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Interleukin-17 plays a role in dental pulp inflammation mediated by zoledronic acid: a mechanism unrelated to the Th17 immune response?

Abstract

Objective

To evaluate the influence of RORγT inhibition by digoxin on inflammatory changes related to interleukin-17 (IL-17) in the pulp of rats treated with zoledronate (ZOL).

Methodology

Forty male Wistar rats were divided into a negative control group (NCG) treated with saline solution, a positive control group (PCG) treated with ZOL (0.20 mg/kg), and three groups treated with ZOL and co-treated with digoxin 1, 2, or 4 mg/kg (DG1, 2, and 4). After four intravenous administrations of ZOL or saline solution in a 70-day protocol, the right molars were evaluated by histomorphometry (number of blood vessels, blood vessels/µm2, cells/µm2, total blood vessel area, and average blood vessel area) and immunohistochemistry (IL-17, TNF-α, IL-6, and TGF-β). The Kruskal-Wallis/Dunn test was used for statistical analysis.

Results

PCG showed an increase in total blood vessel area (p=0.008) and average blood vessel area (p=0.014), and digoxin treatment reversed these changes. DG4 showed a reduction in blood vessels/µm2 (p<0.001). In PCG odontoblasts, there was an increase in IL-17 (p=0.002) and TNF-α (p=0.002) immunostaining, and in DG4, these changes were reversed. Odontoblasts in the digoxin-treated groups also showed an increase in IL-6 immunostaining (p<0.001) and a reduction in TGF-β immunostaining (p=0.002), and all ZOL-treated groups showed an increase in IL-17 (p=0.011) and TNF-α (p=0.017) in non-odontoblasts cells.

Conclusion

ZOL induces TNF-α- and IL-17-dependent vasodilation and ectasia, and the classical Th17 response activation pathway does not seem to participate in this process.

Zoledronic acid; Dental pulp; Acute-phase reaction; Inflammation

Introduction

Bisphosphonates (BPs) are osteolysis inhibitors commonly used in the treatment of degenerative bone diseases such as osteoporosis and Paget’s disease. Recently, the use of this group of drugs in the treatment of bone metastases has gained momentum due to their relatively low cost and their ability to increase the life expectancy and quality of life of patients with malignancies such as multiple myeloma and breast, prostate, kidney, and lung cancers.11 - Bowden SA, Mahan JD. Zoledronic in pediatric metabolic bone disorders. Transl Pediatr. 2017;6(4):256-68. doi: 10.21037/tp.2017.09.10
https://doi.org/10.21037/tp.2017.09.10...

2 - Goldvaser H, Amir E. Role of bisphosphonates in breast cancer therapy. Curr Treat Options Oncol. 2019;20(4):26. doi: 10.1007/s11864-019-0623-8
-33 - Reyes C, Hitz M, Prieto-Alhambra D, Abrahamsen B. Risks and benefits of bisphosphonate therapies. J Cell Biochem. 2016;117(1):20-8. doi: 10.1002/jcb.25266
https://doi.org/10.1002/jcb.25266...
However, the use of BPs is strongly associated with the development of several immune-inflammatory changes affecting the maxillofacial region.44 - Kalyan S. It may seem inflammatory, but some T cells are innately healing to the bone. J Bone Miner Res. 2016;31(11):1997-2000. doi: 10.1002/jbmr.2875
https://doi.org/10.1002/jbmr.2875...

Zoledronate (ZOL) has a direct toxic effect on many cell groups, including epithelial cells, fibroblasts,55 - Basso FG, Pansani TN, Soares DG, Cardoso LM, Hebling J, Costa CA. Influence of bisphosphonates on the adherence and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces. Clin Oral Investig. 2018;22(2):893-900. doi: 10.1007/s00784-017-2167-2,66 - Basso FG, Soares DG, Pansani TN, Turrioni AP, Scheffel DL, Hebling J, et al. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid. Braz Oral Res. 2016;30(1):e122. doi: 10.1590/1807-3107BOR-2016.vol30.0122 osteoblasts,77 - Zara S, De Colli M, di Giacomo V, Zizzari VL, Di Nisio C, Di Tore U, et al. Zoledronic acid at subtoxic dose extends osteoblastic stage span of primary human osteoblasts. Clin Oral Investig. 2015;19(3):601-11. doi: 10.1007/s00784-014-1280-8 macrophages,88 - Scheller EL, Hankenson KD, Reuben JS, Krebsbach PH. Zoledronic acid inhibits macrophage SOCS3 expression and enhances cytokine production. J Cell Biochem. 2011;112(11):3364-72. doi: 10.1002/jcb.23267
https://doi.org/10.1002/jcb.23267...
,99 - Zhu W, Xu R, Du J, Fu Y, Li S, Zhang P, et al. Zoledronic acid promotes TLR-4-mediated M1 macrophage polarization in bisphosphonate-related osteonecrosis of the jaw. FASEB J. 2019;33(4):5208-19. doi: 10.1096/fj.201801791RR
https://doi.org/10.1096/fj.201801791RR...
neutrophils,1010 - Kuiper JW, Forster C, Sun C, Peel S, Glogauer M. Zoledronate and pamidronate depress neutrophil functions and survival in mice. Br J Pharmacol. 2012;165(2):532-9. doi: 10.1111/j.1476-5381.2011.01592.x
https://doi.org/10.1111/j.1476-5381.2011...
and endothelial cells.1111 - Wang Q, Liu J, Guo T, Liu D, Pan J. Epidermal growth factor reverses the inhibitory effects of the bisphosphonate, zoledronic acid, on human oral keratinocytes and human vascular endothelial cells in vitro via the epidermal growth factor receptor (EGFR)/Akt/phosphoinositide 3-kinase (PI3K) signaling. Med Sci Monit. 2019;25:700-10. doi: 10.12659/MSM.911579
https://doi.org/10.12659/MSM.911579...
Cytotoxic changes in these cellular elements are known to activate the production of several pro-inflammatory cytokines. This leaves the ZOL contact site, which is usually close to bone tissue, with a higher inflammatory potential compared with untreated tissues.

Some evidence of ZOL toxicity in the dental pulp is beginning to accumulate. Alendronate can lead to dental teratogenicity1212 - Hiraga T, Ninomiya T, Hosoya A, Nakamura H. Administration of the bisphosphonate zoledronic acid during tooth development inhibits tooth eruption and formation and induces dental abnormalities in rats. Calcif Tissue Int. 2010;86(6):502-10. doi: 10.1007/s00223-010-9366-z and induces denticle and odontoma formation.1313 - Massa LF, Bradaschia-Correa V, Arana-Chavez VE. Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats. J Histochem Cytochem. 2006;54(6):713-25. doi: 10.1369/jhc.5A6853.2006
https://doi.org/10.1369/jhc.5A6853.2006...
ZOL affects alkaline phosphatase synthesis, changes the cell morphology of odontoblasts,1414 - Basso FG, Turrioni PS, Hebling J, Costa CA. Effects of zoledronic acid on odontoblast-like cells. Arch Oral Biol. 2013;58(5):467-73. doi: 10.1016/j.archoralbio.2012.09.016 and also reduces cell viability, proliferation, and protein synthesis in pulp cells in a time-dependent manner.1515 - Cvikl B, Agis H, Stogerer K, Moritz A, Watzek G, Gruber R. The response of dental pulp-derived cells to zoledronate depends on the experimental model. Int Endod J. 2011;44(1):33-40. doi: 10.1111/j.1365-2591.2010.01792.x In a study conducted by our group, it was observed for the first time that animals treated with ZOL showed a dose-dependent pro-inflammatory state in the dental pulp. In this study, even 70 d after the start of ZOL treatment, the dental pulp cells of rats in the experimental group had a high expression of TNF-α, IL-1β, and iNOS. This change was especially prominent in odontoblasts, which are cells in close contact with mineralized dentin material.1616 - Silva PG, Oliveira CC, Brizeno L, Wong D, Lima R Júnior, Gonçalves RP, et al. Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw. Oral Dis. 2016;22(7):649-57. doi: 10.1111/odi.12513 In a follow-up study, it was recently observed that other mediators are significantly increased in the dental pulp, such as COX-2, and even bacterial recognition molecules, such as toll-like receptor 4, are overexpressed after long-term treatment with ZOL.1717 - Silva PG, Ferreira AE Junior, Oliveira CC, Verde MEQL, Freitas MO, Sousa FB, et al. Chronic treatment with zoledronic acid alters the expression levels of inflammatory, bone, and apoptotic markers and toll-like receptors 2 and 4 in rat dental pulp. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019;128(2):139-45. doi: 10.1016/j.oooo.2019.01.069

Although these are tissues of different embryonic origin and with different molecular properties, if there is overexpression of IL-17 in BF-treated bone tissue, similar changes are likely to occur in the dental pulp, since IL-17 can sustain the transcription stability of some acute-phase cytokines, such as TNF-α,11 - Bowden SA, Mahan JD. Zoledronic in pediatric metabolic bone disorders. Transl Pediatr. 2017;6(4):256-68. doi: 10.21037/tp.2017.09.10
https://doi.org/10.21037/tp.2017.09.10...
,88 - Scheller EL, Hankenson KD, Reuben JS, Krebsbach PH. Zoledronic acid inhibits macrophage SOCS3 expression and enhances cytokine production. J Cell Biochem. 2011;112(11):3364-72. doi: 10.1002/jcb.23267
https://doi.org/10.1002/jcb.23267...
a cytokine overexpressed in the dental pulp after long periods of ZOL treatment.1616 - Silva PG, Oliveira CC, Brizeno L, Wong D, Lima R Júnior, Gonçalves RP, et al. Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw. Oral Dis. 2016;22(7):649-57. doi: 10.1111/odi.12513

Recently, drugs with indications unrelated to the control of inflammation have been described as capable of inhibiting RORγT, leading to a reduction in IL-17 synthesis. Digoxin is a drug that belongs to the glycoside class and is widely used to treat heart failure. Its mechanism of action involves inhibiting the calcium-dependent Na+K+ATPase pump in the cardiac sarcoplasmic reticulum, which raises the intracellular calcium concentration and increases its contraction force.1919 - Katzung BG, Masters SB, Trevor AJ. Farmacologia básica e clínica. 12. ed. Porto Alegre: AMGH; 2014.

Due to the ability of digoxin to block RORγT and IL-17 transcription, digoxin may attenuate the overexpression of cytokines in the dental pulp of rats exposed to ZOL. Therefore, this study aimed to evaluate the role of IL-17 inhibition by digoxin in inflammatory events in the dental pulp of rats chronically treated with ZOL.

Methodology

Animals, groups, and doses

The Ethics Committee on the Use of Animals (CEUA) of the Christus University Center (UNICHRISTUS) approved this project, registered under number 37/18, and 40 male Wistar rats (Rattus novergicus) were used. The rats (180–200 g) were kept with ad libitum access to water and food, on a 12-h light-dark cycle, at a temperature of 20–25°C. They were weighed weekly. The rats were randomly divided (“Random” function, Excel 2010, Microsoft Corporation®) into five groups: a negative control group (NCG) treated with sterile saline solution (0.1 mL/kg), a positive control group (PCG) treated with ZOL (0.20 mg/kg diluted in 0.1 mL/kg of sterile saline solution), and three test groups treated with ZOL (0.20 mg/kg diluted in 0.1 mL/kg of sterile saline solution) and digoxin 1 (DG1), 2 (DG2), or 4 (DG4) mg/kg diluted in 0.1 mL/kg of sterile saline solution.

Sample size calculation

A study on an aortic aneurysm model by Wei, et al.2020 - Wei Z, Wang Y, Zhang K, Liao Y, Ye P, Wu J, et al. Inhibiting the Th17/IL-17A-related inflammatory responses with digoxin confers protection against experimental abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 2014;34(11):2429-38. doi: 10.1161/ATVBAHA.114.304435
https://doi.org/10.1161/ATVBAHA.114.3044...
(2014) showed that animals treated with digoxin had reduced levels of IL-17 (0.70±0.07) compared with untreated animals (0.80±0.04). They estimated that it is necessary to have a sample of seven animals per study group to obtain a sample with 90% power and 95% confidence in rejecting the null hypothesis. Considering the possibility of sample loss during the protocol, 10% was added to the calculated sample, totaling eight animals per study group.

In vivo experimental protocol

We used a previously published experimental protocol in which doses infusion simulated administration in humans. Briefly, after using the software Dose Calculator, Conversion Chemotherapy of Humans to Animals, provided free by the Food and Drug Administration (http://www.accessdata.fda.gov), body weight and surface area were the parameters considered for pharmacological conversion of the human dose of ZA for the experimental rats. The monthly dose (4 mg) used to treat multiple myeloma was estimated at 0.60 mg/kg for the Wistar rats and divided into three weekly administrations of 0.20 mg/kg. This dose proved to be a potent inducer of BRONJ in rats. Then, we performed a dose-response curve with little or no toxicity.2121 - Silva PG, Ferreira AE Junior, Teófilo CR, Barbosa MC, Lima RC Júnior, Sousa FB, et al. Effect of different doses of zoledronic acid in establishing of bisphosphonate-related osteonecrosis. Arch Oral Biol. 2015;60(9):1237-45. doi: 10.1016/j.archoralbio.2015.05.015

The rats underwent three consecutive weekly administrations (days 0, 7, and 14) of ZOL or saline solution (control). On day 49, the fourth dose of the drug was administered and, on day 70, the rats were euthanized. They received sterile saline solution or digoxin at different doses by gavage three times a week from the beginning of the experimental protocol. The first infusion of ZOL was on a Monday (D0), marking the beginning of the experimental protocol. Digoxin administration began on the same Monday and was performed every Monday, Wednesday, and Friday until the end of the experimental protocol proposed by Lee, et al.2222 - Lee J, Baek S, Lee J, Lee J, Lee DG, Park MK, et al. Digoxin ameliorates autoimmune arthritis via suppression of Th17 differentiation. Int Immunopharmacol. 2015;26(1):103-11. doi: 10.1016/j.intimp.2015.03.017
https://doi.org/10.1016/j.intimp.2015.03...
(2015), totaling 30 administrations of digoxin.

After euthanasia, the right hemimandibles were surgically excised, stored in 10% neutral formalin for 24 h, then decalcified (suspension) in 10% EDTA solution (pH 7.3; NaOH, PA) for 30 d for histological slides (hematoxylin & eosin, 3 µm).

Histological analysis of dental pulp: cellularity and vascular events

Histological sections were made using a semi-automatic microtome (Leica®) until the pulp chamber was visible in both specimens. Photomicrographs (U-TV0.63XC, Olympus®) were taken at 400x magnification (BX43, Olympus® plus Olympus Soft Imagining LCMicro software) and mounted to access the entire dental pulp area of the right mandibular first molars. The images were then mounted and the total pulp area measured in µm2 using the Freehand Selection tool in ImageJ® software.

To count the number of cellular elements in the dental pulp, the command Color Deconvolution > Hematoxylin & Eosin was used to segregate the images into three color scales: hematoxylin (nuclei), eosin (cytoplasm and connective tissue), and a residual image. The hematoxylin images were binarized (Image > Binary > Make Binary) to count the number of nuclei (Analyze > Analyze Particles). Cellularity was defined as the number of nuclei/µm2.

Using the same Freehand Selection tool, the area of each blood vessel in the dental pulp of the right mandibular first molars of each animal was measured. The sum of these areas was divided by the total area of the dental pulp and expressed as the relative blood vessel area (%).23 The number of vessels in each tooth of each animal was divided by the total area of the dental pulp and expressed as the number of vessels/µm2. The average area of each blood vessel was estimated and divided by the total area of the dental pulp of each tooth in each animal, and was expressed as the average blood vessel area/µm2.

Tissue microarray technique (TMA) and immunohistochemistry

To study the immunoexpression of cytokines involved in the Th17 response (IL-17, IL-6, and TGF-β) and their influence on the expression of pro-apoptotic proteins (TNF-α) in the dental pulp of rats, we performed histological and histomorphometric analysis of the dental pulp. A 2 mm diameter microfield from each of these areas was selected and marked on the histological slides for subsequent block pairing and sample removal using a tissue microarrayer device. Then, a recipient block containing 70 wells of the same diameter and depth (2 mm) received paraffin-embedded tissue samples, which were organized and mapped.

After placing all the samples in the recipient block, it was incubated for 3 min on a glass slide in an oven at 65°C and removed with a rotational movement to attach the tissue samples to the recipient block. After cooling the recipient block to room temperature (20–25°C), 3 µm sections were prepared and placed on silanized slides for immunohistochemistry and apoptosis assays.

The silanized slides were deparaffinized in an incubator for 1 h at 65°C, immersed in xylene solution, rehydrated in decreasing alcohol solution, washed in tap water, and subjected to antigenic recovery with 0.1 M citrate solution at pH 6.0 in a water bath at 95°C for 40 min. After cooling to room temperature, the sections were washed in 0.1 M phosphate buffer solution at pH 7.3 (PBS) in two baths for 5 min each, and endogenous peroxidase was blocked with a 3% solution of H2O2 (hydrogen peroxide) diluted in PBS for 30 min.

After two 5 min PBS baths, the slides were incubated with 1% albumin solution for nonspecific protein blocking for 1 h. After two 5 min washes in PBS, the slides were incubated overnight with the following antibodies: IL-17 (1:100, ab79056; Abcam), TNF-α (1:100, ab6671; Abcam), IL-6 (1:300, ab19324; Abcam), and TGF-β (1:400, ab92486; Abcam).

The next day, after cooling to room temperature, the sections were washed with two 5 min PBS baths and incubated with anti-rabbit/anti-mouse polymer Dako Envision Dual Link System HRP (Dako®) for 1 h. After two more 5 min PBS baths, the slides were incubated with 3,3-diaminobenzidine (DAB) for 5 min. The reaction was stopped using distilled water.

The slides were counterstained with 7% Harris hematoxylin for 10 s, washed in running water, and dehydrated, diaphanized, and mounted with Enthellam®.

The same method was used to measure the percentage of immunostained cells. Histological sections were made using a semi-automatic microtome (Leica®) until the pulp chamber was visible in both specimens. Photomicrographs (U-TV0.63XC, Olympus®) were taken at 400x magnification (BX43, Olympus® plus Olympus Soft Imagining LCMicro software) and mounted to access the entire area of the dental pulp of the right mandibular first molars. The images were then mounted and, using the Count Cells function, the number of odontoblasts and non-odontoblasts dental pulp cells was counted to measure the percentage of immunostained cells. We consider immunostained cells to be those with brown cytoplasm.2323 - Moreira JE Sobrinho, Aguiar MT, Machado LC, Carlos AC, Alves AP, Mesquita KC, et al. Intense orthodontic force induces the three dental pulp nitric oxide synthase isoforms and leads to orofacial discomfort in rats. Orthod Craniofac Res. 2022;25(4):485-93. doi: 10.1111/ocr.12560
https://doi.org/10.1111/ocr.12560...

Statistical analysis

Data were presented as mean and standard error and analyzed using the Kruskal-Wallis test followed by Dunn’s post-hoc test. All analyses were performed using GraphPad Prism 5.0. statistical software, considering a 95% confidence level (p<0.05).

Results

Treatment with the Th17 response inhibitor digoxin reverses vascular changes in the dental pulp of rats treated with ZOL

There were no significant differences in the pulp area of the groups treated with saline solution (127.974±14.131 µm2), ZOL (145.551±16.016 µm2), or co-treated with digoxin (p=0.866) 1 mg/kg (125.899±17.414 µm2), 2 mg/kg (147.451±19.104 µm2), or 4 mg/kg (124.033±26.424 µm2). Similarly, the cellularity of the dental pulp did not change significantly in the groups treated with saline solution (60±3 nuclei/µm2), ZOL (60±5 nuclei/µm2), or co-treated with digoxin (p=0.617) 1 mg/kg (60±2 nuclei/µm2), 2 mg/kg (60±2 nuclei/µm2), or 4 mg/kg (53±6 nuclei/µm2) (Table 1, Figure 1).

Table 1
Immunostaining score for Th17 response cytokines and associated transcription factors in odontoblasts and non-odontoblasts dental pulp cells from rats chronically treated with ZOL

Figure 1
Cellular profile and vascular events in the dental pulp of rats chronically treated with ZOL and different doses of digoxin.Sal = saline solution

The percentage of the pulp area filled with blood vessels of the rats treated with ZOL (4.16±0.54%) was significantly higher than that of NCG (2.79±0.20%). Rats treated with digoxin 1 mg/kg (2.99±0.24%), 2 mg/kg (2.84±0.28%), or 4 mg/kg (2.44±0.31%) showed an equal percentage reduction in the pulp area filled with blood vessels compared with the group treated with ZOL alone (p=0.008) (Table 1, Figure 1).

There was no significant difference between the number of blood vessels/µm2 in the groups treated with saline solution (95±5) and ZOL (83±5) and co-treated with ZOL and digoxin 1 mg/kg (87±6) or 2 mg/kg (78±4). However, rats co-treated with ZOL and the highest dose of digoxin (40±5) showed a significant reduction in the number of vessels/µm2 compared with the other groups (p<0.001) (Table 1, Figure 1).

The mean blood vessel area showed a significant increase compared with NGC (28.72±1.87 µm2) in the ZOL-treated group (64.56±12.56 µm2), with a significant reduction in DG1 (41.09±6.35 µm2), DG2 (42.18±5.12 µm2), and DG4 (47.61±4.94 µm2) (p=0.014) (Table 1, Figure 1).

Digoxin treatment reduces IL-17-related TNF-α expression in the dental pulp of rats treated with ZOL

ZOL treatment significantly increased IL-17 expression in PCG compared with NCG. Rats co-treated with ZOL and digoxin 4 mg/kg showed significantly reduced immunoexpression scores for IL-17 compared with the ZOL-treated group (p=0.002) (Table 1, Figure 2).

Figure 2
Immunostaining profile for IL-17 and TNF-α in the dental pulp of rats subjected to chronic ZOL treatment.

Similarly, rats treated with ZOL showed higher immunoexpression scores for TNF-α than the control group treated with saline solution. The group treated with the highest dose of digoxin showed significantly reduced immunoexpression scores for TNF-α compared with rats treated with ZOL (p=0.002) (Table 1, Figure 2).

Regarding IL-6, ZOL treatment did not significantly increase immunoexpression of IL-6 compared with NCG, but the groups co-treated with digoxin, regardless of dose, showed increased immunoexpression of IL-6 compared with the group treated with ZOL alone (p<0.001) (Table 1, Figure 3).

Figure 3
Immunostaining profile for IL-6 and TGF-β in the dental pulp of rats subjected to chronic ZOL treatment

Immunoexpression of TGF-β was high in all groups. There was no significant difference between the immunoexpression of the saline-treated control group and the ZOL-treated group, but DG4 showed significantly reduced immunoexpression scores for TGF-β compared with the negative and positive control groups (p=0.002) (Table 1, Figure 3).

Digoxin treatment alters the immunoexpression of IL-6 and TGF-β in the dental pulp in a manner unrelated to the Th17 response

Immunoexpression of IL-17 (p=0.011) and TNF-α (p=0.017) in non-odontoblasts pulp cells was significantly higher in the ZOL-treated group than in the negative control group, with no significant reduction after digoxin treatment (Table 1, Figure 2).

There was no significant difference in the expression of IL-6 (p=0.140) or TGF-β (p=0.850) in non-odontoblasts pulp cells (Table 1, Figure 2 and 3).

Discussion

Inflammatory changes in the dental pulp of rats treated with ZOL have been previously described. Although not culminating in a state of inflammatory pulpitis, Silva, et al.1616 - Silva PG, Oliveira CC, Brizeno L, Wong D, Lima R Júnior, Gonçalves RP, et al. Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw. Oral Dis. 2016;22(7):649-57. doi: 10.1111/odi.12513 (2016) and Silva, et al.1717 - Silva PG, Ferreira AE Junior, Oliveira CC, Verde MEQL, Freitas MO, Sousa FB, et al. Chronic treatment with zoledronic acid alters the expression levels of inflammatory, bone, and apoptotic markers and toll-like receptors 2 and 4 in rat dental pulp. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019;128(2):139-45. doi: 10.1016/j.oooo.2019.01.069 (2019) reported increased expression of TNF-α, IL-1β, iNOS, COX-2, and toll-like receptors in odontoblasts after prolonged exposure to clinical doses of ZOL. Since there is no significant influx of inflammatory cells into the dental pulp, it seems that odontoblast metabolism is mainly involved in this process.

In this study, histological signs of vasodilation were observed with an increase in blood vessel area without changing the cellularity of the dental pulp. In vitro studies have shown that incubation with ZOL leads to cell death of odontoblasts,1515 - Cvikl B, Agis H, Stogerer K, Moritz A, Watzek G, Gruber R. The response of dental pulp-derived cells to zoledronate depends on the experimental model. Int Endod J. 2011;44(1):33-40. doi: 10.1111/j.1365-2591.2010.01792.x and in vivo studies have shown increased caspase-3 expression.1717 - Silva PG, Ferreira AE Junior, Oliveira CC, Verde MEQL, Freitas MO, Sousa FB, et al. Chronic treatment with zoledronic acid alters the expression levels of inflammatory, bone, and apoptotic markers and toll-like receptors 2 and 4 in rat dental pulp. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019;128(2):139-45. doi: 10.1016/j.oooo.2019.01.069 However, the fact that there was no difference in the number of cell nuclei between the groups led to the hypothesis that pulpal inflammation associated with ZOL is not associated with significant cell death. This analysis also showed that there was no significant influx of inflammatory cells. As there is increased expression of inflammatory cytokines in the dental pulp,1616 - Silva PG, Oliveira CC, Brizeno L, Wong D, Lima R Júnior, Gonçalves RP, et al. Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw. Oral Dis. 2016;22(7):649-57. doi: 10.1111/odi.12513 it seems that pulpal inflammation associated with ZOL is linked to the metabolism of the dental pulp cells themselves.

Odontoblasts are the cells responsible for dentinogenesis throughout life and promote the continuous metabolism of calcium and phosphate.2424 - Lundquist P. Odontoblast phosphate and calcium transport in dentinogenesis. Swed Dent J Suppl. 2002;(154):1-52. Although dentin metabolism turnover is considerably slower than that of bone tissue, systemic changes that interfere with bone mineral density also interfere with dentin formation.2525 - Guimarães GN, Cardoso GB, Naves LZ, Correr-Sobrinho L, Line SR, Marques MR. Short-term PTH administration increases dentine apposition and microhardness in mice. Arch Oral Biol. 2012;57(10):1313-9. doi: 10.1016/j.archoralbio.2012.03.007 Since ZOL has a high affinity for calcium and phosphate and also interferes with bone metabolism,2626 - Lawson MA, Xia Z, Barnett BL, Triffitt JT, Phipps RJ, Dunford JE, et al. Differences between bisphosphonates in binding affinities for hydroxyapatite. J Biomed Mater Res B Appl Biomater. 2010;92(1):149-55. doi: 10.1002/jbm.b.31500 it is likely that bisphosphonates directly interfere with odontoblasts, leading to this inflammatory process.

In rats treated with ZOL, there was a significant increase in TNF-α expression, especially in odontoblasts, as previously described.1616 - Silva PG, Oliveira CC, Brizeno L, Wong D, Lima R Júnior, Gonçalves RP, et al. Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw. Oral Dis. 2016;22(7):649-57. doi: 10.1111/odi.12513 TNF-α is a potent vasodilator in the dental pulp, but the primary inflammatory stimuli that lead to the expression of these cytokines in the pulp tissue are transient.2727 - Vaz MM, Lopes LG, Cardoso PC, Souza JB, Batista AC, Costa NL, et al. Inflammatory response of human dental pulp to at-home and in-office tooth bleaching. J Appl Oral Sci. 2016;24(5):509-17. doi: 10.1590/1678-775720160137 As ZOL is pro-inflammatory and spends long periods adhered to mineralized tissues,2626 - Lawson MA, Xia Z, Barnett BL, Triffitt JT, Phipps RJ, Dunford JE, et al. Differences between bisphosphonates in binding affinities for hydroxyapatite. J Biomed Mater Res B Appl Biomater. 2010;92(1):149-55. doi: 10.1002/jbm.b.31500 the chronification of the inflammatory process may alter the cytokine profile in the dental pulp, prolonging the half-life of TNF-α or its production.

No increase in IL-6 or TGF-β expression was observed in ZOL-treated odontoblasts, which is a necessary precondition for the activation of the Th17 response in lymphocytes.2828 - McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, et al. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol. 2007;8(12):1390-7. doi: 10.1038/ni1539
https://doi.org/10.1038/ni1539...
ZOL-dependent pulpal inflammation was not associated with the presence of inflammatory cells. Chronic exposure to ZOL is directly associated with IL-17 synthesis2929 - Xiong H, Wei L, Peng B. IL-17 stimulates the production of the inflammatory chemokines IL-6 and IL-8 in human dental pulp fibroblasts. Int Endod J. 2015;48(6):505-11. doi: 10.1111/iej.12339 via activation of STAT1/IL-6.3030 - Zhang Q, Atsuta I, Liu S, Chen C, Shi S, Shi S, et al. IL-17-mediated M1/M2 macrophage alteration contributes to pathogenesis of bisphosphonate-related osteonecrosis of the jaws. Clin Cancer Res. 2013;19(12):3176-88. doi: 10.1158/1078-0432.CCR-13-0042 However, in the dental pulp, this process seems to use undescribed pathways, since IL-17 expression in the pulp is independent of standard inflammation cascades.3131 - Horst OV, Horst JA, Samudrala R, Dale BA. Caries induced cytokine network in the odontoblast layer of human teeth. BMC Immunol. 2011;12:9. doi: 10.1186/1471-2172-12-9

IL-17 synthesis can change the natural course of several diseases, as the perpetuation of pro-inflammatory cytokine production is its most reported effect. By interacting with its receptors (IL-17R), IL-17 can activate TNFR synthesis. TNFR is TNF-α receptor and, by stabilizing the mRNA of this cytokine, it prolongs its action and increases the synthesis of this mediator. We observed that blocking IL-17 synthesis using digoxin also reduced TNF-α expression and vasodilation, but paradoxically increased IL-6 expression.3232 - Gaffen SL, Jain R, Gard AV, Cua DJ. IL-23-IL-17 immune axis: discovery, mechanistic understanding, and clinical testing. Nat Rev Immunol. 2014;14(9):585-600. doi: 10.1038/nri3707
https://doi.org/10.1038/nri3707...

Digoxin, besides being anti-inflammatory, has anti-vascular properties. Cardiac glycosides, including digoxin, ouabain, and proscillaridin A, inhibit hypoxia-inducible factor 1 (HIF-1α) protein synthesis and expression,3333 - Zhang H, Qian DZ, Tan YS, Lee K, Gao P, Ren YR, et al. Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth. Proc Natl Acad Sci U S A. 2008;105(50):19579-86. doi: 10.1073/pnas.0809763105
https://doi.org/10.1073/pnas.0809763105...
which leads to vascular endothelial growth factor (VEGF) downregulation.3434 - Wei D, Peng JJ, Gao H, Li H, Li D, Tan Y, et al. Digoxin downregulates NDRG1 and VEGF through the inhibition of HIF-1α under hypoxic conditions in human lung adenocarcinoma A549 cells. Int J Mol Sci. 2013;14(4):7273-85. doi: 10.3390/ijms14047273
https://doi.org/10.3390/ijms14047273...
The inhibition of other growth factors contributes to the antiangiogenic properties of digoxin, such as fibroblast growth factor (FGF) and epidermoid growth factor (EGF), both in vivo and ex vivo,3535 - Trenti A, Zulato E, Pasqualini L, Indraccolo S, Bolego C, Trevisi L. Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors. Br J Pharmacol. 2017;174(18):3094-106. doi: 10.1111/bph.13944
https://doi.org/10.1111/bph.13944...
which explains the reduction in TGF-β immunostaining showed in high doses of digoxin treatment and when added to the antiangiogenic action of ZOL, leading to considerable damage to the dental pulp.

The role of IL-17-ZOL-dependent pulp inflammation seems to be crucial, since inhibition by digoxin attenuated vasodilation, the microscopic change that indicates inflammation. Digoxin is a glycoside used to treat congestive heart failure and strongly inhibits RORγT, the main IL-17 transcription factor.3636 - Huh JR, Leung MW, Huang P, Ryan DA, Krout MR, Malapaka RR, et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature. 2011;472(7344):486-90. doi: 10.1038/nature09978
https://doi.org/10.1038/nature09978...
The inhibitory action of digoxin on the Th17 response significantly improves autoimmune arthritis,2222 - Lee J, Baek S, Lee J, Lee J, Lee DG, Park MK, et al. Digoxin ameliorates autoimmune arthritis via suppression of Th17 differentiation. Int Immunopharmacol. 2015;26(1):103-11. doi: 10.1016/j.intimp.2015.03.017
https://doi.org/10.1016/j.intimp.2015.03...
and chronic use of this drug significantly reduces the risk of bone fractures due to osteoporosis.3737 - Rejnmark L, Vestergaard P, Mosekilde L. Fracture risk in patients treated with amiodarone or digoxin for cardiac arrhythmias: a nation-wide case-control study. Osteoporos Int. 2007;18(4):409-17. doi: 10.1007/s00198-006-0250-7
https://doi.org/10.1007/s00198-006-0250-...
In this study, IL-17 is strongly associated with impaired mineralized tissue health2222 - Lee J, Baek S, Lee J, Lee J, Lee DG, Park MK, et al. Digoxin ameliorates autoimmune arthritis via suppression of Th17 differentiation. Int Immunopharmacol. 2015;26(1):103-11. doi: 10.1016/j.intimp.2015.03.017
https://doi.org/10.1016/j.intimp.2015.03...
,3737 - Rejnmark L, Vestergaard P, Mosekilde L. Fracture risk in patients treated with amiodarone or digoxin for cardiac arrhythmias: a nation-wide case-control study. Osteoporos Int. 2007;18(4):409-17. doi: 10.1007/s00198-006-0250-7
https://doi.org/10.1007/s00198-006-0250-...
and the maintenance of chronic inflammation in the dental pulp.

The main limitation of this study is the lack of evaluation of the Th1 response in the dental pulp. Although digoxin preferentially inhibits the Th17 response compared with the Th1 response, this aminoglycoside reduces the overproduction of some cytokines related to both immune responses, such as IL-1β, IL-6, IL-17, and IL-23.3838 - Saeed H, Mateen S, Moin S, Khan AQ, Owais M. Cardiac glycoside digoxin ameliorates pro-inflammatory cytokines in PBMCs of rheumatoid arthritis patients in vitro. Int Immunopharmacol. 2020;82:106331. doi: 10.1016/j.intimp.2020.106331.
https://doi.org/10.1016/j.intimp.2020.10...
There is an overlap between the Th1 and Th17 immune responses during the development of immunity. However, toll-like receptors 2 and 4, which are overstained in odontoblasts after ZOL treatment,1717 - Silva PG, Ferreira AE Junior, Oliveira CC, Verde MEQL, Freitas MO, Sousa FB, et al. Chronic treatment with zoledronic acid alters the expression levels of inflammatory, bone, and apoptotic markers and toll-like receptors 2 and 4 in rat dental pulp. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019;128(2):139-45. doi: 10.1016/j.oooo.2019.01.069 play an important role that favors the Th17 response, since while the Th1 response related to toll-like receptors is more important for intracellular microorganisms, the Th17 response related to toll-like receptors is more important for extracellular microorganisms.3939 - Kaiko GE, Horvat JC, Beagley KW, Hansbro PM. Immunological decision-making: how does the immune system decide to mount a helper T-cell response? Immunology. 2008;123(3):326-38. doi: 10.1111/j.1365-2567.2007.02719.x The main focus of these toll-like receptors is on odonoblasts.4040 - Veerayutthwilai O, Byers MR, Pham TT, Darveau RP, Dale BA. Differential regulation of immune responses by odontoblasts. Oral Microbiol Immunol. 2007;22(1):5-13. doi: 10.1111/j.1399-302X.2007.00310.x

The reduction in IL-17 in digoxin-treated odontoblasts was not followed by reduced IL-17 immunoexpression in non-odontoblasts pulp cells. Thus, ZOL exerts a direct effect on odontoblasts via pathways independent of the classical activation by T helper lymphocytes, prolonging activation of IL-6 and TGF-β. However, it is still impossible to assess whether ZOL is incorporated into dentin as part of this change.

Conclusion

Thus, we conclude that chronic ZOL administration induces IL-17-dependent vasodilation and pulp ectasia, and treatment with anti-RORyT digoxin partially reverted these outcomes. The anti-angiogenic effects of digoxin and the activity of odontoblasts seem to be responsible for this process. Since IL-17 expression in odontoblasts occurs independently of the classic IL-6 and TGF-β expression and the Th1 response overlaps with the Th17 response, further studies are needed to evaluate the pathways of activation of Th cells and IL-17 in odontoblasts and other dental pulp cells.

Acknowledgements

We thank the UNICHRISTUS Institutional Vivarium and its staff for providing the means for animal experimentation.

References

  • 1
    - Bowden SA, Mahan JD. Zoledronic in pediatric metabolic bone disorders. Transl Pediatr. 2017;6(4):256-68. doi: 10.21037/tp.2017.09.10
    » https://doi.org/10.21037/tp.2017.09.10
  • 2
    - Goldvaser H, Amir E. Role of bisphosphonates in breast cancer therapy. Curr Treat Options Oncol. 2019;20(4):26. doi: 10.1007/s11864-019-0623-8
  • 3
    - Reyes C, Hitz M, Prieto-Alhambra D, Abrahamsen B. Risks and benefits of bisphosphonate therapies. J Cell Biochem. 2016;117(1):20-8. doi: 10.1002/jcb.25266
    » https://doi.org/10.1002/jcb.25266
  • 4
    - Kalyan S. It may seem inflammatory, but some T cells are innately healing to the bone. J Bone Miner Res. 2016;31(11):1997-2000. doi: 10.1002/jbmr.2875
    » https://doi.org/10.1002/jbmr.2875
  • 5
    - Basso FG, Pansani TN, Soares DG, Cardoso LM, Hebling J, Costa CA. Influence of bisphosphonates on the adherence and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces. Clin Oral Investig. 2018;22(2):893-900. doi: 10.1007/s00784-017-2167-2
  • 6
    - Basso FG, Soares DG, Pansani TN, Turrioni AP, Scheffel DL, Hebling J, et al. Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid. Braz Oral Res. 2016;30(1):e122. doi: 10.1590/1807-3107BOR-2016.vol30.0122
  • 7
    - Zara S, De Colli M, di Giacomo V, Zizzari VL, Di Nisio C, Di Tore U, et al. Zoledronic acid at subtoxic dose extends osteoblastic stage span of primary human osteoblasts. Clin Oral Investig. 2015;19(3):601-11. doi: 10.1007/s00784-014-1280-8
  • 8
    - Scheller EL, Hankenson KD, Reuben JS, Krebsbach PH. Zoledronic acid inhibits macrophage SOCS3 expression and enhances cytokine production. J Cell Biochem. 2011;112(11):3364-72. doi: 10.1002/jcb.23267
    » https://doi.org/10.1002/jcb.23267
  • 9
    - Zhu W, Xu R, Du J, Fu Y, Li S, Zhang P, et al. Zoledronic acid promotes TLR-4-mediated M1 macrophage polarization in bisphosphonate-related osteonecrosis of the jaw. FASEB J. 2019;33(4):5208-19. doi: 10.1096/fj.201801791RR
    » https://doi.org/10.1096/fj.201801791RR
  • 10
    - Kuiper JW, Forster C, Sun C, Peel S, Glogauer M. Zoledronate and pamidronate depress neutrophil functions and survival in mice. Br J Pharmacol. 2012;165(2):532-9. doi: 10.1111/j.1476-5381.2011.01592.x
    » https://doi.org/10.1111/j.1476-5381.2011.01592.x
  • 11
    - Wang Q, Liu J, Guo T, Liu D, Pan J. Epidermal growth factor reverses the inhibitory effects of the bisphosphonate, zoledronic acid, on human oral keratinocytes and human vascular endothelial cells in vitro via the epidermal growth factor receptor (EGFR)/Akt/phosphoinositide 3-kinase (PI3K) signaling. Med Sci Monit. 2019;25:700-10. doi: 10.12659/MSM.911579
    » https://doi.org/10.12659/MSM.911579
  • 12
    - Hiraga T, Ninomiya T, Hosoya A, Nakamura H. Administration of the bisphosphonate zoledronic acid during tooth development inhibits tooth eruption and formation and induces dental abnormalities in rats. Calcif Tissue Int. 2010;86(6):502-10. doi: 10.1007/s00223-010-9366-z
  • 13
    - Massa LF, Bradaschia-Correa V, Arana-Chavez VE. Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats. J Histochem Cytochem. 2006;54(6):713-25. doi: 10.1369/jhc.5A6853.2006
    » https://doi.org/10.1369/jhc.5A6853.2006
  • 14
    - Basso FG, Turrioni PS, Hebling J, Costa CA. Effects of zoledronic acid on odontoblast-like cells. Arch Oral Biol. 2013;58(5):467-73. doi: 10.1016/j.archoralbio.2012.09.016
  • 15
    - Cvikl B, Agis H, Stogerer K, Moritz A, Watzek G, Gruber R. The response of dental pulp-derived cells to zoledronate depends on the experimental model. Int Endod J. 2011;44(1):33-40. doi: 10.1111/j.1365-2591.2010.01792.x
  • 16
    - Silva PG, Oliveira CC, Brizeno L, Wong D, Lima R Júnior, Gonçalves RP, et al. Immune cellular profile of bisphosphonate-related osteonecrosis of the jaw. Oral Dis. 2016;22(7):649-57. doi: 10.1111/odi.12513
  • 17
    - Silva PG, Ferreira AE Junior, Oliveira CC, Verde MEQL, Freitas MO, Sousa FB, et al. Chronic treatment with zoledronic acid alters the expression levels of inflammatory, bone, and apoptotic markers and toll-like receptors 2 and 4 in rat dental pulp. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019;128(2):139-45. doi: 10.1016/j.oooo.2019.01.069
  • 18
    - Lauridsen HM, Pellowe AS, Ramanathan A, Liu R, Miller-Jensen K, McNiff JM, Pober JS, Gonzalez AL. Tumor Necrosis Factor-α and IL-17A Activation Induces Pericyte-Mediated Basement Membrane Remodeling in Human Neutrophilic Dermatoses. Am J Pathol. 2017;187(8):1893-906. doi: 10.1016/j.ajpath.2017.04.008
    » https://doi.org/10.1016/j.ajpath.2017.04.008
  • 19
    - Katzung BG, Masters SB, Trevor AJ. Farmacologia básica e clínica. 12. ed. Porto Alegre: AMGH; 2014.
  • 20
    - Wei Z, Wang Y, Zhang K, Liao Y, Ye P, Wu J, et al. Inhibiting the Th17/IL-17A-related inflammatory responses with digoxin confers protection against experimental abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 2014;34(11):2429-38. doi: 10.1161/ATVBAHA.114.304435
    » https://doi.org/10.1161/ATVBAHA.114.304435
  • 21
    - Silva PG, Ferreira AE Junior, Teófilo CR, Barbosa MC, Lima RC Júnior, Sousa FB, et al. Effect of different doses of zoledronic acid in establishing of bisphosphonate-related osteonecrosis. Arch Oral Biol. 2015;60(9):1237-45. doi: 10.1016/j.archoralbio.2015.05.015
  • 22
    - Lee J, Baek S, Lee J, Lee J, Lee DG, Park MK, et al. Digoxin ameliorates autoimmune arthritis via suppression of Th17 differentiation. Int Immunopharmacol. 2015;26(1):103-11. doi: 10.1016/j.intimp.2015.03.017
    » https://doi.org/10.1016/j.intimp.2015.03.017
  • 23
    - Moreira JE Sobrinho, Aguiar MT, Machado LC, Carlos AC, Alves AP, Mesquita KC, et al. Intense orthodontic force induces the three dental pulp nitric oxide synthase isoforms and leads to orofacial discomfort in rats. Orthod Craniofac Res. 2022;25(4):485-93. doi: 10.1111/ocr.12560
    » https://doi.org/10.1111/ocr.12560
  • 24
    - Lundquist P. Odontoblast phosphate and calcium transport in dentinogenesis. Swed Dent J Suppl. 2002;(154):1-52.
  • 25
    - Guimarães GN, Cardoso GB, Naves LZ, Correr-Sobrinho L, Line SR, Marques MR. Short-term PTH administration increases dentine apposition and microhardness in mice. Arch Oral Biol. 2012;57(10):1313-9. doi: 10.1016/j.archoralbio.2012.03.007
  • 26
    - Lawson MA, Xia Z, Barnett BL, Triffitt JT, Phipps RJ, Dunford JE, et al. Differences between bisphosphonates in binding affinities for hydroxyapatite. J Biomed Mater Res B Appl Biomater. 2010;92(1):149-55. doi: 10.1002/jbm.b.31500
  • 27
    - Vaz MM, Lopes LG, Cardoso PC, Souza JB, Batista AC, Costa NL, et al. Inflammatory response of human dental pulp to at-home and in-office tooth bleaching. J Appl Oral Sci. 2016;24(5):509-17. doi: 10.1590/1678-775720160137
  • 28
    - McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, et al. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol. 2007;8(12):1390-7. doi: 10.1038/ni1539
    » https://doi.org/10.1038/ni1539
  • 29
    - Xiong H, Wei L, Peng B. IL-17 stimulates the production of the inflammatory chemokines IL-6 and IL-8 in human dental pulp fibroblasts. Int Endod J. 2015;48(6):505-11. doi: 10.1111/iej.12339
  • 30
    - Zhang Q, Atsuta I, Liu S, Chen C, Shi S, Shi S, et al. IL-17-mediated M1/M2 macrophage alteration contributes to pathogenesis of bisphosphonate-related osteonecrosis of the jaws. Clin Cancer Res. 2013;19(12):3176-88. doi: 10.1158/1078-0432.CCR-13-0042
  • 31
    - Horst OV, Horst JA, Samudrala R, Dale BA. Caries induced cytokine network in the odontoblast layer of human teeth. BMC Immunol. 2011;12:9. doi: 10.1186/1471-2172-12-9
  • 32
    - Gaffen SL, Jain R, Gard AV, Cua DJ. IL-23-IL-17 immune axis: discovery, mechanistic understanding, and clinical testing. Nat Rev Immunol. 2014;14(9):585-600. doi: 10.1038/nri3707
    » https://doi.org/10.1038/nri3707
  • 33
    - Zhang H, Qian DZ, Tan YS, Lee K, Gao P, Ren YR, et al. Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth. Proc Natl Acad Sci U S A. 2008;105(50):19579-86. doi: 10.1073/pnas.0809763105
    » https://doi.org/10.1073/pnas.0809763105
  • 34
    - Wei D, Peng JJ, Gao H, Li H, Li D, Tan Y, et al. Digoxin downregulates NDRG1 and VEGF through the inhibition of HIF-1α under hypoxic conditions in human lung adenocarcinoma A549 cells. Int J Mol Sci. 2013;14(4):7273-85. doi: 10.3390/ijms14047273
    » https://doi.org/10.3390/ijms14047273
  • 35
    - Trenti A, Zulato E, Pasqualini L, Indraccolo S, Bolego C, Trevisi L. Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors. Br J Pharmacol. 2017;174(18):3094-106. doi: 10.1111/bph.13944
    » https://doi.org/10.1111/bph.13944
  • 36
    - Huh JR, Leung MW, Huang P, Ryan DA, Krout MR, Malapaka RR, et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature. 2011;472(7344):486-90. doi: 10.1038/nature09978
    » https://doi.org/10.1038/nature09978
  • 37
    - Rejnmark L, Vestergaard P, Mosekilde L. Fracture risk in patients treated with amiodarone or digoxin for cardiac arrhythmias: a nation-wide case-control study. Osteoporos Int. 2007;18(4):409-17. doi: 10.1007/s00198-006-0250-7
    » https://doi.org/10.1007/s00198-006-0250-7
  • 38
    - Saeed H, Mateen S, Moin S, Khan AQ, Owais M. Cardiac glycoside digoxin ameliorates pro-inflammatory cytokines in PBMCs of rheumatoid arthritis patients in vitro Int Immunopharmacol. 2020;82:106331. doi: 10.1016/j.intimp.2020.106331.
    » https://doi.org/10.1016/j.intimp.2020.106331
  • 39
    - Kaiko GE, Horvat JC, Beagley KW, Hansbro PM. Immunological decision-making: how does the immune system decide to mount a helper T-cell response? Immunology. 2008;123(3):326-38. doi: 10.1111/j.1365-2567.2007.02719.x
  • 40
    - Veerayutthwilai O, Byers MR, Pham TT, Darveau RP, Dale BA. Differential regulation of immune responses by odontoblasts. Oral Microbiol Immunol. 2007;22(1):5-13. doi: 10.1111/j.1399-302X.2007.00310.x
  • Data availability statement

    All data generated and analyzed during this study are included in this published article.
  • Funding: This study was funded by the National Council for Scientific and Technological Development (CNPq – Conselho Nacional de Desenvolvimento Científico e Tecnológico), under protocol 429851/2018-9, and the Cearense Foundation for Scientific and Technological Development Support (FUNCAP – Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico), under protocol 00164-00078.01.00/19.

Edited by

Editor: Linda Wang
Associate Editor: Renato Menezes Silva

Data availability

All data generated and analyzed during this study are included in this published article.

Publication Dates

  • Publication in this collection
    09 Oct 2023
  • Date of issue
    2023

History

  • Received
    4 July 2023
  • Reviewed
    14 Aug 2023
  • Accepted
    21 Aug 2023
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