003e###\u003c/sup\u003e, p\u0026lt;0,001 vs TQ; \u003csup\u003eƐ\u003c/sup\u003e, p\u0026lt;0,05 \u003csup\u003eƐƐ\u003c/sup\u003e, p\u0026lt;0,01 vs LPS \u003csup\u003eƐƐƐ\u003c/sup\u003e, p\u0026lt;0,001 vs LPS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: Trp, \u003c/strong\u003eTryptophan; \u003cstrong\u003eKYN,\u003c/strong\u003e Kynurenine; \u003cstrong\u003eKMO, \u003c/strong\u003eKynurenine 3-monooxygenase.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/c4cd2f4e93eb9976a3a79c73.png"},{"id":82922303,"identity":"b889b5b8-c03a-4d0f-92b1-c97b1adfbf90","added_by":"auto","created_at":"2025-05-16 18:05:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological changes in the pancreas with hematoxylin and eosin stain (magnification × 40\u003c/strong\u003e). \u003cstrong\u003eA:\u003c/strong\u003e Photomicrograph of the pancreas from control rats showing normal parenchyma cells with collagen fibrils. \u003cstrong\u003eB:\u003c/strong\u003e Photomicrograph of the pancreas from TQ control rats showing typical tissue architecture. \u003cstrong\u003eC:\u003c/strong\u003e Photomicrograph of the pancreas from LPS groups showing abnormal acinar cells with mononuclear cell infiltration, inflammation, and hemorrhage. \u003cstrong\u003eD:\u003c/strong\u003e Photomicrograph of the pancreas from rats from the LPS+ TQ groups showing many areas of typical tissue architecture with mild inflammatory changes.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/0fe2f193bf241ffe5a21ee75.jpg"},{"id":82923148,"identity":"a7bf3ff1-f648-4a71-87a0-d4bc084712b0","added_by":"auto","created_at":"2025-05-16 18:29:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1296359,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/cac490ce-1231-41f5-816f-702022a6cc47.pdf"},{"id":82922300,"identity":"63713afe-758c-4971-8185-a87b60c65151","added_by":"auto","created_at":"2025-05-16 18:05:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18819,"visible":true,"origin":"","legend":"","description":"","filename":"TABLE1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/ce6be54970d65f1347a718b5.docx"},{"id":82922722,"identity":"48921756-4fb6-47f3-8854-6980e40f27dd","added_by":"auto","created_at":"2025-05-16 18:13:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17298,"visible":true,"origin":"","legend":"","description":"","filename":"TABLE2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/3b5d537677dabb24a4d5f032.docx"},{"id":82922723,"identity":"daff0151-b456-4a84-a4bf-bfeb59316d21","added_by":"auto","created_at":"2025-05-16 18:13:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14299,"visible":true,"origin":"","legend":"","description":"","filename":"TABLE3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/99f79bf2a89fede72ccc6310.docx"},{"id":82922125,"identity":"7c34e2bd-0b11-42a7-9f3e-47ee06834056","added_by":"auto","created_at":"2025-05-16 17:57:31","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":212723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eThe effect of P.gingivalis-LPS injection on pancreatic KP and glucose homeostasis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB: \u003c/strong\u003eThe effect of TQ administration on changes of P.gingivalis-LPS-Induced pancreatic KP and glucose homeostasis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations: P.gingivalis-LPS, \u003c/strong\u003ePorphyromonas gingivalis-lipopolysaccharide\u003cstrong\u003e; Trp, \u003c/strong\u003eTryptophan; \u003cstrong\u003eKYN,\u003c/strong\u003e Kynurenine. \u003cstrong\u003eKP,\u003c/strong\u003e Kynurenine pathway;\u003cstrong\u003e KYNA, \u003c/strong\u003ekynurenic acid;\u003cstrong\u003e QA, \u003c/strong\u003equinolinic acid;\u003cstrong\u003e KMO, \u003c/strong\u003eKynurenine 3-monooxygenase, \u003cstrong\u003eIDO\u003c/strong\u003e, indoleamine 2,3-dioxygenase \u003cstrong\u003eTDO\u003c/strong\u003e, tryptophan-2,3-dioxygenase.\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6641065/v1/4623d8d7c319de98b696deb6.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Does Zn-mediated regulation of the kynurenine pathway provide the link between periodontal disease and diabetes?","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMicrobes highly colonize the gingival tissues in the oral cavity surrounding each tooth. Correlations have been found between oral health and cardiovascular disease (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), pregnancy outcomes (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), Rheumatoid Arthritis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), and type II diabetes (T2DM) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Porphyromonas gingivalis (P. gingivalis) is a gram-negative anaerobic bacterium found in the mouth. This species plays a crucial role as a keystone pathogen in the progression of periodontal disease (PD) and contributes to various systemic diseases (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). P. gingivalis or virulence factors such as lipopolysaccharide (LPS) can spread systemically through active periodontal lesions or by invading cells directly (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Therefore, inflammatory conditions caused by LPS of P. gingivalis may be the cause of the molecular link between periodontal health and diabetes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Previous studies showed the relationship between P. gingivalis and diabetes (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, many unknown points exist about the molecular connections between oral health impaired by P. gingivalis and the pathogenesis of T2DM.\u003c/p\u003e \u003cp\u003eThe disruption of zinc may lead to insulin resistance and the development of diabetic complications (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In addition, recent studies have revealed that there may be a causal relationship between periodontitis pathology and Zn (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Therefore, Zn disregulation may provide molecular connections between PD and T2DM.\u003c/p\u003e \u003cp\u003ePrevious studies showed that PD and T2DM mellitus share many pathological pathways, including the kynurenine pathway (KP) (\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Tryptophan (Trp) is an essential amino acid metabolized by the serotonin or the KP to various bioactive molecules. Pro-inflammatory cytokines, such as interferon-γ (IFN-γ), upregulate the enzyme indoleamine 2,3-dioxygenase (IDO), causing decreases in L-Trp and increasing the metabolite kynurenine (KYN) levels (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Then, KYN is converted to kynurenic acid (KYNA) by kynurenine aminotransferase (KAT) or quinolinic acid (QA) by two different pathways: 1) KYN can be converted to anthranilic acid by kynureninase and then converted to QA, or 2) KYN can be converted to 3-hydroxyKYN (3-HK) by KYN 3-monooxygenase (KMO) and then converted to QA by kynureninase-mediated pathway. It is evident from the previous study that increasing concentrations of Zn\u003csup\u003e2+\u003c/sup\u003e inhibit the kynureninase, whereas they activate the KAT (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, it is unknown how the change in Zn levels caused by P. gingivalis alters the KP pathway and whether it contributes to the link between PD and diabetes.\u003c/p\u003e \u003cp\u003eThymoquinone (TQ) is one of the main active components of the essential oil obtained from black cumin (Nigella sativa) seeds. Many studies have demonstrated TQ's antibacterial, hypoglycemic, anti-inflammatory, and anti-oxidative activities (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Thus, this study investigated the effect of Zn on KP metabolism in the pancreas on glucose regulation in impaired oral health rat models created by P. gingivalis-LPS injection and whether TQ affects this change.\u003c/p\u003e \u003cp\u003eOur study is the first to examine the molecular link between P. gingivalis-specific periodontal health deterioration and diabetes in pancreatic tissue. It may contribute to understanding the pathogen-specific molecular link between periodontal diseases and diabetes.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eAll experimental protocols conducted on male rats were performed by the standards established by the Institutional Animal Care and Use Committee at Aksaray University (2024/9\u0026ndash;56). The rats were obtained from the Aksaray University Animal Care Unit. Male albino Wistar rats aged 3 months, weighing 200\u0026ndash;300 g, were used throughout all experiments. Standard rat, cow, and tap water were given ad libitum, and the animals were housed in stainless steel cages at standard conditions (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and humidity 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) with 12:12 h light-dark cycles at all times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Establishment of animal model and treatment\u003c/h2\u003e \u003cp\u003eP. gingivalis is one of the pathogens that mediates PD; for this reason, we injected P. gingivalis-LPS into the palatal gingiva to create a model of impaired periodontal health according to previous studies (\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). TQ was supplied from Sigma Aldrich (Saint Louis, MO, USA), with purity\u0026thinsp;\u0026ge;\u0026thinsp;98%. TQ administration was performed using the dose previously reported in the literature. The water solubility of TQ is reported to be \u0026gt;\u0026thinsp;0,5 mg/mL, which is enough to exert pharmacologic effects (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Animals were randomly divided into four groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 for each group). Oral injections were done 6 times at 48-hour intervals into the palatal gingiva between the right and left side first and second upper molars with 10 \u0026micro;l P. gingivalis-LPS or saline; tap water or TQ solution (10 mg/kg per day) was given by oral gavage for 2 weeks. TQ administration was started on the day of injection and continued for 14 days. \u003cb\u003eThe Control group\u003c/b\u003e received saline injections and tap water; \u003cb\u003ethe LPS group (LPS)\u003c/b\u003e received P. gingivalis-LPS injections and tap water; the TQ group (TQ) received saline injections and TQ; and the LPS\u0026thinsp;+\u0026thinsp;TQ group (LPS\u0026thinsp;+\u0026thinsp;TQ) received P. gingivalis-LPS injections and TQ. At the end of the experimental period, the rats were examined orally, and their clinical features of periodontitis, such as swelling, redness, and gingival bleeding, were evaluated [2]. After rats were anesthetized intraperitoneally with a mixture of ketamine (80 mg/kg) and xylazine hydrochloride (5 mg/kg), the animals were sacrificed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Tissue Collection and Preparation\u003c/h2\u003e \u003cp\u003eThe maxillary jawbone was separated for X-ray analysis, and the other was separated for biochemical analysis. Some pancreatic tissues were stored at -80\u0026deg;C for biochemical analysis, and some were separated for hematoxylin and eosin staining. The excised pancreas and gingival tissues were homogenized in cold PBS and centrifuged at 10,000\u0026times;g for 5 min for biochemical analysis. The plasma samples were separated by centrifugation at 1,500\u0026times;g for 15 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Biochemical Analysis\u003c/h2\u003e \u003cp\u003eKYN, TRP, KYNA, QA, IFN-γ, caspase-3 levels, KMO, and ZIP10 levels were measured in the pancreas tissue, and insulin levels were also measured in plasma samples using the enzyme-linked immunosorbent assay technique, employing commercial kits from Bioassay Technology Laboratory (BT Lab) according to the manufacturer's instructions. Zinc levels in the pancreas tissue and plasma samples were measured using a colorimetric method (Fully Automatic Clinical Biochemistry Analyzer, Mindray BS400).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eFasting glucose levels assessment\u003c/strong\u003e \u003cp\u003eThe evening before the measurement day, 8\u0026ndash;10 hours before the measurement, animals were restricted from food access, and the tail region was bled and measured by glucometer (plusMED Blood GlucoseMeter, Accuro, pM1-300, Bionime Corporation, Taiwan).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHomeostatic model (HOMA) assessment\u003c/strong\u003e \u003cp\u003eInsulin resistance was assessed using the Homeostatic Model Assessment (HOMA-IR) (insulin (mIU/L) multiplied by fasting blood glucose (mM) divided by 22.5) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). β cell function was assessed using the analysis of HOMA-β levels, calculated by the Eq.\u0026nbsp;(360 x fasting plasma insulin)/(fasting plasma glucose \u0026minus;\u0026thinsp;63) (%) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDetermination of Kyn/Trp ratio (KTR)\u003c/strong\u003e \u003cp\u003eThe Kyn/Trp ratio change reflects the degradation rate of Trp. Therefore, the Trp/Kynurenine ratio determined IDO activity (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProtein measurements\u003c/strong\u003e \u003cp\u003eProtein concentrations of tissue samples were measured at 595 nm by a modified Bradford assay using Coomassie Plus reagent with bovine serum albumin as a standard (Pierce Chemical Company, Rockford, IL) (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histopathologic examinations of pancreatic tissues\u003c/h2\u003e \u003cp\u003eThe pancreas tissues were fixed in a 10% paraformaldehyde/phosphate-buffered saline solution and then subjected to a standard paraffin-embedding process, obtaining 5\u0026ndash;6 \u0026micro;m sections. Finally, the sections were stained with Hematoxylin-eosin (H\u0026amp;E) and evaluated under light microscopy (Leica DFC450, Almanya) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 X-ray films of the periodontal tissue\u003c/h2\u003e \u003cp\u003eThe dissected maxillary bone was examined with a periapical X-ray device (KaVo Focus (KaVo Dental, Bieberich, Germany) at 10x magnification to determine the alveolar bone level. The distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) between the alveolar bone was measured medially and distally. The CEJ was connected with a line drawn in silico and indicated on the tooth's mesial and distal surface projections. Then, perpendicular lines were drawn to the alveolar bone crest along the CEJ and the distance was automatically measured by the computer-aided system (CLINIVIEW\u0026trade; software, Instrumentarium Dental, Tuusula, Finland) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analyses of the obtained data were performed using SPSS 23.0 (SPSS, Chicago, IL, USA) software for Windows. Biochemical parameters were analyzed by a one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s Post Hoc Test for normally distributed variables, and Kruskal-Wallis followed by the Mann-Whitney U test for non-normally distributed variables. Results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Oral examination and radiography results of the experimental groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe observed that the rats' gum tissues became red and swollen, displaying a dark red color and experiencing spontaneous bleeding after two weeks of LPS injection. These preliminary findings indicated the successful induction of impaired oral health. Oral findings of rats in the TQ group were similar to those of the healthy groups. In addition, the gingival tissues of the LPS+TQ group appeared pink, while their gingival margins became red and swollen. No bleeding was detected.\u0026nbsp;X-ray films revealed slightly alveolar bone resorption\u0026nbsp;in the second maxillary molar in the\u0026nbsp;rats injected with LPS, in contrast to the control and TQ-administered rats. Also, alveolar bone resorption was alleviated in the LPS+TQ group (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Analysis of Zn-related parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 1, ZIP10 levels in the pancreatic tissue of the control groups were significantly higher than in the TQ and LPS+TQ groups (p=0.001, 0.000), and significantly lower than in the LPS+TQ group (p=0.042). Also, TQ administration significantly decreased ZIP10 levels in the pancreas of the LPS+TQ group compared to the LPS group.\u003c/p\u003e\n\u003cp\u003eThe control group's plasma Zn levels were significantly higher than those of the TQ and LPS groups (p=0.017, p=0.000) and significantly lower than those of the LPS+TQ group (p=0.000). Also, the LPS+TQ group's plasma Zn levels significantly increased compared to the LPS group (p=0.000). Pancreas Zn levels of the control group were significantly higher than those of the TQ and LPS+TG groups (p=0.000, p=0.000), and significantly lower than those of the LPS group (p=0.003). Also, pancreas Zn levels of the LPS+TQ group significantly decreased compared to the LPS group (p=0.000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Analysis of Cytokine and Caspase-3 Levels in Pancreas Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePro-inflammatory cytokine levels in various groups are shown in Table 1. IFN-γ levels in pancreas samples of the control group were lower than those of the TQ group (p=0.055), the LPS group (p=0.025), and the LPS+TQ group (p=0.037). Also, IFN-γ levels in pancreas samples of the LPS+TQ group were higher than those of the LPS group. However, it is not significant. Caspase-3 levels in pancreas samples of the LPS group significantly decreased compared to the control group (p=0.026) and markedly reduced compared to the LPS+TQ group (p=0.055).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Analysis Of Glucose Homeostasis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, we investigated the fasting glucose and insulin levels in various groups. As seen in Table 2, the fasting glucose levels of the control group were significantly lower than those of the LPS group and the LPS+TQ group (p=0.000, p=0.000), and the TQ group compared to the LPS group and the LPS+TQ group (p=0.000, p=0.000). Plasma insülin levels of the TQ group were significantly lower than those of the control, LPS, and LPS+TQ groups (p=0.030, p=0.000, p=0.000). TQ administration markedly increased plasma insülin levels of the LPS+TQ group compared to LPS; however, this trend did not reach significant levels.\u003c/p\u003e\n\u003cp\u003eThen, we determined HOMA-IR and\u0026nbsp;HOMA-β levels. The HOMA-IR levels of the control groups were lower than those of the LPS and LPS+TQ groups (p=0.000, p=0.000), and those of the TQ group were lower than those of the LPS and LPS+TQ groups (p=0.000, p=0.000).TQ administration slightly decreased HOMA-IR levels of the TQ group compared to the control group and increased somewhat LPS+TQ group compared to the LPS group; however, these trends did not reach significant levels. Additionally,\u0026nbsp;HOMA-β levels were higher in the control groups compared to TQ, LPS, and LPS+TQ groups (p=0.002, \u0026nbsp;p=0.001, p=0.006). HOMA-β Levels of\u0026nbsp;LPS+TQ groups markedly increased compared to the LPS group. However, this increasing trend did not reach significant levels\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalysis of Change in KYN Pathways\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Analysis of Trp, KYN,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKYNA, and QA levels in the pancreas tissue and plasma samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated differences in the various groups' Trp and KYN levels of the pancreas tissue. There was no difference in the Trp and KYN levels of pancreas tissue (p =0.567; p = 0.071). TRP levels determined for the control group 99.727 ± 47.40 ng/g protein, the LPS group 43.495 ± 8.16 ng/g protein, the LPS+TQ group 9.369 ± 31.75 ng/g protein, and KYN levels determined for the control group 0.510±0.132 ng/mg protein, the LPS group 0.871 ± 0.151 ng/mg protein, TQ group 2.156 ± 0.770 ng/mg protein, LPS+TQ group 2.737 ± 0.868 ng/mg protein (Figure 2. A and Figure 2.C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePancreatic KYNA levels of the LPS group\u0026nbsp;(27.328 ± 3.553 ng/mg protein) were higher than\u0026nbsp;those of the control group (18.601 ± 3.319 ng/mg protein); however, with this increasing trend, there was no significant difference (p=0.078).KYNA levels of the\u0026nbsp;LPS+TQ group (5.679 ± 0.643 ng/mg protein) were decreased compared to the control group, the TQ group (38.023 ± 9.697 ng/mg protein), and\u0026nbsp;the LPS group (p=0.016, p=0.004, p=0.004) (Figure 2B).\u0026nbsp;QA levels of the LPS group (1,029±0,207 ng/mg protein) were lower than those of the control group (2,585±0,317 ng/mg protein) and LPS+TQ group (11,109±2,400 ng/mg protein) (P=0.004, p=0.004)\u0026nbsp;(Figure 2. D ).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2.\u0026nbsp; \u0026nbsp; \u0026nbsp;Analysis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eKyn/Trp ratio\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003eKMO, and Kynureninase levels in the pancreas tissue\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant differences in the\u0026nbsp;Kyn/Trp ratio\u0026nbsp;of various groups in the pancreas tissue. The pancreas\u0026nbsp;Kyn/Trp ratio\u0026nbsp;is lower in the control group (0.0110 ± 0.0013 µg/ng) compared to the LPS group (0,0213±0,0024 µg/ng) and the LPS+TQ group (0.0301±0.0026 µg/ng) (respectively p=0.010, p=0.004). TQ administration increased the\u0026nbsp;Kyn/Trp ratio\u0026nbsp;in the pancreas of LPS+TQ group rats compared to the LPS group. However, this increasing trend does not reach significant levels (p=0.078) (Figure 3.A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pancreatic KMO levels of the control group (144.022 ± 25.075 ng/g protein) were higher than those of the LPS group (54.046 ± 11.874 ng/g protein) and the LPS+TQ group (48.342 ± 8.306 ng/g protein) (p=0.048, p=0.033). Also, KMO levels of the TQ group (156.276 ± 34.649 ng/g protein) were higher than those of the LPS group (p=0.021, p=0.015)\u0026nbsp;(Figure 3.B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt was observed that the pancreatic kynureninase levels of the LPS group (1.399 ± 0.059 ng/mg protein) decreased compared to the control group (3.136 ± 0.712 ng/mg protein) and compared to the LPS+TQ group (6.401 ± 2,848 ng/mg protein) (p=0.025, p=0.004). TQ administration increased kynureninase levels of the TQ group (7.328 ± 3.171 ng/mg protein). However, with this increasing trend, there was no significant (Figure 3.C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHistological analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of HE of the pancreatic tissues were evaluated regarding edema, inflammatory cell infiltration, acinar cell degeneration, and hemorrhage parameters (Figure 4). The severity of each criterion was graded from 0 to 3: 0: absent or rare, 1: mild, 2: moderate, 3: severe (28, 29). As seen in Table 3, the damage score scores of the LPS group were statistically higher than those of the control group (p=0.05). The TQ group scores were similar to those of the control group. The damage score of the LPS+TQ group decreased statistically significantly compared to the LPS group but increased significantly compared to the control and TQ groups (p=0.05).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePeriodontal health and diabetes have long been known to have reciprocal relationships (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e); however, how these connections are achieved remains unclear. In this study, we investigated the effect of oral pathogen P. gingivalis-LPS on glucose homeostasis by changes in Zn-mediated KYN metabolism and whether TQ affects these conditions.\u003c/p\u003e \u003cp\u003eOur results showed that P. gingivalis-LPS injection impaired glucose homeostasis by causing peripheral insulin resistance and deterioration of beta cell functions. Insulin resistance and defects in pancreatic beta cells are the two significant pathophysiologic abnormalities that underlie type 2 diabetes (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), resulting in hyperglycemia (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Additionally, reduced insulin signalling and/or insulin resistance and the associated diminution in glucose transport promote a compensatory increase in pancreatic insulin production, resulting in hyperinsulinaemia. However, even before a diagnosis of type 2 diabetes mellitus (T2DM), excess insulin levels can indicate who is likely to develop the disease (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Therephore, increased insulin synthesis, which was observed in the present study, may be an adaptation mechanism against LPS-treatment. Abdelmageed et al. showed that T2D rats exhibited significant increases in fasting glucose and insulin levels, higher HOMA-IR, and lower HOMA-β than the control group (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Therefore, our findings are consistent with other studies that show the relationship between PD and DM (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, Zn levels decreased in the plasma of the LPS group, which is consistent with reduced plasma Zn levels in the T2DM and (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) periodontitis patient (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Also, ZIP10 and Zn levels were increased in the pancreas tissue of the LPS group.\u003c/p\u003e \u003cp\u003eZn is an essential component for the normal function of the pancreas, and deficiency and overload of Zn are linked to diverse disorders, including diabetes and obesity (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Therephore, the physiological and cellular Zn concentrations are regulated by Zn transporters (ZnTs), and Zn importers (ZIPs, Zrt- and Irt-like proteins) (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). ZIP10 is part of the ZIP (SLC39) class of transporters that enhance Zn concentration in the cytoplasm by facilitating its influx from the extracellular space or promoting its efflux from intracellular vesicles. Research has shown that ZIP10 transcripts are expressed in pancreatic alpha (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) and beta cells (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), suggesting that ZIP10 may regulate glucose homeostasis (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). This view supports the idea that in isolated breast cancer cells treated with glucose concentrations equivalent to hyperglycemia in humans, the expression of ZIP10 is reported to be up-regulated with a concomitant increase in cellular Zn concentrations (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Moreover, Zn is crucial in insulin synthesis, crystallization, storage, secretion, and signaling in the pancreatic β-cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our results suggest that increased ZIP10 may lead to transport of Zn into the β cell and increased ins\u0026uuml;lin syntesis. Thus, our results also indicate that changing Zn levels in the pancreas may affect the KP pathway.\u003c/p\u003e \u003cp\u003eA study supporting this view demonstrated that Zn triggered the degradation of tryptophan by IDO and the production of kynurenine by dendritic cells (DCs) while also significantly suppressing the pro-inflammatory response to stimulation by Toll-like receptor (TLR) ligands (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Similarly, inflammatory cytokine IFN-γ levels and IDO activity in the pancreas tissue of the LPS group were increased in our study. These results may depend on the pathogen type because multiple pathogens are involved in PD pathogenesis (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Furthermore, our findings revealed that the effect of P. gingivalis-LPS activates the KP pathway and changes metabolite levels in the pancreas.\u003c/p\u003e \u003cp\u003eThis may be related to increased pancreatic Zn levels. It is demonstrated that Zn2\u0026thinsp;+\u0026thinsp;inhibits the kynureninase, which is responsible for converting KYN to QA, whereas it activates the KAT, which converts KYN to KYNA (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Consistent with these findings in our study, kynureninase levels decreased in the plasma of the LPS group compared to the control group. Also, our results show that caspase-3 levels decreased in the pancreas of the LPS group, suggesting increased apoptosis. Caspase-3 is a key zymogen in cell apoptosis. It is not activated until it cleaves by initiator caspases during apoptotic flux (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Therephore, decreasing zymogen form caspase-3 levels may increase cleaved caspase-3, active caspase form. Previous in vivo and in vitro studies show that QA has apoptotic properties (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) while KYNA has anti-apoptotic properties (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). However, Arya et al. showed that the combination dose of quercetin (QE) and QA (50 mg/kg) exhibited maximum inhibition of the pro-apoptotic protein Bax expression and enhanced the anti-apoptotic protein Bcl-2 expression, suggesting a protective role in the kidneys of diabetic rats (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Therephore, our results indicate that decreased QA levels in the pancreas of the LPS group may be related to the decrease in HOMA β levels and pancreatic beta cell structure. İmportantly, increased ZIP10-mediated Zn levels may be responsible for these changes. Supporting this view, TQ administration decreased ZIP10 and Zn levels in the pancreas tissue of the LPS-TQ group. Also, IDO activity increased more in the LPS\u0026thinsp;+\u0026thinsp;TQ group than in the LPS group, while QA levels increased and KYNA levels decreased, contrary to the LPS group. Decreased Zn levels and increased IDO activity may be related to Zn's dose-dependent effects. Moreover, our results suggest that QA levels in the pancreas tissue of the LPS\u0026thinsp;+\u0026thinsp;TQ group, due to the suppressive effect on kynureninase, may be eliminated by decreasing the Zn level, as mentioned earlier (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Also, results showed that insulin levels in the plasma and zymogen form of caspase-3 levels in the pancreas were increased, and pancreatic morphology was improved in the LPS\u0026thinsp;+\u0026thinsp;TQ group.\u003c/p\u003e \u003cp\u003eQA is an agonist of neuronal N-methyl-D-aspartate receptors (NMDARs) [45], and KYNA is a competitive NMDAR antagonist (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Lockridge et al. showed that D-serine can have acute antidiabetic effects in mice and potentiates insulin secretion through excitatory β-cell NMDAR co-agonism (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Thus, our results suggest that changes in QA and KYNA levels may be responsible for decreasing β-cell number and function in the LPS group's pancreas. TQ administration may improve this by regulating Zn levels through ZIP10. Our findings were consistent with previous studies, which showed that TQ protects against STZ-induced diabetes by repressing apoptosis of β-cells, ameliorating β-cell ultrastructure, and leading to ins\u0026uuml;lin secretion (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e); KYNA, which has potentiated hyperglycemic effects by inhibiting proinsulin synthesis and insulin secretion in rat pancreatic islet cells (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) and an apoptotic effect on cancer cells [55, 56].\u003c/p\u003e \u003cp\u003eAdditionally, TQ administration decreased plasma and pancreatic Zn levels and ZIP10 levels in the pancreas tissue of the TQ group. Our result showed decreased HOMA-IR and plasma ins\u0026uuml;lin levels in the TQ group. Zn is crucial in insulin synthesis, crystallization, storage, secretion, and signaling in the pancreatic β-cells. In the process of ınsulın synthesis, ins\u0026uuml;lin is transformed into an insoluble crystalline hexamer, including two Zn ions (Zn\u0026sup2;⁺) and one calcium ion (Ca\u0026sup2;⁺) in granules. As β-cells secrete hexameric insulin into the extracellular space, the hexamers quickly dissociate into active monomers within a few seconds [20]. Therephore, decreased plasma insulin levels may be related to suppressed β-cell function and ins\u0026uuml;lin secterion due to the effect of TQ on insulin sensitivity (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Also, this may be due to the increased KYNA levels in the pancreas of rats administered only TQ because KYNA and QA continue to act oppositely in inflammation and other functions (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Our results suggest that Zn-mediated alteration of the pancreatic KP pathway may provide the molecular link between PD and T2DM. Furthermore, ZIP10's regulation of Zn levels in the pancreas may mediate TQ's anti-diabetic effect. However, the increasing impact of TQ on insulin in the LPS group may be a risk of developing diabetes in the long term. This issue needs to be investigated further.\u003c/p\u003e \u003cp\u003eInterestingly, our results showed that the TQ administration increased the inflammatory cytokines in the pancreas of LPS\u0026thinsp;+\u0026thinsp;TQ groups. Cytokines are central mediators of immune responses, and T helper (Th) cells are professional cytokine-producing cells. Once activated, CD4\u0026thinsp;+\u0026thinsp;T helper cells further differentiate into Th1 cells, which specialize in producing IL-2, IL-6, IL-12, and IFNγ. Therefore, agents that can influence the Th cells' differentiation have the potential to alter the adaptive immune response in various diseases and medical conditions (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Th1 cells are crucial for host defense against intracellular pathogens, including viruses, protozoa, and bacteria. One of their primary functions is to activate macrophages by producing IFN-γ (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Additionally, our findings were consistent with the observation that purified protein extracts of N. sativa seeds significantly enhanced the production of TNF-α from unstimulated and PWM-activated lymphocytes [52] and serum IFN-γ levels in CMV-infected mice (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Also, our results are consistent with the study's findings, which show that TQ reduces bone resorption in an experimental periodontitis model (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Therephore, our results may indicate that activation of the immune response by TQ has a beneficial effect on impaired glucose regulation and bone resorption by P. gingivalis-LPS.\u003c/p\u003e \u003cp\u003eWhen the results obtained from our research are evaluated together, it can be thought that the increase in pancreatic Zn concentration via ZIP10 due to LPS treatment induces the KP pathway and increased insulin synthesis as an adaptation mechanism against disturbance of pancreatic function. TQ treatment reversed the effect of LPS on KP pathways, improved the pancreas morphological structure, and increased ins\u0026uuml;lin syntesis.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, our results suggest that oral application of P. gingivalis-LPS causes an increase\u003c/p\u003e \u003cp\u003ein pancreatic Zn levels by ZIP10, and causes a change in KP metabolites, favoring KYNA production and resulting in apoptosis of pancreatic cells. TQ administration reverses these changes induced by P. gingivalis, alleviating the impairment of β-cell function.\u003c/p\u003e \u003cp\u003eOur study examined the relationship between periodontal diseases and diabetes in the early period when oral health deterioration and bone resorption begin through the Zn-mediated changes in the KP pathway, specifically for P. gingivalis. According to the results of our study, the P. gingivalis pathogen may mediate the formation of diabetogenic conditions via the KP pathway in the deterioration of oral health. The results obtained from our research are expected to contribute to understanding this link.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLPS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) injected group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTQ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThymoquinone administered group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eLPS\u0026thinsp;+\u0026thinsp;TQ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eP. gingivalis-LPS injected and TQ administered group.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatements \u0026amp; Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis work was supported by\u0026nbsp;\u003c/em\u003e\u003cem\u003eKÜN.2024-BAGP-012\u003c/em\u003e\u003cem\u003e\u0026nbsp;Grant numbers and Author E.A. has received research support from\u0026nbsp;\u003c/em\u003ethe Cappodoccia University Research Foundation, Turkey\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEA (Ebru Afşar), EA (Erdem Arslan), and MO did a P.gingivalis injection and oral gavage application. EA (Erdem Arslan) and MO also monitored laboratory animals' living conditions and nutrition. KD and TC sacrificed experimental animals and collected samples. They were also responsible for transferring samples from the experimental animal unit to Cappadocia University, where the analyses would be conducted. NO performed X-ray analyses and evaluated these analyses. IE performed hematoxylin and eosin staining analyses and evaluation of these analyses. SS performed oral examinations of experimental animals before and after the injection procedure. EA (Ebru Afşar) and SS conducted the literature review and designed the study. EA (Ebru Afşar) designed the study, performed biochemical and statistical analyses, and interpreted the analysis results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study's findings are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval provided by the Institutional Animal Care and Use Committee at Aksaray University (2024/9-56).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Qutub MN, Braham PH, Karimi-Naser LM, Liu X, Genco CA, Darveau RP (2006) Hemin-dependent modulation of the lipid A structure of Porphyromonas gingivalis lipopolysaccharide. 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J Periodontal Res 47(1):74\u0026ndash;80\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Kynurenine, Porphyromonas gingivalis, Diabetes, Thymoquinone, Zinc","lastPublishedDoi":"10.21203/rs.3.rs-6641065/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6641065/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIt has long been known that there is a relationship between periodontal diseases and diabetes. The present study aimed to assess the effect of pancreatic zinc (Zn) levels on Kynurenin pathways (KP) and glucose homeostasis and the impact of Thymoquinone (TQ) in the periodontal disease animal model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: 10 µl Porphyromonas gingivalis-Lipopolysaccharide (P. gingivalis-Lps) (1mg/ml) was injected 6 times at 48-hour intervals into the palatal gingiva of rats. TQ was given by oral gavage (10 mg/kg per day) for 2 weeks. Glucose homeostasis was assessed using the Homeostatic Model Assessment (HOMA-IR), and β-cell function (HOMA-β Levels). Kynurenine (KYN), Tryptophan (TRP), kynurenic acid (KYNA), quinolinic acid (QA), KYN 3-monooxygenase (KMO), kynureninase, interferon-γ (IFN-γ), insulin, ZIP10, and caspase-3 levels measured by the enzyme-linked immunosorbent assay (ELISA). Zinc levels in the pancreas tissue and plasma samples were measured using a colorimetric method. Morphological changes in the pancreas were identified by hematoxylin and eosin staining, and X-ray radiography determined bone resorption in the maxillary bone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: In the LPS group, pancreas ZIP10 and Zn levels increased, the KP pathway was changed to favor KYNA, and impaired glucose homeostasis. TQ administration decreased pancreatic Zn levels, changed KP to favor QA, and improved morphological changes in the pancreas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: During the periodontal diseases, KP may be altered by Zn levels through ZIP10 in the pancreas, and impair pancreatic function. Regulation of Zn levels may be key to shared pathways between periodontal diseases and diabetes.\u003c/p\u003e","manuscriptTitle":"Does Zn-mediated regulation of the kynurenine pathway provide the link between periodontal disease and diabetes?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 17:57:26","doi":"10.21203/rs.3.rs-6641065/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-21T01:31:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-20T04:11:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162669356879538886673651352220863624843","date":"2025-07-14T14:08:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-15T03:21:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16655132290166488643224203623058606892","date":"2025-06-09T06:00:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T21:49:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-12T12:07:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-12T05:49:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2025-05-11T18:06:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e1aeb4a0-36c9-49bc-b7b2-5a6ab0c6f618","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-31T16:38:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 17:57:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6641065","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6641065","identity":"rs-6641065","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}