The Role Of Aspirin In H₂O₂-induced Oxidative Injury In Human Melanocytes And Its Machanism

Vitiligo is a common depigmented disease with its pathogenesis not fullyclarified. Nowadays it is still difficult to cure vitiligo. Our team as well as manyother researchers had demonstrated that oxidative stress is closely related tovitiligo. Aspirin is one of the mostly used nonsteroidal anti-inflammatory drugs（NSAIDs）. Recently mounting research showed aspirin has antioxidant propertyand it has demonstrated potient antioxidant effect in many cell and animal study.As a result, we postulate that aspirin may also have therapeutical effect in vitiligo.By literature review, we found that researcher had explored this postulation anddemonstrated that low dose aspirin could promote the proliferation of primarymelanocytes from vitiligo patients, reduce the lipid peroxidation, and arrest thedisease activity. However, there are only four relevant studies about aspirin invitiligo by now, and the mechanism underline the effect of aspirin has not beenwell investigated. Our previously work confirmed that Nrf2-ARE pathway playsan important role in melanocytes against H₂O₂-induced oxidative injury, and itsmain effector is HO-1. Further research demonstrated that the activation of Nrf2-ARE pathway in melanocytes from vitiligo patients is defective, thus themelanocytes is more vulnerable in oxidative stress. Some research confirmed thatantioxidative property of aspirin is related with its induction of HO-1expressionand NO synthesis. HO-1is known to be the downstream molecule of Nrf2-AREpathway, and NO is a stimulator of this pathway. To investigate whether aspirincan protect human melanocytes in vitro from H₂O₂-induced oxidative injury andwhether its antioxidant effect is resulted from activating Nrf2-ARE pathway andinducing the downstream molecule, we designed this research.Part I: The role of aspirin in H₂O₂-induced oxidative injury of normalhuman melanocyte cell line PIG1cells and primary human melanocytesMethod: PIG1cells were treated with different concentrations of aspirin for24h, then the safe concentrations of aspirin were determined by observing themorphology and viability of PIG1cells using photographing and CCK-8assaysrespectively. After pretreatment with aspirin at safe concentrations for24h, PIG1cells were treated with0.75mM H₂O₂to induce oxidative injury, the morphology,viability, apoptosis and intracellular ROS levels as well as LDH release rate ofPIG1cells were measured by photographing, CCK-8assays, Flow cotometryanalysis and spectrophotometer method respectively. Normal primarymelanocytes were isolated from human forekin specimens obtained duringcircumcision surgery and cultured in the same condition with PIG1cells asmentioned above. Before experiment, primary melanocytes were identified byS-100and MelanA staining. The morphology of primary melanocytes wereobserved by photographing after treatment with aspirin at different concentrationsfor24h. After pretreatment with aspirin at safe concentrations for24h, primarymelanocytes were treated with0.75mM H₂O₂to induce oxidative injury, the morphology and viability were measured by photographing and CCK-8assaysrespectively.Result: Treatment of aspirin alone for24h had no significant effect on themorphology and viability of PIG1cells when the concentrations were rangingfrom10to90μM. However, aspirin at the concentration of270μM and810μMresulted in signifant changes of morphology and viability to varying degrees.PIG1cells were incubated with10-90μM aspirin for24h followed by treatmentof0.75mM H₂O₂to induce oxidative injury, the morphology of PIG1cellsrecovered at different degrees when compared to H₂O₂treatment alone, theviability was significantly increased, and the apoptotic cells and LDH release rateof PIG1cells were significantly decreased. The intracellular ROS levelsdecreased at different degrees. The primary melanocytes grow well, and thepurity is very high. Pretreatment of10-90μM aspirin had no significant effect onthe morphology of primary melanocytes. After incubated with10-90μM aspirinfor24h, primary melanocytes were treated with0.75mM H₂O₂to induceoxidative injury, the morphology of primary melanocytes recovered at differentdegrees when compared to H₂O₂treatment alone, the viability was significantlyincreased.Part II: Mechanistic investigation of protection effect of aspirin onhuman melanocytes agaist H₂O₂-induced oxidative injuryMethod: After treating with90μM aspirin for24h, the p-Nrf2and Nrf2ofPIG1cells were measured by western blotting, and the mRNA of HO-1, NQO-1,GCLC, GCLM were determined by RT-PCR. After tranfected with Nrf2-specificsiRNA for48h, PIG1cell were treated with0.75mM H₂O₂to induce oxidativeinjury with pretreatment of90μM aspirin for24h, the morphology, viability, apoptosis and intracellular ROS levels of PIG1cells were measured byphotographing, CCK-8assays and Flow cotometry analysis respectively.Result: After treating with90μM aspirin for24h, the p-Nrf2and Nrf2ofPIG1cells were significantly increased, the mRNA of HO-1significantlyincreased too, while the mRNA of NQO-1, GCLC, GCLM had no significantchanges. After tranfection of Nrf2-specific siRNA into PIG1cells for48h,pretreatment of90μM aspirin had no significant effect on the morphology,viability, apoptosis and intracellular ROS levels when compared with H₂O₂treatalone. Knockdown of Nrf2expression abrogated the protective effect of aspirinon PIG1cellsConlusion:1. Low dose aspirin （approximately equal to the peak plasmaconcentrations after the oral administration of100-1000mg aspirin） had nocytotoxicity to normal human epidermal melanocytes.2Low dose aspirin couldprotect melanocytes from H₂O₂-induced oxidative injury and reduce intracellularROS level.3Low dose aspirin protected melanocytes from oxidative injury byactivating Nrf2-ARE pathway and inducing HO-1expression.