doi:10.1111/iej.12312
Catechins inhibit vascular endothelial growth factor production and cyclooxygenase-2 expression in human dental pulp cells
T. Nakanishi, K. Mukai, Y. Hosokawa, D. Takegawa & T. Matsuo Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
Abstract Nakanishi T, Mukai K, Hosokawa Y, Takegawa D, Matsuo T. Catechins inhibit vascular endothelial growth factor production and cyclooxygenase-2 expression in human dental pulp cells. International Endodontic Journal.
Aim To investigate the effect of catechins on vascular endothelial growth factor (VEGF) production and cyclooxygenase-2 (COX-2) expression in human dental pulp cells (HDPC) stimulated with bacteria-derived factors or pro-inflammatory cytokines. Methodology Morphologically fibroblastic cells established from explant cultures of healthy human dental pulp tissues were used as HDPC. HDPC pretreated with catechins, epigallocatechin-3-gallate (EGCG) or epicatechin gallate (ECG), were exposed to lipopolysaccharide (LPS), peptidoglycan (PG), interlukin-1b (IL-1b) or tumour necrosis factor-a (TNF-a). VEGF production was examined by enzyme-linked immunosorbent assay, and COX-2 expression was assessed by immunoblot.
Introduction Dental caries and subsequent tooth pulp inflammation are caused by bacterial infection, and increased expression of various inflammatory mediators including growth factors (Ohnishi et al. 2000, Artese et al. 2002) and prostaglandins (Cohen et al. 1985, Nakanishi et al. 1995, Miyauchi et al. 1996) are
Correspondence: Tadashi Nakanishi, Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan (e-mail: tnakanishi@tokushima-u. ac.jp).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Results EGCG and ECG significantly reduced LPS- or PG-mediated VEGF production in the HDPC in a dosedependent manner. EGCG also prevented IL-1b-mediated VEGF production. Although TNF-a did not enhance VEGF production in the dental pulp cells, treatment of 20 lg mL 1 of EGCG decreased the level of VEGF. In addition, the catechins attenuated COX-2 expression induced by LPS and IL-1b. Conclusions The up-regulated VEGF and COX-2 expressions in the HDPC stimulated with these bacteria-derived factors or IL-1b were diminished by the treatment of EGCG and ECG. These findings suggest that the catechins may be beneficial as an antiinflammatory tool of the treatment for pulpal inflammation. Keywords: catechin, cyclooxygenase-2, dental pulp, vascular endothelial growth factor. Received 21 September 2013; accepted 18 May 2014
reported. Vascular endothelial growth factor (VEGF), glycoprotein that has the ability to increase the permeability of blood vessels, induces angiogenesis and is normally expressed in the dental pulp (Artese et al. 2002). It has been demonstrated that the increase of VEGF expression is consistent with enhanced vascularization during dental pulp inflammation (TranHung et al. 2006). The dental pulp cells can also produce VEGF in response to lipopolysaccharide (LPS; Matsushita et al. 1999) and pro-inflammatory cytokines such as interleukin (IL)-1 (Chu et al. 2004). These previous reports suggested that dental pulp cells play a role in the aetiology of pulpitis through the production of VEGF.
International Endodontic Journal
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Effect of catechin on pulp cells Nakanishi et al.
Prostaglandins are important mediators of inflammation, the synthesis of which is initiated by release of arachidonic acid from cell membranes. Cyclooxygenases are the key and rate-limiting step in the biosynthesis of prostanoid. In particular, cyclooxygenase-2 (COX-2), which is inducible with pro-inflammatory cytokines and is considered an ‘inflammatory’ isoform (Smith & Dewitt 1996), has been shown to play an important role in the pathogenesis of pulpal inflammation (Nakanishi et al. 2001). Recent evidence has demonstrated that COX-2-transfected cells exhibit enhanced expression of VEGF (Tsujii et al. 1997). Interestingly, an immunohistological study on expression of COX-2 and VEGF in inflamed dental pulp also suggests a possible induction of VEGF via a COX-2 dependent pathway (G€ uven et al. 2007). Catechins are present in various species of plant such as green tea, and they have beneficial properties on human health. ( )-Epigallocatechin-3-gallate (EGCG) and ( )-epicatechin-3-gallate (ECG) are representative constituents of catechins, and some studies reported that these catechins inhibited several proteins involved in inflammation through the activation of nuclear factor kappa B (NF-jB; Lin & Lin 1997, Yang et al. 2001). In particular, it has been shown that EGCG attenuates VEGF production, COX-2 expression and NF-jB activation induced by inflammatory cytokines in human astrocytoma cells (Kim et al. 2007). Previous reports have indicated that the catechins EGCG and ECG inhibited the production of pro-inflammatory cytokines such as IL-6 and IL-8, and the expression of adhesion molecules in dental pulp cells treated with various bacteria-related factors (Nakanishi et al. 2010). However, there are no reports of the effects of catechins on a behaviour of VEGF related to COX-2 in dental pulp inflammation. The aim of this study was to evaluate the effect of catechins on expression of VEGF, and COX-2 protein as well in human dental pulp cells (HDPC) stimulated with bacteria-related factors (LPS or peptidoglycan (PG)) or pro-inflammatory cytokines. The null hypothesis is that catechins do not affect the expressions of VEGF and COX-2 in HDPC.
were obtained from Peprotech (Rocky Hill, NJ, USA). Antibodies against COX-2 and actin were purchased from Cayman chemical (Ann Arbor, MI, USA) and Sigma-Aldrich, respectively.
Cell culture and treatment Human dental pulp cells were established from explant cultures of healthy pulp tissues as described previously (Adachi et al. 2007). In brief, clinically healthy pulp tissue samples (from 22- to 30-year old patients; n = 3) were obtained from noncarious teeth extracted for orthodontic reasons under informed consent at Tokushima University Hospital. The minced pieces of pulp tissues were seeded into 35-mm culture dishes and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 1 mmol sodium pyruvate (Gibco) and 100 U mL 1 penicillin/100 lg mL 1 streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO2. Confluent primary cultures were harvested and subcultured. Morphologically fibroblastic cells obtained in this manner were used as HDPC for experiments at passages four to ten. This study was performed with the approval and compliance of the Tokushima University Ethical Committee (No.329). The experiments were undertaken with the understanding and written consent of each subject and in full accordance with ethical principles, including the World Medical Association Declaration of Helsinki. Human dental pulp cells were seeded in wells of 24-well tissue culture plates and incubated until a confluent monolayer developed (5 9 104 cells/well). The medium was then replaced with DMEM containing 2% FBS. To study the effects of catechins on VEGF production and COX-2 expression, HDPC were pretreated with or without catechins in 2% FBS-containing DMEM for 1 h and then incubated with LPS (1 lg mL 1), PG (10 lg mL 1), IL-1b (10 ng mL 1) or TNF-a (10 ng mL 1) for the designated times (24 h). After incubation, culture supernatants were collected and used to determine the quantities of VEGF, and attached cells were used for protein extraction.
Materials and methods Enzyme-linked immunosorbent assay Reagents Staphylococcus aureus PG, Escherichia coli LPS and catechins (EGCG and ECG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). IL-1b and TNF-a
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The concentration of VEGF in the cell culture supernatants was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kit (Peprotech).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Nakanishi et al. Effect of catechin on pulp cells
Results
Protein extraction and Western blot analysis After the treatment with catechins and various factors described above, HDPC were washed twice with icecold phosphate-buffered saline and then collected with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein concentrations in lysates were quantified with a bicinchoninic acid protein assay kit (Sigma-Aldrich). Equal amount of protein samples were then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes. The membrane was first incubated with a primary antibody for COX-2. After washing, the membrane was reacted with a specific horseradish peroxidaseconjugated secondary antibody (Dako Corporation, Carpinteria, CA, USA). Protein bands were finally visualized on X-ray film using the enhanced chemiluminescence system (GE Healthcare, Little Chalfont, UK). An equal loading of gels was confirmed by immunoblot with anti-actin antibody as an internal control and an appropriate secondary antibody (SigmaAldrich).
Effects of catechins on the VEGF production in HDPC in response to bacteria-derived factors and pro-inflammatory cytokines To demonstrate the potential of catechins on HDPC stimulated with bacterial components or pro-inflammatory cytokines, the effect of EGCG or ECG on VEGF expression in HDPC exposed to LPS, PG, IL-1b or TNF-a using ELISA was examined. Treatment with EGCG effectively inhibited VEGF production in bacterial component-stimulated HDPC in a concentrationdependent manner (P < 0.05; Fig. 1a). ECG also caused a significant inhibition of LPS- or PG-induced VEGF secretion (P < 0.05; Fig. 1b). Up-regulated VEGF production in HDPC incubated with IL-1b was also inhibited by EGCG (P < 0.05; Fig. 2). Although TNF-a did not affect VEGF production, the treatment of 20 lg mL 1 of EGCG decreased the level of VEGF in dental pulp cells (P < 0.05; Fig. 2).
Effects of catechins on the COX-2 induction in HDPC stimulated with LPS or IL-1b Statistical analysis
In order to clarify whether catechins inhibit the induction of COX-2 in HDPC, Western blot analysis was used to examine the effect of EGCG or ECG on COX-2 expression in HDPC exposed to LPS or IL-1b.
Data are expressed as the mean SD. All statistical analyses were performed using unpaired Student’s t-test.
(a)
(b)
Figure 1 Effect of epigallocatechin-3-gallate (EGCG) (a) or epicatechin-3-gallate (ECG) (b) pre-treatment (1 h) on vascular endothelial growth factor (VEGF) production in human dental pulp cells stimulated with lipopolysaccharide (LPS; 1 lg mL 1) or peptidoglycan (PG; 10 lg mL 1) for 24 h. The data are expressed as the mean and standard deviation of triplicate cultures from three independent experiments. *P < 0.05 compared with the control (no catechin pre-treatment).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
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Effect of catechin on pulp cells Nakanishi et al.
Up-regulated COX-2 expression on HDPC stimulated with LPS was diminished by the treatment of EGCG or ECG (Fig. 3). In addition, treatment with EGCG or
ECG inhibited the induction of COX-2 expression in HDPC exposed to IL-1b (Fig. 4).
Discussion
Figure 2 Effect of epigallocatechin-3-gallate (EGCG) pretreatment (1 h) on vascular endothelial growth factor (VEGF) expression in human dental pulp cells stimulated with interleukin-1b (IL-1 b; 10 ng mL 1) or tumour necrosis factor-a (TNF-a; 10 ng mL 1) for 24 h. The data are expressed as the mean and standard deviation of triplicate cultures from three independent experiments. *P < 0.05 compared with the control (no catechin pre-treatment).
In the present study, catechins were shown to inhibit up-regulated expression of VEGF in HDPC stimulated with LPS or PG. In the progression of pulpal disease, the vasculature is affected (Tran-Hung et al. 2006). HDPC express VEGF in both healthy and inflamed situations, such as irreversible pulpitis, a role for dental pulp cells in angiogenesis (Artese et al. 2002). Moreover, it has been shown that the production of VEGF is enhanced in dental pulp cells in response to pathogen-associated molecular patterns (PAMPs) including LPS in vitro (Matsushita et al. 1999). The innate immune system senses microbial infection using pattern recognition receptors (PRRs) that recognize PAMPs. Toll-like receptors (TLRs) generally acts as PRRs; TLR4 is involved in the recognition of most bacterial LPS, and TLR2 recognizes a wide range of PAMPs including PG of Gram-positive bacteria (Takeda & Akira 2005). It has been shown that human dental pulp fibroblasts responds to TLR2- and TLR4-specific agonists (Staquet et al. 2008). A previous study has also shown that TLR2 and TLR4 expressed in HDPC function to up-regulate the expression of various pro-inflammatory mediators such as
(a) (a)
(b) (b)
Figure 3 Effect of epigallocatechin-3-gallate (EGCG) (a) or epicatechin-3-gallate (ECG) (b) pre-treatment (1 h) on cyclooxygenase-2 (COX-2) expression in human dental pulp cells stimulated with lipopolysaccharide (LPS; 1 lg mL 1) for 24 h. Equal loading of gels was confirmed by Western blot with anti-actin antibody. Data are representative of two independent experiments with similar results.
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Figure 4 Effect of epigallocatechin-3-gallate (EGCG) (a) or epicatechin-3-gallate (ECG) (b) pre-treatment (1 h) on cyclooxygenase-2 (COX-2) expression in human dental pulp cells stimulated with interleukin-1b (IL-1 b; 10 ng mL 1) for 24 h. Equal loading of gels was confirmed by Western blot with anti-actin antibody. Data are representative of two independent experiments with similar results.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Nakanishi et al. Effect of catechin on pulp cells
IL-6 and IL-8 (Hirao et al. 2009). In addition, it has been reported that the EGCG and ECG inhibited the production of IL-6 and IL-8 in the dental pulp cells treated with LPS and PG (Nakanishi et al. 2010). Therefore, these catechins might exhibit an antiinflammatory effect on pulpal inflammation through the inhibition of VEGF as well as pro-inflammatory cytokines. Consistent with the present observation, it has been reported that angiogenesis can be inhibited by EGCG through blocking the induction of VEGF in human carcinoma cells (Jung et al. 2001). Other studies also found that VEGF was inhibited by EGCG-treated human cervical carcinoma cells (Zhang et al. 2006), hepatoma cells (Zhang et al. 2006) and human nasal polyp fibroblasts (Lin et al. 2008). Thus, the present study and previous reports suggest that catechins are able to modulate the angiogenesis by their anti-angiogenic activities. On the other hand, it has been reported that EGCG enhanced prostaglandin F2ainduced VEGF synthesis in osteoblast cell line (Tokuda et al. 2007). This discrepancy might reflect differences of cell type and stimulator. It was also observed that EGCG reduced the enhanced-level of VEGF in HDPC in response to IL-1b. However, TNF-a failed to induce the increase of VEGF production. A previous immunohistochemistry study has shown that IL-1 and TNF-a-positive cells were localized in pulpitis lesions, and thus, they are known to be involved in pulpal inflammation (Tani-Ishii et al. 1995). These cytokines were employed to check the effect of catechins on VEGF secretion from HDPC. Chu et al. (2004) demonstrated that not only IL-1 but also TNF-a induced VEGF gene expression in human pulp fibroblasts. This is in contradiction to the present results, which displayed no significant induction of VEGF production by TNFa. This fact may be explained by the difference of analysis level. It has been reported that a statistically significant difference between normal and inflamed pulp was found in the concentration of prostaglandin E2, and COX-2 was mainly expressed in fibroblasts from inflamed pulp tissues (Nakanishi et al. 1995, 2001). These findings indicate that pulpal fibroblasts may participate in prostaglandin E2 production through COX-2 expression in pulpal inflammation, and might be involved in the pathogenesis of irreversible pulpitis. In this study, EGCG and ECG reduced induction of COX-2 protein in HDPC in response to bacteria-related factors or pro-inflammatory cytokines, suggesting that these catechins may mitigate pulpal inflammation. Other
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
recent reports demonstrated that VEGF expression is up-regulated via a COX-2-dependent mechanism, and COX-2-derived prostaglandin E2 has been found to promote angiogenesis (Tsujii et al. 1997, Nie & Honn 2002). Interestingly, a previous report has shown that EGCG attenuates both VEGF production and COX-2 expression through regulation of the NF-jB activation, and phosphorylation of MAPK induced by inflammatory cytokines in human astrocytoma cells (Kim et al. 2007). Previous reports also demonstrated that the inhibitory effect of catechins on pro-inflammatory mediator expression with PAMPs is mediated by blocking the MAPK pathway with subsequent inhibition of NF-jB activity in HDPC (Hirao et al. 2010). Therefore, the findings in this study might be revealed by an antiinflammatory activity of catechins. This research was conceived with the intention of applying the catechins to the surface of exposed pulp. The present results showed that 20 lg mL 1 and 50 lg mL 1 catechins significantly suppressed both VEGF and COX-2 expression. These concentrations of catechins are found to have no cytotoxicity for HDPC in a previous report (Hirao et al. 2010). However, further investigation is necessary to test the use of catechins for application to the exposed pulp in clinical therapies such as pulp capping.
Conclusion Catechins inhibited the expression of VEGF and COX-2 in HDPC treated with PAMPs and IL-1b. These findings suggest that catechins may be useful as anti-inflammatory modulator of pulpal inflammation.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 20592228, 24592871 [Grant-in-Aid for Scientific Research (C)].
References Adachi T, Nakanishi T, Yumoto H et al. (2007) Cariesrelated bacteria and cytokines induce CXCL10 in dental pulp. Journal of Dental Research 86, 1217–22. Artese L, Rubini C, Ferrero G et al. (2002) Vascular endothelial growth factor (VEGF) expression in healthy and inflamed human dental pulps. Journal of Endodontics 28, 20–3. Chu SC, Tsai CH, Yang SF et al. (2004) Induction of vascular endothelial growth factor gene expression by
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proinflammatory cytokines in human pulp and gingival fibroblasts. Journal of Endodontics 30, 704–7. Cohen JS, Reader A, Fertel R, Beck M, Meyers WJ (1985) A radioimmunoassay determination of the concentrations of prostaglandins E2 and F2 alpha in painful and asymptomatic human dental pulps. Journal of Endodontics 11, 330–5. G€ uven G, Altun C, Gunhan O et al. (2007) Co-expression of cyclooxygenase-2 and vascular endothelial growth factor in inflamed human pulp: an immunohistochemical study. Journal of Endodontics 33, 18–20. Hirao K, Yumoto H, Takahashi K et al. (2009) Roles of TLR2, TLR4, NOD2, and NOD1 in pulp fibroblasts. Journal of Dental Research 88, 762–7. Hirao K, Yumoto H, Nakanishi T et al. (2010) Tea catechins reduce inflammatory reactions via mitogen-activated protein kinase pathways in toll-like receptor 2 ligand-stimulated dental pulp cells. Life Sciences 86, 654–60. Jung YD, Kim MS, Shin BA et al. (2001) EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma cells. British Journal of Cancer 84, 844–50. Kim SJ, Jeong HJ, Lee KM et al. (2007) Epigallocatechin-3gallate suppresses NF-kappaB activation and phosphorylation of p38 MAPK and JNK in human astrocytoma U373MG cells. The Journal of Nutritional Biochemistry 18, 587–96. Lin YL, Lin JK (1997) (–)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-kappaB. Molecular Pharmacology 52, 465–72. Lin SK, Shun CT, Kok SH et al. (2008) Hypoxia-stimulated vascular endothelial growth factor production in human nasal polyp fibroblasts: effect of epigallocatechin-3-gallate on hypoxia-inducible factor-1 alpha synthesis. Archives of Otolaryngology–Head & Neck Surgery 134, 522–7. Matsushita K, Motani R, Sakuta T et al. (1999) Lipopolysaccharide enhances the production of vascular endothelial growth factor by human pulp cells in culture. Infection and Immunity 67, 1633–9. Miyauchi M, Takata T, Ito H et al. (1996) Immunohistochemical demonstration of prostaglandins E2, F2 alpha, and 6-keto-prostaglandin F1 alpha in rat dental pulp with experimentally induced inflammation. Journal of Endodontics 22, 600–2. Nakanishi T, Matsuo T, Ebisu S (1995) Quantitative analysis of immunoglobulins and inflammatory factors in human pulpal blood from exposed pulps. Journal of Endodontics 21, 131–6.
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Nakanishi T, Shimizu H, Hosokawa Y, Matsuo T (2001) An immunohistological study on cyclooxygenase-2 in human dental pulp. Journal of Endodontics 27, 385–8. Nakanishi T, Mukai K, Yumoto H et al. (2010) Anti-inflammatory effect of catechin on cultured human dental pulp cells affected by bacteria-derived factors. European Journal of Oral Sciences 118, 145–50. Nie D, Honn KV (2002) Cyclooxygenase, lipoxygenase and tumor angiogenesis. Cellular and Molecular Life Sciences: CMLS 59, 799–807. Ohnishi T, Suwa M, Oyama T et al. (2000) Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation. Journal of Dental Research 79, 748–55. Smith WL, Dewitt DL (1996) Prostaglandin endoperoxide H synthases-1 and -2. Advances in Immunology 62, 167–215. Staquet MJ, Durand SH, Colomb E et al. (2008) Different roles of odontoblasts and fibroblasts in immunity. Journal of Dental Research 87, 256–61. Takeda K, Akira S (2005) Toll-like receptors in innate immunity. International Immunology 17, 1–14. Tani-Ishii N, Wang CY, Stashenko P (1995) Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure. Oral Microbiology and Immunology 10, 213–9. Tokuda H, Takai S, Matsushima-Nishiwaki R et al. (2007) (–)-epigallocatechin gallate enhances prostaglandin F2alpha-induced VEGF synthesis via upregulating SAPK/ JNK activation in osteoblasts. Journal of Cellular Biochemistry 100, 1146–53. Tran-Hung L, Mathieu S, About I (2006) Role of human pulp fibroblasts in angiogenesis. Journal of Dental Research 85, 819–23. Tsujii M, Kawano S, DuBois RN (1997) Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proceedings of the National Academy of Sciences of the United States of America 94, 3336–40. Yang F, Oz HS, Barve S et al. (2001) The green tea polyphenol (–)-epigallocatechin-3-gallate blocks nuclear factorkappa B activation by inhibiting I kappa B kinase activity in the intestinal epithelial cell line IEC-6. Molecular Pharmacology 60, 528–33. Zhang Q, Tang X, Lu Q et al. (2006) Green tea extract and (–)-epigallocatechin-3-gallate inhibit hypoxia- and seruminduced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells. Molecular Cancer Therapeutics 5, 1227–38.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Catechins inhibit vascular endothelial growth factor production and cyclooxygenase-2 expression in human dental pulp cells
T. Nakanishi, K. Mukai, Y. Hosokawa, D. Takegawa & T. Matsuo Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
Abstract Nakanishi T, Mukai K, Hosokawa Y, Takegawa D, Matsuo T. Catechins inhibit vascular endothelial growth factor production and cyclooxygenase-2 expression in human dental pulp cells. International Endodontic Journal.
Aim To investigate the effect of catechins on vascular endothelial growth factor (VEGF) production and cyclooxygenase-2 (COX-2) expression in human dental pulp cells (HDPC) stimulated with bacteria-derived factors or pro-inflammatory cytokines. Methodology Morphologically fibroblastic cells established from explant cultures of healthy human dental pulp tissues were used as HDPC. HDPC pretreated with catechins, epigallocatechin-3-gallate (EGCG) or epicatechin gallate (ECG), were exposed to lipopolysaccharide (LPS), peptidoglycan (PG), interlukin-1b (IL-1b) or tumour necrosis factor-a (TNF-a). VEGF production was examined by enzyme-linked immunosorbent assay, and COX-2 expression was assessed by immunoblot.
Introduction Dental caries and subsequent tooth pulp inflammation are caused by bacterial infection, and increased expression of various inflammatory mediators including growth factors (Ohnishi et al. 2000, Artese et al. 2002) and prostaglandins (Cohen et al. 1985, Nakanishi et al. 1995, Miyauchi et al. 1996) are
Correspondence: Tadashi Nakanishi, Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan (e-mail: tnakanishi@tokushima-u. ac.jp).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Results EGCG and ECG significantly reduced LPS- or PG-mediated VEGF production in the HDPC in a dosedependent manner. EGCG also prevented IL-1b-mediated VEGF production. Although TNF-a did not enhance VEGF production in the dental pulp cells, treatment of 20 lg mL 1 of EGCG decreased the level of VEGF. In addition, the catechins attenuated COX-2 expression induced by LPS and IL-1b. Conclusions The up-regulated VEGF and COX-2 expressions in the HDPC stimulated with these bacteria-derived factors or IL-1b were diminished by the treatment of EGCG and ECG. These findings suggest that the catechins may be beneficial as an antiinflammatory tool of the treatment for pulpal inflammation. Keywords: catechin, cyclooxygenase-2, dental pulp, vascular endothelial growth factor. Received 21 September 2013; accepted 18 May 2014
reported. Vascular endothelial growth factor (VEGF), glycoprotein that has the ability to increase the permeability of blood vessels, induces angiogenesis and is normally expressed in the dental pulp (Artese et al. 2002). It has been demonstrated that the increase of VEGF expression is consistent with enhanced vascularization during dental pulp inflammation (TranHung et al. 2006). The dental pulp cells can also produce VEGF in response to lipopolysaccharide (LPS; Matsushita et al. 1999) and pro-inflammatory cytokines such as interleukin (IL)-1 (Chu et al. 2004). These previous reports suggested that dental pulp cells play a role in the aetiology of pulpitis through the production of VEGF.
International Endodontic Journal
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Effect of catechin on pulp cells Nakanishi et al.
Prostaglandins are important mediators of inflammation, the synthesis of which is initiated by release of arachidonic acid from cell membranes. Cyclooxygenases are the key and rate-limiting step in the biosynthesis of prostanoid. In particular, cyclooxygenase-2 (COX-2), which is inducible with pro-inflammatory cytokines and is considered an ‘inflammatory’ isoform (Smith & Dewitt 1996), has been shown to play an important role in the pathogenesis of pulpal inflammation (Nakanishi et al. 2001). Recent evidence has demonstrated that COX-2-transfected cells exhibit enhanced expression of VEGF (Tsujii et al. 1997). Interestingly, an immunohistological study on expression of COX-2 and VEGF in inflamed dental pulp also suggests a possible induction of VEGF via a COX-2 dependent pathway (G€ uven et al. 2007). Catechins are present in various species of plant such as green tea, and they have beneficial properties on human health. ( )-Epigallocatechin-3-gallate (EGCG) and ( )-epicatechin-3-gallate (ECG) are representative constituents of catechins, and some studies reported that these catechins inhibited several proteins involved in inflammation through the activation of nuclear factor kappa B (NF-jB; Lin & Lin 1997, Yang et al. 2001). In particular, it has been shown that EGCG attenuates VEGF production, COX-2 expression and NF-jB activation induced by inflammatory cytokines in human astrocytoma cells (Kim et al. 2007). Previous reports have indicated that the catechins EGCG and ECG inhibited the production of pro-inflammatory cytokines such as IL-6 and IL-8, and the expression of adhesion molecules in dental pulp cells treated with various bacteria-related factors (Nakanishi et al. 2010). However, there are no reports of the effects of catechins on a behaviour of VEGF related to COX-2 in dental pulp inflammation. The aim of this study was to evaluate the effect of catechins on expression of VEGF, and COX-2 protein as well in human dental pulp cells (HDPC) stimulated with bacteria-related factors (LPS or peptidoglycan (PG)) or pro-inflammatory cytokines. The null hypothesis is that catechins do not affect the expressions of VEGF and COX-2 in HDPC.
were obtained from Peprotech (Rocky Hill, NJ, USA). Antibodies against COX-2 and actin were purchased from Cayman chemical (Ann Arbor, MI, USA) and Sigma-Aldrich, respectively.
Cell culture and treatment Human dental pulp cells were established from explant cultures of healthy pulp tissues as described previously (Adachi et al. 2007). In brief, clinically healthy pulp tissue samples (from 22- to 30-year old patients; n = 3) were obtained from noncarious teeth extracted for orthodontic reasons under informed consent at Tokushima University Hospital. The minced pieces of pulp tissues were seeded into 35-mm culture dishes and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 1 mmol sodium pyruvate (Gibco) and 100 U mL 1 penicillin/100 lg mL 1 streptomycin (Gibco) at 37 °C in a humidified atmosphere of 5% CO2. Confluent primary cultures were harvested and subcultured. Morphologically fibroblastic cells obtained in this manner were used as HDPC for experiments at passages four to ten. This study was performed with the approval and compliance of the Tokushima University Ethical Committee (No.329). The experiments were undertaken with the understanding and written consent of each subject and in full accordance with ethical principles, including the World Medical Association Declaration of Helsinki. Human dental pulp cells were seeded in wells of 24-well tissue culture plates and incubated until a confluent monolayer developed (5 9 104 cells/well). The medium was then replaced with DMEM containing 2% FBS. To study the effects of catechins on VEGF production and COX-2 expression, HDPC were pretreated with or without catechins in 2% FBS-containing DMEM for 1 h and then incubated with LPS (1 lg mL 1), PG (10 lg mL 1), IL-1b (10 ng mL 1) or TNF-a (10 ng mL 1) for the designated times (24 h). After incubation, culture supernatants were collected and used to determine the quantities of VEGF, and attached cells were used for protein extraction.
Materials and methods Enzyme-linked immunosorbent assay Reagents Staphylococcus aureus PG, Escherichia coli LPS and catechins (EGCG and ECG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). IL-1b and TNF-a
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The concentration of VEGF in the cell culture supernatants was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kit (Peprotech).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Nakanishi et al. Effect of catechin on pulp cells
Results
Protein extraction and Western blot analysis After the treatment with catechins and various factors described above, HDPC were washed twice with icecold phosphate-buffered saline and then collected with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein concentrations in lysates were quantified with a bicinchoninic acid protein assay kit (Sigma-Aldrich). Equal amount of protein samples were then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes. The membrane was first incubated with a primary antibody for COX-2. After washing, the membrane was reacted with a specific horseradish peroxidaseconjugated secondary antibody (Dako Corporation, Carpinteria, CA, USA). Protein bands were finally visualized on X-ray film using the enhanced chemiluminescence system (GE Healthcare, Little Chalfont, UK). An equal loading of gels was confirmed by immunoblot with anti-actin antibody as an internal control and an appropriate secondary antibody (SigmaAldrich).
Effects of catechins on the VEGF production in HDPC in response to bacteria-derived factors and pro-inflammatory cytokines To demonstrate the potential of catechins on HDPC stimulated with bacterial components or pro-inflammatory cytokines, the effect of EGCG or ECG on VEGF expression in HDPC exposed to LPS, PG, IL-1b or TNF-a using ELISA was examined. Treatment with EGCG effectively inhibited VEGF production in bacterial component-stimulated HDPC in a concentrationdependent manner (P < 0.05; Fig. 1a). ECG also caused a significant inhibition of LPS- or PG-induced VEGF secretion (P < 0.05; Fig. 1b). Up-regulated VEGF production in HDPC incubated with IL-1b was also inhibited by EGCG (P < 0.05; Fig. 2). Although TNF-a did not affect VEGF production, the treatment of 20 lg mL 1 of EGCG decreased the level of VEGF in dental pulp cells (P < 0.05; Fig. 2).
Effects of catechins on the COX-2 induction in HDPC stimulated with LPS or IL-1b Statistical analysis
In order to clarify whether catechins inhibit the induction of COX-2 in HDPC, Western blot analysis was used to examine the effect of EGCG or ECG on COX-2 expression in HDPC exposed to LPS or IL-1b.
Data are expressed as the mean SD. All statistical analyses were performed using unpaired Student’s t-test.
(a)
(b)
Figure 1 Effect of epigallocatechin-3-gallate (EGCG) (a) or epicatechin-3-gallate (ECG) (b) pre-treatment (1 h) on vascular endothelial growth factor (VEGF) production in human dental pulp cells stimulated with lipopolysaccharide (LPS; 1 lg mL 1) or peptidoglycan (PG; 10 lg mL 1) for 24 h. The data are expressed as the mean and standard deviation of triplicate cultures from three independent experiments. *P < 0.05 compared with the control (no catechin pre-treatment).
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Up-regulated COX-2 expression on HDPC stimulated with LPS was diminished by the treatment of EGCG or ECG (Fig. 3). In addition, treatment with EGCG or
ECG inhibited the induction of COX-2 expression in HDPC exposed to IL-1b (Fig. 4).
Discussion
Figure 2 Effect of epigallocatechin-3-gallate (EGCG) pretreatment (1 h) on vascular endothelial growth factor (VEGF) expression in human dental pulp cells stimulated with interleukin-1b (IL-1 b; 10 ng mL 1) or tumour necrosis factor-a (TNF-a; 10 ng mL 1) for 24 h. The data are expressed as the mean and standard deviation of triplicate cultures from three independent experiments. *P < 0.05 compared with the control (no catechin pre-treatment).
In the present study, catechins were shown to inhibit up-regulated expression of VEGF in HDPC stimulated with LPS or PG. In the progression of pulpal disease, the vasculature is affected (Tran-Hung et al. 2006). HDPC express VEGF in both healthy and inflamed situations, such as irreversible pulpitis, a role for dental pulp cells in angiogenesis (Artese et al. 2002). Moreover, it has been shown that the production of VEGF is enhanced in dental pulp cells in response to pathogen-associated molecular patterns (PAMPs) including LPS in vitro (Matsushita et al. 1999). The innate immune system senses microbial infection using pattern recognition receptors (PRRs) that recognize PAMPs. Toll-like receptors (TLRs) generally acts as PRRs; TLR4 is involved in the recognition of most bacterial LPS, and TLR2 recognizes a wide range of PAMPs including PG of Gram-positive bacteria (Takeda & Akira 2005). It has been shown that human dental pulp fibroblasts responds to TLR2- and TLR4-specific agonists (Staquet et al. 2008). A previous study has also shown that TLR2 and TLR4 expressed in HDPC function to up-regulate the expression of various pro-inflammatory mediators such as
(a) (a)
(b) (b)
Figure 3 Effect of epigallocatechin-3-gallate (EGCG) (a) or epicatechin-3-gallate (ECG) (b) pre-treatment (1 h) on cyclooxygenase-2 (COX-2) expression in human dental pulp cells stimulated with lipopolysaccharide (LPS; 1 lg mL 1) for 24 h. Equal loading of gels was confirmed by Western blot with anti-actin antibody. Data are representative of two independent experiments with similar results.
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Figure 4 Effect of epigallocatechin-3-gallate (EGCG) (a) or epicatechin-3-gallate (ECG) (b) pre-treatment (1 h) on cyclooxygenase-2 (COX-2) expression in human dental pulp cells stimulated with interleukin-1b (IL-1 b; 10 ng mL 1) for 24 h. Equal loading of gels was confirmed by Western blot with anti-actin antibody. Data are representative of two independent experiments with similar results.
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Nakanishi et al. Effect of catechin on pulp cells
IL-6 and IL-8 (Hirao et al. 2009). In addition, it has been reported that the EGCG and ECG inhibited the production of IL-6 and IL-8 in the dental pulp cells treated with LPS and PG (Nakanishi et al. 2010). Therefore, these catechins might exhibit an antiinflammatory effect on pulpal inflammation through the inhibition of VEGF as well as pro-inflammatory cytokines. Consistent with the present observation, it has been reported that angiogenesis can be inhibited by EGCG through blocking the induction of VEGF in human carcinoma cells (Jung et al. 2001). Other studies also found that VEGF was inhibited by EGCG-treated human cervical carcinoma cells (Zhang et al. 2006), hepatoma cells (Zhang et al. 2006) and human nasal polyp fibroblasts (Lin et al. 2008). Thus, the present study and previous reports suggest that catechins are able to modulate the angiogenesis by their anti-angiogenic activities. On the other hand, it has been reported that EGCG enhanced prostaglandin F2ainduced VEGF synthesis in osteoblast cell line (Tokuda et al. 2007). This discrepancy might reflect differences of cell type and stimulator. It was also observed that EGCG reduced the enhanced-level of VEGF in HDPC in response to IL-1b. However, TNF-a failed to induce the increase of VEGF production. A previous immunohistochemistry study has shown that IL-1 and TNF-a-positive cells were localized in pulpitis lesions, and thus, they are known to be involved in pulpal inflammation (Tani-Ishii et al. 1995). These cytokines were employed to check the effect of catechins on VEGF secretion from HDPC. Chu et al. (2004) demonstrated that not only IL-1 but also TNF-a induced VEGF gene expression in human pulp fibroblasts. This is in contradiction to the present results, which displayed no significant induction of VEGF production by TNFa. This fact may be explained by the difference of analysis level. It has been reported that a statistically significant difference between normal and inflamed pulp was found in the concentration of prostaglandin E2, and COX-2 was mainly expressed in fibroblasts from inflamed pulp tissues (Nakanishi et al. 1995, 2001). These findings indicate that pulpal fibroblasts may participate in prostaglandin E2 production through COX-2 expression in pulpal inflammation, and might be involved in the pathogenesis of irreversible pulpitis. In this study, EGCG and ECG reduced induction of COX-2 protein in HDPC in response to bacteria-related factors or pro-inflammatory cytokines, suggesting that these catechins may mitigate pulpal inflammation. Other
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
recent reports demonstrated that VEGF expression is up-regulated via a COX-2-dependent mechanism, and COX-2-derived prostaglandin E2 has been found to promote angiogenesis (Tsujii et al. 1997, Nie & Honn 2002). Interestingly, a previous report has shown that EGCG attenuates both VEGF production and COX-2 expression through regulation of the NF-jB activation, and phosphorylation of MAPK induced by inflammatory cytokines in human astrocytoma cells (Kim et al. 2007). Previous reports also demonstrated that the inhibitory effect of catechins on pro-inflammatory mediator expression with PAMPs is mediated by blocking the MAPK pathway with subsequent inhibition of NF-jB activity in HDPC (Hirao et al. 2010). Therefore, the findings in this study might be revealed by an antiinflammatory activity of catechins. This research was conceived with the intention of applying the catechins to the surface of exposed pulp. The present results showed that 20 lg mL 1 and 50 lg mL 1 catechins significantly suppressed both VEGF and COX-2 expression. These concentrations of catechins are found to have no cytotoxicity for HDPC in a previous report (Hirao et al. 2010). However, further investigation is necessary to test the use of catechins for application to the exposed pulp in clinical therapies such as pulp capping.
Conclusion Catechins inhibited the expression of VEGF and COX-2 in HDPC treated with PAMPs and IL-1b. These findings suggest that catechins may be useful as anti-inflammatory modulator of pulpal inflammation.
Acknowledgements This work was supported by JSPS KAKENHI Grant Number 20592228, 24592871 [Grant-in-Aid for Scientific Research (C)].
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