Dental Traumatology 2014; 30: 362–367; doi: 10.1111/edt.12096
Propolis decreases lipopolysaccharideinduced inflammatory mediators in pulp cells and osteoclasts Kathleen G. Neiva1, Dana L. Catalfamo2, Shannon Holliday3, Shannon M. Wallet2, Roberta Pileggi1 1 Department of Endododntics, University of Florida College of Dentistry; 2Department of Periodontology and Oral Biology, University of Florida College of Dentistry; 3Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL, USA
Key words: propolis; inflammation; pulp cells; osteoclasts; intracanal medicament Correspondence to: Kathleen G. Neiva, Department of Endodontics, University of Florida College of Dentistry, 1395 Center Drive, Room D10-41, PO Box 100436, Gainesville, FL 32610-0436, USA Tel.: (352) 273 5435 Fax: (352) 273 5446 e-mail:
[email protected] Accepted 15 December, 2013
Abstract – Background: Intracanal medicaments are used to disinfect the root canal system, reduce interappointment pain and inflammation, and prevent resorption. Bacterial components such as lipopolysaccharide (LPS) are implicated in the development of pulpal and periapical inflammation and inducing osteoclastogenesis. Propolis is a natural, non-toxic substance collected from bee’s wax that has been used for many years in folk medicine. Propolis has been demonstrated to have antibacterial and anti-inflammatory properties. Our previous studies have shown that propolis inhibits osteoclast maturation. However, the effect of propolis on the inflammatory response of pulp cells and osteoclasts has not been explored. Aim: The purpose of this study was to evaluate whether propolis alters the inflammatory response of three endodontically relevant cell lines: mouse odontoblast-like cells (MDPC23), macrophages (RAW264.7), and osteoclasts. Material and methods: Cells were exposed to 0–20 ug ml 1 LPS to induce an inflammatory response, in the presence of propolis or vehicle control. Culture supernatants were collected after 6 and 24 h, and expression of multiple soluble mediators was determined using Luminexâ multiplex technology. Results: Propolis was effective in reducing secretion of the LPS-induced inflammatory cyto/chemokines: IL-1a, IL-6, IL-12(p70), IL-15, G-CSF, TNF-a, MIP-1a, MCP-1, and IP-10. Conclusion: Our results demonstrate that propolis suppresses the LPSinduced inflammatory response of key cells within the root canal system.
An intracanal medicament may be necessary during root canal therapy in order to eliminate bacteria (1), to reduce pain and inflammation in pulp and periapical tissues in cases of pulpectomy (2), and to prevent inflammatory and replacement resorption in cases of trauma (3). Biocompatibility and stability are essential properties for all intracanal medicaments. Propolis is a natural, non-toxic substance collected from bee’s wax that has been used for many years in folk medicine. It contains amino acids, flavonoids, terpenes, and cinnamic acid derivatives (4, 5). Propolis has been demonstrated to have antibacterial properties along with anti-inflammatory effects (6, 7). Notably, propolis’ significant antibacterial activity against oral pathogens has been demonstrated, including Enterococcus faecalis, Staphylococcus aureus, Candida albicans, Streptococcus sanguis, Streptococcus mutans, Streptococcus sobrinus, Streptococcus cricetus, Actinomyces naeslundii, Actinomyces viscosus, Porphyromonas gingivalis, Porphyromonas endodontalis, and Prevotella denticola (6, 8–10). Interestingly, one study has demonstrated that propolis was more effective than calcium hydroxide and as effective as a triantibiotic mixture in inhibiting the growth of E. faecalis (11). 362
In vitro experiments have shown that propolis exhorts its anti-inflammatory properties by inhibiting the cyclo-oxygenase pathway and eicosanoid synthesis (7, 12, 13). Similarly, we have observed that propolis can decrease lipopolysaccharide (LPS)-stimulated iNOS activity (unpublished data). LPS is the major component of the outer membrane in Gram-negative bacteria and is largely responsible for the pathogenicity of these organisms (14, 15). Gram-negative bacteria are prevalent in deep caries and pulpitis (16–18). These bacteria play a significant role in the development of clinical symptoms through the production of pro-inflammatory cytokines and chemokines such as tumor necrosis factor-(TNF)a, interleukin (IL)-1a (19, 20), IL-1b (21), IL-6 (22), IL-8 (23), and vascular endothelial growth factor (VEGF) (24). Although propolis has only been investigated in dental research to a minimal extent, we have observed its biocompatibility with the oral tissues, including the pulp and periodontium (25, 26). In addition, studies have observed that propolis promotes regeneration of dental pulp, enhancement of pulp healing, and greater dentinal bridge formation (27, 28). Finally, we have demonstrated that propolis is also able to inhibit osteoclast maturation, suggesting it could also contribute to © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Propolis decreases inflammatory mediators preventing replacement resorption (29). However, the effect of propolis on osteoclasts and pulpal cell inflammatory responses has not been explored. Therefore, the purpose of this study was to evaluate whether propolis decreases the LPS-induced responsiveness of three endodontically relevant cell lines: murine-derived odontoblast-like cells (MDPC-23), macrophages (RAW264.7), and RAW264.7-derived osteoclasts. Material and methods
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murine soluble RANK-L (rmsRANK-L) (Peprotech, Rocky Hill, NJ, USA) and allowed to culture for 6 days with complete media refreshed every 3 days to stimulate osteoclast differentiation. After that, odontoblast-like cells, macrophages, and osteoclasts were exposed to 0– 100 ng ml 1 of non-pure Escherichia coli LPS (SigmaAldrich, St. Louis, MO, USA) for 1 h. Cells were then treated with 1:100 dilution of propolis or vehicle control (ethanol) for an additional 6 or 24 h. Supernatants were collected, and cyto/chemokine expression was analyzed using multiplex technology.
Propolis
A single source of propolis from the Baccharis dracunculifolia (the most important botanical source of Southeastern Brazilian propolis) was used throughout the study. It was obtained from the same Brazilian bee species (Africanized Apis mellifera), dissolved, purified, and prepared at 80% concentration using 0.4% ethanol solution as vehicle. Cell culture
Mouse odontoblast-like cells (MDPC-23) and macrophages (RAW264.7) (gift from T. Botero, University of Michigan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Mediatech, Manassas, VA, USA), 1% L-glutamine (Thermo Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin/ amphotericin B (Fisher, Waltham, MA, USA). Cells were cultured in T-75 flasks (Fisher) at 80% confluency. Cells were then seeded at a concentration of 4 9 104 in 24-well plates (Fisher). Alternatively, RAW macrophages were supplemented with 50 ng ml 1 recombinant
Tartrate-resistant acid phosphate (Trap) staining
After 6 days of RANKL stimulation, cells plated on glass coverslips were fixed with 2% paraformaldehyde/ PBS (Fisher). Cells were washed with PBS and permeabilized in 0.5% Triton X-100/PBS (Fisher). Cells were washed and probed for leukocyte acid phosphatase (TRAP) (1:1:1:2:4 Fast Garnet GBC Base Solution: Sodium Nitrite Solution:Naphthol AS-BI Phosphate Solution:Tartrate Solution:Acetate Solution) (SigmaAldrich) after which cells were washed and mounted on glass slides with MOWIOL 4-88 solution (Calbiochem). TRAP-positive cells (stained in purple) were observed using light microscopy at 209 and 409 magnification in order to confirm osteoclast differentiation. Soluble mediator analysis
Cytokines and chemokines from supernatants were detected and quantified using a mouse 22-cyto/ chemokine multiplex (Millipore) according to the manufacturer’s instructions. Briefly, supernatant and antibody-coated beads were allowed to incubate over-
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Fig. 1. Propolis reduced lipopolysaccharide (LPS)-induced expression of cyto/chemokines in odontoblast-like cells. MDPC-23 were exposed to 0 or 20 ng ml 1 LPS for 1 h. Cells were then treated with propolis or vehicle control. After 24 h, supernatants were collected and cyto/ chemokine expression was analyzed using a Luminexâ multiplex assay. Graphs represent expression of (a) IL1a, (b) MIP-1a, (c), IL-12(p70), and (d) IL-15. Asterisks represent statistically significant difference (P < 0.05). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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night at 4°C in a 96-well primed plate. Following three washes, reactivity was probed with biotinylated detection antibodies and streptavidin (SAV)–phycoerythrin (PE). All incubations occurred while gently shaking in the dark. Following three washes, beads were resuspended in sheath fluid and reactivity acquired using a Luminex 200 with XPONENT software (Millipore, Billerica, MA, USA). MILLIPLEXâ ANALYST software (Millipore), 5-parameter logistics, and a standard curve were used to determine pg ml 1 concentrations. Outcome measures were normalized to cell number per well. Statistical analysis
One-way ANOVA with Dunn’s correction was used to analyze and determine statistical significance (P < 0.05). Results
Pilot studies were conducted to determine the ideal concentration and timing of E. coli LPS stimulation to induce inflammatory mediators in all cell types. Here, a 20 ng ml 1 concentration of LPS induced significant
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upregulation of soluble mediators at 24 h poststimulation for odontoblast-like cells, while upregulation of these mediators was observed at both 6 and 24 h for macrophages and osteoclasts (data not shown). Bacterial LPS induced upregulation of several cyto/ chemokines in odontoblast-like cells at 24 h. In addition, treatment with propolis alone or vehicle control had no effect on the baseline expression of inflammatory cytokines measured (Fig. 1a–d). However, treatment with propolis significantly reduced the LPSinduced expression of interleukin (IL)-1a (Fig. 1a), MIP-1a (Fig. 1b), IL-12(p70) (Fig. 1c), and IL-15 (Fig. 1d) in odontoblast-like cells. Lipopolysaccharide stimulation upregulated the expression of multiple cyto/chemokines by RAW264.7 macrophages, and propolis was again able to inhibit this LPS-induced expression (Fig. 2a–f). Specifically, 6 h post-treatment, MIP-1a (Fig. 2a), G-CSF (Fig. 2b), tumor necrosis factor (TNF)-a (Fig. 2c), and IL-6 (Fig. 1d) were significantly inhibited by treatment with propolis as compared to LPS treatment alone. Similarly, 24 h following propolis administration, monocyte chemoattractant protein (MCP)-1 (Fig. 2e) expression was significantly reduced. The expression level of IL-6
Fig. 2. Propolis reduced lipopolysaccharide (LPS)-induced expression of cyto/chemokines in macrophages. RAW264.7 macrophages were exposed to 0 or 20 ng ml 1 LPS for 1 h. Cells were then treated with propolis or vehicle control. After 6 or 24 h, supernatants were collected and cyto/chemokine expression was analyzed using a Luminexâ multiplex assay. Graphs represent the expression of (a) MIP-1a, (b) G-CSF, (c) TNF-a, and (d) IL-6 at 6 h after stimulation; (e) MCP-1 and (f) IL-6 at 24 h after stimulation. Asterisks represent statistically significant difference (P < 0.05). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Propolis decreases inflammatory mediators was higher at 24 h than at 6 h, and propolis continued to inhibit the LPS-induced IL-6 (Fig. 2f) expression. As seen in odontoblast-like cells, treatment with propolis or vehicle control alone did not change baseline expression level of cyto/chemokines (Fig. 2a–f). Tartrate-resistant acid phosphate staining was used to confirm that macrophages differentiated into osteoclasts (Fig. 3a,b). TRAP-positive cells showed mononuclear (Fig. 3a) and multinucleated osteoclasts (Fig. 3b). Lastly, LPS also induced the expression of inflammatory cyto/chemokines by osteoclasts (Fig. 3c–f), which was again reduced by propolis treatment. Propolis treatment or vehicle alone had no effect on expression of inflammatory cyto/chemokines (Fig. 3c–f). Six hours following propolis treatment, MIP-1a (Fig. 3c) expression was significant lower when compared to LPS treatment alone. Similarly, the expression of IL-6 (Fig. 3d), IP-10 (Fig. 3e), and MCP-1 (Fig. 3f) was significantly reduced after 24 h of propolis treatment. Discussion
The identification of an intracanal medicament which can not only control infection but also minimize inflammation would be beneficial for more effective healing and success of root canal therapies. Here, we demonstrated that bacterial LPS induces a panel of
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Fig. 3. Propolis reduced lipopolysaccharide (LPS)-induced expression of cyto/chemokines in osteoclasts. (a, b) Tartrate-resistant acid phosphate-positive cells. RAW264.7 macrophages were supplemented with 50 ng ml 1 rmsRANK-L and cultured for 6 days to stimulate osteoclast differentiation. Representative images of (a) mononuclear and (b) multinucleated osteoclasts. (c–f) RAW264.7-derived osteoclasts were exposed to 0 or 20 ng ml 1 LPS for 1 h. Cells were then treated with propolis or vehicle control. After 6 or 24 h, supernatants were collected and cyto/chemokine expression was analyzed using a Luminexâ multiplex assay. Graphs represent expression of (c) MIP-1a at 6 h after stimulation, and (d) IL-6, (e) IP-10, and (f) MCP-1 at 24 h after stimulation. Asterisks represent statistically significant difference (P < 0.05).
inflammatory cytokines and/or chemokines in odontoblast-like cells, macrophages, and osteoclasts. Several studies (19–24) have shown that LPS induces expression of inflammatory mediators in cells associated with endodontic lesions. Here, we demonstrated that propolis decreased this LPS-induced cyto/chemokine expression in odontoblast-like cells, macrophages, and osteoclasts. Concentrated natural ethanol extracts have been shown to have anti-inflammatory effects (30, 31). In this study, we used ethanol to dilute propolis and as vehicle control. Propolis stock solution was diluted before use, and the final concentration of ethanol was not enough to cause cytotoxic or anti-inflammatory effect on the cells (data not shown). Thus, the reduction in LPS-induced inflammatory mediators is attributed to the effect of propolis itself and not of ethanol. The cell types tested here, odontoblast-like cells, macrophages, and osteoclasts, are particularly relevant in endodontics. Odontoblasts are the first pulp cells to encounter external stimulation, playing an important role in innate immunity and inflammatory events in response to cariogenic bacteria (32). Odontoblasts sense pathogens by Toll-like receptors (TLRs) and produce cyto/chemokines upon cell stimulation with microbial by-products (33). Macrophages are the most predominant immune cells of the dental pulp (34, 35) and are activated in the early stages of pulpitis. They
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have three major functions: antigen presentation, phagocytosis, and immunomodulation through the production of various cyto/chemokines and growth factors. Activated macrophages produce TNF-a, IL-1, IL-12, IL-10, chemokines, and short-lived lipid mediators such as platelet-activating factor (PAF), prostaglandins, and leukotrienes to orchestrate a local inflammation (36). Studies have shown significant increase in TNF-a and IL-1 in irreversible pulpitis (37, 38). Similarly, while osteoclasts are involved in maintenance of bone homeostasis, they are also involved in tooth resorption following traumatic injury or irritation of the pulp. Osteoclast precursors are recruited and activated in the presence of inflammatory mediators which can result in inflammatory resorption. Root resorption is the main challenge to clinicians when dealing with traumatized teeth (39–41). Studies have attempted to find a medicament that would ameliorate this problem, and to date, calcium hydroxide is the treatment of choice. Several studies have demonstrated its efficacy as antimicrobial agent (42–47). However, the anti-inflammatory and antiresorptive properties of calcium hydroxide are not evident. Here, we demonstrated that LPS induces the expression of pro-inflammatory cytokines (IL-1a, IL-12), chemokines (MIP-1a), and growth factors (IL-15) by odontoblast-like cells, and that this response can be inhibited by propolis. Similarly, we demonstrated that propolis can inhibit LPS-induced expression of proinflammatory cytokines (TNF-a and IL-6), chemokines (MIP-1a and MCP-1), and growth factors (G-CSF) from macrophages as well as from osteoclasts [IL-6 (cytokine), MIP-1a, MCP-1, and IP-10 (chemokines)]. While IL-1a, IL-12, and IL-15 support the survival, activation, and proliferation of adaptive immune cells (T cells and B cells), G-CSF supports the differentiation and proliferation of innate immune cell populations (neutrophils and macrophages). On the other hand, MIP-1a and MCP-1 are best known for their chemotactic and pro-inflammatory (i.e. induction of cytokines) properties, while IL-6 and TNF-a are more pleiotropic in nature, affecting chemoattractants, induction of cytokine expression, and angiogenic properties. Thus inhibition of these molecules would significantly dampen pulpal and periapical inflammation and enhance the endodontic wound healing processes. Indeed, inhibition of these cytokines has provided potent therapeutic effects in the treatment of multiple inflammatory diseases (48, 49). Here, we demonstrated that propolis, which has successfully been used in the medical field as an antiinflammatory and antimicrobial agent (50–52), had similar efficacy in suppressing the induction of cyto/ chemokines in key cells within the root canal system. Acknowledgement
The authors thank Dr. Tatiana Botero (University of Michigan) for the cells used in this study. Conflict of interest
The authors deny any conflicts of interest.
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