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Davallialactone Reduces Inflammation and Repairs Dentinogenesis on Glucose Oxidase–induced Stress in Dental Pulp Cells
Basic Research—Biology
Davallialactone Reduces Inflammation and Repairs Dentinogenesis on Glucose Oxidase–induced Stress in Dental Pulp Cells Young-Hee Lee, PhD,*‡ Go-Eun Kim, MS,*‡ Yong-Beom Song, DDS, PhD,† Usha Paudel, BS,* Nan-Hee Lee, MS,* Bong-Sik Yun, PhD,‡ Mi-Kyung Yu, DDS, PhD,† and Ho-Keun Yi, PhD* Abstract Introduction: The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. Methods: The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells’ vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. Results: The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. Conclusions: The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy. (J Endod 2013;39:1401–1406)
Key Words Davallialactone, dental pulp cells, dentin mineralization, diabetes mellitus, glucose oxidase, glucose oxidative stress, inflammation
D
ental pulp is a loose connective tissue consisting of various types of cells like ground substance and neural and vascular supplies. Damage to dental pulp can harm pulp vitality. The vitality of pulp during tissue homeostasis, after injury, and in aging is dependent on pulp cell activity. The decrease of pulp cell activity leads to pathological conditions including inflammation (1). Although many studies have examined the pathological condition of pulp cells, the pathogenesis is poorly understood. DM is a common metabolic disease characterized by hyperglycemia resulting from the insufficiency of insulin secretion or action. The chronic hyperglycemia of diabetes is associated with long-term systemic dysfunction (2). The presentation of DM is related to oral manifestations such as xerostomia and taste impairment, and its progression exacerbates various oral diseases such as dental caries, periodontal disease, soft-tissue lesions, and fungal infection (3). In addition, oxidative stress and inflammation are physiopathological mechanisms causing diabetes (4). The enzyme glucose oxidase catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-d-laltone. In cells, it aids in breaking the sugar down into its metabolites. However, the chronic hyperglycemia of diabetes elevates the production of free radicals by glucose oxidation. Therefore, it is assumed that the state of diabetes generates continuous glucose oxidase to manage glucose and then it reacts with D-glucose substrate to yield increasingly higher levels of hydrogen peroxide. DM alters the antioxidant system of dental pulp cells, but supplementation with antioxidant treatment (ie, astaxanthin) can restore the cells (5). In addition, longterm supplementation with vitamin C can markedly prevent the diabetes-induced reduction in pulp blood flow (6). Similarly, many studies have reported careful management of the association between diabetes and chronic periodontal disease (7, 8). Indeed, patients with poorly controlled DM have a low response to endodontic treatment (9). However, little information is available describing the pulp pathogenesis influenced by DM. Davallialactone, a hispidin analog from the mushroom Inonotus xeranticus belonging to the family Hymenochaetaceae, possesses immunosuppressive and potent anti-inflammatory activity via its antioxidant effect in pulp cells (10, 11). The purpose of this study was to evaluate the cellular state of dental pulp cells with pathogenic
From the *Department of Oral Biochemistry and Institute of Oral Bioscience, BK21 Program; †Department of Conservative Dentistry, School of Dentistry; and Division of Biotechnology, College of Environmental and Biosource Science, Chonbuk National University, Jeonju, Korea. Supported by a grant from the Korea Forest Service (grant no. S121010L090120). Young-Hee Lee and Go-Eun Kim contributed equally to this work. Address requests for reprints to Dr Ho-Keun Yi, Department of Oral Biochemistry, School of Dentistry, Chonbuk National University, 634-18, Deokjin-dong, Deokjingu, Jeonju, Jeonbuk 561-712, Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.06.033 ‡
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fluorescein diacetate (DCFH-DA) (Sigma-Aldrich). After a complete reaction, the cells were incubated with 5 mmol/L DCFH-DA for 30 minutes in the dark at 37 C, harvested, and followed by FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ).
Materials and Methods
Assay of ALP Activity After a complete reaction in 1, 7, and 15 days, pulp cells were scraped into cold PBS and then sonicated with a cell disruptor (Heat System-Ultrasonics, Plainview, NJ) in an ice-cold bath. Alkaline phosphatase (ALP) activity in the supernatant was determined according to the SensoLyte p-NPP Alkaline Phosphatase Assay Kit (AnaSpec, Inc, Fremont, CA) using a previously described method (11).
Materials Dexamethasone, b-glycerophosphate disodium salt hydrate, alizarin red S, glucose oxidase, L-ascorbic acid, D-glucose, and actin were purchased from Sigma-Aldrich (St Louis, MO). Antibodies against cyclooxygenase-2 (COX-2), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) were supplied by Cell Signaling (Beverly, MA). Antibodies against intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and heme oxygenase-1 (HO-1) were acquired from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Antibodies against BMP-2 and -7 and Rux-2 were purchased from Bioworld Technology, Inc (St Louis Park, MN). Davallialactone was extracted from the mushroom Inonotus xeranticus (Berk). Imaz ET Aoshi (Hymenochaetaceae) is a species of mushroom (11). Human Pulp Cell Culture and Induction of Glucose Oxidase–treated Oxidative Stress Human dental pulp cells (HDPCs) were isolated and cultured as previously reported (11). The cells at 80% confluence were seeded in 100-mm culture dishes (SPL Life Science, Gyeonggi-do, Korea) and subcultured at a 1:4 ratio. The experiments were performed using the HDPCs between the third and eighth passages. For induced glucose oxidative stress, 80% confluence cells were supplemented with 0.5% fetal bovine serum RPMI 1640 media (Gibco, Invitrogen, Grand Island, NY) before being exposed to various concentrations of glucose oxidase with D-glucose concentration for 15 days. For the glucose-free state, the cells were supplemented with 0.5% RPMI 1640 medium before the induction of glucose oxidative stress. For mineralization experiments, the cells were cultured in osteogenic media (OM) including 50 mg/mL ascorbic acid, 100 nmol/L dexamethasone, and 10 mmol/L b-glycerophosphate as previously described (12). MTT Assay The viability of the cultured cells was determined by examining the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. After a 24-hour incubation period, the cells (1 104/well) in 96-well plates were washed twice with phosphate-buffered saline (PBS). MTT (100 mg/0.1 mL PBS) was added to each well. The cells were incubated at 37 C for 3 hours, and dimethyl sulfoxide (DMSO) (100 mL) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm with an enzyme-linked immunosorbent assay reader (Synergy 2; Bio-TeK, Winooski, VT). Measurement of H2O2 and Reactive Oxygen Species Formation The quantity of H2O2 produced by glucose oxidase in culture medium at the indicated time was determined using the Biovision hydrogen peroxide assay kit (Biovision Research Products, Milpitas, CA) following the manufacturer’s instructions. The absorbance was measured at 570 nm with an enzyme-linked immunosorbent assay reader (Bio-TeK). The level of reactive oxygen species (ROS) formation was assessed using an oxidation-sensitive fluorescent probe 20 ,70 -dichlorodihydro1402
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Alizarin Red S Stain After 15 days of differentiation induction, the cells were rinsed with PBS, air dried, and fixed in ice-cold 95% ethanol for 30 minutes at 20 C. The cells were stained with 40 mmol/L alizarin red S (pH = 4.2) for 1 hour at room temperature, washed extensively 5 times with deionized water, and rinsed with PBS (without magnesium and calcium) for 15 minutes. Western Blot Analysis Western blot analysis was performed as previously described (11). The samples were separated by 8%–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under denaturing conditions and electroblotted onto nitrocellulose membranes. The membranes were incubated with the specific primary antibody and the horseradish peroxidase–conjugated secondary antibody. The signals were visualized by chemiluminescent detection according to the manufacturer’s protocol (Amersham Pharmacia Biotech, London, UK). The membranes were reprobed with antiactin antibody to confirm an equal protein loading. RNA Isolation and Reverse-transcription Polymerase Chain Reaction Total RNA was extracted from the cells by using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Then, 1 mg RNA was reverse transcribed for first-strand complementary DNA synthesis (Invitrogen, Carlsbad, CA). The complementary DNA was amplified by using rTaq Plus 5 PCR master mix (ELPis, Daejeon, Korea) and 1 mmol/L specific primers. The sequences of the specific primers used are human dentin sialophosphoprotein (hDSPP) (sense: 50 -TTCCGATGGGAGTCCTAGTG-30 , antisense: 50 -TGAGCTTCTGGG TGTCCTCT-30 ) and human dentin matrix protein1 (hDMP-1) (sense: 5-CAGGA GCACAGGAAAAGGAG-3, antisense: 5-CTGGTGGTATCTTCCCCCAGGAG-3). The reverse-transcription polymerase chain reaction products were electrophoresed on 1.5% agarose gel with ethidium bromide. Statistical Analysis At least 3 independent experiments were performed, and the mean value was used for further analysis. The statistical significance of the groups was assessed by analysis of variance and the Duncan test. Null hypotheses of no difference were rejected if P values were <.05.
Results Glucose Oxidase Generates H2O2 and Cellular Toxicity and Davallialactone Removes ROS Formation Davallialactone has anti-inflammatory properties through ROS removal activity in HDPCs (11). Accordingly, the antioxidant JOE — Volume 39, Number 11, November 2013
Basic Research—Biology characteristics of davallialactone were evaluated for the vitality of pulp cells against glucose oxidative stress. In this study, pulp cells were cultured in free glucose to high glucose (25 mmol/L). The glucose oxidase treatment continuously increased H2O2 at 24 hours in a glucose oxidase concentration–dependent manner (Fig. 1A). Furthermore, when exposed with low (5 mmol/L) and high (25 mmol/L) glucose with glucose oxidase significantly increased production of H2O2 at each glucose oxidase concentration–dependent manner (Fig. 1A). The effect of glucose oxidase with glucose concentration on cell viability was determined using the MTT assay. When steady state of high glucose, the production of H2O2 over 50 mmol/L after glucose oxidase (5 mU/mL) treated appeared to induce cellular toxicity at the indicated time (Fig. 1B). Furthermore, glucose oxidase in a high glucose state ensures increased ROS formation compared with 150 mmol/L H2O2 (Fig. 1C). However, davallialactone and N-acetylcysteine (NAC) protected cellular cytotoxicity and ROS formation against glucose oxidase–induced oxidative stress (Fig. 1B and D).
Effect of Davallialactone on Odontoblast Mineralization against Glucose Oxidase–induced Oxidative Stress To identify odontoblast mineralization by davallialactone under glucose oxidase treatment, we analyzed the ALP activity and alizarin red S stain in HDPCs. Pulp cells treated with glucose (25 mmol/L) alone markedly raised ALP activity, whereas cotreatment with glucose oxidase and high glucose gradually reduced ALP activity more than control cells. However, the antioxidants davallialactone and NAC increased ALP activity even under glucose oxidase stress on the indicated day (Fig. 4A). Next, we performed the formation of mineralization by davallialactone using alizarin red S stain. Pulp cells were cultured in the presence or absence of davallialactone with glucose oxidase and high glucose containing OM for a 15-day period. Cotreatment with glucose oxidase and high glucose in pulp cells lowered the extent of mineralizationlike, noncontaining OM mock cells. However, the presence of davallialactone and NAC augmented the extent of mineralization to a higher degree even with glucose oxidase–induced stimuli (Fig. 4B).
Glucose Oxidase Disturbs Inflammation and Dentinogenesis, but Davallialactone Helps Recovery from Glucose Oxidase–induced Oxidative Stress To determine whether oxidative stress by glucose oxidase with high glucose was responsible for inflammatory molecules and dentinogenesis-related molecules, pulp cells were treated with glucose oxidase (5 mU/mL) for 15 days in a high glucose (25 mmol/L) state. The duration of glucose oxidase treatment on the indicated day revealed a gradual aggravation of inflammatory molecules (ICAM-1, VCAM-1, Cox-2, and HO-1; Fig. 2A), angiogenesis molecules (VEGF, EGF, and PDGF; Fig. 2B), dentinogenesis messenger RNA levels (DMP-1 and DSPP; Fig. 2C), and protein expression (BMP-2 and -7 and Runx-2; Fig. 2D). However, davallialactone in the same condition reduces inflammatory action (Fig. 3A), induces angiogenesis expression (Fig. 3B), and recovers dentinogenesis molecule expression of messenger RNA and protein levels (Fig. 3C and D).
The vitality of the pulp complex is dependent on the activity of the pulp cells. Thus, pathogenesis to decrease pulp cell activity is better described for healthy teeth. Recently, studies of pulp complex have focused on DM as well. DM is a complicated disease resulting in ROS production in the oral cavity (3, 13). The effects of diabetes on the metabolism of dental pulp tissue have already been studied in an in vivo system (13–15). However, the response of pulp cells to glucose oxidase with high glucose state–induced stress is unknown. Thus, we showed that the generation of H2O2 by glucose oxidase in the presence of substrate glucose may act to stimulate the decline of pulp cell activity and the response to pathological conditions including inflammation. Also, this study helps to understand the associated pathogenesis and elucidated therapeutic strategy to oral disease using an antioxidant like davallialactone for pulp vitality.
Discussion
Figure 1. The determination of H2O2 and ROS produced by glucose oxidase, the protection of cellular toxicity, and the removal of ROS formation by davallialactone in HDPCs. (A) The cells were exposed to glucose oxidase (5 and 10 mU/mL) with D-glucose (5 and 25 mmol/L) and the amount of H2O2 in the culture medium was measured at 24 hours. The cells were pretreated with davallialactone (10 mmol/L) and NAC (1 mmol/L) before glucose oxidase exposed with D-glucose in HDPCs. (B) The cell viability was determined using an MTT assay at the indicated time. (C and D) The level of ROS formation was determined by flow cytometry after DCFHDA treatment at 24 hours. Each value is reported as the mean and standard error of the mean of 3 independent experiments.
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Figure 2. The influence effect of glucose oxidase on the expression of inflammatory and dentinogenesis in HDPCs. The cells were exposed to 5 mU/mL glucose oxidase with a high glucose level and to continuously expose oxidative stress and allow more treated glucose oxidase at interval of 3 days for 15 days. (A) The expression of inflammatory molecules such as ICAM-1, VCAM-1, and COX-2 and the HO-1 protein level in the total cell lysate was determined by Western blot analysis at the indicated time. (B) Angiogenic factors such as VEGF and EGF and the PDGF protein level were determined by using Western blot analysis. (C) Odontoblast differentiation markers such as DMP-1 and DSPP messenger RNA levels were determined by using reverse-transcription polymerase chain reaction using a specific primer. (D) Osteoblast differentiation markers such as BMP-2 and -7 and Runx-2 were determined by Western blot analysis. For loading control, the blots were reprobed with actin antibody. All data are representative of 3 separate experiments.
In this study, the treatment of glucose oxidase in pulp cells revealed the production of H2O2 in the glucose-free state at 24 hours. However, this dose did not affect oxidative stress. Interestingly, the state of high glucose with glucose oxidase increased H2O2 production more compared with glucose oxidase alone. In general, glucose oxidase
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Figure 3. The effect of davallialactone on the expression of inflammatory and dentinogenesis-related molecule expression by glucose oxidase treatment. The cells were pretreated with davallialactone (10 mmol/L) before glucose oxidase exposed with high glucose to continuously expose oxidative stress and allow treatment with davallialactone at an interval of 3 days for 15 days. (A) The expression of inflammatory molecules such as ICAM-1, VCAM-1, COX-2 and the HO-1 protein level in the total cell lysate were determined by using Western blot analysis at the indicated time. (B) Angiogenic factors such as VEGF and EGF and the PDGF protein level were determined by using Western blot analysis. (C) Odontoblast differentiation markers such as DMP-1 and DSPP messenger RNA levels were determined by reverse-transcription polymerase chain reaction using a specific primer. (D) Osteoblast differentiation markers such as BMP-2 and -7 and Runx-2 were determined by using Western blot analysis. For loading control, the blots were reprobed with actin antibody. All data are representative of 3 separate experiments.
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Figure 4. The effect of davallialactone on odontoblast mineralization against glucose oxidase stress. To perform odontoblast mineralization, the cells were pretreated with davallialactone before glucose oxidase exposed with or without glucose concentration to continuously expose oxidative stress and allow treatment with davallialactone at an interval of 3 days for 15 days. Mineralization was determined by (A) ALP activity at the indicated time and (B) alizarin red S stain at 15 days. For alizarin red S stain, cells were cultured in OM as described in the Methods section. Each value is reported as the mean and standard error of the mean of 3 independent experiments.
investigated the state of oxidative stress via the production of H2O2 by means of glucose oxidase. H2O2 is an index of oxidative stress to mediators of cell damage that cause damage to cellular proteins, membrane lipids, and DNA and lead to cell death (17, 18). In this study, H2O2 production by glucose oxidase with a high glucose state exacerbated the cell viability in pulp cells. In addition, even at the same concentration of glucose oxidase, ROS formation was accumulated by means of glucose concentration in pulp cells. Thus, this phenomenon may show the steady state of oxidative stress in pulp cells, which is similar to diabetes-mediated oxidative stress. Also, these results suggested that DM influences pathological conditions damaging pulp vitality via oxidative stress. However, the presence of davallialactone and NAC as antioxidants significantly protects against cellular toxicity under those conditions. Additionally, davallialactone is able to remove ROS formation like NAC. The overproduction of ROS can be attenuated by antioxidant enzymes via enhanced drug treatment (5, 18). Thus, the antioxidant effect of davallialactone and NAC might be enhanced by an oxidative stress defense enzyme against glucose oxidative stress for pulp cell activity. Therefore, it is able to improve the pathogenesis of complicated diseases such as diabetes in pulp cells. The chronic nature of diabetes causes irreversible damage and induces metabolic alteration by oxidative stress in the dental pulp leading to inflammation (3, 13, 18, 19). In addition, in uncontrolled or poorly controlled diabetes, it affects impaired angiopathy and a thickened basement membrane in dental pulp vessels and a reduction in pulpal blood flow in streptozotocin-induced diabetic rats (6, 20). The high glucose state revealed an increase of ALP activity in rat pulp cells and vascular smooth muscle cells that appear with pathological conditions including DM (14, 21, 22). In this study, long-term exposure to glucose oxidase with a high glucose state in pulp cells resulted in the accelerated induction of inflammatory molecules such as ICAM-1, VCAM-1, COX-2, and HO-1. Also, this study found a decreased expression of angiogenic factors such as VEGF, FGF, and PDGF in the same conditions. In particular, the popularly used bimolecular markers of odontoblast and osteoblast differentiation in dental JOE — Volume 39, Number 11, November 2013
pulp cells DMP-1, DSPP, BMP-2 and -7, and Runx-2 attenuated the expression of messenger RNA and the protein level. They are crucial in dentin mineralization (23). Moreover, a high glucose level state significantly accelerates ALP activity compared with the glucose-free state over a long duration. On the contrary, the state of oxidative stress by glucose oxidase decreased ALP activity. Many studies in pulp cells reported that long-term oxidative conditions also disturb dentin mineralization via ALP activity (12, 24). Therefore, these results suggest that the long-term treatment of glucose oxidase with a high glucose state produces a state of oxidative stress similar to DM-mediated oxidative stress. These results are consistent with previous studies implying a pathological condition to pulp cell activity driven by high glucose levels and oxidative stress. Accordingly, these results contribute to considerable evidence for pulp cell pathogenesis from complicated diseases such as DM in cellular and molecular levels. In particular, oxidative stress in diabetes plays a role in the development of complications such as tissue damage, cell death leading to increased free radical production, and compromised free radical scavenger systems that exacerbated aging and inflammation (25). Hence, extensive studies of antioxidant therapies suggested success in the treatment of complications caused by diabetes (6, 18, 26). The natural compound davallialactone isolated from mushroom extract possesses an antioxidant capacity that improved dental pulpitis via the removal of ROS formation in HDPCs (11). Thus, we focused on davallialactone because it may be useful in the treatment of oral pathogenesis or oral diseases. The results of this study showed that davallialactone attenuated inflammatory molecules such as ICMA-1, VCAM-1, COX-2, and HO-1. In addition, davallialactone showed the recovered expression of angiogenic factors such as VEGF, EGF, and PDFG. Furthermore, dentin mineralization appears up-regulated via DMP-1, DSPP, BMP-2 and -7, and Runx-2 expression and ALP activity by davallialactone even with the long-term state of glucose oxidative stress. The mineralization of the extracellular matrix in vitro by alizarin red S stain for mineral deposition augmented the extent of mineralization to a higher degree in davallialactone. Similarly, NAC showed similar effects as davallialactone. The defense properties of antioxidants protect or recover dentin mineralization and pulp cell activity
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Basic Research—Biology (12, 27). Consequently, davallialactone may protect from pathological conditions and improve complicated diseases such as diabetes. In brief, treatment with glucose oxidase with a high glucose state increases the production of H2O2, cellular toxicity, and inflammatory molecules and disturbs pulp cell activity via dentin mineralization. These results can explain how the treatment of glucose oxidase with high glucose is a suitable pathological condition like the steady state of glucose oxidative stress and DM in pulp cells. Although this study is an in vitro study, this phenomenon should be kept in mind when patients with DM are treated with vital pulp therapy for healthy teeth. Also, it will aid in the understanding of changes at the cellular and molecular level by DM in pulp cells. Conversely, davallialactone prevented H2O2 production, cellular cytotoxicity, and ROS formation and suppressed inflammatory molecules and dentin mineralization disturbances. Currently, an ongoing study addresses the treatment of DM with antioxidant therapy (5,6,26). Therefore, davallialactone, a natural antioxidant, may be useful for the treatment of complicated oral diseases and the improvement of pulp vital therapy. Also, this study may increase the understanding of the causes of the development of oral complications related to irreversible damages by pathogenesis, leading to therapeutic strategies for pulp vitality.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Tranasi M, Sberna MT, Zizzari V, et al. Microarray evaluation of age-related changes in human dental pulp. J Endod 2009;35:1211–7. 2. Garber AJ. Vascular disease and lipids in diabetes. Med Clin North Am 1998;82: 931–48. 3. Manfredi M, McCullough MJ, Vescovi P, et al. Update on diabetes mellitus and related oral diseases. Oral Dis 2004;10:187–200. 4. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996;19:257–67. 5. Leite MF, De Lima A, Massuyama MM, et al. In vivo astaxanthin treatment partially prevents antioxidant alterations in dental pulp from alloxan-induced diabetic rats. Int Endod J 2010;43:959–67. 6. Amatyakul S, Chakraphan D, Chotpaibulpan S, et al. The effect of long-term supplementation of vitamin C on pulpal blood flow in streptozotocin-induced diabetic rats. Clin Hemorheol Microcirc 2003;29:313–9. 7. Grossi SG, Genco RJ. Periodontal disease and diabetes mellitus: a two-way relationship. Ann Periodontol 1998;3:51–61.
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8. Lalla E, Lamster IB, Stern DM, et al. Receptor for advanced glycation end products, inflammation, and accelerated periodontal disease in diabetes: mechanisms and insights into therapeutic modalities. Ann Periodontol 2001;6:113–8. 9. Bender IB, Seltzer S, Freedland J. The relationship of systemic diseases to endodontic failures and treatment procedures. Oral Surg Oral Med Oral Pathol 1963;16:1102–15. 10. Lull C, Wichers HJ, Savelkoul HF. Antiinflammatory and immunomodulating properties of fungal metabolites. Mediators Inflamm 2005;2:63–80. 11. Lee NH, Lee YH, Bhattari G, et al. Reactive oxygen species removal activity of davallialactone reduces lipopolysaccharide-induced pulpal inflammation through inhibition of the extracellular signal-regulated kinase 1/2 and nuclear factor kappa b pathway. J Endod 2011;37:491–5. 12. Lee YH, Lee NH, Bhattarai G, et al. Anti-inflammatory effect of pachymic acid promotes odontoblastic differentiation via HO-1 in dental pulp cells. Oral Dis 2012 Jul 7 [Epub ahead of print]. 13. Leite MF, Ganzerla E, Marques MM, et al. Diabetes induces metabolic alterations in dental pulp. J Endod 2008;34:1211–4. 14. Inagaki Y, Yoshida K, Ohba H, et al. High glucose levels increase osteopontin production and pathologic calcification in rat dental pulp tissues. J Endod 2010; 36:1014–20. 15. Garber SE, Shabahang S, Escher AP, et al. The effect of hyperglycemia on pulpal healing in rats. J Endod 2009;35:60–2. 16. Ma X, Liu L, Liu F, et al. Biocatalytically induced growth of gold nanoshells: using enzyme reaction for the controllable fabrication of nanomaterials. J Nanosci Nanotechnol 2012;12:870–8. 17. Halliwell B, Clement MV, Long LH. Hydrogen peroxide in the human body. FEBS Lett 2000;486:10–3. 18. Maritim AC, Sanders RA, Watkins JB 3rd. Diabetes, oxidative stress, and antioxidants: a review. J Biochem Mol Toxicol 2003;17:24–38. 19. Catanzaro O, Dziubecki D, Lauria LC, et al. Diabetes and its effects on dental pulp. J Oral Sci 2006;48:195–9. 20. Russell BG. The dental pulp in diabetes mellitus. Acta Pathol Microbiol Scand 1967; 70:319–20. 21. Chen NX, Duan D, O’Neill KD, et al. High glucose increases the expression of Cbfa1 and BMP-2 and enhances the calcification of vascular smooth muscle cells. Nephrol Dial Transplant 2006;21:3435–42. 22. Goga R, Chandler NP, Oginni AO. Pulp stones: a review. Int Endod J 2008;41: 457–68. 23. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–30. 24. Lee DH, Lim BS, Lee YK, et al. Effects of hydrogen peroxide (H2O2) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines. Cell Biol Toxicol 2006;22:39–46. 25. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 1991;40:405–12. 26. Dene BA, Maritim AC, Sanders RA, et al. Effects of antioxidant treatment on normal and diabetic rat retinal enzyme activities. J Ocul Pharmacol Ther 2005; 21:28–35. 27. Minamikawa H, Yamada M, Deyama Y, et al. Effect of N-acetylcysteine on rat dental pulp cells cultured on mineral trioxide aggregate. J Endod 2011;37: 637–41.
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Davallialactone Reduces Inflammation and Repairs Dentinogenesis on Glucose Oxidase–induced Stress in Dental Pulp Cells Young-Hee Lee, PhD,*‡ Go-Eun Kim, MS,*‡ Yong-Beom Song, DDS, PhD,† Usha Paudel, BS,* Nan-Hee Lee, MS,* Bong-Sik Yun, PhD,‡ Mi-Kyung Yu, DDS, PhD,† and Ho-Keun Yi, PhD* Abstract Introduction: The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. Methods: The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells’ vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. Results: The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. Conclusions: The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy. (J Endod 2013;39:1401–1406)
Key Words Davallialactone, dental pulp cells, dentin mineralization, diabetes mellitus, glucose oxidase, glucose oxidative stress, inflammation
D
ental pulp is a loose connective tissue consisting of various types of cells like ground substance and neural and vascular supplies. Damage to dental pulp can harm pulp vitality. The vitality of pulp during tissue homeostasis, after injury, and in aging is dependent on pulp cell activity. The decrease of pulp cell activity leads to pathological conditions including inflammation (1). Although many studies have examined the pathological condition of pulp cells, the pathogenesis is poorly understood. DM is a common metabolic disease characterized by hyperglycemia resulting from the insufficiency of insulin secretion or action. The chronic hyperglycemia of diabetes is associated with long-term systemic dysfunction (2). The presentation of DM is related to oral manifestations such as xerostomia and taste impairment, and its progression exacerbates various oral diseases such as dental caries, periodontal disease, soft-tissue lesions, and fungal infection (3). In addition, oxidative stress and inflammation are physiopathological mechanisms causing diabetes (4). The enzyme glucose oxidase catalyzes the oxidation of glucose to hydrogen peroxide and D-glucono-d-laltone. In cells, it aids in breaking the sugar down into its metabolites. However, the chronic hyperglycemia of diabetes elevates the production of free radicals by glucose oxidation. Therefore, it is assumed that the state of diabetes generates continuous glucose oxidase to manage glucose and then it reacts with D-glucose substrate to yield increasingly higher levels of hydrogen peroxide. DM alters the antioxidant system of dental pulp cells, but supplementation with antioxidant treatment (ie, astaxanthin) can restore the cells (5). In addition, longterm supplementation with vitamin C can markedly prevent the diabetes-induced reduction in pulp blood flow (6). Similarly, many studies have reported careful management of the association between diabetes and chronic periodontal disease (7, 8). Indeed, patients with poorly controlled DM have a low response to endodontic treatment (9). However, little information is available describing the pulp pathogenesis influenced by DM. Davallialactone, a hispidin analog from the mushroom Inonotus xeranticus belonging to the family Hymenochaetaceae, possesses immunosuppressive and potent anti-inflammatory activity via its antioxidant effect in pulp cells (10, 11). The purpose of this study was to evaluate the cellular state of dental pulp cells with pathogenic
From the *Department of Oral Biochemistry and Institute of Oral Bioscience, BK21 Program; †Department of Conservative Dentistry, School of Dentistry; and Division of Biotechnology, College of Environmental and Biosource Science, Chonbuk National University, Jeonju, Korea. Supported by a grant from the Korea Forest Service (grant no. S121010L090120). Young-Hee Lee and Go-Eun Kim contributed equally to this work. Address requests for reprints to Dr Ho-Keun Yi, Department of Oral Biochemistry, School of Dentistry, Chonbuk National University, 634-18, Deokjin-dong, Deokjingu, Jeonju, Jeonbuk 561-712, Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.06.033 ‡
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fluorescein diacetate (DCFH-DA) (Sigma-Aldrich). After a complete reaction, the cells were incubated with 5 mmol/L DCFH-DA for 30 minutes in the dark at 37 C, harvested, and followed by FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ).
Materials and Methods
Assay of ALP Activity After a complete reaction in 1, 7, and 15 days, pulp cells were scraped into cold PBS and then sonicated with a cell disruptor (Heat System-Ultrasonics, Plainview, NJ) in an ice-cold bath. Alkaline phosphatase (ALP) activity in the supernatant was determined according to the SensoLyte p-NPP Alkaline Phosphatase Assay Kit (AnaSpec, Inc, Fremont, CA) using a previously described method (11).
Materials Dexamethasone, b-glycerophosphate disodium salt hydrate, alizarin red S, glucose oxidase, L-ascorbic acid, D-glucose, and actin were purchased from Sigma-Aldrich (St Louis, MO). Antibodies against cyclooxygenase-2 (COX-2), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) were supplied by Cell Signaling (Beverly, MA). Antibodies against intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and heme oxygenase-1 (HO-1) were acquired from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Antibodies against BMP-2 and -7 and Rux-2 were purchased from Bioworld Technology, Inc (St Louis Park, MN). Davallialactone was extracted from the mushroom Inonotus xeranticus (Berk). Imaz ET Aoshi (Hymenochaetaceae) is a species of mushroom (11). Human Pulp Cell Culture and Induction of Glucose Oxidase–treated Oxidative Stress Human dental pulp cells (HDPCs) were isolated and cultured as previously reported (11). The cells at 80% confluence were seeded in 100-mm culture dishes (SPL Life Science, Gyeonggi-do, Korea) and subcultured at a 1:4 ratio. The experiments were performed using the HDPCs between the third and eighth passages. For induced glucose oxidative stress, 80% confluence cells were supplemented with 0.5% fetal bovine serum RPMI 1640 media (Gibco, Invitrogen, Grand Island, NY) before being exposed to various concentrations of glucose oxidase with D-glucose concentration for 15 days. For the glucose-free state, the cells were supplemented with 0.5% RPMI 1640 medium before the induction of glucose oxidative stress. For mineralization experiments, the cells were cultured in osteogenic media (OM) including 50 mg/mL ascorbic acid, 100 nmol/L dexamethasone, and 10 mmol/L b-glycerophosphate as previously described (12). MTT Assay The viability of the cultured cells was determined by examining the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. After a 24-hour incubation period, the cells (1 104/well) in 96-well plates were washed twice with phosphate-buffered saline (PBS). MTT (100 mg/0.1 mL PBS) was added to each well. The cells were incubated at 37 C for 3 hours, and dimethyl sulfoxide (DMSO) (100 mL) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm with an enzyme-linked immunosorbent assay reader (Synergy 2; Bio-TeK, Winooski, VT). Measurement of H2O2 and Reactive Oxygen Species Formation The quantity of H2O2 produced by glucose oxidase in culture medium at the indicated time was determined using the Biovision hydrogen peroxide assay kit (Biovision Research Products, Milpitas, CA) following the manufacturer’s instructions. The absorbance was measured at 570 nm with an enzyme-linked immunosorbent assay reader (Bio-TeK). The level of reactive oxygen species (ROS) formation was assessed using an oxidation-sensitive fluorescent probe 20 ,70 -dichlorodihydro1402
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Alizarin Red S Stain After 15 days of differentiation induction, the cells were rinsed with PBS, air dried, and fixed in ice-cold 95% ethanol for 30 minutes at 20 C. The cells were stained with 40 mmol/L alizarin red S (pH = 4.2) for 1 hour at room temperature, washed extensively 5 times with deionized water, and rinsed with PBS (without magnesium and calcium) for 15 minutes. Western Blot Analysis Western blot analysis was performed as previously described (11). The samples were separated by 8%–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under denaturing conditions and electroblotted onto nitrocellulose membranes. The membranes were incubated with the specific primary antibody and the horseradish peroxidase–conjugated secondary antibody. The signals were visualized by chemiluminescent detection according to the manufacturer’s protocol (Amersham Pharmacia Biotech, London, UK). The membranes were reprobed with antiactin antibody to confirm an equal protein loading. RNA Isolation and Reverse-transcription Polymerase Chain Reaction Total RNA was extracted from the cells by using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Then, 1 mg RNA was reverse transcribed for first-strand complementary DNA synthesis (Invitrogen, Carlsbad, CA). The complementary DNA was amplified by using rTaq Plus 5 PCR master mix (ELPis, Daejeon, Korea) and 1 mmol/L specific primers. The sequences of the specific primers used are human dentin sialophosphoprotein (hDSPP) (sense: 50 -TTCCGATGGGAGTCCTAGTG-30 , antisense: 50 -TGAGCTTCTGGG TGTCCTCT-30 ) and human dentin matrix protein1 (hDMP-1) (sense: 5-CAGGA GCACAGGAAAAGGAG-3, antisense: 5-CTGGTGGTATCTTCCCCCAGGAG-3). The reverse-transcription polymerase chain reaction products were electrophoresed on 1.5% agarose gel with ethidium bromide. Statistical Analysis At least 3 independent experiments were performed, and the mean value was used for further analysis. The statistical significance of the groups was assessed by analysis of variance and the Duncan test. Null hypotheses of no difference were rejected if P values were <.05.
Results Glucose Oxidase Generates H2O2 and Cellular Toxicity and Davallialactone Removes ROS Formation Davallialactone has anti-inflammatory properties through ROS removal activity in HDPCs (11). Accordingly, the antioxidant JOE — Volume 39, Number 11, November 2013
Basic Research—Biology characteristics of davallialactone were evaluated for the vitality of pulp cells against glucose oxidative stress. In this study, pulp cells were cultured in free glucose to high glucose (25 mmol/L). The glucose oxidase treatment continuously increased H2O2 at 24 hours in a glucose oxidase concentration–dependent manner (Fig. 1A). Furthermore, when exposed with low (5 mmol/L) and high (25 mmol/L) glucose with glucose oxidase significantly increased production of H2O2 at each glucose oxidase concentration–dependent manner (Fig. 1A). The effect of glucose oxidase with glucose concentration on cell viability was determined using the MTT assay. When steady state of high glucose, the production of H2O2 over 50 mmol/L after glucose oxidase (5 mU/mL) treated appeared to induce cellular toxicity at the indicated time (Fig. 1B). Furthermore, glucose oxidase in a high glucose state ensures increased ROS formation compared with 150 mmol/L H2O2 (Fig. 1C). However, davallialactone and N-acetylcysteine (NAC) protected cellular cytotoxicity and ROS formation against glucose oxidase–induced oxidative stress (Fig. 1B and D).
Effect of Davallialactone on Odontoblast Mineralization against Glucose Oxidase–induced Oxidative Stress To identify odontoblast mineralization by davallialactone under glucose oxidase treatment, we analyzed the ALP activity and alizarin red S stain in HDPCs. Pulp cells treated with glucose (25 mmol/L) alone markedly raised ALP activity, whereas cotreatment with glucose oxidase and high glucose gradually reduced ALP activity more than control cells. However, the antioxidants davallialactone and NAC increased ALP activity even under glucose oxidase stress on the indicated day (Fig. 4A). Next, we performed the formation of mineralization by davallialactone using alizarin red S stain. Pulp cells were cultured in the presence or absence of davallialactone with glucose oxidase and high glucose containing OM for a 15-day period. Cotreatment with glucose oxidase and high glucose in pulp cells lowered the extent of mineralizationlike, noncontaining OM mock cells. However, the presence of davallialactone and NAC augmented the extent of mineralization to a higher degree even with glucose oxidase–induced stimuli (Fig. 4B).
Glucose Oxidase Disturbs Inflammation and Dentinogenesis, but Davallialactone Helps Recovery from Glucose Oxidase–induced Oxidative Stress To determine whether oxidative stress by glucose oxidase with high glucose was responsible for inflammatory molecules and dentinogenesis-related molecules, pulp cells were treated with glucose oxidase (5 mU/mL) for 15 days in a high glucose (25 mmol/L) state. The duration of glucose oxidase treatment on the indicated day revealed a gradual aggravation of inflammatory molecules (ICAM-1, VCAM-1, Cox-2, and HO-1; Fig. 2A), angiogenesis molecules (VEGF, EGF, and PDGF; Fig. 2B), dentinogenesis messenger RNA levels (DMP-1 and DSPP; Fig. 2C), and protein expression (BMP-2 and -7 and Runx-2; Fig. 2D). However, davallialactone in the same condition reduces inflammatory action (Fig. 3A), induces angiogenesis expression (Fig. 3B), and recovers dentinogenesis molecule expression of messenger RNA and protein levels (Fig. 3C and D).
The vitality of the pulp complex is dependent on the activity of the pulp cells. Thus, pathogenesis to decrease pulp cell activity is better described for healthy teeth. Recently, studies of pulp complex have focused on DM as well. DM is a complicated disease resulting in ROS production in the oral cavity (3, 13). The effects of diabetes on the metabolism of dental pulp tissue have already been studied in an in vivo system (13–15). However, the response of pulp cells to glucose oxidase with high glucose state–induced stress is unknown. Thus, we showed that the generation of H2O2 by glucose oxidase in the presence of substrate glucose may act to stimulate the decline of pulp cell activity and the response to pathological conditions including inflammation. Also, this study helps to understand the associated pathogenesis and elucidated therapeutic strategy to oral disease using an antioxidant like davallialactone for pulp vitality.
Discussion
Figure 1. The determination of H2O2 and ROS produced by glucose oxidase, the protection of cellular toxicity, and the removal of ROS formation by davallialactone in HDPCs. (A) The cells were exposed to glucose oxidase (5 and 10 mU/mL) with D-glucose (5 and 25 mmol/L) and the amount of H2O2 in the culture medium was measured at 24 hours. The cells were pretreated with davallialactone (10 mmol/L) and NAC (1 mmol/L) before glucose oxidase exposed with D-glucose in HDPCs. (B) The cell viability was determined using an MTT assay at the indicated time. (C and D) The level of ROS formation was determined by flow cytometry after DCFHDA treatment at 24 hours. Each value is reported as the mean and standard error of the mean of 3 independent experiments.
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Figure 2. The influence effect of glucose oxidase on the expression of inflammatory and dentinogenesis in HDPCs. The cells were exposed to 5 mU/mL glucose oxidase with a high glucose level and to continuously expose oxidative stress and allow more treated glucose oxidase at interval of 3 days for 15 days. (A) The expression of inflammatory molecules such as ICAM-1, VCAM-1, and COX-2 and the HO-1 protein level in the total cell lysate was determined by Western blot analysis at the indicated time. (B) Angiogenic factors such as VEGF and EGF and the PDGF protein level were determined by using Western blot analysis. (C) Odontoblast differentiation markers such as DMP-1 and DSPP messenger RNA levels were determined by using reverse-transcription polymerase chain reaction using a specific primer. (D) Osteoblast differentiation markers such as BMP-2 and -7 and Runx-2 were determined by Western blot analysis. For loading control, the blots were reprobed with actin antibody. All data are representative of 3 separate experiments.
In this study, the treatment of glucose oxidase in pulp cells revealed the production of H2O2 in the glucose-free state at 24 hours. However, this dose did not affect oxidative stress. Interestingly, the state of high glucose with glucose oxidase increased H2O2 production more compared with glucose oxidase alone. In general, glucose oxidase
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Figure 3. The effect of davallialactone on the expression of inflammatory and dentinogenesis-related molecule expression by glucose oxidase treatment. The cells were pretreated with davallialactone (10 mmol/L) before glucose oxidase exposed with high glucose to continuously expose oxidative stress and allow treatment with davallialactone at an interval of 3 days for 15 days. (A) The expression of inflammatory molecules such as ICAM-1, VCAM-1, COX-2 and the HO-1 protein level in the total cell lysate were determined by using Western blot analysis at the indicated time. (B) Angiogenic factors such as VEGF and EGF and the PDGF protein level were determined by using Western blot analysis. (C) Odontoblast differentiation markers such as DMP-1 and DSPP messenger RNA levels were determined by reverse-transcription polymerase chain reaction using a specific primer. (D) Osteoblast differentiation markers such as BMP-2 and -7 and Runx-2 were determined by using Western blot analysis. For loading control, the blots were reprobed with actin antibody. All data are representative of 3 separate experiments.
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Figure 4. The effect of davallialactone on odontoblast mineralization against glucose oxidase stress. To perform odontoblast mineralization, the cells were pretreated with davallialactone before glucose oxidase exposed with or without glucose concentration to continuously expose oxidative stress and allow treatment with davallialactone at an interval of 3 days for 15 days. Mineralization was determined by (A) ALP activity at the indicated time and (B) alizarin red S stain at 15 days. For alizarin red S stain, cells were cultured in OM as described in the Methods section. Each value is reported as the mean and standard error of the mean of 3 independent experiments.
investigated the state of oxidative stress via the production of H2O2 by means of glucose oxidase. H2O2 is an index of oxidative stress to mediators of cell damage that cause damage to cellular proteins, membrane lipids, and DNA and lead to cell death (17, 18). In this study, H2O2 production by glucose oxidase with a high glucose state exacerbated the cell viability in pulp cells. In addition, even at the same concentration of glucose oxidase, ROS formation was accumulated by means of glucose concentration in pulp cells. Thus, this phenomenon may show the steady state of oxidative stress in pulp cells, which is similar to diabetes-mediated oxidative stress. Also, these results suggested that DM influences pathological conditions damaging pulp vitality via oxidative stress. However, the presence of davallialactone and NAC as antioxidants significantly protects against cellular toxicity under those conditions. Additionally, davallialactone is able to remove ROS formation like NAC. The overproduction of ROS can be attenuated by antioxidant enzymes via enhanced drug treatment (5, 18). Thus, the antioxidant effect of davallialactone and NAC might be enhanced by an oxidative stress defense enzyme against glucose oxidative stress for pulp cell activity. Therefore, it is able to improve the pathogenesis of complicated diseases such as diabetes in pulp cells. The chronic nature of diabetes causes irreversible damage and induces metabolic alteration by oxidative stress in the dental pulp leading to inflammation (3, 13, 18, 19). In addition, in uncontrolled or poorly controlled diabetes, it affects impaired angiopathy and a thickened basement membrane in dental pulp vessels and a reduction in pulpal blood flow in streptozotocin-induced diabetic rats (6, 20). The high glucose state revealed an increase of ALP activity in rat pulp cells and vascular smooth muscle cells that appear with pathological conditions including DM (14, 21, 22). In this study, long-term exposure to glucose oxidase with a high glucose state in pulp cells resulted in the accelerated induction of inflammatory molecules such as ICAM-1, VCAM-1, COX-2, and HO-1. Also, this study found a decreased expression of angiogenic factors such as VEGF, FGF, and PDGF in the same conditions. In particular, the popularly used bimolecular markers of odontoblast and osteoblast differentiation in dental JOE — Volume 39, Number 11, November 2013
pulp cells DMP-1, DSPP, BMP-2 and -7, and Runx-2 attenuated the expression of messenger RNA and the protein level. They are crucial in dentin mineralization (23). Moreover, a high glucose level state significantly accelerates ALP activity compared with the glucose-free state over a long duration. On the contrary, the state of oxidative stress by glucose oxidase decreased ALP activity. Many studies in pulp cells reported that long-term oxidative conditions also disturb dentin mineralization via ALP activity (12, 24). Therefore, these results suggest that the long-term treatment of glucose oxidase with a high glucose state produces a state of oxidative stress similar to DM-mediated oxidative stress. These results are consistent with previous studies implying a pathological condition to pulp cell activity driven by high glucose levels and oxidative stress. Accordingly, these results contribute to considerable evidence for pulp cell pathogenesis from complicated diseases such as DM in cellular and molecular levels. In particular, oxidative stress in diabetes plays a role in the development of complications such as tissue damage, cell death leading to increased free radical production, and compromised free radical scavenger systems that exacerbated aging and inflammation (25). Hence, extensive studies of antioxidant therapies suggested success in the treatment of complications caused by diabetes (6, 18, 26). The natural compound davallialactone isolated from mushroom extract possesses an antioxidant capacity that improved dental pulpitis via the removal of ROS formation in HDPCs (11). Thus, we focused on davallialactone because it may be useful in the treatment of oral pathogenesis or oral diseases. The results of this study showed that davallialactone attenuated inflammatory molecules such as ICMA-1, VCAM-1, COX-2, and HO-1. In addition, davallialactone showed the recovered expression of angiogenic factors such as VEGF, EGF, and PDFG. Furthermore, dentin mineralization appears up-regulated via DMP-1, DSPP, BMP-2 and -7, and Runx-2 expression and ALP activity by davallialactone even with the long-term state of glucose oxidative stress. The mineralization of the extracellular matrix in vitro by alizarin red S stain for mineral deposition augmented the extent of mineralization to a higher degree in davallialactone. Similarly, NAC showed similar effects as davallialactone. The defense properties of antioxidants protect or recover dentin mineralization and pulp cell activity
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Basic Research—Biology (12, 27). Consequently, davallialactone may protect from pathological conditions and improve complicated diseases such as diabetes. In brief, treatment with glucose oxidase with a high glucose state increases the production of H2O2, cellular toxicity, and inflammatory molecules and disturbs pulp cell activity via dentin mineralization. These results can explain how the treatment of glucose oxidase with high glucose is a suitable pathological condition like the steady state of glucose oxidative stress and DM in pulp cells. Although this study is an in vitro study, this phenomenon should be kept in mind when patients with DM are treated with vital pulp therapy for healthy teeth. Also, it will aid in the understanding of changes at the cellular and molecular level by DM in pulp cells. Conversely, davallialactone prevented H2O2 production, cellular cytotoxicity, and ROS formation and suppressed inflammatory molecules and dentin mineralization disturbances. Currently, an ongoing study addresses the treatment of DM with antioxidant therapy (5,6,26). Therefore, davallialactone, a natural antioxidant, may be useful for the treatment of complicated oral diseases and the improvement of pulp vital therapy. Also, this study may increase the understanding of the causes of the development of oral complications related to irreversible damages by pathogenesis, leading to therapeutic strategies for pulp vitality.
Acknowledgments The authors deny any conflicts of interest related to this study.
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