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STAT signaling in diabetic mice
Journal Pre-proofs 25-Hydroxyvitamin D3 positively regulates periodontal inflammaging via SOCS3/STAT signaling in diabetic mice Qian Wang, Xinyi Zhou, Peng Zhang, Pengfei Zhao, Lulingxiao Nie, Ning Ji, Yi Ding, Qi Wang PII: DOI: Reference:
S0039-128X(19)30260-0 https://doi.org/10.1016/j.steroids.2019.108570 STE 108570
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Steroids
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Please cite this article as: Wang, Q., Zhou, X., Zhang, P., Zhao, P., Nie, L., Ji, N., Ding, Y., Wang, Q., 25Hydroxyvitamin D3 positively regulates periodontal inflammaging via SOCS3/STAT signaling in diabetic mice, Steroids (2019), doi: https://doi.org/10.1016/j.steroids.2019.108570
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25-Hydroxyvitamin D3 positively regulates periodontal inflammaging via SOCS3/STAT signaling in diabetic mice Qian Wang1,2, Xinyi Zhou1,2, Peng Zhang1,2, Pengfei Zhao1,2, Lulingxiao Nie1,2, Ning Ji1, Yi Ding1,3 and Qi Wang1,2,* 1 State
Key Laboratory of Oral Diseases, National Clinical Research
Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University 2 Department
of Prosthodontics, West China Hospital of Stomatology, Sichuan University
3Department
of Periodontology, West China Hospital of Stomatology, Sichuan University
*Correspondence: Dr. Qi Wang, Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, 3rd Section S Renmin Road, 14#, Chengdu, China, 610041 [email protected] Fax: +86 28 85503503 Email: [email protected]
Abbreviations: VD3, Vitamin D3; 25(OH)D3, 25-Hydroxyvitamin D3; P.g., Porphyromonas gingivalis; SASP, senescence-associated secretory phenotype; CS, cellular senescence; VDR, Vitamin D Receptor; SOCS3, suppressor of cytokine signaling-3; JAK, Janus kinase; STAT, signal transducer and activator of transcription; p-STAT3, phosphorylated STAT3; p-STAT5, phosphorylated STAT5; T2DM, type 2 diabetes mellitus; FBG, fasting blood glucose; OPG, osteoprotegerin, Adi, adiponectin; PP, pancreatic polypeptide; RAGE, receptor for advanced glycation end product; 1,25(OH)2D3, 1α,25-Dihydroxyvitamin D3; BMD, bone mineral density; PDL, periodontal ligament; Ab, antibody.
Abstract Background: Diabetes is a known age-related disease. Inflammaging has recently been shown to result in diabetic complications. Vitamin D3 is related to aging in the latest study but little is known about the underlying mechanism. Here, we investigated the effects of 25-Hydroxyvitamin D3 (25(OH)D3) on inflammaging in diabetic periodontitis, a common chronic inflammatory diabetic complication. Experimental design: A model of Porphyromonas gingivalis-infected db/db mice as experimental type 2 diabetic periodontitis was adopted in the whole study. A range of techniques, including microCT, western blotting, ELISA, histological and immunohistochemical analysis, were carried out in this study. The distinctive senescence-associated secretory phenotype (SASP) in serum was measured by Luminex technology. Results: We found an archetypal inflammaging status occurred in db/db mice. An increased SASP, senescent enhancement, and periodontal destruction were observed in periodontitis-db/db mice. Upon administration with 25(OH)D3, periodontitis-db/db mice presented increased levels of serum 25(OH)D3, 1α,25-Dihydroxyvitamin D3 and calcium. Moreover, decreased p16/p21-positive cells, relieved periodontal conditions and ameliorated serum SASP secretion were found in the periodontitis-db/db mice after treatment. Gingival tissue exhibited increased level of VDR and decreased expression of SOCS3, p-STAT3/STAT3, p-STAT5/STAT5, NF-κB and IL-1β, which were consistent with the change of p16/p21 expression. Conclusion: Diabetic periodontitis appeared to develop an inflammaging status resulted in periodontal infection. 25(OH)D3 could inhibit SASP secretion through reducing SOCS3 expression in experimental diabetic periodontitis, dependently inactivating NF-κB pro-inflammatory signaling.
The reversible effect further documented that the inflammaging might be a highly likely contributor in diabetic periodontitis.
Graphical abstract 25(OH)D3 played the protective role in gingival tissue of db/db mice with periodontitis. SOCS3 is an endogenous negative regulator of the inflammaging. Diabetic periodontitis developed an inflammaging status with up-regulated SOCS3 in gingival tissue. Black arrows indicate pathogenic mechanism, wherein solid arrows indicate directly active effects and black dashed arrows indicate the indirectly active effects. Green solid arrows indicate the functions of 25(OH)D3, green barheaded lines indicate inhibit effects.
Keywords: 25-Hydroxyvitamin D3, inflammaging, senescence-associated secretory phenotypes, senescence, SOCS3, diabetic periodontitis
1. Introduction Diabetes mellitus is an age-related disease [1] that poses a major challenge to the longevity and life quality of humans [2, 3]. Diabetic periodontitis, as a mainly inflammatory diabetic complication affects about 11.2% of the global adult population [4, 5]. Patients often suffer from severe gingival inflammation, extensive alveolar bone destruction and tooth loss. Gingival tissue constitutes the first barrier of periodontal protection [6]. Diabetes increase the susceptibility to opportunistic oral infections, leading to severe periodontal outcomes [4]. However, the underlying pathogenic mechanisms and therapeutic treatment are still not fully understood.
Recently, inflammaging has been suggested to be a major contributor to type 2 diabetes mellitus (T2DM) [7, 8]. Inflammaging is a secondary senescent pattern that can be induced chronically in response to chronic low-grade inflammation independent of replicative senescence [7, 9]. It causes aging-associated pathological changes and increases vulnerability to death primarily through enhancing cellular senescence (CS) and promoting a senescence-associated secretory phenotype (SASP) [7-10]. CS restricts cell proliferation and deteriorates tissue microenvironment [3, 9]. The SASP involves the secretion of a variety of molecules disrupting the surrounding normal cell functions, including cytokines, chemokines, growth factors and proteases [10].
Nutrients are theoretically modulators to senescence [11, 12]. Vitamin D3 (VD3) has been highly recommended as a complementary nutrient for diabetes [12]. It is currently considered as an immunomodulator in aging and inflammation [12-14]. 25-Hydroxyvitamin D3 (25(OH)D3), a main circulatory form of VD3, has been found an excellent anti-inflammatory effect on diabetic
periodontitis in our previous studies [15]. 25(OH)D3 was shown to improve alveolar bone loss and suppress the expression of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, the key players in inflammaging [7, 10]. VD3 has been found to suppress the expression of suppressor of cytokine signaling (SOCS)-3, which in turn decreases pro-inflammatory cytokine synthesis [16]. SOCS3 attenuates proinflammatory signaling mediated by the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway [17]. Previous studies demonstrated that enhanced SOCS3 was associated with aging [18]. Therefore, we propose that VD3 supplements linked with inflammaging in diabetes. Wherein 25(OH)D3 has a certain therapeutic effect on these SASP factors by regulating SOCS3/STAT pathway in diabetic periodontitis.
2. Experimental design 2.1 Animal model Both male db/db mice (C57BLKS/J-leprdb/leprdb*) and male C57BL/6 wild-type mice kept in the State Key Laboratory of Oral Diseases at Sichuan University under standard housing conditions, were studied at the age of 4 weeks. The point mutation of the leptin receptor in db/db mice results in the disorder of leptin signaling in the hypothalamus with characteristics of hyperphagia and obesity, which develop a spontaneous T2DM [19, 20]. All diabetic mice developed sustained hyperglycemia (fasting blood glucose (FBG) > 18 mmol/L) but experienced no incident of severe hyperglycemia. db/db mice (n = 10 per group) were blindly randomly divided into diabetes (D), diabetic periodontitis (DP), and diabetic periodontitis with 25(OH)D3 treatment (DPV) groups. C57BL/6 mice were recognized as a normal control (N). All animals were included in data analysis.
*
Model Animal Resource Information Platform, Nanjing, Jiangsu, China
Sample size was selected on the basis of previous publications. Mice were sacrificed at the age of 18 weeks. In vivo studies were performed with approval of the institutional committee for animal use and care at Sichuan University (Accession Number: WCCSIRB-D-2015-075).
2.2 Bacteria and oral inoculation Porphyromonas gingivalis (P.g.) strain ATCC 33277* was provided by the State Key Laboratory of Oral Diseases of Sichuan University and cultured in sheep-blood agar with hemin/vitamin K1 under anaerobic conditions. At 5 weeks of age, the mice in the DP and DPV groups were infected with P.g. as follows: 109 colony-forming units of live bacteria and 2% carboxymethylcellulose in 100 μL of phosphate-buffered vehicle were orally administered 3 times at 2 d intervals and then the vehicle with 2% carboxymethylcellulose was administered to the control mice.
2.3 25(OH)D3 treatment From 7 weeks of age, mice in the DPV group received an intraperitoneal injection of 25(OH)D3† (5 μg/kg) within 5 min every other day, which prior was dissolved in refined peanut oil vehicle. Control mice (N, D and DP groups) received the vehicle alone similar to that of the DPV group by intraperitoneal injection. 25(OH)D3 supplementation was sufficient with the vitamin D requirements for mice (mean analyzed concentration: 14.6 ± 1.4 ug/kg) [21]. The administration was continued to 1 d before sacrifice.
2.4 FBG and body weight measurements
* †
ATCC®33277™, Chengdu, Sichuan, China CAS:63283-36-3, Sigma-Aldrich Co., Santa Clara, CA, USA
Mouse tail vein blood was collected following a 10-h fast. A glucometer* was subsequently used to determine the FBG level. The body weight was monitored by a scale with 0.01 g accuracy, and the measurements were implemented every other week from 4 weeks old.
2.5 Biochemical analysis The tail vein blood was collected in units of each mouse by the capillary pipette when determining FBG at 4 and 18 weeks old, then serum was directly isolated (centrifuging at 3000 g for 12 min; High speed centrifuge†) and stored at –80°C for subsequent determination. The levels of serum SASP factors, including IL-1β, IL-4, IL-6, IL-10, TNF-α, intercellular adhesion molecule (ICAM)-1, macrophage colony stimulating factor (M-CSF), matrix metalloproteinases (MMP)-2, MMP-8, osteoprotegerin (OPG), adiponectin (Adi), glucagon, pancreatic polypeptide (PP), leptin and receptor for advanced glycation end product (RAGE), were determined using Luminex multiplefactor assay kits‡ according to the manufacturer’s specifications. The concentration was expressed in pg/mL. Serum 25(OH)D3 and 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3)§ were quantified by ELISA according to manufacturer’s instructions. Blood levels of serum calcium was detected with an Enzyme Marker Detection kit** and results are presented as mmol/L.
2.6 MicroCT analysis After disassociating the attached soft tissue, the mandibular jaws of all groups were scanned by a
OneTouch Glucometer; LifeScan, Milpitas, CA HC-3012, USTC Ltd., CA ‡ Luminex 200, R&D Systems, USA § 25-OH-Vitamin-D ELISA, REA300/96; 1,25(OH) Vitamin D Total ELISA, RIS021R; BioVendor Co., CZE 2 ** Serum Calcium Assay Kit, ZC-S0445, ZCI BIO, shanghai, China; THERMO VARIOSKAN FLASH, ThermoFisher, USA * †
µCT50* at 10 μm intervals. The resorption of the alveolar bone was quantified by morphometric analysis of micro-CT images of the mandibles. Alveolar bone loss level was defined by measuring the area bordered by the cemento-enamel junction, the alveolar bone crest, and the mesial and distal line angles on the lingual side of the mandibular first molars. Attachment loss was measured by calculating the vertical distance of the alveolar bone crest and the cemento-enamel junction of the second molar. For the analysis of bone mineral density (BMD), the volume of interest was defined as a rectangular area covering the alveolar bone loss. The blinded measurements were randomly repeated 3 times.
2.7 Histological and immunohistochemical analysis Mandibular jaw samples were collected and stained for hematoxylin-eosin (HE) and immunohistochemical (IHC) staining (anti-p16, anti-p21, diluted 1:100; secondary antibody, diluted 1:1000). Images were obtained using Aperio ImageScope†. All of the antibodies were acquired from Santa Cruz Biotechnology‡. HE staining was performed for periodontal tissue and alveolar bone loss between the cement-enamel junction and the bone crest. The periodontal ligament (PDL) width was measured as the apical height segments from the PDL coronal section. The PDL attachment height was measured between the base of the sulcus and the alveolar bone crest. These two indicators represent the degree of PDL attachment loss and alveolar bone absorption.
2.8 Western blotting
SCANCO Medical, Bruettisellen, Switzerland ScanScope; Aperio; Kenilworth, NJ, USA ‡ Santa Cruz, CA, USA * †
After separated by dispase I, the proteins extracted from mouse mandible gingival tissues were subjected to 5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, and transferred to nitrocellulose membranes by electro-blotting. The membranes were incubated overnight at 4°C with primary antibodies against β-actin, Vitamin D Receptor (VDR)*, SOCS3† , STAT3‡ , p-STAT3§ , STAT5**, p-STAT5†† , NF-κB‡‡ , IL-1⧧ , p16*** or p21††† , and then with secondary antibodies for 1 hour at room temperature. The signals were detected by electrochemiluminescence using BioRad system‡‡‡.
2.9 Statistical analysis Error bars on all graphs showed the standard error of multiple independent measurements. Statistical analyses were done using Graphpad Prism software Version 8.0.0. In each group of statistical data, largest or smallest deviations were removed during analysis. Significance was determined using Student’s t-test or ANOVA, as appropriate. A level of P < 0.05 was considered significant.
3. Results 3.1 The inflammaging status occurred in db/db mice The SASP (associated with chronic inflammation, proteinases, bone metabolism and glycolipid
1:500 mouse mAb, sc-13133, Santa Cruz Biotechnology, Inc, CA, USA 1:1000 rabbit pAb, ab16030, Abcam, Abcam Trading Co. Ltd., Shanghai, China ‡ 1:1000 mouse mAb, #9139, Cell Signaling Technology, Massachusetts, USA § 1:2000 rabbit mAb, #9145, Cell Signaling Technology, Massachusetts, USA ** 1:1000 rabbit pAb, ab209544, Abcam, Abcam Trading Co. Ltd., Shanghai, China †† 1:1000 rabbit mAb, ab32364, Abcam, Abcam Trading Co. Ltd., Shanghai, China ‡‡ 1:1000 mouse mAb, sc-514451, Santa Cruz Biotechnology, Inc, CA, USA §§ 1:1000 rabbit mAb, #31202, Cell Signaling Technology, Massachusetts, USA *** 1:1000 mouse mAb, sc-166760, Santa Cruz Biotechnology, Inc, CA, USA ††† 1:1000 mouse mAb, sc-166630, Santa Cruz Biotechnology, Inc, CA, USA ‡‡‡ Bio-Rad Laboratories, Shanghai, China * †
metabolism) were examined to investigate the inflammaging condition in diabetes (Tab. 1). The positive cells of p16 and p21 increased obviously in the periodontal tissue of db/db mice compared with the normal mice (Fig. 1A). As the results shown (Fig. 1B), the pro-inflammatory factors and the matrix metalloproteinases were significantly higher in the D group (P < 0.05), comprising IL1β, IL-6, TNF-α, ICAM-1, M-CSF, MMP-2 and MMP-8. The levels of IL-4 and IL-10 were lower statistically in the D group compared with the N group (P < 0.05). Likewise, the secretion of OPG in serum remarkably decreased (P < 0.05). We also measured the level of glycolipid metabolism related to diabetes, another common feature of metabolic disorder disease. Glycogen, PP and RAGE increased, leptin level accumulated, but Adi decreased in the db/db mice (P < 0.05 for all). In addition, the FBG level was obviously higher than that of the normal mice (Fig. 1C).
3.2 A senescent-like state with an increased expression of the SASP factors was performed in db/db mice with periodontitis To determine if the inflammaging was associated with periodontal injury of diabetic periodontitis, periodontal tissues were examined by immunohistochemistry for p16/p21 staining for senescence detection and HE staining to quantify the extent of periodontal destruction, and Luminex analysis for serum SASP as an inflammaging status marker. Results showed that the percentage of p16positive cells was increased dramatically, meanwhile the percentage of p21-positive cells was also increased significantly in the DP group compared with the D group (Fig. 2A). The expression of IL1β, IL-6, TNF-α, MMP-2, MMP-8 and ICAM-1 at protein levels increased significantly from baseline to 18 weeks after infection (P < 0.05), also were significantly higher than those of the D group (P < 0.05) (Fig. 2B). In contrast, IL-4, IL-10, M-CSF and OPG behaved a decrease obviously
in the DP group. IL-10, not IL-4, was significantly lower than that in the D group at the time of killing (P < 0.05). As observed in the D group, glycogen, PP, leptin and RAGE remained increase within the DP group, but Adi was reduced (Fig. 2B). Importantly, the differences of them were markedly between the two groups (DP vs. D P < 0.05). Besides, the differences in the PDL attachment width and height was statistically significant between the DP and the D groups (P < 0.01 for both) (Fig. 2C, D, E), suggesting that oral inoculation could lead to severe alveolar bone loss and inflammaging status in diabetes.
3.3 25(OH)D3 improved the impaired periodontal condition in db/db mice with periodontitis To investigate whether 25(OH)D3 could relieve the outcomes of diabetic periodontitis, weight and FBG were measured every 2 weeks in all groups after the baseline record. Notably, the uncontrollable hyperglycemia occurred in the DP group and significantly improved after 7-week 25(OH)D3 treatment (Fig. 3A, B). The FBG levels of both the DP and DPV groups were significantly higher than that of the D group from the 10th week to the 14th week, suggesting that periodontitis could aggravate diabetes and rise blood glucose. The weight graph (Fig. 3C) showed that the DPV and DP mice were close after oral inoculation, and eventually the DPV group exhibited a lower weight. Meanwhile, The DPV group shared a slight alveolar bone loss, whereas the vehicle groups had a much more severe attachment loss and bone loss (P < 0.05 for both) (Fig. 3D-F). In addition, we established measurements of the BMD (Fig. 3D, G), showing the difference between the DPV and DP group was not significant, although the DP group was remarkably higher than the D group (P < 0.05).
3.4 25(OH)D3 suppressed gingival tissue senescence and the serum SASP factors in db/db mice with periodontitis To elucidate if 25(OH)D3 improved the inflammaging in diabetic periodontitis, we measured the levels of the SASP and the expression of p16 and p21. In the DPV group, significant decreases were observed in the percentages of positive areas for activated p16 and p21 compared with the DP group (Fig. 4A). Continuous treatment with 25(OH)D3 significantly lowered IL-1β, IL-6, TNF-α, ICAM-1, M-CSF, MMP-2 and MMP-8 secretions in serum (P < 0.05) (Fig. 4B). IL-4, IL-10 and OPG had an obvious increase after treatment (P < 0.05), as expected. Glycogen, leptin and RAGE downregulated, suggesting a decrease of some pathways activation in diabetic periodontitis mediates the reduced the secretion level of SASP factors. Instead, Adi increased significantly after treatment. The level of PP was the highest in all groups (P < 0.05). These results demonstrated that 25(OH)D3 ameliorated the SASP caused by periodontal inflammaging.
3.5 25(OH)D3 downregulated SOCS3/STAT signaling in gingival tissue of db/db mice with periodontitis To further determine the potential molecular mechanism that 25(OH)D3 ameliorates periodontal inflammaging, the proteins expression of SOCS3 and VDR were detected using western blotting. The results showed that although they were expressed in the periodontal tissue from all groups, the expression level of SOCS3 was up-regulated significantly in db/db mice with periodontitis and returned to the normal levels in the DPV group (P < 0.05) (Fig. 5A, B). The level of VDR in the DP group was significantly lower than those of the N and the DPV groups (P < 0.05) (Fig. 5A, C). The expression of p16 and p21 increased consistently in the DP group and decreased after 25(OH)D3
treatment (Fig. 5A, D, E). In addition, when the phosphorylation levels of STAT3 and STAT5 were reduced significantly upon 25(OH)D3 treatment, the expression of NF-κB and IL-1β in protein levels was down-regulated subsequently compared with the DP group (P < 0.05 for all) (Fig. 5F-J). These results suggest that 25(OH)D3 delayed periodontal inflammaging might be related to regulate the SOCS3/STAT signaling.
Consistent with the western blot results of VDR, serum concentrations of VD3 metabolites decreased in diabetic and periodontitis-diabetic mice at the end of study (P < 0.01) (Fig. 5K, L). At baseline, all db/db mice had lower levels of serum 25(OH)D3, 1,25(OH)2D3 and calcium compared with the N group (P < 0.05) (Fig. 5K-M). Within the observation period, the non-treatment groups as well as the N group presented a gradual and accordant decline in 25(OH)D3 and 1,25(OH)2D3 levels (Fig. 5K, L). Serum calcium level manifested irregular fluctuation, but a notable decline appeared in the D and DP group over time (Fig. 5M). These results indicated that VD3 deficiency correlated not only with aging, but also with the progress of disease. After 10-week treatment, the level of 25(OH)D3 was apparently higher in the DPV group than that of the DP group (P < 0.05). Meanwhile, increased 1,25(OH)2D3 and calcium levels in the DPV group was found and there was no significant difference relative to the N group (Fig. 5K-M).
4. Discussion This study confirmed the inflammaging status occurred in diabetes using db/db mouse as a type 2 diabetic model. The experimental diabetic periodontitis mice demonstrated impaired periodontal structure, enhancement of senescence burden and development of SASP levels. 25(OH)D3
ameliorated senescence and the SASP in mice with diabetic periodontitis by downregulating SOCS3/STAT signaling. These findings indicated that 25(OH)D3 supplement-treatment could be a critical event in preventing periodontal injury and the excessive inflammaging, a likely intervention target in diabetic periodontitis.
Previous evidence suggested that db/db mice are typical T2DM model [19]. Due to hyperglycemic impair and imbalanced metabolism, the persistent accumulation of pro-inflammatory SASP in serum are sufficient to induce senescence [8, 11]. The cell cycle regulator p16 is transcriptionally activated in cells undergoing irreversible senescence, which leads to aging-associated impaired function and regenerative capacity [22, 23]. The activation of p21 is important in the initiation of senescence [24]. In this study, the percentage of p16 and p21-positive cells were greatly increased in gingival tissues from the D group to DP group, suggesting that the chronic inflammation could give impetus to tissue senescence. Moreover, inflammatory cell infiltration in gingival tissue, characterized by inflammatory SASP secretion, maybe a major pathological change of periodontal senescence.
The role of nutrition in inflammaging process is of great importance. The low-grade nutrition metabolic state present inside the chronic phase is strongly related to inflammaging [8, 11, 13]. Most of the existing evidence claims that increasing the intake of 25(OH)D3, having anti-inflammatory properties, contributes to restore a normal nutrition synthesis [25]. VD3 deficiency increases the risk of developing chronic diseases and the rate of aging [14, 26]. In contrast, chronic diseases, highly common in aging, are thought to increase the risk for VD3 deficiency, as these would reduce its
synthesis and enhance its catabolism [12, 13, 26]. One of the important actions of VD3 is to reduce the expression of inflammatory cytokines, which are such a prominent feature of how inflammatory responses alter cellular activity accelerating aging [27]. The fact that the ability of the skin to make VD3 declines as aging progresses is one reason why cognition tends to decline during aging [28]. After three-month observation, VD3 metabolites accordantly decreased in the N group, which proved the presence of a close relationship between age and VD3 levels [14]. Thus, it has been suggested that the rate of aging is regulated by vitamin D. This study showed that the number of senescent cells declined and SASP was ameliorated upon 25(OH)D3 treatment, indicating that 25(OH)D3 supplementation is beneficial to mitigation of inflammaging in diabetic periodontitis.
Lower levels of VD3 are associated with diabetes and periodontitis [29, 30]. In this study, genetically diabetic mice suffered VD3 deficiency and hypocalcemia, namely negative calcium homeostasis [31], throughout the observation period. As the disease progresses, the decrease of 25(OH)D3 is more obvious in the DP group compared with the D group. Changes of serum calcium and VD3 metabolites at least partly due to the decrease of 1,25(OH)2D3 receptor in intestine and kidney of diabetic mice [32], similar to the manifestation of gingival tissue that VDR downregulated in the DP group. Studies have found that when serum 25(OH)D3 levels are lower than 20 ng/mL, most of vitamin D uptake or skin production immediately turns to bone formation, which is insufficient to provide advanced functions comprising immune system, brain or gene regulation [33]. It is predicted that the significant decrease of 25(OH)D3 may be related to the severity of tissue injury with aging. Upon 25(OH)D3 treatment, serum 25(OH)D3, 1,25(OH)2D3 and calcium levels presented raising trend and were equivalent to that of the N group terminally. The inflammaging
process can be delayed by adjusting the concentration of 25(OH)D3 or 1,25(OH)2D3 in the body. Furthermore, we inferred that the VD3 metabolites can be used as biomarkers of inflammaginginduced diseases.
Like many age-related diseases, diabetes is mainly fostered by nutrient and metabolic surplus to trigger a higher level of “basal” inflammation [3, 11]. When subjects sustained a complicated chronic periodontitis, even higher levels of SASP were observed compared to those without periodontitis [34, 35]. It is mostly because the overall inflammatory state was aggravated by inoculation with p.g. in T2DM [34]. As reported previously, the elevated IL-6 and TNF-α in aging tissues is one of the reasons for senescence-driven tissue dysfunction [36, 37], explaining that the uncontrollable hyperglycemia and severe alveolar bone loss were observed in the DP group. In addition, our results showed BMD of the DP group was higher than that of the D group, further supporting that OPG is reduced with inflammatory status. Therefore, this study demonstrated that the inflammaging was excessive induced by these several senescent-like phenotypes in diabetic periodontitis.
Our results indicate that significantly reduced IL-1β, IL-6, TNF-α, MMP-2, MMP-8, ICAM-1 and M-CSF secretion in serum of the DPV mice, suggesting 25(OH)D3 may slow down inflammaging by attenuating the pro-inflammatory SASP [38]. In addition, 25(OH)D3 suppressed leptin secretion from fat tissue, which has been reported previously [39]. Adi has an anti-inflammatory effect and is attenuated leading to the other SASP up-regulation by suppressing NF-κB signaling and TNF-α release [40, 41], suggesting that 25(OH)D3 could up-regulate Adi by inflammatory pathway. There
is no direct evidence explaining the regulatory effect between 25(OH)D3 and glycolipid metabolism. VD3 stimulates the production of insulin growth factor (IGF)-1, the key player in the crossroad linking nutrition with inflammatory signaling, whose levels are positively regulated by diet and anabolic hormonal systems and negatively affected by inflammation and oxidative stress [42]. IGF1 levels tend to decline with aging, in conjunction with the increase in subclinical inflammatory status [43]. Thus, 25(OH)D3 improves the metabolic SASP factors, possibly involving IGF-1.
In this study, we found significantly decreased protein expression of VDR and increased SOCS3 in the gingival tissues of the DP mice, whereas an opposite change in the 25(OH)D3-supplement mice. A study has been reported that the aging process may influence the expression of VDR genes, wherein circulating 25(OH)D3 levels did not appear to affect [44]. In addition to regulating the expression of inflammatory genes, genetic variance in VDR gene influences the susceptibility to age-related changes [45]. These suggested that VDR might play a key role in inflammaging. Previous studies about the linking between VD3 and SOCS3 mainly focused on inflammatory signaling [16, 46], but little on aging. Our study indicated that the periodontal inflammaging might be associated with increased SOCS3 and decreased VDR. It has been found that aging increases SOCS3 expression in hypothalamus [18]. Previous studies also demonstrated that decreased expression of SOCS3 involved STAT3 and NF-κB interaction [47]. The activation of VDR inhibits NF-κB resulting in decreased pro-inflammatory signaling and induced hypo-responsiveness to antigenic stimulation [48, 49]. NF-κB is the pivot regulating the transcription of inflammatory factors [50]. Its knockdown significantly decreases the levels of 75% of SASP factors [51]. We found that the expression level of NF-κB was lower in diabetic periodontitis compared with the D
group and increased distinctly after 25(OH)D3 treatment, which is consistent with that VD3 downregulates the expression and production of pro-inflammatory cytokines by suppressing NF-κB activation [52]. In this study, 25(OH)D3 inhibited SASP through down-regulating NF-κB proinflammatory signaling to improve periodontal inflammaging, might be dependent on the VDR/SOCS3/STAT signaling. Further experiments in vitro are needed to reveal the precise mechanism.
Connectively, inflammaging may be the particular reason to increase the adverse outcomes in diabetic periodontitis, leading to considerable difficulties in curing its effect. Notably, diabetic periodontitis, as a verified chronic inflammatory disease [4], could be ameliorated by 25(OH)D3 altering the inflammaging through SOCS3/STAT signaling. VD3 also inhibits IL-6 synthesis at both the protein kinase C pathway and the protein kinase A pathway. According to a number of reports, SASP is also initiated by p38 MAPK signaling, except for NF-κB [36, 53]. Future research should better address all these issues, clarifying the molecular and clinical rationale of combined nutritional interventions especially with VD3.
5. Conclusion Our study showed that diabetic periodontitis performed a senescent-like state with a secretory of the SASP (inflammaging). The inflammaging might play a crucial role in diabetic periodontitis. 25(OH)D3 treatment performed a delay in senescent cell enrichment and alleviated the elevated serum SASP. Furthermore, it reveals the potential utility of VD3 as therapeutic agents, and its antiinflammaging therapies makes it a promising candidate in preventing age-related diseases. The
clinicians should also pay attention to the levels of 25(OH)D3 and calcium in patients with agerelated diseases, and routinely detect and give appropriate treatment with nutritional intervention.
6. Data availability All data generated or analyzed during this study are included in the published article and supplementary information files.
7. Conflicts of interest The authors declare that they have no potential conflicts of interest relevant to this study.
8. Author contributions Q.W. performed the experiments, analyzed the data, and wrote the manuscript; X.Z. contributed to the experimental work; P.Z. and P.F Z. provided intellectual contribution on the research design; L.N. helped prepare the manuscript and optimize image analysis; N.J. and Y.D. provided intellectual contribution on experimental techniques and related knowledge; Q.W.* contributed to the research design and takes primary responsibility for the final content; and all authors discussed the results and commented on the manuscript.
9. Acknowledgements We thank Qiang Guo (State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University) for assisting with
microCT analysis strategies.
10. Funding statement This work was supported by the National Natural Science Foundation of China [grant numbers 81870779], the International Cooperation Project of Chengdu Municipal Science and Technology Bureau [grant number 2015-GH02-00035-HZ] and the International Scientific Cooperation and Exchanges Project of Sichuan Province [grant number 2017HH0078].
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Figure legends Fig. 1 (A) Immunohistochemical staining for p16 and p21 in mandibular gingival tissue. Black arrows were drawn to point out positive cells, and the upper right corner box is the enlarged area indicated by the arrow. (B) The serum levels of SASP (about inflammatory cytokines, metalloproteases, chemokines, bone metabolism and glycolipid metabolism). (C) Fasting blood glucose. Data were obtained at the age of 18 weeks. Results are shown as mean ± SEM; n = 10; the value about D vs. N by analysis of variance (ANOVA) or Student’s t-test; * P < 0.05, ** P < 0.01 and *** P < 0.001. N= normal control mice, D= diabetic mice.
Fig. 2 (A) Immunohistochemical staining for p16 and p21 in mandibular gingival tissue. Black arrows were drawn to point out positive cells, and the upper right corner box is the enlarged area indicated by the arrow. (B) The secretory levels of SASP factors in circulatory serum at 4 and 18 weeks old of mice. (C) Hematoxylin and eosin staining of the periodontal tissue. Black arrows indicated the epithelial spikes in the D and DP groups. Periodontal ligament (PDL) width was measured by the apical height segments from the PDL coronal section, repeated 3 times; PDL attachment height was measured between the base of the sulcus and the alveolar bone crest, repeated 3 times. (D)&(E) Quantification of PDL width and attachment height in the D and DP groups. Results are shown as mean ± SEM; n = 10; * P < 0.05 and ** P < 0.01. D= diabetic mice, DP= diabetic periodontitis mice.
Fig. 3 (A) Experimental diabetic periodontitis mouse model and all protocols were performed strictly according to the procedure. (B) Fasting blood glucose levels. (C) Weight levels. (D) Three-
dimensional reconstructions of the mandibular alveolar bones. (E) The attachment loss among groups. (F) The alveolar bone loss area among groups. (G) The bone mineral density (BMD) of the volume of interest (alveolar bone of the mandibular first molars) in the mandibular jaws of mice. D= diabetic mice, DP= diabetic periodontitis mice, DPV= diabetic periodontitis mice treated with 25(OH)D3. Results are shown as mean ± SEM; n = 10; the value about DP vs. D and DPV vs. DP by ANOVA; * P < 0.05 and ** P < 0.01; BMD, DPV vs. DP P > 0.05.
Fig. 4 (A) Immunohistochemical staining for p16 and p21 in mandibular gingival tissue. Black arrows were drawn to point out positive cells, and the upper right corner box is the enlarged area indicated by the arrow. (B) The serum levels of SASP after receiving 25(OH)D3 treatment at 18 weeks old of mice. Results are shown as mean ± SEM; n = 10; * P < 0.05 and ** P < 0.01.
Fig. 5 Protein expression in the mouse gingival tissue of N, D, DP and DPV mice and serum concentrations of VD3 metabolites were detected. (A-E) Protein expression of SOCS3, VDR, p16 and p21. (F-J) Protein expression of STAT3, p-STAT3, STAT5, p-STAT5, NF-κB and IL-1β. 25(OH)D3-treated mice with diabetic periodontitis inhibited SOCS3, p16, p21, p-STAT3/STAT3, p-STAT5/STAT5, NF-κB and IL-1β expression, and increased the expression level of VDR. (K) Serum 25(OH)D3 levels form the 4th to the 18th week. (L) Serum 1,25(OH)2D3 levels. (M) Blood levels of serum calcium. N= normal control mice, D= diabetic mice, DP= diabetic periodontitis mice, DPV= diabetic periodontitis mice treated with 25(OH)D3. Results are shown as mean ± SEM; n = 10; * P < 0.05, ** P < 0.01 and *** P < 0.001.
Author contributions
Qian Wang1, Xinyi Zhou, Peng Zhang, Pengfei Zhao, Lulingxiao Nie, Ning Ji, Yi Ding and Qi Wang*.
Q.W. performed the experiments, analyzed the data, and wrote the manuscript; X.Z. contributed to the experimental work; P.Z. and P.F Z. provided intellectual contribution on the research design; L.N. helped prepare the manuscript and optimize image analysis; N.J. and Y.D. provided intellectual contribution on experimental techniques and related knowledge; Q.W.* contributed to the research design and takes primary responsibility for the final content; and all authors discussed the results and commented on the manuscript.
Highlights
25-Hydroxyvitamin D3 (25(OH)D3) inhibited serum senescence-associated secretory phenotype (SASP) in diabetic periodontitis. 25(OH)D3 decreased the expression of p16/p21 in the gingival tissue of the diabetic periodontitis mice. 25(OH)D3 suppressed the gingival inflammaging via SOCS3/STAT signaling in diabetes. 25(OH)D3 supplement could be a potential intervention for diabetic periodontitis through relieving the excessive inflammaging.
The List of Chemical compounds
Chemical compounds
Roles
1
Vitamin D3
a steroid hormone with immunoregulatory effects
2
25-Hydroxyvitamin D3
the storage form of VD3; injected to the db/db mice in this study
3
1α,25-Dihydroxyvitamin D3
4
Hemin
5
Vitamin K1
the bioactive form of VD3; promoting calcium absorption; the blood level was monitored in this study added to agar medium for the culture of porphyromonas gingivalis added to agar medium for the culture of p.g.
6
Carboxymethylcellulose
7
Hematoxylin
8
Eosin
9
sodium dodecyl sulfate
10
polyacrylamide
11
nitrocellulose
dissolved with live bacteria for oral inoculation histological staining reagent histological staining reagent preparation of electrophoretic gels preparation of electrophoretic gels raw material membranes
for
immunoblotting
Table 1 The typical SASP response in diabetes related to chronic periodontitis Factors*
Inflammatory cytokines
Metalloproteases
Chemokines
Bone metabolism
Roles
IL-1β
an early component and an upstream regulator of the SASP
IL-4
an anti-inflammatory SASP in early-onset inflammation
IL-6
promoting malignant and senescent phenotypes
IL-10
a signature SASP in pathological senescence
TNF-α
mediating senescence-driven periodontal tissue dysfunction
MMP-2
contributing to periodontal tissue destruction
MMP-8
participating in tissue repair
ICAM-1
recruiting macrophages to promote inflammation
M-CSF
inducing osteoclast differentiation
OPG
a SASP inhibiting osteoclast formation in alveolar bone absorption
Glycolipid metabolism
Glucagon PP Leptin Adi RAGE
exacerbating glucose toxicity aging-related metabolic hormone controlling food intake and increasing energy metabolism an anti-inflammatory adipokine accelerating senescence
* interleukin (IL)-1β; tumor necrosis factor (TNF)-α; matrix metalloproteinases (MMP)-2; intercellular adhesion molecule (ICAM)-1; macrophage colony stimulating factor (M-CSF); OPG, osteoprotegerin; PP, pancreatic polypeptide; Adi, adiponectin; RAGE, receptor for advanced glycation end product.
S0039-128X(19)30260-0 https://doi.org/10.1016/j.steroids.2019.108570 STE 108570
To appear in:
Steroids
Received Date: Revised Date: Accepted Date:
1 June 2019 16 November 2019 21 December 2019
Please cite this article as: Wang, Q., Zhou, X., Zhang, P., Zhao, P., Nie, L., Ji, N., Ding, Y., Wang, Q., 25Hydroxyvitamin D3 positively regulates periodontal inflammaging via SOCS3/STAT signaling in diabetic mice, Steroids (2019), doi: https://doi.org/10.1016/j.steroids.2019.108570
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25-Hydroxyvitamin D3 positively regulates periodontal inflammaging via SOCS3/STAT signaling in diabetic mice Qian Wang1,2, Xinyi Zhou1,2, Peng Zhang1,2, Pengfei Zhao1,2, Lulingxiao Nie1,2, Ning Ji1, Yi Ding1,3 and Qi Wang1,2,* 1 State
Key Laboratory of Oral Diseases, National Clinical Research
Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University 2 Department
of Prosthodontics, West China Hospital of Stomatology, Sichuan University
3Department
of Periodontology, West China Hospital of Stomatology, Sichuan University
*Correspondence: Dr. Qi Wang, Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, 3rd Section S Renmin Road, 14#, Chengdu, China, 610041 [email protected] Fax: +86 28 85503503 Email: [email protected]
Abbreviations: VD3, Vitamin D3; 25(OH)D3, 25-Hydroxyvitamin D3; P.g., Porphyromonas gingivalis; SASP, senescence-associated secretory phenotype; CS, cellular senescence; VDR, Vitamin D Receptor; SOCS3, suppressor of cytokine signaling-3; JAK, Janus kinase; STAT, signal transducer and activator of transcription; p-STAT3, phosphorylated STAT3; p-STAT5, phosphorylated STAT5; T2DM, type 2 diabetes mellitus; FBG, fasting blood glucose; OPG, osteoprotegerin, Adi, adiponectin; PP, pancreatic polypeptide; RAGE, receptor for advanced glycation end product; 1,25(OH)2D3, 1α,25-Dihydroxyvitamin D3; BMD, bone mineral density; PDL, periodontal ligament; Ab, antibody.
Abstract Background: Diabetes is a known age-related disease. Inflammaging has recently been shown to result in diabetic complications. Vitamin D3 is related to aging in the latest study but little is known about the underlying mechanism. Here, we investigated the effects of 25-Hydroxyvitamin D3 (25(OH)D3) on inflammaging in diabetic periodontitis, a common chronic inflammatory diabetic complication. Experimental design: A model of Porphyromonas gingivalis-infected db/db mice as experimental type 2 diabetic periodontitis was adopted in the whole study. A range of techniques, including microCT, western blotting, ELISA, histological and immunohistochemical analysis, were carried out in this study. The distinctive senescence-associated secretory phenotype (SASP) in serum was measured by Luminex technology. Results: We found an archetypal inflammaging status occurred in db/db mice. An increased SASP, senescent enhancement, and periodontal destruction were observed in periodontitis-db/db mice. Upon administration with 25(OH)D3, periodontitis-db/db mice presented increased levels of serum 25(OH)D3, 1α,25-Dihydroxyvitamin D3 and calcium. Moreover, decreased p16/p21-positive cells, relieved periodontal conditions and ameliorated serum SASP secretion were found in the periodontitis-db/db mice after treatment. Gingival tissue exhibited increased level of VDR and decreased expression of SOCS3, p-STAT3/STAT3, p-STAT5/STAT5, NF-κB and IL-1β, which were consistent with the change of p16/p21 expression. Conclusion: Diabetic periodontitis appeared to develop an inflammaging status resulted in periodontal infection. 25(OH)D3 could inhibit SASP secretion through reducing SOCS3 expression in experimental diabetic periodontitis, dependently inactivating NF-κB pro-inflammatory signaling.
The reversible effect further documented that the inflammaging might be a highly likely contributor in diabetic periodontitis.
Graphical abstract 25(OH)D3 played the protective role in gingival tissue of db/db mice with periodontitis. SOCS3 is an endogenous negative regulator of the inflammaging. Diabetic periodontitis developed an inflammaging status with up-regulated SOCS3 in gingival tissue. Black arrows indicate pathogenic mechanism, wherein solid arrows indicate directly active effects and black dashed arrows indicate the indirectly active effects. Green solid arrows indicate the functions of 25(OH)D3, green barheaded lines indicate inhibit effects.
Keywords: 25-Hydroxyvitamin D3, inflammaging, senescence-associated secretory phenotypes, senescence, SOCS3, diabetic periodontitis
1. Introduction Diabetes mellitus is an age-related disease [1] that poses a major challenge to the longevity and life quality of humans [2, 3]. Diabetic periodontitis, as a mainly inflammatory diabetic complication affects about 11.2% of the global adult population [4, 5]. Patients often suffer from severe gingival inflammation, extensive alveolar bone destruction and tooth loss. Gingival tissue constitutes the first barrier of periodontal protection [6]. Diabetes increase the susceptibility to opportunistic oral infections, leading to severe periodontal outcomes [4]. However, the underlying pathogenic mechanisms and therapeutic treatment are still not fully understood.
Recently, inflammaging has been suggested to be a major contributor to type 2 diabetes mellitus (T2DM) [7, 8]. Inflammaging is a secondary senescent pattern that can be induced chronically in response to chronic low-grade inflammation independent of replicative senescence [7, 9]. It causes aging-associated pathological changes and increases vulnerability to death primarily through enhancing cellular senescence (CS) and promoting a senescence-associated secretory phenotype (SASP) [7-10]. CS restricts cell proliferation and deteriorates tissue microenvironment [3, 9]. The SASP involves the secretion of a variety of molecules disrupting the surrounding normal cell functions, including cytokines, chemokines, growth factors and proteases [10].
Nutrients are theoretically modulators to senescence [11, 12]. Vitamin D3 (VD3) has been highly recommended as a complementary nutrient for diabetes [12]. It is currently considered as an immunomodulator in aging and inflammation [12-14]. 25-Hydroxyvitamin D3 (25(OH)D3), a main circulatory form of VD3, has been found an excellent anti-inflammatory effect on diabetic
periodontitis in our previous studies [15]. 25(OH)D3 was shown to improve alveolar bone loss and suppress the expression of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α, the key players in inflammaging [7, 10]. VD3 has been found to suppress the expression of suppressor of cytokine signaling (SOCS)-3, which in turn decreases pro-inflammatory cytokine synthesis [16]. SOCS3 attenuates proinflammatory signaling mediated by the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway [17]. Previous studies demonstrated that enhanced SOCS3 was associated with aging [18]. Therefore, we propose that VD3 supplements linked with inflammaging in diabetes. Wherein 25(OH)D3 has a certain therapeutic effect on these SASP factors by regulating SOCS3/STAT pathway in diabetic periodontitis.
2. Experimental design 2.1 Animal model Both male db/db mice (C57BLKS/J-leprdb/leprdb*) and male C57BL/6 wild-type mice kept in the State Key Laboratory of Oral Diseases at Sichuan University under standard housing conditions, were studied at the age of 4 weeks. The point mutation of the leptin receptor in db/db mice results in the disorder of leptin signaling in the hypothalamus with characteristics of hyperphagia and obesity, which develop a spontaneous T2DM [19, 20]. All diabetic mice developed sustained hyperglycemia (fasting blood glucose (FBG) > 18 mmol/L) but experienced no incident of severe hyperglycemia. db/db mice (n = 10 per group) were blindly randomly divided into diabetes (D), diabetic periodontitis (DP), and diabetic periodontitis with 25(OH)D3 treatment (DPV) groups. C57BL/6 mice were recognized as a normal control (N). All animals were included in data analysis.
*
Model Animal Resource Information Platform, Nanjing, Jiangsu, China
Sample size was selected on the basis of previous publications. Mice were sacrificed at the age of 18 weeks. In vivo studies were performed with approval of the institutional committee for animal use and care at Sichuan University (Accession Number: WCCSIRB-D-2015-075).
2.2 Bacteria and oral inoculation Porphyromonas gingivalis (P.g.) strain ATCC 33277* was provided by the State Key Laboratory of Oral Diseases of Sichuan University and cultured in sheep-blood agar with hemin/vitamin K1 under anaerobic conditions. At 5 weeks of age, the mice in the DP and DPV groups were infected with P.g. as follows: 109 colony-forming units of live bacteria and 2% carboxymethylcellulose in 100 μL of phosphate-buffered vehicle were orally administered 3 times at 2 d intervals and then the vehicle with 2% carboxymethylcellulose was administered to the control mice.
2.3 25(OH)D3 treatment From 7 weeks of age, mice in the DPV group received an intraperitoneal injection of 25(OH)D3† (5 μg/kg) within 5 min every other day, which prior was dissolved in refined peanut oil vehicle. Control mice (N, D and DP groups) received the vehicle alone similar to that of the DPV group by intraperitoneal injection. 25(OH)D3 supplementation was sufficient with the vitamin D requirements for mice (mean analyzed concentration: 14.6 ± 1.4 ug/kg) [21]. The administration was continued to 1 d before sacrifice.
2.4 FBG and body weight measurements
* †
ATCC®33277™, Chengdu, Sichuan, China CAS:63283-36-3, Sigma-Aldrich Co., Santa Clara, CA, USA
Mouse tail vein blood was collected following a 10-h fast. A glucometer* was subsequently used to determine the FBG level. The body weight was monitored by a scale with 0.01 g accuracy, and the measurements were implemented every other week from 4 weeks old.
2.5 Biochemical analysis The tail vein blood was collected in units of each mouse by the capillary pipette when determining FBG at 4 and 18 weeks old, then serum was directly isolated (centrifuging at 3000 g for 12 min; High speed centrifuge†) and stored at –80°C for subsequent determination. The levels of serum SASP factors, including IL-1β, IL-4, IL-6, IL-10, TNF-α, intercellular adhesion molecule (ICAM)-1, macrophage colony stimulating factor (M-CSF), matrix metalloproteinases (MMP)-2, MMP-8, osteoprotegerin (OPG), adiponectin (Adi), glucagon, pancreatic polypeptide (PP), leptin and receptor for advanced glycation end product (RAGE), were determined using Luminex multiplefactor assay kits‡ according to the manufacturer’s specifications. The concentration was expressed in pg/mL. Serum 25(OH)D3 and 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3)§ were quantified by ELISA according to manufacturer’s instructions. Blood levels of serum calcium was detected with an Enzyme Marker Detection kit** and results are presented as mmol/L.
2.6 MicroCT analysis After disassociating the attached soft tissue, the mandibular jaws of all groups were scanned by a
OneTouch Glucometer; LifeScan, Milpitas, CA HC-3012, USTC Ltd., CA ‡ Luminex 200, R&D Systems, USA § 25-OH-Vitamin-D ELISA, REA300/96; 1,25(OH) Vitamin D Total ELISA, RIS021R; BioVendor Co., CZE 2 ** Serum Calcium Assay Kit, ZC-S0445, ZCI BIO, shanghai, China; THERMO VARIOSKAN FLASH, ThermoFisher, USA * †
µCT50* at 10 μm intervals. The resorption of the alveolar bone was quantified by morphometric analysis of micro-CT images of the mandibles. Alveolar bone loss level was defined by measuring the area bordered by the cemento-enamel junction, the alveolar bone crest, and the mesial and distal line angles on the lingual side of the mandibular first molars. Attachment loss was measured by calculating the vertical distance of the alveolar bone crest and the cemento-enamel junction of the second molar. For the analysis of bone mineral density (BMD), the volume of interest was defined as a rectangular area covering the alveolar bone loss. The blinded measurements were randomly repeated 3 times.
2.7 Histological and immunohistochemical analysis Mandibular jaw samples were collected and stained for hematoxylin-eosin (HE) and immunohistochemical (IHC) staining (anti-p16, anti-p21, diluted 1:100; secondary antibody, diluted 1:1000). Images were obtained using Aperio ImageScope†. All of the antibodies were acquired from Santa Cruz Biotechnology‡. HE staining was performed for periodontal tissue and alveolar bone loss between the cement-enamel junction and the bone crest. The periodontal ligament (PDL) width was measured as the apical height segments from the PDL coronal section. The PDL attachment height was measured between the base of the sulcus and the alveolar bone crest. These two indicators represent the degree of PDL attachment loss and alveolar bone absorption.
2.8 Western blotting
SCANCO Medical, Bruettisellen, Switzerland ScanScope; Aperio; Kenilworth, NJ, USA ‡ Santa Cruz, CA, USA * †
After separated by dispase I, the proteins extracted from mouse mandible gingival tissues were subjected to 5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, and transferred to nitrocellulose membranes by electro-blotting. The membranes were incubated overnight at 4°C with primary antibodies against β-actin, Vitamin D Receptor (VDR)*, SOCS3† , STAT3‡ , p-STAT3§ , STAT5**, p-STAT5†† , NF-κB‡‡ , IL-1⧧ , p16*** or p21††† , and then with secondary antibodies for 1 hour at room temperature. The signals were detected by electrochemiluminescence using BioRad system‡‡‡.
2.9 Statistical analysis Error bars on all graphs showed the standard error of multiple independent measurements. Statistical analyses were done using Graphpad Prism software Version 8.0.0. In each group of statistical data, largest or smallest deviations were removed during analysis. Significance was determined using Student’s t-test or ANOVA, as appropriate. A level of P < 0.05 was considered significant.
3. Results 3.1 The inflammaging status occurred in db/db mice The SASP (associated with chronic inflammation, proteinases, bone metabolism and glycolipid
1:500 mouse mAb, sc-13133, Santa Cruz Biotechnology, Inc, CA, USA 1:1000 rabbit pAb, ab16030, Abcam, Abcam Trading Co. Ltd., Shanghai, China ‡ 1:1000 mouse mAb, #9139, Cell Signaling Technology, Massachusetts, USA § 1:2000 rabbit mAb, #9145, Cell Signaling Technology, Massachusetts, USA ** 1:1000 rabbit pAb, ab209544, Abcam, Abcam Trading Co. Ltd., Shanghai, China †† 1:1000 rabbit mAb, ab32364, Abcam, Abcam Trading Co. Ltd., Shanghai, China ‡‡ 1:1000 mouse mAb, sc-514451, Santa Cruz Biotechnology, Inc, CA, USA §§ 1:1000 rabbit mAb, #31202, Cell Signaling Technology, Massachusetts, USA *** 1:1000 mouse mAb, sc-166760, Santa Cruz Biotechnology, Inc, CA, USA ††† 1:1000 mouse mAb, sc-166630, Santa Cruz Biotechnology, Inc, CA, USA ‡‡‡ Bio-Rad Laboratories, Shanghai, China * †
metabolism) were examined to investigate the inflammaging condition in diabetes (Tab. 1). The positive cells of p16 and p21 increased obviously in the periodontal tissue of db/db mice compared with the normal mice (Fig. 1A). As the results shown (Fig. 1B), the pro-inflammatory factors and the matrix metalloproteinases were significantly higher in the D group (P < 0.05), comprising IL1β, IL-6, TNF-α, ICAM-1, M-CSF, MMP-2 and MMP-8. The levels of IL-4 and IL-10 were lower statistically in the D group compared with the N group (P < 0.05). Likewise, the secretion of OPG in serum remarkably decreased (P < 0.05). We also measured the level of glycolipid metabolism related to diabetes, another common feature of metabolic disorder disease. Glycogen, PP and RAGE increased, leptin level accumulated, but Adi decreased in the db/db mice (P < 0.05 for all). In addition, the FBG level was obviously higher than that of the normal mice (Fig. 1C).
3.2 A senescent-like state with an increased expression of the SASP factors was performed in db/db mice with periodontitis To determine if the inflammaging was associated with periodontal injury of diabetic periodontitis, periodontal tissues were examined by immunohistochemistry for p16/p21 staining for senescence detection and HE staining to quantify the extent of periodontal destruction, and Luminex analysis for serum SASP as an inflammaging status marker. Results showed that the percentage of p16positive cells was increased dramatically, meanwhile the percentage of p21-positive cells was also increased significantly in the DP group compared with the D group (Fig. 2A). The expression of IL1β, IL-6, TNF-α, MMP-2, MMP-8 and ICAM-1 at protein levels increased significantly from baseline to 18 weeks after infection (P < 0.05), also were significantly higher than those of the D group (P < 0.05) (Fig. 2B). In contrast, IL-4, IL-10, M-CSF and OPG behaved a decrease obviously
in the DP group. IL-10, not IL-4, was significantly lower than that in the D group at the time of killing (P < 0.05). As observed in the D group, glycogen, PP, leptin and RAGE remained increase within the DP group, but Adi was reduced (Fig. 2B). Importantly, the differences of them were markedly between the two groups (DP vs. D P < 0.05). Besides, the differences in the PDL attachment width and height was statistically significant between the DP and the D groups (P < 0.01 for both) (Fig. 2C, D, E), suggesting that oral inoculation could lead to severe alveolar bone loss and inflammaging status in diabetes.
3.3 25(OH)D3 improved the impaired periodontal condition in db/db mice with periodontitis To investigate whether 25(OH)D3 could relieve the outcomes of diabetic periodontitis, weight and FBG were measured every 2 weeks in all groups after the baseline record. Notably, the uncontrollable hyperglycemia occurred in the DP group and significantly improved after 7-week 25(OH)D3 treatment (Fig. 3A, B). The FBG levels of both the DP and DPV groups were significantly higher than that of the D group from the 10th week to the 14th week, suggesting that periodontitis could aggravate diabetes and rise blood glucose. The weight graph (Fig. 3C) showed that the DPV and DP mice were close after oral inoculation, and eventually the DPV group exhibited a lower weight. Meanwhile, The DPV group shared a slight alveolar bone loss, whereas the vehicle groups had a much more severe attachment loss and bone loss (P < 0.05 for both) (Fig. 3D-F). In addition, we established measurements of the BMD (Fig. 3D, G), showing the difference between the DPV and DP group was not significant, although the DP group was remarkably higher than the D group (P < 0.05).
3.4 25(OH)D3 suppressed gingival tissue senescence and the serum SASP factors in db/db mice with periodontitis To elucidate if 25(OH)D3 improved the inflammaging in diabetic periodontitis, we measured the levels of the SASP and the expression of p16 and p21. In the DPV group, significant decreases were observed in the percentages of positive areas for activated p16 and p21 compared with the DP group (Fig. 4A). Continuous treatment with 25(OH)D3 significantly lowered IL-1β, IL-6, TNF-α, ICAM-1, M-CSF, MMP-2 and MMP-8 secretions in serum (P < 0.05) (Fig. 4B). IL-4, IL-10 and OPG had an obvious increase after treatment (P < 0.05), as expected. Glycogen, leptin and RAGE downregulated, suggesting a decrease of some pathways activation in diabetic periodontitis mediates the reduced the secretion level of SASP factors. Instead, Adi increased significantly after treatment. The level of PP was the highest in all groups (P < 0.05). These results demonstrated that 25(OH)D3 ameliorated the SASP caused by periodontal inflammaging.
3.5 25(OH)D3 downregulated SOCS3/STAT signaling in gingival tissue of db/db mice with periodontitis To further determine the potential molecular mechanism that 25(OH)D3 ameliorates periodontal inflammaging, the proteins expression of SOCS3 and VDR were detected using western blotting. The results showed that although they were expressed in the periodontal tissue from all groups, the expression level of SOCS3 was up-regulated significantly in db/db mice with periodontitis and returned to the normal levels in the DPV group (P < 0.05) (Fig. 5A, B). The level of VDR in the DP group was significantly lower than those of the N and the DPV groups (P < 0.05) (Fig. 5A, C). The expression of p16 and p21 increased consistently in the DP group and decreased after 25(OH)D3
treatment (Fig. 5A, D, E). In addition, when the phosphorylation levels of STAT3 and STAT5 were reduced significantly upon 25(OH)D3 treatment, the expression of NF-κB and IL-1β in protein levels was down-regulated subsequently compared with the DP group (P < 0.05 for all) (Fig. 5F-J). These results suggest that 25(OH)D3 delayed periodontal inflammaging might be related to regulate the SOCS3/STAT signaling.
Consistent with the western blot results of VDR, serum concentrations of VD3 metabolites decreased in diabetic and periodontitis-diabetic mice at the end of study (P < 0.01) (Fig. 5K, L). At baseline, all db/db mice had lower levels of serum 25(OH)D3, 1,25(OH)2D3 and calcium compared with the N group (P < 0.05) (Fig. 5K-M). Within the observation period, the non-treatment groups as well as the N group presented a gradual and accordant decline in 25(OH)D3 and 1,25(OH)2D3 levels (Fig. 5K, L). Serum calcium level manifested irregular fluctuation, but a notable decline appeared in the D and DP group over time (Fig. 5M). These results indicated that VD3 deficiency correlated not only with aging, but also with the progress of disease. After 10-week treatment, the level of 25(OH)D3 was apparently higher in the DPV group than that of the DP group (P < 0.05). Meanwhile, increased 1,25(OH)2D3 and calcium levels in the DPV group was found and there was no significant difference relative to the N group (Fig. 5K-M).
4. Discussion This study confirmed the inflammaging status occurred in diabetes using db/db mouse as a type 2 diabetic model. The experimental diabetic periodontitis mice demonstrated impaired periodontal structure, enhancement of senescence burden and development of SASP levels. 25(OH)D3
ameliorated senescence and the SASP in mice with diabetic periodontitis by downregulating SOCS3/STAT signaling. These findings indicated that 25(OH)D3 supplement-treatment could be a critical event in preventing periodontal injury and the excessive inflammaging, a likely intervention target in diabetic periodontitis.
Previous evidence suggested that db/db mice are typical T2DM model [19]. Due to hyperglycemic impair and imbalanced metabolism, the persistent accumulation of pro-inflammatory SASP in serum are sufficient to induce senescence [8, 11]. The cell cycle regulator p16 is transcriptionally activated in cells undergoing irreversible senescence, which leads to aging-associated impaired function and regenerative capacity [22, 23]. The activation of p21 is important in the initiation of senescence [24]. In this study, the percentage of p16 and p21-positive cells were greatly increased in gingival tissues from the D group to DP group, suggesting that the chronic inflammation could give impetus to tissue senescence. Moreover, inflammatory cell infiltration in gingival tissue, characterized by inflammatory SASP secretion, maybe a major pathological change of periodontal senescence.
The role of nutrition in inflammaging process is of great importance. The low-grade nutrition metabolic state present inside the chronic phase is strongly related to inflammaging [8, 11, 13]. Most of the existing evidence claims that increasing the intake of 25(OH)D3, having anti-inflammatory properties, contributes to restore a normal nutrition synthesis [25]. VD3 deficiency increases the risk of developing chronic diseases and the rate of aging [14, 26]. In contrast, chronic diseases, highly common in aging, are thought to increase the risk for VD3 deficiency, as these would reduce its
synthesis and enhance its catabolism [12, 13, 26]. One of the important actions of VD3 is to reduce the expression of inflammatory cytokines, which are such a prominent feature of how inflammatory responses alter cellular activity accelerating aging [27]. The fact that the ability of the skin to make VD3 declines as aging progresses is one reason why cognition tends to decline during aging [28]. After three-month observation, VD3 metabolites accordantly decreased in the N group, which proved the presence of a close relationship between age and VD3 levels [14]. Thus, it has been suggested that the rate of aging is regulated by vitamin D. This study showed that the number of senescent cells declined and SASP was ameliorated upon 25(OH)D3 treatment, indicating that 25(OH)D3 supplementation is beneficial to mitigation of inflammaging in diabetic periodontitis.
Lower levels of VD3 are associated with diabetes and periodontitis [29, 30]. In this study, genetically diabetic mice suffered VD3 deficiency and hypocalcemia, namely negative calcium homeostasis [31], throughout the observation period. As the disease progresses, the decrease of 25(OH)D3 is more obvious in the DP group compared with the D group. Changes of serum calcium and VD3 metabolites at least partly due to the decrease of 1,25(OH)2D3 receptor in intestine and kidney of diabetic mice [32], similar to the manifestation of gingival tissue that VDR downregulated in the DP group. Studies have found that when serum 25(OH)D3 levels are lower than 20 ng/mL, most of vitamin D uptake or skin production immediately turns to bone formation, which is insufficient to provide advanced functions comprising immune system, brain or gene regulation [33]. It is predicted that the significant decrease of 25(OH)D3 may be related to the severity of tissue injury with aging. Upon 25(OH)D3 treatment, serum 25(OH)D3, 1,25(OH)2D3 and calcium levels presented raising trend and were equivalent to that of the N group terminally. The inflammaging
process can be delayed by adjusting the concentration of 25(OH)D3 or 1,25(OH)2D3 in the body. Furthermore, we inferred that the VD3 metabolites can be used as biomarkers of inflammaginginduced diseases.
Like many age-related diseases, diabetes is mainly fostered by nutrient and metabolic surplus to trigger a higher level of “basal” inflammation [3, 11]. When subjects sustained a complicated chronic periodontitis, even higher levels of SASP were observed compared to those without periodontitis [34, 35]. It is mostly because the overall inflammatory state was aggravated by inoculation with p.g. in T2DM [34]. As reported previously, the elevated IL-6 and TNF-α in aging tissues is one of the reasons for senescence-driven tissue dysfunction [36, 37], explaining that the uncontrollable hyperglycemia and severe alveolar bone loss were observed in the DP group. In addition, our results showed BMD of the DP group was higher than that of the D group, further supporting that OPG is reduced with inflammatory status. Therefore, this study demonstrated that the inflammaging was excessive induced by these several senescent-like phenotypes in diabetic periodontitis.
Our results indicate that significantly reduced IL-1β, IL-6, TNF-α, MMP-2, MMP-8, ICAM-1 and M-CSF secretion in serum of the DPV mice, suggesting 25(OH)D3 may slow down inflammaging by attenuating the pro-inflammatory SASP [38]. In addition, 25(OH)D3 suppressed leptin secretion from fat tissue, which has been reported previously [39]. Adi has an anti-inflammatory effect and is attenuated leading to the other SASP up-regulation by suppressing NF-κB signaling and TNF-α release [40, 41], suggesting that 25(OH)D3 could up-regulate Adi by inflammatory pathway. There
is no direct evidence explaining the regulatory effect between 25(OH)D3 and glycolipid metabolism. VD3 stimulates the production of insulin growth factor (IGF)-1, the key player in the crossroad linking nutrition with inflammatory signaling, whose levels are positively regulated by diet and anabolic hormonal systems and negatively affected by inflammation and oxidative stress [42]. IGF1 levels tend to decline with aging, in conjunction with the increase in subclinical inflammatory status [43]. Thus, 25(OH)D3 improves the metabolic SASP factors, possibly involving IGF-1.
In this study, we found significantly decreased protein expression of VDR and increased SOCS3 in the gingival tissues of the DP mice, whereas an opposite change in the 25(OH)D3-supplement mice. A study has been reported that the aging process may influence the expression of VDR genes, wherein circulating 25(OH)D3 levels did not appear to affect [44]. In addition to regulating the expression of inflammatory genes, genetic variance in VDR gene influences the susceptibility to age-related changes [45]. These suggested that VDR might play a key role in inflammaging. Previous studies about the linking between VD3 and SOCS3 mainly focused on inflammatory signaling [16, 46], but little on aging. Our study indicated that the periodontal inflammaging might be associated with increased SOCS3 and decreased VDR. It has been found that aging increases SOCS3 expression in hypothalamus [18]. Previous studies also demonstrated that decreased expression of SOCS3 involved STAT3 and NF-κB interaction [47]. The activation of VDR inhibits NF-κB resulting in decreased pro-inflammatory signaling and induced hypo-responsiveness to antigenic stimulation [48, 49]. NF-κB is the pivot regulating the transcription of inflammatory factors [50]. Its knockdown significantly decreases the levels of 75% of SASP factors [51]. We found that the expression level of NF-κB was lower in diabetic periodontitis compared with the D
group and increased distinctly after 25(OH)D3 treatment, which is consistent with that VD3 downregulates the expression and production of pro-inflammatory cytokines by suppressing NF-κB activation [52]. In this study, 25(OH)D3 inhibited SASP through down-regulating NF-κB proinflammatory signaling to improve periodontal inflammaging, might be dependent on the VDR/SOCS3/STAT signaling. Further experiments in vitro are needed to reveal the precise mechanism.
Connectively, inflammaging may be the particular reason to increase the adverse outcomes in diabetic periodontitis, leading to considerable difficulties in curing its effect. Notably, diabetic periodontitis, as a verified chronic inflammatory disease [4], could be ameliorated by 25(OH)D3 altering the inflammaging through SOCS3/STAT signaling. VD3 also inhibits IL-6 synthesis at both the protein kinase C pathway and the protein kinase A pathway. According to a number of reports, SASP is also initiated by p38 MAPK signaling, except for NF-κB [36, 53]. Future research should better address all these issues, clarifying the molecular and clinical rationale of combined nutritional interventions especially with VD3.
5. Conclusion Our study showed that diabetic periodontitis performed a senescent-like state with a secretory of the SASP (inflammaging). The inflammaging might play a crucial role in diabetic periodontitis. 25(OH)D3 treatment performed a delay in senescent cell enrichment and alleviated the elevated serum SASP. Furthermore, it reveals the potential utility of VD3 as therapeutic agents, and its antiinflammaging therapies makes it a promising candidate in preventing age-related diseases. The
clinicians should also pay attention to the levels of 25(OH)D3 and calcium in patients with agerelated diseases, and routinely detect and give appropriate treatment with nutritional intervention.
6. Data availability All data generated or analyzed during this study are included in the published article and supplementary information files.
7. Conflicts of interest The authors declare that they have no potential conflicts of interest relevant to this study.
8. Author contributions Q.W. performed the experiments, analyzed the data, and wrote the manuscript; X.Z. contributed to the experimental work; P.Z. and P.F Z. provided intellectual contribution on the research design; L.N. helped prepare the manuscript and optimize image analysis; N.J. and Y.D. provided intellectual contribution on experimental techniques and related knowledge; Q.W.* contributed to the research design and takes primary responsibility for the final content; and all authors discussed the results and commented on the manuscript.
9. Acknowledgements We thank Qiang Guo (State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University) for assisting with
microCT analysis strategies.
10. Funding statement This work was supported by the National Natural Science Foundation of China [grant numbers 81870779], the International Cooperation Project of Chengdu Municipal Science and Technology Bureau [grant number 2015-GH02-00035-HZ] and the International Scientific Cooperation and Exchanges Project of Sichuan Province [grant number 2017HH0078].
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Figure legends Fig. 1 (A) Immunohistochemical staining for p16 and p21 in mandibular gingival tissue. Black arrows were drawn to point out positive cells, and the upper right corner box is the enlarged area indicated by the arrow. (B) The serum levels of SASP (about inflammatory cytokines, metalloproteases, chemokines, bone metabolism and glycolipid metabolism). (C) Fasting blood glucose. Data were obtained at the age of 18 weeks. Results are shown as mean ± SEM; n = 10; the value about D vs. N by analysis of variance (ANOVA) or Student’s t-test; * P < 0.05, ** P < 0.01 and *** P < 0.001. N= normal control mice, D= diabetic mice.
Fig. 2 (A) Immunohistochemical staining for p16 and p21 in mandibular gingival tissue. Black arrows were drawn to point out positive cells, and the upper right corner box is the enlarged area indicated by the arrow. (B) The secretory levels of SASP factors in circulatory serum at 4 and 18 weeks old of mice. (C) Hematoxylin and eosin staining of the periodontal tissue. Black arrows indicated the epithelial spikes in the D and DP groups. Periodontal ligament (PDL) width was measured by the apical height segments from the PDL coronal section, repeated 3 times; PDL attachment height was measured between the base of the sulcus and the alveolar bone crest, repeated 3 times. (D)&(E) Quantification of PDL width and attachment height in the D and DP groups. Results are shown as mean ± SEM; n = 10; * P < 0.05 and ** P < 0.01. D= diabetic mice, DP= diabetic periodontitis mice.
Fig. 3 (A) Experimental diabetic periodontitis mouse model and all protocols were performed strictly according to the procedure. (B) Fasting blood glucose levels. (C) Weight levels. (D) Three-
dimensional reconstructions of the mandibular alveolar bones. (E) The attachment loss among groups. (F) The alveolar bone loss area among groups. (G) The bone mineral density (BMD) of the volume of interest (alveolar bone of the mandibular first molars) in the mandibular jaws of mice. D= diabetic mice, DP= diabetic periodontitis mice, DPV= diabetic periodontitis mice treated with 25(OH)D3. Results are shown as mean ± SEM; n = 10; the value about DP vs. D and DPV vs. DP by ANOVA; * P < 0.05 and ** P < 0.01; BMD, DPV vs. DP P > 0.05.
Fig. 4 (A) Immunohistochemical staining for p16 and p21 in mandibular gingival tissue. Black arrows were drawn to point out positive cells, and the upper right corner box is the enlarged area indicated by the arrow. (B) The serum levels of SASP after receiving 25(OH)D3 treatment at 18 weeks old of mice. Results are shown as mean ± SEM; n = 10; * P < 0.05 and ** P < 0.01.
Fig. 5 Protein expression in the mouse gingival tissue of N, D, DP and DPV mice and serum concentrations of VD3 metabolites were detected. (A-E) Protein expression of SOCS3, VDR, p16 and p21. (F-J) Protein expression of STAT3, p-STAT3, STAT5, p-STAT5, NF-κB and IL-1β. 25(OH)D3-treated mice with diabetic periodontitis inhibited SOCS3, p16, p21, p-STAT3/STAT3, p-STAT5/STAT5, NF-κB and IL-1β expression, and increased the expression level of VDR. (K) Serum 25(OH)D3 levels form the 4th to the 18th week. (L) Serum 1,25(OH)2D3 levels. (M) Blood levels of serum calcium. N= normal control mice, D= diabetic mice, DP= diabetic periodontitis mice, DPV= diabetic periodontitis mice treated with 25(OH)D3. Results are shown as mean ± SEM; n = 10; * P < 0.05, ** P < 0.01 and *** P < 0.001.
Author contributions
Qian Wang1, Xinyi Zhou, Peng Zhang, Pengfei Zhao, Lulingxiao Nie, Ning Ji, Yi Ding and Qi Wang*.
Q.W. performed the experiments, analyzed the data, and wrote the manuscript; X.Z. contributed to the experimental work; P.Z. and P.F Z. provided intellectual contribution on the research design; L.N. helped prepare the manuscript and optimize image analysis; N.J. and Y.D. provided intellectual contribution on experimental techniques and related knowledge; Q.W.* contributed to the research design and takes primary responsibility for the final content; and all authors discussed the results and commented on the manuscript.
Highlights
25-Hydroxyvitamin D3 (25(OH)D3) inhibited serum senescence-associated secretory phenotype (SASP) in diabetic periodontitis. 25(OH)D3 decreased the expression of p16/p21 in the gingival tissue of the diabetic periodontitis mice. 25(OH)D3 suppressed the gingival inflammaging via SOCS3/STAT signaling in diabetes. 25(OH)D3 supplement could be a potential intervention for diabetic periodontitis through relieving the excessive inflammaging.
The List of Chemical compounds
Chemical compounds
Roles
1
Vitamin D3
a steroid hormone with immunoregulatory effects
2
25-Hydroxyvitamin D3
the storage form of VD3; injected to the db/db mice in this study
3
1α,25-Dihydroxyvitamin D3
4
Hemin
5
Vitamin K1
the bioactive form of VD3; promoting calcium absorption; the blood level was monitored in this study added to agar medium for the culture of porphyromonas gingivalis added to agar medium for the culture of p.g.
6
Carboxymethylcellulose
7
Hematoxylin
8
Eosin
9
sodium dodecyl sulfate
10
polyacrylamide
11
nitrocellulose
dissolved with live bacteria for oral inoculation histological staining reagent histological staining reagent preparation of electrophoretic gels preparation of electrophoretic gels raw material membranes
for
immunoblotting
Table 1 The typical SASP response in diabetes related to chronic periodontitis Factors*
Inflammatory cytokines
Metalloproteases
Chemokines
Bone metabolism
Roles
IL-1β
an early component and an upstream regulator of the SASP
IL-4
an anti-inflammatory SASP in early-onset inflammation
IL-6
promoting malignant and senescent phenotypes
IL-10
a signature SASP in pathological senescence
TNF-α
mediating senescence-driven periodontal tissue dysfunction
MMP-2
contributing to periodontal tissue destruction
MMP-8
participating in tissue repair
ICAM-1
recruiting macrophages to promote inflammation
M-CSF
inducing osteoclast differentiation
OPG
a SASP inhibiting osteoclast formation in alveolar bone absorption
Glycolipid metabolism
Glucagon PP Leptin Adi RAGE
exacerbating glucose toxicity aging-related metabolic hormone controlling food intake and increasing energy metabolism an anti-inflammatory adipokine accelerating senescence
* interleukin (IL)-1β; tumor necrosis factor (TNF)-α; matrix metalloproteinases (MMP)-2; intercellular adhesion molecule (ICAM)-1; macrophage colony stimulating factor (M-CSF); OPG, osteoprotegerin; PP, pancreatic polypeptide; Adi, adiponectin; RAGE, receptor for advanced glycation end product.