Thymoquinone exhibits anti-inflammatory, antioxidant, and immunomodulatory effects on allergic airway inflammation

Abstract

Aim: Asthma is an allergic disease causing mucus secretion, release various pro-inflammatory mediators such as tumor necrosis factor- α (TNF-α) and interleukins. The aim of this study was to evaluate the effect of thymoquinone (TQ) on allergic airway inflammation in rats.

Methods: Allergic airway inflammation induced by ovalbumin (OVA) challenge in sensitized-rats and effect of TQ were studied. Inflammatory cells, interleukin (IL)-6 and TNF-α in bronchoalveolar lavage (BAL) fluid, and lipid peroxidation (LPO) in lung tissue were measured. Microvascular leakage was detected by Evans blue dye leakage in airway tissues.

Results: Tidal volume was significantly lower in OVA group (1.4± 0.07 ml) than control group (1.9±0.04 ml) (p = 0.002), while breathing frequency was significantly higher in OVA group (135.3±12.9 min^-1) than control group (p=0.017). In terms of tidal volume, statistical significance between TQ30 and OVA groups was found (1.8±0.07 ml) (p=0.008), while in terms of breathing frequency, no significance was found between both of them (126.7±7.3). Total white blood cell count was significantly higher in OVA group (1,376.8±136.4 x10³/ml) than control group (545.0±106.7 x10³/ml) (p<0.001). Statistical significance was found in TQ10 (824.7±4.5 x10³/ml) group when compared OVA group (p=0.036), while statistical significance was not found in TQ1 group (1,282.2±137.7 x10³/ml). When compared OVA group (60.3±4.9 pg/ml) with control group in terms of the TNF-α level, statistical significance was found (36.7± 4.7 pg/ml) (p=0.011). The Evans blue dye level was significantly higher in OVA group (31.8±3.6 ng/mg of tissue) than control (12.5±1.1 ng/mg of tissue) group (p<0.001), and TQ10 group (16.3±6.7 ng/mg of tissue) (p=0.002), and TQ30 (13.5±1.0 ng/mg of tissue) group (p<0.001).

Conclusion: These findings reveal that TQ could be beneficial in asthma pathophysiology due to its immunomodulatory, anti-inflammatory, and antioxidant effects.

Keywords

• Allergic airway inflammation
• TNF-α
• microvascular leakage
• ovalbumin
• thymoquinone

Timokinon allerjik solunum yolu inflamasyonu üzerine anti-inflamatuar, antioksidan ve immünomodülatör etkiler gösterir

Öz

Amaç: Astım, mukus sekresyonuna neden olan, TNF-α, IL'ler gibi çeşitli proinflamatuar mediatörleri salgılayan alerjik bir hastalıktır. Timokinon’un (TQ) sıçanlarda alerjik solunum yolu inflamasyonu üzerindeki etkisini değerlendirmektir.

Yöntemler: Duyarlılaştırılmış sıçanlarda ovalbumin (OVA) ile indüklen alerjik solunum yolu enflamasyonu ve TQ etkisi çalışıldı. Bronkoalveoler lavaj (BAL) sıvısında enflamatuar hücre sayıları, interlökin (IL)-6 ve tümör nekroz faktör alfa (TNF-α) düzeyleri ve akciğer dokusunda lipit peroksidasyonu (LPO) seviyeleri ölçüldü. Mikrovasküler sızıntı, solunum yolu dokusunda Evans mavisi ile tespit edildi.

Bulgular: Bu çalışmada, OVA grubunda tidal hacim (1,4±0,07 ml), kontrol grubuna göre önemli ölçüde düşük bulunmuştur (1,9±0,04 ml) (p = 0,002). OVA grubunda (135,3±12,9 dk^-1) solunum sıklığı kontrol grubuna (p=0,017) göre anlamlı bulunmuştur. Tidal hacim açısından TQ30 (1,8±0,07 ml) grubu ile OVA grubu karşılaştırıldığında istatistiksel anlamlılık bulunurken (p=0,008), solunum frekansı açısından gruplar arasında istatistiksel anlamlılık bulunamamıştır (126,7±7,3 dk^-1). OVA grubunda (1.376,8±136,4 x10³/ml) toplam beyaz küre sayısı, kontrol grubuna göre anlamlıydı (545,0±106,7 x10³/ml) (p<0,001). TQ10 (824,7±4,5 x10³/ml) grubu ile OVA grubu arasında istatistiksel anlamlılık bulunurken (p=0,036),  TQ1 (1.282,2±137,7 x10³/ml) ile OVA grubu arasında istatistiksel anlamlılık yoktu. TNF-α yönünden OVA grubu (60,3±4,9 pg/ml) ile kontrol grubu (36,7±4,7 pg/ml) karşılaştırıldığında, aralarında istatistiksel anlamlılık bulundu (p=0,011). OVA grubunda Evans mavi düzeyi (31,8±3,6 ng/mg doku), kontrol (12,5±1,1 ng/mg doku) (p<0,001), TQ10 (16,3±6,7 ng/mg doku) (p=0,002) ve TQ30 (13,5±1,0 ng/mg doku) (p<0,001) gruplarına göre önemli derecede yüksekti.

Sonuç: Bu bulgular, timokinonun immünomodülatör, antienflamatuar ve antioksidan etkileri nedeniyle astım patofizyolojisinde yararlı olabileceğini ortaya koymaktadır.

Anahtar Kelimeler

• TNF-α
• Alerjik solunum yolu enflamasyonu
• mikrovasküler sızıntı
• ovalbumin
• timokinon

Kaynakça

1. 1. Kim SY, Kim T-B, Moon K, Kim TJ, Shin D, Cho YS, et al. Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo. Exp Mol Med. 2008;40:461.
2. 2. Halwani R, Sultana A, Vazquez-Tello A, Jamhawi A, Al-Masri AA, Al-Muhsen S. Th-17 regulatory cytokines IL-21, IL-23, and IL-6 enhance neutrophil production of IL-17 cytokines during asthma. J Asthma. 2017;54:893–904.
3. 3. Roussel L, Houle F, Chan C, Yao Y, Berube J, Olivenstein R, et al. IL-17 Promotes p38 MAPK-Dependent Endothelial Activation Enhancing Neutrophil Recruitment to Sites of Inflammation. J Immunol. 2010;184:4531–7.
4. 4. Boskabady MH, Javan H, Sajady M, Rakhshandeh H. The possible prophylactic effect of Nigella sativa seed extract in asthmatic patients. Fundam Clin Pharmacol. 2007;21:559–66.
5. 5. Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi AK, Siddique NA, et al. A review on therapeutic potential of Nigella sativa: A miracle herb. Asian Pac J Trop Biomed. 2013;3:337–52.
6. 6. el Tahir KE, Ashour MM, al-Harbi MM. The respiratory effects of the volatile oil of the black seed (Nigella sativa) in guinea-pigs: elucidation of the mechanism(s) of action. Gen Pharmacol. 1993;24:1115–22.
7. 7. Salem ML. Immunomodulatory and therapeutic properties of the Nigella sativa L. seed. Int. Immunopharmacol. 2005;5:1749–70.
8. 8. Khader M, Bresgen N, Eckl PM. In vitro toxicological properties of thymoquinone. Food Chem Toxicol 2009;47:129–33.

9. 9. Isik AF, Kati I, Bayram I, Ozbek H. A new agent for treatment of acute respiratory distress syndrome: thymoquinone. An experimental study in a rat model. Eur J Cardio-Thoracic Surg. 2005;28:301–5.
10. 10. Houghton P, Zarka R, de las Heras B, Hoult J. Fixed Oil of Nigella sativa and Derived Thymoquinone Inhibit Eicosanoid Generation in Leukocytes and Membrane Lipid Peroxidation. Planta Med. 1995;61:33–6.
11. 11. Arslan SO, Gelir E, Armutcu F, Coskun O, Gurel A, Sayan H, et al. The protective effect of thymoquinone on ethanol-induced acute gastric damage in the rat. Nutr Res. 2005;25:673–80.
12. 12. El Mezayen R, El Gazzar M, Nicolls MR, Marecki JC, Dreskin SC, Nomiyama H. Effect of thymoquinone on cyclooxygenase expression and prostaglandin production in a mouse model of allergic airway inflammation. Immunol Lett. 2006;106:72–81.
13. 13. El Gazzar M, El Mezayen R, Marecki JC, Nicolls MR, Canastar A, Dreskin SC. Anti-inflammatory effect of thymoquinone in a mouse model of allergic lung inflammation. Int. Immunopharmacol. 2006;6:1135–42.
14. 14. Vuolo F, Petronilho F, Sonai B, Ritter C, Hallak JEC, Zuardi AW, et al. Evaluation of Serum Cytokines Levels and the Role of Cannabidiol Treatment in Animal Model of Asthma. Mediators Inflamm. 2015;2015:538670.
15. 15. Drevytska T, Morhachov R, Tumanovska L, Portnichenko G, Nagibin V, Boldyriev O, et al. shRNA-Induced Knockdown of a Bioinformatically Predicted Target IL10 Influences Functional Parameters in Spontaneously Hypertensive Rats with Asthma. J Integr Bioinform. 2018. 2018;15(4). doi: 10.1515/jib-2018-0053.
16. 16. Shinagawa K, Kojima M. Mouse Model of Airway Remodeling. Am J Respir Crit Care Med. 2003;168:959–67.
17. 17. Campos MG, Toxqui E, Tortoriello J, Oropeza M V, Ponce H, Vargas MH, et al. Galphimia glauca organic fraction antagonizes LTD(4)-induced contraction in guinea pig airways. J Ethnopharmacol. 2001;74:7–15.
18. 18. Chen Y-H, Wu R, Geng B, Qi Y-F, Wang P-P, Yao W-Z, et al. Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma. Cytokine. 2009;45:117–23.
19. 19. Escott KJ, Belvisi MG, Birrell MA, Webber SE, Foster ML, Sargent CA. Effect of the p38 kinase inhibitor, SB 203580, on allergic airway inflammation in the rat. Br J Pharmacol. 2000;131:173–6.
20. 20. Mukherjee AA, Kandhare AD, Rojatkar SR, Bodhankar SL. Ameliorative effects of Artemisia pallens in a murine model of ovalbumin-induced allergic asthma via modulation of biochemical perturbations. Biomed Pharmacother. 2017;94:880–9.
21. 21. Li X, Hufnagel S, Xu H, Valdes SA, Thakkar SG, Cui Z, et al. Aluminum (Oxy)Hydroxide Nanosticks Synthesized in Bicontinuous Reverse Microemulsion Have Potent Vaccine Adjuvant Activity. ACS Appl Mater Interfaces. 2017;9:22893–901.
22. 22. Xie Q-M, Chen J-Q, Shen W-H, Bian R-L. Correlative changes of interferon-gamma and interleukin-4 between cortical layer and pulmonary airway of sensitized rats. Acta Pharmacol Sin. 2002;23:248–52.
23. 23. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351–8.
24. 24. Oktay ARSLAN S. Morphine modulates microvascular leakage dose-dependently in the airway of ovalbumin-sensitized rats. Turk J Med Sci. 2010;40:279–86.
25. 25. Aun M, Bonamichi-Santos R, Arantes-Costa FM, Kalil J, Giavina-Bianchi P. Animal models of asthma: utility and limitations. J. Asthma Allergy. 2017;10:293–301.
26. 26. Wenzel SE, Szefler SJ, Leung DYM, Sloan SI, Rex MD, Martin RJ. Bronchoscopic Evaluation of Severe Asthma. Am J Respir Crit Care Med. 1997;156:737–43.
27. 27. Dong F, Wang C, Duan J, Zhang W, Xiang D, Li M. Puerarin attenuates ovalbumin-induced lung inflammation and hemostatic unbalance in rat asthma model. Evid. Based Complement Alternat Med. 2014;2014:726740.
28. 28. Marefati N, Eftekhar N, Kaveh M, Boskabadi J, Beheshti F, Boskabady MH. The Effect of Allium cepa Extract on Lung Oxidant, Antioxidant, and Immunological Biomarkers in Ovalbumin-Sensitized Rats. Med Princ Pract. 2018;27:122–8.
29. 29. Olivenstein R, Du T, Xu LJ, Martin JG. Microvascular Leakage in the Airway Wall and Lumen During Allergen Induced Early and Late Responses in Rats. Pulm. Pharmacol Ther. 1997;10:223–30.
30. 30. Ikezono K, Kamata M, Mori T. Adrenal Influences on the Inhibitory Effects of Procaterol, a Selective Beta-Two-Adrenoceptor Agonist, on Antigen-Induced Airway Microvascular Leakage and Bronchoconstriction in Guinea Pigs. Pharmacology. 2005;73:209–15.
31. 31. Underwood SL, Lewis SA, Raeburn D. RP 58802B, a long-acting beta 2-adrenoceptor agonist: assessment of antiasthma activity in the guinea-pig in vivo. Pulm Pharmacol. 1992;5:203–12.
32. 32. Ikemura T, Sasaki Y, Ohmori K. Inhibitory effect of NPC-17731 on BK-induced and antigen-induced airway reactions in guinea-pigs. Clin Exp Allergy. 1998;28:635–43.
33. 33. Birrell MA, Crispino N, Hele DJ, Patel HJ, Yacoub MH, Barnes PJ, et al. Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD. Br J Pharmacol. 2002;136:620–8.
34. 34. Rodger IW, Tousignant C, Young D, Savoie C, Chan CC. Neurokinin receptors subserving plasma extravasation in guinea pig airways. Can J Physiol Pharmacol. 1995;73:927–31.
35. 35. Mauser PJ, House A, Jones H, Correll C, Boyce C, Chapman RW. Pharmacological characterization of the late phase reduction in lung functions and correlations with microvascular leakage and lung edema in allergen-challenged Brown Norway rats. Pulm Pharmacol Ther. 2013;26:677–84.
36. 36. Tokuyama K, Nishimura H, Iizuka K, Kato M, Arakawa H, Saga R, et al. Effects of Y-27632, a Rho/Rho Kinase Inhibitor, on Leukotriene D(4)- and Histamine-Induced Airflow Obstruction and Airway Microvascular Leakage in Guinea Pigs in vivo. Pharmacology. 2002;64:189–95.
37. 37. Ramalho R, Almeida J, Beltrão M, Pirraco A, Costa R, Sokhatska O, et al. Neurogenic inflammation in allergen-challenged obese mice: a missing link in the obesity-asthma association? Exp Lung Res. 2012;38:316–24.
38. 38. Tamaoki J, Yamawaki I, Taira M, Nagano Y, Nakata J, Nagai A. Effect of cromolyn on adenosine-induced airway microvascular leakage in sensitized rats. Eur Respir J. 1999;14:1082–7.
39. 39. Planquois JM, Mottin G, Artola M, Lagente V, Payne A, Dahl S. Effects of phosphodiesterase inhibitors and salbutamol on microvascular leakage in guinea-pig trachea. Eur J Pharmacol. 1998;344:59–66.
40. 40. Vermeersch S, Benschop RJ, Van Hecken A, Monteith D, Wroblewski VJ, Grayzel D, et al. Translational Pharmacodynamics of Calcitonin Gene-Related Peptide Monoclonal Antibody LY2951742 in a Capsaicin-Induced Dermal Blood Flow Model. J Pharmacol Exp Ther. 2015;354:350–7.
41. 41. Giuffrida MJ, Valero N, Mosquera J, Duran A, Arocha F, Chacín B, et al. Increased Systemic Cytokine/Chemokine Expression in Asthmatic and Non-asthmatic Patients with Bacterial, Viral or Mixed Lung Infection. Scand J Immunol. 2017;85:280–90.
42. 42. Wu W, Li Y, Jiao Z, Zhang L, Wang X, Qin R. Phyllanthin and hypophyllanthin from Phyllanthus amarus ameliorates immune-inflammatory response in ovalbumin-induced asthma: role of IgE, Nrf2, iNOs, TNF-α, and IL’s. Immunopharmacol Immunotoxicol. 2018:1–13.