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iNOS inhibition for vitiligo Eur J Transl Myol 29 (3): 251-260, 2019 Therapeutic effects of iNOS inhibition against vitiligo in an animal model Hamid Mansourpour (1), Katayoun Ziari (2), Sahar Kalantar Motamedi (3), Amin Hassan Poor (4) (1) Shahid Beheshti University of Medical Science, Tehran, Iran and AJA University of Medical Science, Tehran, Iran; (2) Department of Pathology, AJA University of Medical Science, Tehran, Iran; (3) Shahid Beheshti University of Medical Science, Tehran, Iran; (4) Tehran University of Medical Science, Tehran, Iran This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (CC BY-NC 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. Abstract Nitric oxide (NO) is involved in several biological processes, but its role in human melanogenesis and vitiligo need further studies. Previous studies revealed that exposure to UVA and UVB were capable of the inducing nitric oxide production in keratinocytes and melanocytes through the activation of constitutive nitric oxide synthase, whereas onlyinducible nitric oxide synthase overexpression has been reported to play an important role in hyperpigmentary disorders. The aim of this study was to evaluate iNOS inhibitor aminoguanidine (AG) as a therapeutic agent in our mouse model of vitiligo. In this study, male C57BL/6J Ler-vit/vit mice were purchased to evaluate the effect of iNOS inhibitor (aminoguanidine) (50 anduse 100 mg/kg) and L-arginine (100 mg/kg) in a mouse model of vitiligo induced by monobenzone 40%. Moreover, we used phototherapy device to treat the mice with NBUVB as a gold standard.The findings revealed that monobenzone was capable of inducing depigmentation after 6 weeks. However, aminoguanidine in combination with monobenzone was decrease the effect of monobenzone, while L-arginine play a key role in promoting the effect of monobenzone (P<0.001). Based on the phototherapy, the efficacy of phototherapy significantly increased by adding L-arginine (P<0.05). Taken together, we suggest that iNOS inhibitor can be a novel treatment for the prevention and treatment of vitiligo by combination of NBUVB therapy, furthermore; NO agents like L-arginine could also increase the effectiveness of phototherapy. Taken together, this pilot study showed significant repigmentation of vitiligous lesions treated with iNOS inhibitor plus NBUVB therapy, where other aspect including expression of an inducible iNOS, NO and TNF levels remained to be evaluated in mice model. commercial Key Words: Vitiligo, Nitric oxide, iNOS, Monobenzone, Treatment. Eur J Transl Myol 29 (3): 251-260, 2019 Non itiligo, a skin disease, is a common progressive role in development of disease when detected in V 8 depigmentation of epidermis that results in the perilesional skin of actively spreading vitiligo. Several destruction of melanocytes in the depigmented parts of cell lines has been described to play their role in vitiligo the skin, mucous membrane and retinal wall. As a result, development including lymphocytes, keratinocytes, and endothelial cells, involving in melanocyte migration, irregular white patches appear in different areas of the 2, 9-11 body. Vitiligo has been estimated to be one of the most proliferation, and differentiation. Increasing evidence supports that vitiligo could be a disorder of the frequent pigmentary diseases, accounting for about 0.1– 12 2% of the worldwide population.1-3 The pathophysiology entire epidermal melanin unit. Especially, keratinocytes of this disease is not well understood; however, several has been revealed to contribute to the melanocyte homeostasis and its changes affecting melanocyte theories about the pathomechanism of vitiligo have been 1,13,14 previously defined, such as genetic, biochemical, dysfunction in vitiligo epidermis. Vitiligo has been immunological, and environmental theories.4,5 On the defined as a multifactorial disease with autocytotoxic, neural, and autoimmune susceptibilities as well as other hand, increasing evidence indicated that there has a 15,16 correlation between vitiligo and other autoimmune environmental factors. Nitric oxide as an intracellular disease of the endocrine glands,6,7 as well as a messenger plays an important role in the synthesis of an lymphocytic infiltrate was found to play an important enzyme called NO synthase, which consists of three - 251 - iNOS inhibition for vitiligo Eur J Transl Myol 29 (3): 251-260, 2019 Fig 1. Monobenzone 40% induced depigmentation in exposed regions. isoforms: inducible NOS (iNOS), neuronal NOS the pathophysiologyonly of this disease using a (nNOS), and endothelial NOS (eNOS).17-30 Nitric oxide pharmacological inhibition of the iNOS, where a mouse has been suggested as a potential factor in inhibiting model of vitiligo was induced by monobenzone. cell proliferation, differentiation, and apoptosis. This free radical gas can be considered as a great challenge, when Materialsuse and Method a key role plays in pathogenesis of a lot of kind of Ethics Approvals autoimmune diseases such as vitiligo. Additionally, it is All procedures were performed in accordance with the likely to be involved, not only in melanocyte dysfunction National Institutes of Health (NIH) Guide for the Care and/or destruction, but also in the accumulating toxic and Use of Laboratory Animals (NIH publication # 80- intermediates of melanin synthesis and in elevating 23) and institutional guidelines for animal care and use reactive oxygen species.17-20,22-27 A growing body of (Department of Pharmacology, School of Medicine, evidence highlights that exposure to UVA and UVB may TUMS). Each experimental group consisted of 6 to 8 be capable of producing nitric oxide, especially in animals. keratinocytes and melanocytes, where it is involved, not only in increasing tyrosinase activity via activating Animals constitutive NOS, but also in the synthesiscommercial melanin.21, In this study, male C57BL/6J Ler-vit/vit mice were 31 The use of NBUVB can improve treatment of vitiligo obtained from Pasteur Institute, Tehran, Iran (weighing by stimulating the proliferation and migration of 25-30 g). Animals were then housed in Plexiglas boxes melanocytes, as well as stimulating the differentiation of (25 × 25 × 15 cm), under standard conditions melanocyte stem cells. UVA and NBUVB have been (temperature: 22 ± 2 °C, humidity: 50 ± 10%, 12-h light– introduced as common typesNon of phototherapy in vitiligo. dark cycle, and free access to food and water). Despite the similar effect of both interventions, Phototherapy device narrowband ultraviolet B (NBUVB) phototherapy can be more effective for patients suffering from vitiligo due to BIOSKIN®, a phototherapy device is equipped with a the lack of side effects of Psoralen.32,33 The topical use of short arc generator that provides a beam of visible and immunosuppressive agents, including glucocorticoids ultraviolet radiations, and is also filtered by an and calcineurin inhibitors, have been introduced as the interference filter for obtaining UV-B narrow band. The first line treatment, while the use of phototherapy has operator is capable of regulating emission time, and plays been considered as the second line for treating these its role in regulating emission time by accompanying a patients.32 iNOS is involved in the production of NO, time-controlled shutter. Furthermore, BIOSKIN® is where promotor polymorphisms of iNOS gene and NO equipped to prepare a spectrum of intensity up to 400 2 overproduction has been found to be associated with the mW/cm , where provide coverage from 300 to 320 nm pathogenesis of vitiligo, leading to melanocyte for emission spectrum, with an emission peak of 311 nm. destruction.34-38 Based on above-mentioned data, the Mice were then irradiated three times a week for 4 weeks. current study was aimed to demonstrate the association The narrow band UV-B (NBUVB) irradiation was of vitiligo with iNOS in phototherapy and to investigate performed using a BIOSKIN® device. It is worth noting - 252 - iNOS inhibition for vitiligo Eur J Transl Myol 29 (3): 251-260, 2019 Fig 2. Effect of aminiguanidine (100 mg/kg) plus monobenzone 40% on depigmentation that vitiligo patches were next irradiated by excluding investigated. In the sixthonly group, injectable NO inducer mucous membrane and genital part for each micro- was daily used at dose of 100 mg/kg (L-arginine) and the phototherapy session. Although BIOSKIN® equipment combined effect of this drug was investigated by is able to provide a large spectrum of intensity (0–400 phototherapy. As a matter of fact, the effects of L- mW/cm2), the intensity of 50 mW/cm2 was employed in arginine anduse AG on development of vitiligo were the present study for all animals during all sessions. The assessed by the vitiligo-inducing compound initial dose of irradiation was 20% less than minimum monobenzone. Optimal treatment dose of erythema dose (MED) that was applied to vitiligo area at aminoguanidine was determined by implementing a least 3 days before the beginning of the treatment. During dose-response model, assessing increasing doses (50 mg the subsequent sessions, the dose in each session and 100 mg). All drugs were purchased from Sigma, St increased to 20% until the development of erythema. Louis, MO, USA and afterward dissolved in saline. Additional dose of the next session was diminished by Animals received drugs intraperitoneally (i.p.), except 20% only in the erythematous area

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