Role of L-arginine/NO/cGMP/KATP channel signaling pathway in the central and peripheral antinociceptive effect of thymoquinone in rats

Document Type : Original Article


1 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran


Objective(s): Growing evidence demonstrates that L-arginine/NO/cGMP/KATP channel pathway has a modulatory role in pain perception. Previous studies have shown that thymoquinone exerts antinociceptive effects; however, the mechanisms underlying antinociception induced by thymoquinone have not been fully clarified. The aim of the present study was to evaluate the role of L-arginine/NO/cGMP/KATP channel pathway in the central and peripheral antinociceptive effect of thymoquinone in rats.
Materials and Methods: Rats were pretreated intraplantarly (IPL) or intracerebroventricularly (ICV) with L-arginine (the NO precursor), l-NAME (an NO synthase inhibitor), SNAP (an NO donor), methylene blue (a guanylyl cyclase inhibitor), glibenclamide (the blocker of KATP channel), and tetraethylammonium (TEA, a Kv channel blocker) before the injection of thymoquinone.
Results: Local ipsilateral (20 and 40 μg, IPL) but not contralateral and ICV (4 and 8 μg) administration of thymoquinone caused a dose-dependent and significant antinociception in both early and late phases of the formalin test. Pretreatment of rats with L-arginine (100 μg, IPL or ICV) and SNAP (200 μg, IPL or ICV) increased while l-NAME (100 μg, IPL or 1 μg, ICV) and methylene blue (400 μg, IPL or ICV) decreased the antinociceptive effects of thymoquinone in the formalin test. The administration of TEA (IPL or ICV) did not modify but glibenclamide (50 μg, IPL or ICV) significantly abolished the peripheral and central antinociceptive effects of thymoquinone in both phases of the formalin test.
Conclusion: The results of the present study indicate that L-arginine/NO/cGMP/KATP channel pathway participates in the central and peripheral antinociceptive effect of thymoquinone.


Main Subjects

1.Khader M, Eckl PM. Thymoquinone: an emerging natural drug with a wide range of medical applications. Iran J Basic Med Sci 2014; 17:950–957.
2.Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils- a review. Food Chem Toxicol 2008; 46:446–475.
3.Hosseinzadeh H, Parvardeh S, Nassiri-Asl M, Mansouri MT. Intracerebroventricular administration of thymoquinone, the major constituent of Nigella sativa seeds, suppresses epileptic seizures in rats. Med Sci Monit 2005; 11:BR106–110.
4.Hosseinzadeh H, Parvardeh S. Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine 2004; 11:56–64.
5.Parvardeh S, Fatehi M. Effects of thymoquinone, the major constituent of Nigella sativa seeds, on the contractile responses of rat vas deferens. Pharm Biol 2003; 41:616–621.
6.Parvardeh S, Fatehi M. Inhibitory effects of thymoquinone, the major component of Nigella sativa L. seeds, on spontaneous and evoked contractions of guinea pig isolated ileum. J Med Plants 2007; 3:29–39.
7.Parvardeh S, Moghimi M. Skeletal muscle relaxant effects of thymoquinone, the major constituent of Nigella sativa. J Med Plants 2015; 2:122–133.
8.Hosseinzadeh H, Parvardeh S, Asl MN, Sadeghnia HR, Ziaee T. Effect of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus. Phytomedicine 2007; 14:621–627.
9.Hosseinzadeh H, Parvardeh S, Masoudi A, Moghimi M, Mahboobifard F. Attenuation of morphine tolerance and dependence by thymoquinone in mice. Avicenna J Phytomed 2016; 6:55–66.
10.Khan A, Vaibhav K, Javed H, Khan MM, Tabassum R, Ahmed ME. Attenuation of Aβ-induced neurotoxicity by thymoquinone via inhibition of mitochondrial dysfunction and oxidative stress. Mol Cell Biochem 2012; 369:55–65.
11.Mehri S, Shahi M, Razavi BM, Hassani FV, Hosseinzadeh H. Neuroprotective effect of thymoquinone in acrylamide-induced neurotoxicity in Wistar rats. Iran J Basic Med Sci 2014; 17:1007–1011.
12.Abdel-Fattah AM, Matsumoto K, Watanabe H. Antinociceptive effects of Nigella sativa oil and its major component, thymoquinone, in mice. Eur J Pharmacol 2000; 400:89–97.
13.De Sousa DP, Nóbrega FF, Santos CC, Benedito RB, Vieira YW, Uliana MP, et al. Antinociceptive activity of thymoquinone and its structural analogues: A structure-activity relationship study. Trop J Pharma Res 2012; 11:605–610.
14.Ocaña M, Cendán CM, Cobos EJ, Entrena JM, Baeyens JM. Potassium channels and pain: present realities and future opportunities. Eur J Pharmacol 2004; 500:203–219.
15.Yalcin I, Aksu F. Involvement of potassium channels and nitric oxide in tramadol antinociception. Pharmacol Biochem Behav 2005; 80:69–75.
16.Tsantoulas C. Emerging potassium channel targets for the treatment of pain. Curr Opin Support Palliat Care 2015; 9:147–154.
17.Galeotti N, Ghelardini C, Bartolini A. The role of potassium channels in antihistamine analgesia. Neuropharmacology 1999; 38:1893–1901.
18.Alves DP, Soares AC, Francischi JN, Castro MS, Perez AC, Duarte ID. Additive antinociceptive effect of the combination of diazoxide, an activator of ATP-sensitive K+ channels, and sodium nitroprusside and dibutyryl-cGMP. Eur J Pharmacol 2004; 489:59–65.
19.Alves DP, Tatsuo MA, Leite R, Duarte ID. Diclofenac-induced peripheral antinociception is associated with ATP-sensitive K+ channels activation. Life Sci 2004; 74:2577–2591.
20.Rodrigues AR, Duarte ID. The peripheral antinociceptive effect induced by morphine is associated with ATP‐sensitive K+ channels. Br J Pharmacol 2000; 129:110–114.
21.Ortiz MI, Granados-Soto V, Castañeda-Hernández G. The NO-cGMP-K+ channel pathway participates in the antinociceptive effect of diclofenac, but not of indomethacin. Pharmacol Biochem Behav 2003; 76:187–195.
22.Ferreira SH, Duarte ID, Lorenzetti BB. The molecular mechanism of action of peripheral morphine analgesia: stimulation of the cGMP system via nitric oxide release. Eur J Pharmacol 1991; 201:121–122.
23.Dal D, Salman MA, Salman AE, Iskit AB, Aypar Ü. The involvement of nitric oxide on the analgesic effect of tramadol. Eur J Anaesthesiol 2006; 23:175–177.
24.Miclescu A, Gordh T. Nitric oxide and pain:‘Something old, something new’. Acta Anaesthesiol Scand 2009; 53:1107–1120.
25.Lázaro-Ibáñez GG, Torres-López JE, Granados-Soto V. Participation of the nitric oxide-cyclic GMP-ATP-sensitive K+ channel pathway in the antinociceptive action of ketorolac. Eur J Pharmacol 2001; 426:39–44.
26.Luger TJ, Hayashi T, Weiss CG, Hill HF. The spinal potentiating effect and the supraspinal inhibitory effect of midazolam on opioid-induced analgesia in rats. Eur J Pharmacol 1995; 275:153–162.
27.Granados-Soto V, Argüelles CF, Ortiz MI. The peripheral antinociceptive effect of resveratrol is associated with activation of potassium channels. Neuropharmacology 2002; 43:917–23.
28.Ortiz MI, Medina-Tato DA, Sarmiento-Heredia D, Palma-Martínez J, Granados-Soto V. Possible activation of the NO-cyclic GMP-protein kinase G-K+ channels pathway by gabapentin on the formalin test. Pharmacol Biochem Behav 2006; 83:420–427.
29.Ghorbanzadeh B, Mansouri MT, Hemmati AA, Naghizadeh B, Mard SA, Rezaie A. Involvement of L-arginine/NO/cGMP/K ATP channel pathway in the peripheral antinociceptive actions of ellagic acid in the rat formalin test. Pharmacol Biochem Behav 2014; 126:116–121.
30.Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Elsevier Academic Press; 2005.
31.Lima DK, Ballico LJ, Lapa FR, Gonçalves HP, de Souza LM, Iacomini M, et al. Evaluation of the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oil from Piper aleyreanum C. DC in rodents. J Ethnopharmacol 2012; 142:274–82.
32.Lin DT, Fretier P, Jiang C, Vincent SR. Nitric oxide signaling via cGMP-stimulated phosphodiesterase in striatal neurons. Synapse 2010; 64:460–466.
33.Parvardeh S, Moghimi M, Eslami P, Masoudi A. α-Terpineol attenuates morphine-induced physical dependence and tolerance in mice: role of nitric oxide. Iran J Basic Med Sci 2016; 19:201–208.
34.Ocaña M, Del Pozo E, Baeyens J. ATP-dependent K+ channel blockers antagonize morphine-but not U-504, 88H-induced antinociception. Eur J Pharmacol 1993; 230:203–207.
35.Kawabata A, Manabe S, Manabe Y, Takagi H. Effect of topical administration of l‐arginine on formalin‐induced nociception in the mouse: a dual role of peripherally formed NO in pain modulation. Br J Pharmacol 1994; 112:547–550.
36.Kawabata A, Fukuzumi Y, Fukushima Y, Takagi H. Antinociceptive effect of L-arginine on the carrageenin-induced hyperalgesia of the rat: possible involvement of central opioidergic systems. Eur J Pharmacol 1992; 218:153–158.
37.Déciga-Campos M, López-Muñoz FJ. Participation of the l-arginine-nitric oxide-cyclic GMP-ATP-sensitive K+ channel cascade in the antinociceptive effect of rofecoxib. Eur J Pharmacol 2004; 484:193–199.
38.Bulutcu F, Dogrul A, Güç MO. The involvement of nitric oxide in the analgesic effects of ketamine. Life Sci 2002; 71:841–853.
39.Xu JY, Tseng LF. Increase of nitric oxide by l-arginine potentiates β-endorphin-but not μ-, δ-or κ-opioid agonist-induced antinociception in the mouse. Eur J Pharmacol 1993; 236:137–142.
40.Safaripour S, Nemati Y, Parvardeh S, Ghafghazi S, Fouladzadeh A, Moghimi M. Role of l‐arginine/SNAP/NO/cGMP/KATP channel signaling pathway in antinociceptive effect of α‐terpineol in mice. J Pharm Pharmacol 2018; DOI: 10.1111/jphp.12864.
41.Paoloni JA, Appleyard RC, Nelson J, Murrell GA. Topical nitric oxide application in the treatment of chronic extensor tendinosis at the elbow a randomized, double-blinded, placebo-controlled clinical trial. Am J Sports Med 2003; 31:915–920.
42.Ventura-Martínez R, Déciga-Campos M, Díaz-Reval MI, González-Trujano ME, López-Muñoz FJ. Peripheral involvement of the nitric oxide-cGMP pathway in the indomethacin-induced antinociception in rat. Eur J Pharmacol 2004; 503:43–48.
43.Jesse CR, Savegnago L, Nogueira CW. Role of nitric oxide/cyclic GMP/K+ channel pathways in the antinociceptive effect caused by 2, 3-bis (mesitylseleno) propenol. Life Sci 2007; 81:1694–1702.
44.Srebro DP, Vučković SM, Vujović KR, Prostran MŠ. TRPA1, NMDA receptors and nitric oxide mediate mechanical hyperalgesia induced by local injection of magnesium sulfate into the rat hind paw. Physiol Behav 2015; 139:267–273.
45.Gilhotra N, Dhingra D. Thymoquinone produced antianxiety-like effects in mice through modulation of GABA and NO levels. Pharmacol Rep 2011; 63:660–669.
46.Yamazumi I, Okuda T, Koga Y. Involvement of potassium channels in spinal antinociceptions induced by fentanyl, clonidine and bethanechol in rats. Jpn J Pharmacol 2001; 87:268–276.
47.Tsantoulas C, McMahon SB. Opening paths to novel analgesics: the role of potassium channels in chronic pain. Trends Neurosci 2014; 37:146–158.
48.Tahrani AA, Barnett AH, Bailey CJ. Pharmacology and therapeutic implications of current drugs for type 2 diabetes mellitus. Nat Rev Endocrinol 2016; 12:566–592.
49.Suddek GM. Thymoquinone-induced relaxation of isolated rat pulmonary artery. J Ethnopharmacol 2010; 127:210–214.