737 Opsin as a photoreceptor after UVA irradiation to regulate MITF signal pathway and influence melanogenesis in the cultured human melanocyte

      Background: It is well known that ultraviolet radiation can cause skin pigment darkening. MITF plays an important role as a key transcription factor in the signal pathways of melanin production. Opsin is a photosensitive receptor protein belonging to the G protein coupled receptor superfamily (GPCRs) , which can be detected in the skin and melanocytes. However, it is not clear whether it as a photoreceptor to conduct ultraviolet enter the skin to cause melanogenesis. Objective: To investigate whether the MITF transcription factor can be regulated by the opsin after ultraviolet irradiation on melanocytes and then affect melanin production. Method: Suspended human melanocytes were obtained from human foreskin with two-step enzyme-digestion method and then cultured in M254 medium supplied with human melanocyte growth supplement and for three passages. CCK8 determined the appropriate dose of UVA irradiated melanocytes and used 3J/cm2UVA irradiation. The cells were collected after irradiation 0h, 4h, 12h, 24h and 48h, and then the supernatant was collected after RIPA lysis and the protein quantity was measured. Western blot detected OPN1, OPN2, OPN3, OPN4, OPN5, MITF and p-MITF. Cellular sediment solubilized in 400μl of 1M NaOH in 70°C for 3h to dissolve melanin and the absorbance was measured spectrophotometrically at 405nm using a plate reader. Melanin production was calculated by normalizing the total melanin values with protein content. Result : 48 hours after 3J/cm2 UVA irradiation, the survival rate of melanocytes was 141.87±17.70%.Opsin1,Opsin2 and Opsin5 were significantly increased at 24 h and 48 h compared with control group, MITF did not change significantly, but p-MITF increased at all time points after UVA irradiation, and was significantly increased at 24h and 48 h after irradiation. UVA induced a high increase in melanin content in melanocyte after 24h and 48h of irradiation. Conclusion: After irradiation of melanocytes with UVA, the Opsin may regulate the MITF signal pathway to affect the production of melanin.