The Inhibitory Effects of Nigella sativa Nanocapsules on Increased NNK-induced Inflammation, Oxidative Stress, and Tumor-associated Macrophage Responses in Wistar Rats

Document Type : Research Paper


1 Department of Physical Education and Sports, Payam Noor University, Tehran, Iran.

2 Department of Sport Sciences, University of Mazandaran, Babolsar, Iran

3 Department of Pathology, Babol University of Medical Sciences, Babol, Iran


Introduction: Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent cancer-causing agent in cigarettes and is also associated with the induction of lung tumors by the stimulation of malondialdehyde (MDA) levels and expression of tumor-associated macrophages (TAMs). The present study aimed to examine the variations of MDA levels and TAM expression in the lung tissues of rats exposed to NNK following 12 weeks of Nigella sativa nanocapsule injection. Methods: In this study, 48 Wistar rats were randomly divided into five groups of supplement, supplement with NNK, NNK, control, and saline. For 12 weeks, NNK was injected subcutaneously per kilogram of the animals' body weight with the weekly dose of 12.5 milligrams. In addition, the nanocapsules were subcutaneously injected once a week per kilogram of the body weight with the weekly dose of 12.5 milligrams. The MDA levels and CD68-TAM expression in lungs were determined using the enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Results: The injection of Nigella sativa nanocapsules for 12 weeks significantly decreased the MDA levels and CD68-TAM expression in the NNK group (p <0.001). Additionally, the injection of Nigella sativa nanocapsules along with the consumption of NNK significantly decreased the MDA levels and CD68-TAM expression in the lung tissues of the NNK group (p <0.001). Conclusion: According to the results, the orderly injection of Nigella sativa nanocapsules could significantly deter lung tissue inflammation induced by NNK through the reduction of MDA levels and CD68-TAM expression in rats.


1. Ge GZ, Xu TR, Ceshi Chen. Tobacco carcinogen NNK-induced lung cancer animal models and associated carcinogenic mechanisms. Acta Biochim Biophys Sin. 2015; 47(7):477-87.
2. Alberg AJ, Brock MV, Samet JM. Epidemiology of lung cancer: looking to the future. J Clin Oncol. 2005; 23(14):3175-85.
3. Barzegari A, Mirdar S. Effect of a 12-week submaximal swimming training in rats exposed to tobacco- derived nitrosamine ketone. Caspian J Intern Med. 2018; 9(2):158-63.
4. Barta P, Van Pelt C, Men T, Dickey BF, Lotan R, Moghaddam SJ. Enhancement of lung tumorigenesis in a Gprc5a knockout mouse by chronic extrinsic airway inflammation. Mol Cancer. 2012; 11(1):1-11.
5. Barzegari A, Mirdar S, Ranayi M. Modulation of Vascular Endothelial Growth Factor and Annexin A2 in Response to 4-(Methylnitrosamino)-1-(3-pyridyl)-1-Butanone -Induced Inflammation via Swimming Training. Iran J Allergy Asthma Immunol. 2018; 17(5):418-27.
6. Noy R, Pollard JW. Tumor-Associated Macrophages from Mechanisms to Therapy. Immunity. 2014; 41(1):49-61.
7. Mei J, Xiao Z, Gu C, Pu Q, Ma L, Liu C, et al. Prognostic impact of tumor-associated macrophage infiltration in non-small cell lung cancer: A systemic review and meta-analysis. Oncotarget. 2016; 7(23): 34217-28. 
8. Sun S, Pan X,  Zhao L, Zhou J, Wang H,  Sun Y. The expression and relationship of cd68-tumor-associated macrophages and micro vascular density with the prognosis of patients with laryngeal squamous cell carcinoma. Clin Exp Otorhinolaryngol. 2016; 9(3):270-77.
9. Kopčinović LM, Domijan AM, Posavac K, Čepelak I, Grubišić TZ,  Rumora L. Systemic redox imbalance in ˇstable chronic obstructive pulmonary disease. Biomarkers. 2016; 28(8):692-8.
10. Goenence A, Ozkan Y, Torun M, Seimseek B. Plasma malondialdehyde (MDA) levels in breast and lung cancer patients. J Clin Pharm Ther. 2001; 26(2):141-4.
11. Hamza RZ, El-Shenawy NS. Anti-inflammatory and antioxidant role of resveratrol on nicotine-induced lung changes in male rats. Toxicol Rep. 2017; 4:399-407.
12. Koutsokera A, Kiagia M, Saif MW, Souliotis K, Syrigos KN. Nutrition habits, physical activity, and lung cancer: an authoritative review. Clin Lung Cancer. 2013; 14(4):342-50.
13. Rahmani AH, Alzohairy MA, Khan MA, Aly SM. Therapeutic implications of black seed and its constituent thymoquinone in the prevention of cancer through inactivation and activation of molecular pathways. Hindawi Publishing Corporation. 2014; 1-13.
14. Fadda LM, Al-Rasheed NM, Hasan IH, Ali HM, AlRasheed NM, Al-Fayez M, et al. Bax and CD68 expression in response to liver injury induced by acetaminophen: the hepatoprotective role of thymoquinone and curcumin. Pakistan J Zool. 2016; 49(1):85-93.
15. Wilson AJ, Saskowski J, Barham W, Khabele D, Yull F. Microenvironmental effects limit efficacy of thymoquinone treatment in a mouse model of ovarian cancer. Mol Cancer. 2015; 14(1):1-4.
16. Mabrouk GM, Moselhy SS, Zohny SF, Ali E, Helal TEA, Amin AA, et al. Inhibition of methylnitrosourea (MNU) induced oxidative stress and carcinogenesis by orally administered bee honey and Nigella grains in Sprague Dawely rats. J Exp Clin Cancer Res. 2002; 21(3):341-46. 
17. Assayed ME. Radioprotective effects of black seed (Nigella sativa) oil against hemopoietic damage and immunosuppression in gamma-irradiated rats. Immunopharmacol Immunotoxicol. 2010; 32 (2):284-96.
18. Rafienia M, Orang F, Emamy SH. Preparation and characterization of polyurethane microspheres containing theophylline. Journal of Bioactive and Compatible Polymers. 2006; 21(4):341-49.
19. Belinsky SA, Foley JF, White CM, Anderson MW, Maronpot R. Dose-response relationship between O6-methylguanine formation in Clara cells and induction of pulmonary neoplasia in the rat by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Research. 1990; 50(12):3772-80.
20. Khan MA, Ashfaq MK, Zuberi HS Saeed M, Gilani AUH. The in vivo antifungal activity of the aqueous extract from Nigella sativa seeds. Phytother Res. 2003; 17(2):183-86.
21. Keshavarzi M, Shakeri S, Kiani K. Preparation and in vitro investigation of antigastric cancer activities of carvacrol-loaded human serum albumin nanoparticles. IET Nanobiotechnology. 2015; 9(5):294-9.
22. Verma SK, Rastogi S, Javed K, Akhtar M, Arorac I, Samim M. Nanothymoquinone, a novel hepatotargeted delivery system for treating CCl4 mediated hepatotoxicity in rats. J Mater Chem. 2013; 1:2956-66.
23. Barzegari A, Mirdar S, Ranayi M. Modulation of vascular endothelial growth factor and annexin a2 in response to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone -induced inflammation via swimming training. Iran J Allergy Asthma Immunol. 2018; 17(5):418-27.
24. Nagababu E, Rifkind JM, Boindala S, Nakka L. Assessment of antioxidant activity of eugenol in vitro and in vivo. Methods Mol Biol. 2010; 610:165-80.
25. Barzegari A, Miradar s, Ranaee M. Physiology of exercise and physical activity. 2019; 12(2): 67-79.
26. Sun S, Pan X, Zhao L, Zhou J, Wang H, Sun Y. The expression and relationship of cd68-tumor-associated macrophages and microvascular density with the prognosis of patients with laryngeal squamous cell carcinoma. Clin Exp Otorhinolaryngol. 2016; 9(3): 270-7.
27. Ahmed AM. The dual role of oxidative stress in lung cancer. In: Chakraborti S, Chakraborti T, Das S, Chattopadhyay D. (eds) Oxidative stress in lung diseases. Springer, Singapore; 2019.
28. Nachiappan V, Mufti SI, Chakravarti A, Eskelson CD, Rajasekharan R. Lipid peroxidation and ethanol-related tumor promotion in Fischer-344 rats treated with tobacco-specific nitrosamines. Alcohol and Alcohol. 1994; 29(5):565-74.
29. Marnett LJ. Oxy radicals, lipid peroxidation and DNA damage. Toxicology. 2002; 181(182): 219-22.
30. Ayaori M, Hisada T, Suzukawa M, Yoshida H, Nishiwaki M, Ito T, et al. Plasma levels and redox status of ascorbic acid and levels of lipid peroxidation products in active and passive smokers. Environ Health Perspect. 2000; 108(2):105-8.
31. Proulx LI, Castonguay A, Bissonnette EY. Cytokine production by alveolar macrophages is down regulated by the alpha-methylhydroxylation pathway of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carcinogenesis. 2004; 25(6):997-1003.
32. Wang R, Zhang J, Chen S, Lu M, Luo X, Yao S, et al. Tumor-associated macrophages provide a suitable microenvironment for non-small lung cancer invasion and progression. Lung Cancer. 2011; 74 (2):188-96.
33. Avti PK, Kumar S, Pathak CM, Vaiphei K, Kl K. Smoke-less tobacco impairs the antioxidant defense in liver, lung, andkidney of rats. Toxicol Sci. 2006; 89(2):547-53.
34. Velayutharaj A, Ramesh R, Niranjan G, Kala C. Biochemical assessment of liver damage in smokeless tobacco users. Int J Curr Res Rev. 2013; 5(23):63-9.
35. Bouasla I, Bouasla A, Boumendjel A, Messarah M, Abdennour C, Boulakoud MS, et al. Nigella sativa oil reduces aluminiumchloride-induced oxidative injury in liver and erythrocytes of rats. Biol Trace Elem Res. 2014; 162:252-61.
36. Wilson AJBarham WSaskowski J, Tikhomirov O, Chen L, Lee HJ, et al. Tracking NF-κB activity in tumor cells during ovarian cancer progression in a syngeneic mouse model. J Ovarian Res. 2013; 6:63.
37. Ben-Shaul V, Lomnitski L, Nyska A, Zurovsky Y, Bergman M, Grossman S. Search articles by 'S Grossman' Grossman S et al. The effect of natural antioxidants, NAO and apocynin, on oxidative stress in the rat heart following LPS challenge. Toxicol Lett. 2001; 123(1): 1-10.
38. Entok E, Ustuner MC, Ozbayer C, Tekin N, Akyuz F, Yangi B, et al. Anti-inflammatuar and anti-oxidative effects of Nigella sativa L.: 18FDG-PET imaging of inflammation. Mol Biol Rep. 2014; 41(5):827-34.
Volume 9, Issue 1
March 2021
Pages 82-90
  • Receive Date: 20 December 2020
  • Revise Date: 18 January 2021
  • Accept Date: 31 January 2021
  • First Publish Date: 31 January 2021