Article Versions
Export Article
Cite this article
  • Normal Style
  • MLA Style
  • APA Style
  • Chicago Style
Research Article
Open Access Peer-reviewed

Anti-inflammatory Activity of Indonesian Propolis in Zebrafish (Danio rerio) Larvae

Indra Wibowo , Fauzi Ramadhani Nasution, Intan Taufik, Roya Suffah Zain, Nissa Marlinda, Nuruliawaty Utami, Putri Yunitha Wardiny, Ramadhani Eka Putra, Marselina Irasonia Tan, Sony Heru Sumarsono
Journal of Food and Nutrition Research. 2020, 8(5), 225-230. DOI: 10.12691/jfnr-8-5-2
Received May 02, 2020; Revised June 04, 2020; Accepted June 11, 2020

Abstract

One candidate for anti-inflammatory agents, polyphenol-rich Indonesian propolis, has been rarely studied. This study was conducted to confirm the presence of its anti-inflammatory activity. Zebrafish larvae, as a model, were divided into four groups consisting of a control group, a lipopolysaccharide (LPS)-treated group, an LPS-treated group followed by treatment in propolis solution for 24 hours, and a propolis-treated group. Myeloid leukocytes migrating into the intestine and intestinal goblet cells were counted. The expression level of pro-inflammatory (tnf-α, il-1β, il-8, and il-6) and anti-inflammatory (il-10) cytokine genes were determined using quantitative reverse transcription polymerase chain reaction (RT-PCR). It was shown that Indonesian propolis administration to LPS-induced zebrafish larvae resulted in reduced myeloid leukocytes in the intestine, increased intestinal goblet cells, and decreased the expression level of tnf-α (P<0.05). Overall, these results suggest that Indonesian propolis could be a potential agent to protect against inflammatory damage.

1. Introduction

Inflammation, a part of innate immune system, defends host from pathogen attack as well as tissue damage. Inflammation involves recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through various family of pattern recognition receptors (PRRs). Recognition process triggers synthesis of molecular mediators called cytokines, pro-inflammatory cytokines (such as il-1β, tnf-α, il-6, il-8, and ifn-γ) that stimulate inflammation and anti-inflammatory cytokines (such as il-10) that suppress inflammation by controlling those pro-inflammatory cytokines 1.

Systemic and long-term inflammation could lead to tissue damage, fever, auto-inflammatory disease, and septic shock. These adverse effects can be prevented by means of anti-inflammatory agents, such as aspirin that have been widely used 2. However, most of these agents exhibit toxicity and side effects, i.e. aspirin can cause hypertension, edema, and heart failure 3. Therefore, relatively non-toxic anti-inflammatory agents are needed as alternative. Burdock stated that propolis is relative non-toxic, thus has a potency for being used as anti-inflammatory agent 4.

Propolis is a resinous mixture of plant-derived material collected by honey bees and has been widely used as traditional medicine since antiquity 5. Its chemical components are responsible for its characteristics and biological activities and influenced by geographic location, bee species, plant source, season, and extraction method 6. Compared to other types of propolis, Indonesian propolis has been rarely studied especially at cellular and molecular level 7. According to various studies on other types of propolis, polyphenols and terpenoids are allegedly responsible for anti-inflammatory activity 8.

In this research, zebrafish larvae were used as model to confirm the presence of anti-inflammatory activity of Indonesian propolis, because their immune system is highly similar to its mammalian counterpart, at molecular, cellular, and even physiological level 9. The parameters included were: (1) expression level of pro-inflammatory cytokine genes (tnf-α, il-1β, il-8, and il-6), (2) expression level of anti-inflammatory cytokine gene (il-10), (3) number of myeloid leukocytes migrating into intestine, and (4) number of intestinal goblet cells. Indonesian propolis is expected to suppress the expression level of pro-inflammatory cytokine genes, promote the expression level of anti-inflammatory cytokine genes, decrease the number of myeloid leukocytes migrating into intestine, and increase the number of intestinal goblet cells.

2. Materials and Methods

2.1. Indonesian Propolis Preparation

Propolis used in this research was obtained from stingless bee (Trigona sp.) farmer from Subang, Indonesia. Raw propolis was extracted using ethanol and its chemical constituents were analyzed using gas chromatography-mass spectrometry (GC-MS).

2.2. Zebrafish Maintenance

Zebrafish were maintained in accordance with standard protocol 10. Zebrafish embryos, obtained from natural spawning, and larvae were kept in Petri dish containing E3 medium (5 mM of NaCl, 0.17 mM of KCl, 0.33 mM of CaCl2, 0.33 mM of MgSO4, without methylene blue) at 26-30°C.

2.3. Treatment of Zebrafish Larva

Treatment of zebrafish larvae was performed by immersion in 6-well plate for 2 × 24 hours. Thirty 5-dpf-larvae per group were placed in well containing 6 mL of E3 medium. After the first 24 hours, E3 medium was changed with fresh medium. Larvae were divided into four groups: control/CON (untreated), LPS group (exposed to 50-ppm-LPS for 24 hours), LPS-PRO group (treated with 50 ppm of LPS for 24 hours followed by 14 ppm of Indonesian propolis for 24 hours), and PRO group (treated only with 14 ppm of Indonesian propolis for 24 hours). LPS was obtained from phenol-extraction of Escherichia coli serotype O111:B4 (Sigma-Aldrich), and prepared according to Bates et al. 11. Subsequent to treatment, larvae were sacrificed using an overdose of tricaine methane-sulfonate 12.

2.4. Expression Level Analysis Using Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from euthanized larvae (30 larvae per sample) and the gene-specific primer used were mentioned in Table 1 (obtained from Genetika Science Jakarta, Indonesia). Relative gene expression analysis was performed using method proposed by Livak and Schmittgen 13.

2.5. Myeloperoxidase (MPO) Staining

First, pigmentation was prevented to facilitate visualization, by using a tyrosinase inhibitor N-Phenylthiourea (PTU) 14. MPO staining was performed in accordance with Cordero-Maldonado et al. in five larvae per group 15. Quantification of black-brown-stained myeloid leukocytes at mid and posterior portion of larval gut were carried out manually under stereomicroscope at 90 × magnification.

2.6. Histology Analysis

Euthanized larvae were fixed in freshly prepared Bouin’s solution at room temperature for at least 24 hours, dehydrated in alcohol series, embedded in molten paraffin, and sagittally cut at 5 μm of width. Larval sections were stained using alcian blue and counterstained with hematoxylin-eosin (HE) and enhanced using Scott’s tap water substitute 16, according to Hedrera et al. with modifications 17. Quantification of goblet cell at mid and hind gut was carried out manually by light microscopy (AO American Optical MicroStar One-Ten) at 200 × magnification.

2.7. Statistical Analysis

Data were analyzed using either IBM SPSS Statistics 19 (IBM) or R-3.5.1 (The R Foundation) with significance level (α) of 0.05.

3. Results and Discussion

3.1. Chemical Analysis

The chemical analysis by CG-MS revealed 25 distinct compounds in chemical composition of Indonesian propolis. Most of these compounds are phenolic compounds with recognized therapeutic properties. The most abundant chemical compounds are 3-methoxy- (CAS) m-guaiacol (phenol) and phenyl ester (CAS) phenyl carbamate (carbamic acid) guaiacol, respectively 14.07% and 10.15%.

3.2. Indonesian Propolis Reduces Relative Expression Level of tnf-α

As shown in Figure 1, in general, expression level of pro-inflammatory cytokines genes examined in this study (tnf-α, il-1β, il-8, and il-6) shows similar pattern. Larvae from the LPS group has a higher expression level of pro-inflammatory cytokine genes compared to control (CON) group. It is consistent with the result of a research conducted by Yang et al. that showed increases of il-1β, il-6, and tnf-α expression levels in the LPS-injected larvae 18. Non-significant differences in expression level of tnf-α, il-1β, and il-6 between larvae from the control and from the LPS group (P>0.05) could be due to LPS tolerance to the LPS concentration used, as teleost fishes, including zebrafish, have a high tolerance to LPS 11.

The expression level of pro-inflammatory cytokine genes in larvae from the LPS-PRO group was lower than in larvae from LPS group, though significant difference only observed in the expression level of tnf-α (P<0.05). tnf-α is one of the early genes expressed at an early stage of infection in fish and important for regulating inflammation 19. Based on the similar pattern, Indonesian propolis tends to repress the expression of pro-inflammatory cytokine genes in LPS-induced zebrafish larvae. By comparing to other organism model, such as that conducted by Neiva et al., propolis were shown to decrease LPS-induced pro-inflammatory cytokines in human pulp cells and osteoclasts 20.

3.3. Indonesian Propolis Insignificantly Raises Relative Expression Level of il-10

As shown in Figure 2, the expression level of il-10 in zebrafish larvae from the LPS group was significantly higher than in larvae from the control group (P<0.05). This result is consistent with previous study using adult zebrafish induced by LPS 21. il-10 has a protection capacity from toxic effects of tnf-α induced earlier by LPS, by triggering secondary signaling pathways, which modulate the expression of direct LPS target genes.

Indonesian propolis tends to reduce the expression level of il-10 in LPS-induced zebrafish larvae, but not for the larvae that are not induced by LPS. This is denoted by the result obtained in this study. Zebrafish larvae from the LPS-PRO group and Propolis group are relatively similar in their expression level of il-10 and also similar with larvae from the control group (P>0.05). These results are consistent with the result of a previous study that shows inhibition of the expression of il-10 in RAW 264.7 cell line after propolis treatment 22.

3.4. Indonesian Propolis Reduces the Number of Migrating Myeloid Leukocytes

Representative zebrafish larvae stained with myeloperoxidase are shown in Figure 3A. The number of myeloid leukocytes in larvae from the LPS group was significantly higher than in larvae from the control group (Figure 3B). This suggests that LPS increases myeloid leukocyte migration into intestine which is the site of mucosal inflammation. Migration of myeloid leukocytes is mediated by chemokines, such as IL-8, which are secreted by the endothelial cells induced by TNF-α and macrophages 23.

Zebrafish larvae from the LPS-PRO group (P>0.05) and from the PRO group (P<0.001) had a lower number of myeloid leukocytes compared to larvae from the LPS group. This result suggest that Indonesian propolis tends to inhibit myeloid leukocytes migration into intestine in LPS-induced zebrafish larvae. It was consistent with relative expression of il-8 shown in Figure 1C because il-8 is a pro-inflammatory chemokine produced by various cell types to recruit leukocytes to sites of infection or tissue injury. Also, the TNF-α protein enhances the phagocytic activity of fish leukocyte, by getting involved in the regulation of leukocyte homing, proliferation, and migration 19. These results also consistent with the study done by Franchin et al. where LPS-induced mouse was exposed to vestitol (compound isolated from propolis) 24.

3.5. Indonesian Propolis Increases the Number of Intestinal Goblet Cells

Figure 4A shows the sagittal sections of zebrafish larvae from all groups stained with alcian blue-HE. Larvae from the LPS group had the lowest number of intestinal goblet cells (Figure 4B). It differed significantly with the number of intestinal goblet cells from other groups’ larvae. This result suggests that LPS decreased the number of intestinal goblet cells in zebrafish larvae. Previous study performed by Zapata et al. which utilized LPS-induced weaned pig shows reduction in the number of intestinal goblet cells 25.

The number of intestinal goblet cells in zebrafish larvae from LPS-PRO group was significantly higher than in larvae from the LPS group (Figure 4B). It suggests that Indonesian propolis causes an increase in the number of intestinal goblet cells in LPS-induced zebrafish larvae. It was consistent with the study conducted by Oehlers et al. that showed administration of propolis in mice with damaged nasal mucosa and colitis-induced rat with 2,4,6-trinitrobenzensulfonat acid (TNBS) increased the number of goblet cells in nasal and gastrointestinal mucosa 26. TNF-α and LPS play a role in the regulation of the number of intestinal goblet cells by strengthening the IFN-γ signaling pathways 27, 28.

As the main components of propolis that was used in this study, phenolic compounds are presumably responsible for anti-inflammatory activity of Indonesian propolis. Various studies have proved the existence of anti-inflammatory activity in phenolic compounds and its mechanism that involves inhibition of pro-inflammatory enzymes activity (lipoxygenase, cyclooxygenase, and iNOS) and inhibition of transcription factors (PI 3-kinase, NF-κB, c-JUN, and tyrosine kinase) 29. The effect of Indonesan Propolis suggests its potential as source of new compounds with pharmacological properties and its use in the control of inflammation.

4. Conclusion

Based on the results that have been described, it can be concluded that Indonesian propolis exerts anti-inflammatory activities in the zebrafish larvae stimulated with LPS. The activities include down-regulating pro-inflammatory gene, i.e. tnf-α (P<0.05), reducing the number of myeloid leukocytes that migrate into intestine, and increasing the number of intestinal goblet cells, though the other pro-inflammatory genes’ expression level did not increase and anti-inflammatory gene’s did not decrease in this study. However, Indonesian propolis alone did not show any significant anti-inflammatory activities. Nevertheless, the effect of Indonesan propolis suggests its potential as source of new compounds with pharmacological properties and could be a potential agent to protect against inflammatory damage.

Acknowledgements

This research was supported by the Institute for Research and Community Service (LPPM) ITB under the Research, Community Service, and Innovation of ITB Research Groups Program (P3MI) number 21a/SK/I1.C02/PL/2018.

References

[1]  Opal, S.M. and DePalo, V.A., “Anti-inflammatory cytokines”, Chest, 117. 1167-1172. 2000.
In article      View Article  PubMed
 
[2]  Dinarello, C.A., “Anti-inflammatory agents: present and future”, Cell, 140. 935-950. 2010.
In article      View Article  PubMed
 
[3]  Meek, I.L., Van De Laar, M.A.F.J., and Vonkeman, H.E., “Non-steroidal anti-inflammatory drugs: an overview of cardiovascular risks”, Pharmaceuticals, 3. 2146-2162. 2010.
In article      View Article  PubMed
 
[4]  Burdock, G., “A Review of the biological properties and toxicity of bee propolis (propolis)”, Food Chem. Toxicol., 36(4). 347-363. 1998.
In article      View Article
 
[5]  Sforcin, J.M., “Propolis and immune system: a review”, J. Ethnopharmacol., 113(1). 1-14. 2007.
In article      View Article  PubMed
 
[6]  Huang, S., Zhang, C.P., Wang, K., Li, G.Q., and Hu, F.L., “Recent advances in the chemical composition of propolis”, Molecules, 19. 19610-19632. 2014.
In article      View Article  PubMed
 
[7]  Syamsudin, Wiryowidagdo, S., Simanjuntak, P., and Heffen, W.L., “Chemical composition of propolis from different regions in Java and their cytotoxic activity”, Am. J. Biochem. Biotechnol., 5(4). 180-183. 2009.
In article      View Article
 
[8]  Araujo, M.A.R., Liberio, S.A., Guerra, R.N.M., Ribeiro, M.N.S., and Nascimento, F.R.F., “Mechanisms of action underlying the anti-inflammatory and immunomodulatory effects of propolis: a brief review”, Rev. Bras. Farmacogn., 22(1). 208-219. 2012.
In article      View Article
 
[9]  Brugman, S., “The zebrafish as a model to intestinal inflammation”. Dev. Comp. Immunol., 64. 82-92. 2016.
In article      View Article  PubMed
 
[10]  Westerfield, M., The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio) 4th ed., University of Oregon Press, Eugene, 2000.
In article      
 
[11]  Bates, J.M., Akerlund, J., Mittge, E., and Guillemin, K., “Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota”, Cell Host Microbe, 2. 371-382. 2007.
In article      View Article  PubMed
 
[12]  Caruffo, M., Navarrete, N.C., Salgado, O.A., Faundez, N.B., Gajardo, M.C., Feijoo, C.G., Reyes-Jara, A., Garcia, K., and Navarrete P., “Protective yeasts control V. anguillarum pathogenicity and modulate the innate immune response of challenged zebrafish (Danio rerio) larvae”, Front. Cell. Infect. Microbiol. 6. 127. 2016.
In article      View Article  PubMed
 
[13]  Livak, K. and Schmittgen, T., “Analysis of relative gene expression data using real-time quantitative PCR and the 2-ddCt method”, Methods, 25. 402-408. 2001.
In article      View Article  PubMed
 
[14]  Thisse, C. and Thisse, B., “High-resolution in situ hybridization to whole-mount zebrafish embryos”, Nat. Protoc., 3(1). 59-69. 2008.
In article      View Article  PubMed
 
[15]  Cordero-Maldonado, M.L., Siverio-Mota, D., Vicet-Muro, L., Wilches-Arizábala, I.M., Esguerra, E.M., de Witte, P.A., and Crawford, A.D., “Optimization and pharmacological validation of a leukocyte migration assay in zebrafish larvae for the rapid in vivo bioactivity analysis of anti-inflammatory secondary metabolites”, PLoS ONE, 8(10). e75404. 2013.
In article      View Article  PubMed
 
[16]  Sullivan-Brown, J., Bisher M.E., and Burdine, R.D., “Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin”. Nat. Protoc., 6(1). 46-55. 2011.
In article      View Article  PubMed
 
[17]  Hedrera, M.I., Galdames, J.A., Jimenez-Reyes, M.F., Reyes, A.E., Avendaño-Herrera, R., and Romero, J., “Soybean meal induces intestinal inflammation in zebrafish larvae”, PLoS ONE, 8(7). e69983. 2013.
In article      View Article  PubMed
 
[18]  Yang, L.L., Wang, G.Q., Yang, L.M., Huang, Z.B., Zhang, W.Q., and Yu, L.Z., “Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs”, Molecules, 19(2). 2390-2409. 2014.
In article      View Article  PubMed
 
[19]  Zou, J. and Secombes, C. J., “The function of fish cytokines”, Biology (Basel), 5(2). 23. 2016.
In article      View Article  PubMed
 
[20]  Neiva, K.G., Catalfamo, D.L., Holliday, S., Wallet, S.M., and Pileggi, R., “Propolis decreases lipopolysaccharide-induced inflammatory mediators in pulp cells and osteoclasts”, Dent. Traumatol., 30(5). 362-367. 2014.
In article      View Article  PubMed
 
[21]  Zhang, D.C., Shao, Y.Q., Huang, Y.Q., and Jiang, S.G., “Cloning, characterization, and expression analysis of interleukin-10 from the zebrafish (Danio rerio)”, J. Biochem. Mol. Biol., 38(5). 571-576. 2005.
In article      View Article  PubMed
 
[22]  Bueno-Silva, B., Kawamoto, D., Ando-Suguimoto, E.S., Alencar, S.M., Rosalen, P.L., and Mayer, M.P.A., “Brazilian red propolis attenuates inflammatory signaling cascade in lps-activated macrophages”, PLoS ONE, 10(12). e0144954. 2015.
In article      View Article  PubMed
 
[23]  de Oliveira, S., Reyes-Aldasoro, C.C., Candel, S., Renshaw, S.A., Mulero, V., and Calado, A., “Cxcl8 (interleukin-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response”, J. Immunol., 190(8). 4349-4359. 2013.
In article      View Article  PubMed
 
[24]  Franchin, M., Colon, D.F., Castanheira, F.V.S., da Cunha, M.G., Bueno-Silva, B., Alencar, S.M., Cunha, T.M., and Rosalen, P.L., “Vestitol isolated from brazilian red propolis inhibits neutrophils migration in the inflammatory process: elucidation of the mechanism of action”, J. Nat. Prod., 79(4). 954-960. 2016.
In article      View Article
 
[25]  Zapata, D.J., Rodriguez, B.J., Ramirez, M.C., Lopez, A., and Parra, J., “Escherichia coli lipopolysaccharide affects intestinal mucin secretion in weaned pigs”, Rev. Colomb. Cienc. Pecu., 28. 209-217. 2015.
In article      View Article
 
[26]  Oehlers, S.H., Flores, M.V., Hall, C.J., Okuda, K.S., Sison, J.O., Crosier, K.E., and Crosier, P.S., “Chemically induced intestinal damage models in zebrafish larvae”, Zebrafish, 10(2). 184-193. 2013.
In article      View Article  PubMed
 
[27]  Kim, J.J. and Ho, S.B., “Intestinal goblet cells and mucins in health and disease: Recent insights and progress”. Curr. Gastroenterol. Rep., 12(5). 319-330. 2013.
In article      View Article  PubMed
 
[28]  Schroder, K., Hertzog, P.J., Ravasi, T., and Hume, D.A., “Interferon-gamma: an overview of signals, mechanisms and functions”, J. Leukoc. Biol., 75(2). 163-189. 2004.
In article      View Article  PubMed
 
[29]  Santangelo, C., Vari, R., Scazzocchio, B., Benedetto, R., Filesi, C., and Masella, R., “Polyphenols, intracellular signaling and inflammation”, Ann. Ist. Super. Sanita., 43(4). 394-405. 2007.
In article      
 

Published with license by Science and Education Publishing, Copyright © 2020 Indra Wibowo, Fauzi Ramadhani Nasution, Intan Taufik, Roya Suffah Zain, Nissa Marlinda, Nuruliawaty Utami, Putri Yunitha Wardiny, Ramadhani Eka Putra, Marselina Irasonia Tan and Sony Heru Sumarsono

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Cite this article:

Normal Style
Indra Wibowo, Fauzi Ramadhani Nasution, Intan Taufik, Roya Suffah Zain, Nissa Marlinda, Nuruliawaty Utami, Putri Yunitha Wardiny, Ramadhani Eka Putra, Marselina Irasonia Tan, Sony Heru Sumarsono. Anti-inflammatory Activity of Indonesian Propolis in Zebrafish (Danio rerio) Larvae. Journal of Food and Nutrition Research. Vol. 8, No. 5, 2020, pp 225-230. http://pubs.sciepub.com/jfnr/8/5/2
MLA Style
Wibowo, Indra, et al. "Anti-inflammatory Activity of Indonesian Propolis in Zebrafish (Danio rerio) Larvae." Journal of Food and Nutrition Research 8.5 (2020): 225-230.
APA Style
Wibowo, I. , Nasution, F. R. , Taufik, I. , Zain, R. S. , Marlinda, N. , Utami, N. , Wardiny, P. Y. , Putra, R. E. , Tan, M. I. , & Sumarsono, S. H. (2020). Anti-inflammatory Activity of Indonesian Propolis in Zebrafish (Danio rerio) Larvae. Journal of Food and Nutrition Research, 8(5), 225-230.
Chicago Style
Wibowo, Indra, Fauzi Ramadhani Nasution, Intan Taufik, Roya Suffah Zain, Nissa Marlinda, Nuruliawaty Utami, Putri Yunitha Wardiny, Ramadhani Eka Putra, Marselina Irasonia Tan, and Sony Heru Sumarsono. "Anti-inflammatory Activity of Indonesian Propolis in Zebrafish (Danio rerio) Larvae." Journal of Food and Nutrition Research 8, no. 5 (2020): 225-230.
Share
  • Figure 1. The expression level of pro-inflammatory cytokine genes: (A) tnf-α, (B) il-1b, (C) il-8, and (D) il-6. Significant differences are shown by * (P<0.05)
  • Figure 3. The number of migrating myeloid leukocytes into intestine. (A) Zebrafish larvae intestine. Area confined by the red line was for quantifying the number of myeloid leukocytes (black dots). Horizontal bar = 100 μm; Magnification = 90X. (B) Quantification result. Significant differences are shown by * (P<0.001)
  • Figure 4. The number of intestinal goblet cells (A) Sagittal section of zebrafish larvae. White triangle pinpoints goblet cell. Horizontal bar = 100 μm; Magnification = 200X. (B) Quantification result. Significant differences are shown by ** (P<0.01 against LPS group; P<0.001 against other groups) and *** (P<0.001 against other groups).
[1]  Opal, S.M. and DePalo, V.A., “Anti-inflammatory cytokines”, Chest, 117. 1167-1172. 2000.
In article      View Article  PubMed
 
[2]  Dinarello, C.A., “Anti-inflammatory agents: present and future”, Cell, 140. 935-950. 2010.
In article      View Article  PubMed
 
[3]  Meek, I.L., Van De Laar, M.A.F.J., and Vonkeman, H.E., “Non-steroidal anti-inflammatory drugs: an overview of cardiovascular risks”, Pharmaceuticals, 3. 2146-2162. 2010.
In article      View Article  PubMed
 
[4]  Burdock, G., “A Review of the biological properties and toxicity of bee propolis (propolis)”, Food Chem. Toxicol., 36(4). 347-363. 1998.
In article      View Article
 
[5]  Sforcin, J.M., “Propolis and immune system: a review”, J. Ethnopharmacol., 113(1). 1-14. 2007.
In article      View Article  PubMed
 
[6]  Huang, S., Zhang, C.P., Wang, K., Li, G.Q., and Hu, F.L., “Recent advances in the chemical composition of propolis”, Molecules, 19. 19610-19632. 2014.
In article      View Article  PubMed
 
[7]  Syamsudin, Wiryowidagdo, S., Simanjuntak, P., and Heffen, W.L., “Chemical composition of propolis from different regions in Java and their cytotoxic activity”, Am. J. Biochem. Biotechnol., 5(4). 180-183. 2009.
In article      View Article
 
[8]  Araujo, M.A.R., Liberio, S.A., Guerra, R.N.M., Ribeiro, M.N.S., and Nascimento, F.R.F., “Mechanisms of action underlying the anti-inflammatory and immunomodulatory effects of propolis: a brief review”, Rev. Bras. Farmacogn., 22(1). 208-219. 2012.
In article      View Article
 
[9]  Brugman, S., “The zebrafish as a model to intestinal inflammation”. Dev. Comp. Immunol., 64. 82-92. 2016.
In article      View Article  PubMed
 
[10]  Westerfield, M., The zebrafish book: A guide for the laboratory use of zebrafish (Danio rerio) 4th ed., University of Oregon Press, Eugene, 2000.
In article      
 
[11]  Bates, J.M., Akerlund, J., Mittge, E., and Guillemin, K., “Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota”, Cell Host Microbe, 2. 371-382. 2007.
In article      View Article  PubMed
 
[12]  Caruffo, M., Navarrete, N.C., Salgado, O.A., Faundez, N.B., Gajardo, M.C., Feijoo, C.G., Reyes-Jara, A., Garcia, K., and Navarrete P., “Protective yeasts control V. anguillarum pathogenicity and modulate the innate immune response of challenged zebrafish (Danio rerio) larvae”, Front. Cell. Infect. Microbiol. 6. 127. 2016.
In article      View Article  PubMed
 
[13]  Livak, K. and Schmittgen, T., “Analysis of relative gene expression data using real-time quantitative PCR and the 2-ddCt method”, Methods, 25. 402-408. 2001.
In article      View Article  PubMed
 
[14]  Thisse, C. and Thisse, B., “High-resolution in situ hybridization to whole-mount zebrafish embryos”, Nat. Protoc., 3(1). 59-69. 2008.
In article      View Article  PubMed
 
[15]  Cordero-Maldonado, M.L., Siverio-Mota, D., Vicet-Muro, L., Wilches-Arizábala, I.M., Esguerra, E.M., de Witte, P.A., and Crawford, A.D., “Optimization and pharmacological validation of a leukocyte migration assay in zebrafish larvae for the rapid in vivo bioactivity analysis of anti-inflammatory secondary metabolites”, PLoS ONE, 8(10). e75404. 2013.
In article      View Article  PubMed
 
[16]  Sullivan-Brown, J., Bisher M.E., and Burdine, R.D., “Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin”. Nat. Protoc., 6(1). 46-55. 2011.
In article      View Article  PubMed
 
[17]  Hedrera, M.I., Galdames, J.A., Jimenez-Reyes, M.F., Reyes, A.E., Avendaño-Herrera, R., and Romero, J., “Soybean meal induces intestinal inflammation in zebrafish larvae”, PLoS ONE, 8(7). e69983. 2013.
In article      View Article  PubMed
 
[18]  Yang, L.L., Wang, G.Q., Yang, L.M., Huang, Z.B., Zhang, W.Q., and Yu, L.Z., “Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs”, Molecules, 19(2). 2390-2409. 2014.
In article      View Article  PubMed
 
[19]  Zou, J. and Secombes, C. J., “The function of fish cytokines”, Biology (Basel), 5(2). 23. 2016.
In article      View Article  PubMed
 
[20]  Neiva, K.G., Catalfamo, D.L., Holliday, S., Wallet, S.M., and Pileggi, R., “Propolis decreases lipopolysaccharide-induced inflammatory mediators in pulp cells and osteoclasts”, Dent. Traumatol., 30(5). 362-367. 2014.
In article      View Article  PubMed
 
[21]  Zhang, D.C., Shao, Y.Q., Huang, Y.Q., and Jiang, S.G., “Cloning, characterization, and expression analysis of interleukin-10 from the zebrafish (Danio rerio)”, J. Biochem. Mol. Biol., 38(5). 571-576. 2005.
In article      View Article  PubMed
 
[22]  Bueno-Silva, B., Kawamoto, D., Ando-Suguimoto, E.S., Alencar, S.M., Rosalen, P.L., and Mayer, M.P.A., “Brazilian red propolis attenuates inflammatory signaling cascade in lps-activated macrophages”, PLoS ONE, 10(12). e0144954. 2015.
In article      View Article  PubMed
 
[23]  de Oliveira, S., Reyes-Aldasoro, C.C., Candel, S., Renshaw, S.A., Mulero, V., and Calado, A., “Cxcl8 (interleukin-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response”, J. Immunol., 190(8). 4349-4359. 2013.
In article      View Article  PubMed
 
[24]  Franchin, M., Colon, D.F., Castanheira, F.V.S., da Cunha, M.G., Bueno-Silva, B., Alencar, S.M., Cunha, T.M., and Rosalen, P.L., “Vestitol isolated from brazilian red propolis inhibits neutrophils migration in the inflammatory process: elucidation of the mechanism of action”, J. Nat. Prod., 79(4). 954-960. 2016.
In article      View Article
 
[25]  Zapata, D.J., Rodriguez, B.J., Ramirez, M.C., Lopez, A., and Parra, J., “Escherichia coli lipopolysaccharide affects intestinal mucin secretion in weaned pigs”, Rev. Colomb. Cienc. Pecu., 28. 209-217. 2015.
In article      View Article
 
[26]  Oehlers, S.H., Flores, M.V., Hall, C.J., Okuda, K.S., Sison, J.O., Crosier, K.E., and Crosier, P.S., “Chemically induced intestinal damage models in zebrafish larvae”, Zebrafish, 10(2). 184-193. 2013.
In article      View Article  PubMed
 
[27]  Kim, J.J. and Ho, S.B., “Intestinal goblet cells and mucins in health and disease: Recent insights and progress”. Curr. Gastroenterol. Rep., 12(5). 319-330. 2013.
In article      View Article  PubMed
 
[28]  Schroder, K., Hertzog, P.J., Ravasi, T., and Hume, D.A., “Interferon-gamma: an overview of signals, mechanisms and functions”, J. Leukoc. Biol., 75(2). 163-189. 2004.
In article      View Article  PubMed
 
[29]  Santangelo, C., Vari, R., Scazzocchio, B., Benedetto, R., Filesi, C., and Masella, R., “Polyphenols, intracellular signaling and inflammation”, Ann. Ist. Super. Sanita., 43(4). 394-405. 2007.
In article