An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells

Abstract Objective To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1β, in cultured human dental pulp cells. Methodology Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1β in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. Results Stimulation of the pulp cells with IL-1β resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1β (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1β was also blocked by incubation with the extract. Conclusions Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1β in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.


Introduction
Dental pulp infection is a common event, often found in deep dental caries and complicated crown fractures that allow microbial invasion into the pulp via direct exposure to the external milieu. 1  In case of a deep cavity, if the pulp is diagnosed as reversible pulpitis, dental lining materials are recommended for pulp protection to maintain its healthy status. Many studies have suggested the use of calcium hydroxide paste and mineral trioxide aggregate (MTA) due to their beneficial effects on promotion of pulpal wound healing and induction of reparative dentine formation. 8-10 Other lining agents have later been studied for use in pulp capping, such as propolis and bee glue. The use of propolis as a pulp capping agent may hold therapeutic purposes due to its reported antimicrobial and anti-inflammatory properties 11 that could possibly enhance the pulpal healing process. Accordingly, a study demonstrated that the exposed pulp in rats' teeth, when capped with propolis flavonoids, exhibited mild inflammation in the pulp tissue with reparative dentine formation by the fourth week. 12 Moreover, the magnitude of inflammatory cell infiltration and dentine bridge formation at the exposure site of human pulp lined with propolis extract were found to be comparable to those lined with Dycal. 13 Our previous studies have revealed the potential therapeutic benefits of Thai propolis extract as an alternative storage medium for avulsed teeth.
The viability of periodontal ligament (PDL) cells in experimentally avulsed premolars was preserved by the propolis extract, similar to Hank's balanced salt solution and milk; 14 additionally, mRNA expression of periostin in PDL cells was also maintained by the extract up to 12 h. 15 Regarding another study, this extract could promote pulpal wound healing with little inflammation and formation of dentine bridge with well-organized dentinal tubules in partial pulpotomy of rabbits' teeth. 16 Therefore, Thai propolis extract is biocompatible with both PDL and pulp cells, in addition to possibly exert an anti-inflammatory activity, promoting pulpal wound healing. Although an earlier study demonstrated an anti-inflammatory effect of Indonesian propolis against COX-2 expression in rat dental pulp cells in vivo, 17 it has not been shown whether Thai propolis also exerts its anti-inflammatory action in human dental pulp cells via suppression of COX-2/PGE2 induction upon treatment with a proinflammatory cytokine, IL-1β, or whether the 5-LOX pathway is involved in the anti-inflammatory effect in these cells. Therefore, this study aimed to examine the anti-inflammatory effect of Thai propolis extract on the arachidonic acid pathway activated by IL-1β in cultured human dental pulp cells, in addition to elucidate the signaling molecules mediating this effect.

Methodology Preparation of Thai propolis extract
Thai propolis extract was prepared as described elsewhere. 14 Briefly, propolis from Nong Khai province of Thailand was extracted with 95% ethanol, lyophilized, and then reconstituted in dimethyl sulfoxide (DMSO), which was used as a solvent to make a master stock at  min at room temperature in the dark. The isotype of these five antibodies against human cell surface CD markers is IgG1. Furthermore, human peripheral blood mononuclear cells (hPBMC) were isolated from a whole blood sample obtained from a healthy donor, using Ficoll-Paque TM density gradient centrifugation (GE Healthcare BioSciences, Uppsala, Sweden), as previously described. 18 Then, hPBMC from the buffy layer were transferred into a 50-ml tube, washed with PBS twice, and centrifuged at 2,000 rpm for 5 min.
The supernatant was discarded, and the cell pellet was resuspended in the staining buffer for the final cell density at 1x10 7 cells/ml. The hPBMC at 1x10 6 cells were reacted with 10 µl of each aforementioned antibody. After being washed with PBS, the stained cells were resuspended with 500 µl of sheath fluid, and at least 10,000 stained cells were acquired and analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, Inc.). The unstained cells served as a negative control.

Cytotoxic analysis of Thai propolis extract
The PrestoBlue cytotoxic assay was used to determine non-toxic concentrations of Thai propolis extract for cultured dental pulp cells, as previously described. 15 In brief, the pulp cells were seeded into a 96-well plate (Corning Incorporated, NY, USA) and cultured in the growth medium for 24 h. On the following day, the cells were treated with the extract at 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, or 0.01 mg/ml, using serum-free DMEM to serially dilute consisted of denaturation at 95°C 20 sec, annealing at 60°C 20 sec, and extension at 72°C 10 sec, for 40 cycles. GAPDH was used as a housekeeping gene control to normalize the degree of COX-2 or 5-LOX mRNA expression in each sample. Three independent assays using three dental pulp cell lines derived from three different donors were performed in triplicate.
Using the ΔΔCt method, the mRNA expression of COX-2 or 5-LOX in each experimental sample was calculated in relation to that in an untreated control, set to 1.

Immunoblot analysis
Total protein was extracted from both treated and untreated dental pulp cells using the RIPA buffer (Thermo Fisher Scientific). Cell lysate was sonicated on ice for 12 min. The concentration of total protein was determined using the Bio-Rad protein assay kit

Results
Due to heterogeneous cell populations found within the dental pulp, cultured human dental pulp cells were morphologically and molecularly characterized. The spindle fibroblast-like cell shape was observed for most of these cultured cells ( Figure   1A). Consistently, approximately 99% of these cells were found to express three classical mesenchymal stromal cell (MSC) surface markers, including CD73, CD90, and CD105, whereas expression of CD34, a marker of hematopoietic and endothelial stem cells, or that of CD45, a marker of white blood cells, was virtually absent (Figure 1B), indicating an MSC type of the cultured human dental pulp cells, as previously defined. 24 In contrast to human dental pulp cells, almost all hPBMC (97.54%) were positively stained for CD45, while they were virtually unstained for the other three mesenchymal CD markers or CD34 ( Figure   1B). Since the isotype of all five conjugated antibodies against cell surface CD markers used in this study is the same, i.e., IgG1, the negative staining with anti-CD45 antibody in human dental pulp cells can, thus, be regarded as an isotype antibody control for the positive staining with anti-CD73, anti-CD90, and anti-CD105 antibodies in the same cell type. Note that the anti-CD45 antibody was still found to be effective for staining hPBMC. By immunoblot analysis, induction of COX-2 protein, detected at 72 kDa upon IL-1b treatment, was evidently inhibited by the Thai propolis extract at the doses from 0.08 to 0.31 mg/ml ( Figure 4A).
Protein expression of b-actin, used as a housekeeping gene control, was found to be equivalent among different samples. By densitometry, the inhibition of COX-2 protein induction upon IL-1b treatment was found to be significant at the extract doses ranging from 0.08 to 1.25 mg/ml (p<0.05; Figure 4B), which was confirmed by significant decreases in elevated PGE2 levels upon IL-1b stimulation by treatment with the extract at the doses from 0.08 to 1.25 mg/ ml (p<0.05; Figure 4C). Note that an inverse dosedependent inhibition of COX-2 protein expression and PGE2 levels was found ( Figure 4B and C). As a positive control, treatment with indomethacin at 10 µM significantly inhibited COX-2 protein induction and, thus, reduced PGE2 levels upon IL-1b treatment (p<0.05; Figure 4B and C, respectively). When human dental pulp cells were incubated with various doses of the extract alone, a slight increase in COX-2 protein expression upon treatment was observed by the 1.25 mg/ml dose ( Figure 4D and E), probably as a result of mild cytotoxicity that may have accounted for a lower reduction in upregulated COX-2 protein expression ( Figure 4B) and in raised PGE2 levels ( Figure 4C) by this dose than a remarkable COX-2/PGE2 decrease by the low doses at µg/ml. Interestingly, the extract at 5 mg/ml that was found to be cytotoxic (Figure 2) upregulated, substantially and significantly, COX-2 protein expression (p<0.05; Figure 4D and E). Thus, the inhibitory effects against COX-2 mRNA and protein inductions as well as against enhanced PGE2 synthesis in response to IL-1b treatment in cultured human dental pulp cells seemed to be more pronounced by Thai propolis extract at lower doses (Figure 3 and 4).
Involvement of the p50 and the p65 subunits of NF-kB in the anti-inflammatory effect of Thai propolis extract Because induction of COX-2 expression upon IL-1β treatment was demonstrated to involve NF-kB activation in human gingival fibroblasts, 25 nuclear translocation of the p50 and the p65 subunits of NF-kB was, therefore, examined in human dental pulp cells. In addition, since the most effective dose of Thai propolis extract that inhibits COX-2 and PGE2 was found to be at 0.08 mg/ml (Figure 4), this dose was only selected for an immunofluorescence analysis.
Both p50 and p65 subunits were found to be localized in the cytoplasm of control untreated dental pulp cells, but they were translocated into the nuclei of these cells upon treatment with IL-1b at 10 ng/ml ( Figure 5A and B). Co-incubation with 0.08 mg/ml of the extract shown to inhibit both COX-2 mRNA and protein inductions, in addition to elevate PGE2 levels ( Figure 3 and 4) and block nuclear translocation of both subunits ( Figure 5A and B). By a quantitative analysis, a significant inhibition for the percentages of nuclear translocation of both subunits was found by treatment with the extract at 0.08 mg/ml (p<0.01; Figure 5C), suggesting that the extract is likely to exert its inhibitory effect on COX-2 induction and enhanced PGE2 synthesis upon IL-1b treatment via blockade of nuclear translocation of NF-kB.

Discussion
Pulpal inflammation and pain are major challenges in Endodontics, for which mediation of the COX-2/ PGE 2 pathway is reported to be responsible. 26,27 In this study, the non-toxic doses of Thai propolis extract were investigated for its anti-inflammatory effect against COX-2/PGE2 induction upon treatment with IL-1β in cultured human dental pulp cells. Our results showed that the expression of COX-2, but not 5-LOX, was upregulated in response to treatment with IL-1β in cultured human dental pulp cells, resulting in elevated PGE2 levels, suppressed upregulation of COX-2 and PGE2 in IL-1β-treated human dental pulp cells, and blocked nuclear translocation of both p50 and p65 subunits of NF-kB, suggesting that Thai propolis extract is likely to target both pulpal inflammation and pain via the COX-2/PGE2 pathway and NF-kB signaling.
Vital pulp therapy aims to maintain pulp vitality by eliminating bacteria from the dentine-pulp complex, and to establish an environment to promote pulpal wound healing. Some materials have been used for vital pulp treatment, including the traditional material of choice, calcium hydroxide, and hydraulic calcium silicate cements, i.e., MTA. 10 A recent study has suggested that Thai propolis extract could be another promising alternative material used for pulp capping procedures because its application on the mechanically exposed pulp induces the formation of reparative dentine with orderly arranged dentinal tubules. 16 In addition to its reparative dentine induction 16 and anti-inflammatory properties shown in this study, the Thai propolis extract has antimicrobial properties against the growth of Streptococcus and Lactobacillus species, 28,29 making this extract a good candidate material for vital pulp therapy.
After treatment of human dental pulp cells with IL-1β, mostly secreted by odontoblasts and dental pulp cells in response to cariogenic infection, 3,30 a significant induction of COX-2, but not 5-LOX, was found, Figure 5-Representative immunofluorescence images from three independent experiments using three different human dental pulp cell lines. Immunoreaction with the mouse monoclonal antibody to the p50 (A) or to the p65 (B) subunit of NF-kB, followed by reaction with the anti-mouse IgG conjugated with NorthernLight557 (red) demonstrates the localization of each subunit. DAPI staining (blue) indicates the location of nuclei. Scale bars = 50 μm. (C) A quantitative analysis for the percentages of nuclear staining of both p50 and p65 subunits from (A) and (B), respectively. **p<0.01, as compared with the mean percentages of nuclear staining for both p50 and p65 subunits in the IL-1b-treated sample J Appl Oral Sci. 2023;31:e20230006 10/12 suggesting that the 5-LOX pathway of arachidonic acid metabolism is not involved in an IL-1β-mediated inflammatory pulpal response. This finding agrees with previous ex vivo studies, showing that COX-2 expression was considerably induced in inflamed dental pulp tissues, in which it was found to be at the dental pulp fibroblasts. 7,31 Upon treatment with Thai propolis extract in human dental pulp cells treated with IL-1β, the PGE2, a potent inflammatory mediator generated by COX-2, was significantly decreased, proposing that the inhibitory effect of the extract against inflammatory responses in human dental pulp cells is via the COX-2/PGE2 pathway. Our current study was conducted using non-toxic doses of Thai propolis extract, ranging from 0.08 to 1.25 mg/ml. It was found that upregulated COX-2 expression and raised PGE2 levels in response to the treatment with IL-1β were remarkably dampened by the extract, although the inhibition of COX-2 and PGE2 induction by the extract at 1.25 mg/ml was not as pronounced at lower concentrations, possibly due to mild cytotoxicity. It is noteworthy that the cytotoxic dose of Thai propolis extract at 5 mg/ml considerably induced COX-2 protein expression, which is consistent with the cytotoxicity of dental restorative monomers accompanied by COX-2 induction observed in human dental pulp cells. 37,38 Even though previous investigations have addressed a potential use of propolis as a pulp capping material, 12,13 formulation of the propolis extract to improve its physical characteristics is needed for more practical clinical applications with sustained therapeutic effects and a longer shelf life than its sole active chemical. A previous study has revealed that the release of propolis extract incorporated into polyvinyl alcohol, aimed at a prolonged release, reached its plateau level within 48 h. 39 Correspondingly, the 48-h incubation of the Thai propolis extract at 0.08 mg/ ml in close contact with cultured dental pulp cells has shown the remarkably suppressive effects on COX-2 and PGE2 induction. Given the significance of the dose-and time-dependent responses of dental pulp cells to Thai propolis extract, studies of an appropriate pharmaceutical formulation are, therefore, required to apply this extract for future clinical use as a pulp capping agent that can exert long lasting effects on dental pulp repair and reparative dentine formation.

Conclusions
Thai propolis extract exerts an anti-inflammatory activity against COX-2 induction and enhanced PGE2 synthesis upon IL-1β treatment in human dental pulp cells. The anti-inflammatory effect of this extract is involved with blockade of nuclear translocation of the p50 and the p65 subunits of NF-kB upon IL-1β treatment. Therefore, the anti-inflammatory properties of this extract could be beneficial for therapeutic use as an alternative pulp capping material. An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells