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. 2023 Jun 5:31:e20230006.
doi: 10.1590/1678-7757-2023-0006. eCollection 2023.

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

Nutthapong Kantrong  1 , Jittranut Kumtawee  1 , Teerasak Damrongrungruang  2 , Subin Puasiri  3 , Anupong Makeudom  4 , Suttichai Krisanaprakornkit  5 , Pattama Chailertvanitkul  1
Affiliations

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

Nutthapong Kantrong et al. J Appl Oral Sci. .

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.

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Conflict of interest statement

Conflict of interest

The authors declare no conflicts of interests associated with this study.

Figures

Figure 1
Figure 1. Characterization of human dental pulp cells (hDPC). (A) The fibroblast-like morphology of cultured hDPC, isolated from pulp tissue explants. This image was taken at the 50X magnification power. Scale bar = 100 μm. (B) By flow cytometry, expressions of three mesenchymal stromal cell markers, including CD73 (PB450), CD90 (APC), and CD105 (PE) were found in hDPC, whereas expression of CD34 (FITC) and CD45 (KO525) was not found. Results are representative of three different dental pulp cell lines from three healthy donors with the same finding. On the contrary, expression of CD45 (KO525) was only detected in human peripheral blood mononuclear cells (hPBMC)
Figure 2
Figure 2. Determination of the cytotoxicity of Thai propolis extract by the PrestoBlue assay. Cultured human dental pulp cells were treated with indicated doses (0.01-5 mg/ml) of the extract for 48 h. The line graph demonstrates the average percentages of cell viability from three independent experiments, each of which was performed in triplicate, using three different dental pulp cell lines. Error bars = standard deviation; **p<0.01, as compared with the percentage of cell viability in an untreated control, set to 100 (blue circle). 10% DMSO (red circle) = treatment with 10% (vol/vol) of dimethyl sulfoxide for 48 h
Figure 3
Figure 3. IL-1b mediates signaling via COX-2 in human dental pulp cells. (A) Significant induction of COX-2 mRNA, but not 5-LOX mRNA, upon IL-1b treatment in human dental pulp cells. (B) Significant inhibition of COX-2 mRNA induction upon IL-1b treatment by Thai propolis extract (P) at the doses of 0.08, 0.16, 0.31, and 0.63 mg/ml. (C) No significant difference between treatment with IL-1b at 10 ng/ml or with the extract (P) at all tested concentrations and an untreated control in the average degrees of 5-LOX mRNA expression. The three bar graphs demonstrate the mean degrees of COX-2 or 5-LOX mRNA expression from three separate experiments using three different dental pulp cell lines. Error bars = standard deviation; *p<0.05 in (A) and (B), as compared with the mean degree of COX-2 mRNA induction in the pulp cells treated with IL-1b at 10 ng/ml; ns = not significant; IND = treatment with indomethacin at 10 µM
Figure 4
Figure 4. Inhibition of COX-2 protein induction and raised PGE2 levels upon treatment with IL-1b at 10 ng/ml by Thai propolis extract in human dental pulp cells. (A) A representative immunoblot from three independent experiments using three different dental pulp cell lines with a similar finding, showing the absence of COX-2 band, detected at 72 kDa, by treatment with the extract (TPE) at 0.08 and 0.16 mg/ml. (B) Significant inhibition of the mean degrees of COX-2 protein expression by the extract (P) at all doses tested, as quantified by densitometry from protein bands in (A). (C) Significant decreases in the mean concentrations of PGE2 in pg/ml, released into conditioned media of the samples in (A). *p<0.05 in (B) and (C), as compared with the mean degree of COX-2 protein induction and the mean level of elevated PGE2, respectively, in the cells treated with IL-1b at 10 ng/ml; IND = treatment with indomethacin at 10 µM. (D) Immunoblots showing the effects of treatment with various doses of Thai propolis extract (TPE) alone on COX-2 protein expression and its densitometry (E). *p<0.05, a significant increase in the mean degree of COX-2 protein expression, as compared with those of other samples. Error bars in (B), (C), and (E) = standard deviation
Figure 5
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
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Grants and funding

Funding: This study was supported by the Fundamental Fund of Khon Kaen University, has received funding support from the National Science, Research and Innovation Fund (NSRF) to P.C.; the Chiang Mai University Presidential Post-Doctoral Fellowship 2019 to A.M and S.K.; and the Fundamental Fund (#R000029994), Thailand Science Research and Innovation to S.K.
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