The Study On The In Vitro Model Of Human Epidermal Melanocytes And Keratinocytes Co-culture System And On The Effects Of 11 Compounds On Pigmentation

Objective To establish the in vitro co-culture model of Chinese human epidermalmelanocytes and keratinocytes. To compare the effects of some known compounds onpigmentation in B16F10 melanoma cells, melanocytes and the co-culture model ofmelanocytes and keatinocytes. To study the influence of some Chinese herbal monomerson pigmentation in B16F10 melanoma cells, melanocytes and the co-culture model ofmelanocytes and keatinocytes. To further investigate the mechanisms of the effects ofscreened promising monomer on melanogenesis and melanin transfer. To preliminarilyinvestigate the clinical effects of the screened promising monomer on bleaching skin andtreating patients with chloasma.Methods B16F10 melanoma cells, human melanocytes and co-cultures of humanmelanocytes and keratinocytes wee cultured in invitro respevtively. Inverted microscope,confocal microscopy and electron microscope were used to observe the co-culture modeland the melanosome transfer in the model. Some candidates including a few drugs on themarket (hydroquinone, arbutin, 8-MOP, all trans retinoic acid, NO, viaminate andniacinamide) and a few herbal monomers (paeonol, emodin, matrine and oxymatrine) weretested for melanogenesis (melanin synthesis and tyrosianase activity) in B16F10melanoma cells, human melanocytes and co-culture of human melanocytes andkeratinocytes. The influences of these candidates on melanin transfer in co-culture modelwere also investigated quantitately by flow cytometer. RT-PCR assay was used to detectthe effects of paeonol on the tyrosianse mRNA expression levels in human melanocytesand on the PAR-2 mRNA expression levels in co-culture cells. Western-blot assay wasused to investigste the influence of paeonol on the tyrosianse expression levels in humanmelanocytes. The clinical dipigmenting effects of 10% paeonol cream on normal skin andlesional skin of chloasma were also studied. Results 1 The in vitro co-culture model of Chinese human epidermalmelanocytes and keratinocytes was established successfully. On the 3rd day, 5th day andthe 7th day, live melanocytes and keratinocytes were observed in the co-culture system anddendritic contact was observed between them by inverted microscopy. Moreover, thetransfer of melanosomes from the melanocytes to the keratinocytes were observed visuallyby dint of fluorescence CFDA-SE under confocal microscope.2 Hydroquinone, arbutin, paeonol, matrine, oxymatrine and viaminate inhibited themelanin synthesis and tyrosinase activity in B16F10 melanoma cells, human melanocytesand co-cultures in a dose-dependent manner. The results of hydroquinone, arbutin, matrineand viaminate in co-culture cells were more dramatic than those in monoculture cellswhereas the results of paeonol were less evdent than those in monoculture. There were nodifferences of the results of oxymatrine in co-culture cells and in monoculture cells.3 As for emodin, interesting results were obtained. Emodin had no effects on themelanin synthesis and tyrosinase activity in B16F10 melanoma cells whereas it increasedthe melanin synthesis and tyrosinase activity in melanocytes and co-cultures atconcentrations below 2μmol/L. However, emodin exhibited inhibitory effects on melaninsynthesis and tyrosinase activity in all cells at higher concentrations. The results of emodinin co-culture cells were more dramatic than those in monoculture cells at loweconcentrations whereas the results of emodin in monoculture cells were more dramaticthan those in co-culture cells at high concentrations.4 8-MOP and NO increased the melanin synthesis and tyrosinase activity in B16F10melanoma cells, human melanocytes and co-cultures in a dose-dependent manner. Theresults in co-culture cells were less dramatic than those in monoculture cells.5 All-trans retinoic acid increased the melanin formation and tyrosinase activity inB16F10 melanoma cells, whereas it had no significant effects on melanin synthesis in melanocytes and inhibited melanin formation and tyrosinase activity in a dose-dependentmanner in co-culture cells.6 Niacinamide had no effects on melanin synthesis and tyrosinase activity in B16F10melanoma cells and melanocytes whereas it exerted a suppressing effects on melaninsynthesis and tyrosinase activity in co-cultures.7 Hydroquinone, arbutin, paeonol, matrine, all trans retinoic acid, niacinamide andviaminate inhibited the melanin transfer in co-culture model in a dose-dependent manner;8-MOP and NO promoted the melanin transfer whereas oxymatrine and emodin had noeffects on this process.8 Paeonol reduced the tyrosianse mRNA expression level and protein level in humanmelanocytes as well as melanin transfer enhanced by SLIGRL in co-culture system in adose-dependent manner whereas it had no effects on the PAR-2 mRNA expression level inco-culture cells.9 Excellent results were also obtained in the study on the clinical dipigmentingeffects of 10% paeonol cream on normal skin and lesional skin of chloasma.Conclusion 1. The in vitro co-culture model of Chinese human epidermal melanocytes andkeratinocytes was established successfully, in which melanin transfer was observed.2. The co-culture model is a necessary supplement of the usual models on pigmentation, asit is predominant in simulating the epidermal melanin unit and in screening the effects ofthe candidates on both melanogenesis and melanin transfer. The false positive results ornegative results can be avoided in this model. Moreover some known compounds werefound to have new effects on pigmentation, such as that hydroquinone, arbitin, all-transretinoic acid can inhibit the melanin transfer and 8-MOP, NO can increase melanintransfer. 3. The results of screening Chinese herbal monomers showed paeonol is a promisingcandidate in treating hyperpigmentation. Matrine and emodin are to be further investigated.4. The mechanisms of the effects of paeonol on pigmentation involve its inhibitory effectson melanin synthesis and tyrosianse activity, its down-regulating effects on the tyrosiansemRNA and protein expressions, and its suppressing effects on melanin transfer.5. Topical paeonol is effective in bleaching skin and treating patients with chloasma.