Experimental immunology
Effect of ethanol extract of propolis (EEP) on interleukin 8 release by human gastric adenocarcinoma cells (AGS) infected with Helicobacter pylori

Introduction

The Gram-negative bacteria Helicobacter pylori are potent agents of gastritis and peptic ulcer [1-3]. Inflammatory response to this germ activates immunological system and due to infiltration of neutrophils, monocytes and lymphocytes into the gastric mucosa [4, 5]. Interleukin 8 (IL-8) produced by infected epithelial cells [6-10] is a key cytokine responsible for neutrophil migration from mucosal vessels into the gastric epithelium and their activation. Local production of inflammatory mediators by activated cells, not only pro-inflammatory chemokines but also reactive oxygen and nitrogen species, is likely to be responsible for mucosal damage.

Propolis, a resinous hive product collected by honeybees from various plants sources is known for anti-inflammatory and immunomodulatory activities [11-17]. Extracts of propolis show its hepatoprotective [18-20] and anti-oxidative effects both in vivo [21] and in vitro [22, 23]. Recent observations suggest that extracts of propolis might down-regulate inflammatory mediators gene expression, such as inducible nitric oxide synthase and Il-1 genes [24, 25].

The aim of this study was to investigate the effect of ethanolic extracts of propolis from Poland on IL-8 release by AGS cells infected with H. pylori.

Materials and methods

Ethanol extract of propolis

Polish propolis was collected manually in the bee-hive of the University’s farm and kept desiccated pending its processing. Propolis was extracted in 95% (v/v) ethyl alcohol in a hermetically closed glass vessel for 4 days at 37°C, under occasional shaking. The ethanolic extract were then filtrated through a Whatman # 4 filter paper and evaporated under vacuum. Immediately prior to use, ethanol extract of propolis (EEP) samples were weighed, dissolved in Dimethylsulphoxide (DMSO; Sigma Chemical Company, St. Louis, MO, USA) and diluted with culture medium into appropriate concentrations. The final concentration of DMSO was adjusted to 0.2% (v/v). The control AGS cells and bacteria received the same amount of DMSO.

AGS cell culture

The human gastric adenocarcinoma cells (AGS) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in the atmosphere of 5% CO2 at 37°C in F12K medium (Vitacell, ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD, USA), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco BRL Life Technologies, Paisley, UK). The cells were cultured in 50 ml plastic flasks (Nunc A/S Rosklide, Denmark) and passaged 3 times a week (continuous cell culture). Cell viability was determined by Trypan blue (Sigma Chemical Company, St. Louis, MO, USA). The concentration of cell suspension used was 105 cells/ml.

Bacterial culture

Helicobacter pylori (strain CCUG 17874) was obtained from Deutsche Sammlung von Microorganismen und Zellkulturen (Braunschweig, Germany). The bacteria were grown on Columbia agar plates (Bio Merieux, Marcy l’Etoile, France) supplemented with 5% sheep blood at 37°C under microaerophillic conditions (85% N2, 10% CO2, 5% O2) generated with Genbox microaer (Bio Merieux) using an anaerobic chamber.

Antibacterial activity

Helicobacter pylori were suspended in brain-heart infusion (5 × 105 colony forming units/ml) and 1 ml of aliquots was inoculated on Columbia agar plates with appropriate concentrations of EEP preparations (15-90 g/ml). The control plates contained medium alone and medium plus DMSO. After 3 days of incubation at 37°C under microaerophilic conditions, bacteria were harvested and the growth inhibition was determined by an optical density method (OD at 595 nm) using a microplate reader Elx800 (Bio-Tek Instruments, Inc., Winooski, VT, USA).

Procedure of infection AGS cell lines incubated with different concentrations of EEP with



Helicobacter pylori

AGS cells were suspended in antibiotics-free medium (5 × 105 cells/ml), seeded in 24-well plates (Nunc A/S Roskilde, Denmark) and cultured to confluence. Then, monolayers were treated with increased concentrations of tested compounds of EEP (7.5, 15.0, 30.0 and 60.0 g/ml) for 30 min and then infected with H. pylori (bacteria cell to AGS cell ratio of 100 : 1 in a 1 ml volume). A 2-days culture of H. pylori was used. After 16 hours at 37°C in 5% CO2 the culture supernatants were collected after centrifugation and stored at –70°C until measurement. As a control cells not pretreated with EEP preparations were used.

Determination of interleukin-8 release

The concentration of IL-8 in culture supernatants was quantitated by ELISA method (R and D Systems Minneapolis, MN, USA), using human recombinant IL-8 as a standard, following the manufacturer protocol. The absorbance values were measured at 450 nm. The sensitivity of detection for this assay was less than 10 pg/ml.

Statistical analysis

All obtained values are expressed as means ± SD. Student unpaired t-test was used to asses the statistical significance of differences. A P value less than 0.05 was considered significant.

Results

Cytotoxicity and antibacterial activity of EEP

AGS cell viability and H. pylori growth were not affected by polish ethanol extract of propolis (EEPP) at the concentrations of between 7.5 and 60 g/ml. A significant decrease of bacterial growth by a higher dose of EEPP (90 g/ml) was observed (P < 0.05) (Fig. 1). The viability of infected AGS cells was greater than 95% in all performed experiments.

Effect of EEPP on interleukin-8 release

The ability of EEPP to demonstrate an influence on IL-8 synthesis by infected AGS cells was investigated. The concentrations of IL-8 in culture supernatants were measured by ELISA method. Uninfected AGS cells, after 16 hours incubation in medium alone, produced background levels of IL-8 (57.8 ±8.0 pg/ml). Cell incubation with tested preparations of EEPP or DMSO (0.2%) did not increase IL-8 release. After cell infection with H. pylori the concentration of IL-8 markedly increased (894.6 ± 58.9 pg/ml). Pretreatment of infected cells with tested EEPP preparations significantly reduced IL-8 secretion in comparison with the control (p < 0.01) (Table 1, Fig. 2), and more, this effect of EEPP action was dose dependent.

Discussion

The aim of this study was to investigate the effect of ethanolic extracts of propolis from Poland on IL-8 release by AGS cells infected with H. pylori. These cells are useful gastric epithelial cell line in vitro, mimicking IL-8 secretion of normal mucosa stimulated by different damaging agents [26]. This study has shown that EEPP can potently reduce secretion of IL-8 by AGS cells. It is conceivable that the observed action of propolis might be due, at least in part, to the content of flavone derivatives and other flavonoids [23, 27-29], which are able to inhibit inflammatory mediators gene expression in activated cells [30, 31]. Interleukin-8 production is controlled by transcription factor NF-B [9, 10, 32]. This factor also regulates the expression of other pro-inflammatory cytokines such as TNF-, IL-1, IL-6 [33]. NF-B is sequestrated in the cytoplasm as an inactive complex with the inhibitory protein IB family. Upon induction by bacterial products its inhibitory subunit is phosphorylated and degradated. Released active subunit NF-B is translocated to the nucleus [33, 35, 36]. Helicobacter pylori infection greatly increases the number of cells containing active NF-B, and the activation occurs predominantly in cells of epithelium [34, 35]. Moreover, the epithelial NF-B activation correlated with neutrophil influx in H. pylori-associated gastritis [35] and with the histological degree of gastric inflammation [36]. Keats et al. reported that inhibition of NF-B potently decreased IL-8 release by infected epithelial cells [34]. Previous study revealed that propolis significantly suppressed NF-B activation in RAW 264.7 macrophages [24]. This action might be referred to caffeic acid phenethyl ester (CAPE), an active component of propolis. It has been reported that CAPE potently suppressed NF-B activation [37, 38]. Some other flavone derivatives are also able to prevent NF-B activation [39, 40]. Because of its key role in inflammation, NF-B might be a target for new types of anti-inflammatory treatment. Their blocking might prevent the early events in the inflammatory cascade, decreasing H. pylori induced gastric injury. The data obtained from this study has shown that EEPP possess anti-H. pylori activity. Previously it has been reported that propolis preparations are active against Gram-positive bacteria and less against Gram-negative bacteria [34, 41-43, 45]. This activity is dependent on the chemical composition of samples collected in different geographical areas. Antibacterial properties of propolis are mainly attributed to a significant part of flavonoids, phenolic acids and their esters. Our results obtained indicate that EEPP preparations might partially attenuate H. pylori-induced gastric inflammation by decreasing IL-8 synthesis and release. Furthermore, natural substances such as propolis might be suitable for medical purposes.

References

 1. Buck GE (1990): Campylobacter pylori and gastroduodenal disease. Clin Microbiol Rev 3: 1-12.  

2. Haziri A, Juniku-Shkololli A, Gashi Z et al. (2010): Helicobacter pylori infection and precancerous lesions of the stomach. Med Arh 64: 248-249.  

3. Nomura A, Stemmermann GN, Chyou P et al. (1994): Helicobacter pylori infection and the risk for duodenal and gastric ulceration. Ann Inter Med 120: 977-981.  

4. Tan VP, Wong BC (2011): Helicobacter pylori and gastritis: Untangling a complex relationship 27 years on. J Gastroenterol Hepatol 26 Suppl 1: 42-45.  

5. Hatz RA, Brooks WP, Komling HJ et al. (1992): Stomach immunology and Helicobacter pylori infection. Curr Opin Gastroenterol 8: 993-1001.  

6. Sundquist M, Quiding-Järbrink M (2010): Helicobacter pylori and its effect on innate and adaptive immunity: new insights and vaccination strategies. Expert Rev Gastroenterol Hepatol 4: 733-744.  

7. Huang J, O’Toole PW, Doig P, Trust TJ (1995): Stimulation of interleukin-8 production in epithelial cell line by Helicobacter pylori. Infect Immun 63: 1732-1738.  

8. Sharma SA, Tummuru MK, Miller GG et al. (1995): Interleukin-8 response of gastric epithelial cell lines to Helicobacter pylori stimulation in vitro. Infect Immun 63: 1681-1687.  

9. Aihara M, Tsuchimoto D, Takizawa H et al. (1997): Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by gastritis cancer cell Line, MKN45. Infect Immun 65: 3218-3224.

10. Sharma SA, Tummuru MK, Blaser MJ et al. (1998): Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastrin epithelial cell. J Immunol 160: 2401-2407.

11. Girgin G, Baydar T, Ledochowski M et al. (2009): Immunomodulatory effects of Turkish propolis: changes in neopterin release and tryptophan degradation. Immunobiology 214: 129-134.

12. Dimov V, Ivanovska N, Manolova N et al. (1991): Immunomodulatory action of propolis. Infulence on anti-infectious protection and macrophage function. Apidologie 22: 155-162.

13. Paulino N, Abreu SR, Uto Y et al. (2008): Anti-inflammatory effects of a bioavailable compound, Artepillin C, in Brazilian propolis. Eur J Pharmacol 587: 296-301.

14. Mirzoeva OK, Calder PC (1996): The effect of propolis and its components on eicosanoid production during the inflammatory response. Prostaglandins Leukot Essent Fatty Acids 55: 441-449.

15. Korkina LG (2007): Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health. Cell Mol Biol 53: 15-25.

16. Park EH, Kim SH, Park SS (1996): Anti-inflammatory activity of propolis. Arch Pharm Res 19: 337-341.

17. Park EH, Kahng JH (1999): Suppressive effects of propolis in rat adjuvant arthritis. Arch Pharm Res 22: 554-558.

18. Banskota AH, Tezuka Y, Adnyana IK et al. (2000): Cytotoxic, hepatoprotective and free radical scavenging effects of propolis from Brazil, Peru, the Netherlands and China. J Ethno­pharmacol 72: 239-246.

19. Paulino N, Teixeira C, Martins R (2006): Evaluation of the analgesic and anti-inflammatory effects of a Brazilian green propolis. Planta Med 72: 899-906.

20. Banskota AH, Tezuka Y, Adnyana IK et al. (2001): Hepatoprotective and anti-Helicobacter pylori activities of constituents from Brazilian propolis. Phytomedicine 8: 16-23.

21. Sun F, Hayami S, Haruna S et al. (2000): In vivo antioxidative activity of propolis evaluated by the interaction with vitamins C and E and level of lipid hydroperoxides in rats. J Agric Food Chem 48: 1462-1465.

22. Scheller S, Wilczok T, Imielski S et al. (1990): Free radical scavenging by ethanol extract of propolis. Int J Radiat Biol 57: 461-465.

23. Krol W, Scheller S, Czuba Z et al. (1996): Inhibition of neutrophilis’ chemoluminescencje by ethanol extract of propolis (EEP) and its phenolic components. J Ethnopharmacol 55: 19-25.

24. Song YS, Park EH, Hur GM et al. (2002): Ethanol extract of propolis inhibits nitric oxide synthase gene expression and ezyme activity. J Ethnopharmacol 80: 155-161.

25. Park YK, Alencar SM, Aguiar CL (2002): Botanical origin and chemical composition of Brazilian propolis. J Agric Food Chem 50: 2502-2506.

26. Rieder G, Hatz RA, Moran AP et al. (1997): Role of adherence in interleukin-8 induction in Helicobacter pylori-associated gastritis. Infect Immun 65: 3622-3630.

27. Gülçin I, Bursal E, Sehito?lu MH et al. (2010): Polyphenol contents and antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey. Food Chem Toxicol 48: 2227-2238.

28. Bankova VS, Propov SS, Marekov NI (1983): A study on flavonoids of propolis. J Nat Prod 46: 471-474.

29. Marcucci MC (1995): Propolis: chemical composition, biological properties and therapeutic activity. Apidologie 26: 83-99.

30. Chen YC, Yang LL, Lee TJ (2000): Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor KB activation. Biochem Pharmacol 59: 1445-1457.

31. Blonska M, Czuba ZP, Krol W (2003): Effect of flavone derivatives on interleukin-1beta (IL-1beta) mRNA expression and IL-1beta protein synthesis in stimulated RAW 264.7 macrophages. Scand J Immunol 57: 162-166.

32. Mukaida N, Okamoto S, Ishikawa K (1994): Molecular mechanism of interleukin-8 gene expression. J Leukoc Biol 56: 554-558.

33. Baeuerle PA, Henkel T (1994): Function and activation of NF-kB in the immune system. Annu Rev Immunol 12: 141-179.

34. Keats S, Hitii YS, Upton M et al. (1997): Helicobacter pylori infection activates NF-kB in gastrin epithelial cells. Gastroebnterology 113: 1099-1109.

35. Van den Brink GR, Kate FJ, Ponsioen CY et al. (2000): Expression and activation of NF-kB in the antrum of the human stomach. J Immun 164: 3353-3359.

36. Isomoto H, Mizuta Y, Miyazaki M et al. (2000): Implication of NF-kB in Helicobacter pyroli-associated gastritis. Am J Gastroenterol 95: 2768-2776.

37. Natarajan K, Singh S, Burke TR et al. (1996): Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proc Natl Acad Sci USA 93: 9090-9095.

38. Song YS, Park EH, Hur GM et al. (2002): Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity. Cancer Lett 175: 53-61.

39. Tsai SH, Liang YC, Lin-Shiau SY, Lin JK (1999): Suppression of TNFαlpha-mediated NF-kB activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells. J Cell Biochem 74: 606-615.

40. Musonda CA, Chipman JK (1998): Quercetin inhibits hydrogen peroxide (H₂O2)-induced NF-kB DNA binding activity and DNA damage in HepG2 cells. Carcinogenesis 19: 1583-1589.

41. Sforcin JM, Fernandes A Jr, Lopes CA et al. (2000): Seasonal effect on Brazilian propolis antibacterial activity. J Ethno­pharmacol 73: 243-249.

42. Aga H, Shibuya T, Sugimoto T et al. (1994): Isolation and identification of antimicrobial compounds in Brazilian propolis. Biosci Biotech Biochem 58: 945-946.

43. Bankova V, Christov R, Kujumgiev A et al. (1995): Chemical composition and antibacterial activity of Brazilian propolis. Z Naturforsh C 50: 167-172.

44. Banskota AH, Tezuka Y, Prasain JK et al (1998): Chemical constituents of Brazilian propolis and their cytotoxic activity. J Nat Prod 61: 896-900.

45. Hayashi K, Komura S, Isaji N et al. (1999): Isolation of antioxidative compounds from Brazilian propolis: 3,4-dihydroxy-5-prenylcinnamic acid, a novel potent antioxidant. Chem Pharm Bull 47: 1521-1524.