The Mechanism Of Melatonin Reducing Melanogenesis By Inhibiting The Paracrine Effects Of Keratinocytes

Objective:1.To explore whether melatonin affects the melanogenesis of melanocytes by affecting the paracrine effect of keratinocytes;2.To clarify the specific mechanism of melatonin affecting the paracrine effect of keratinocytes.Methods:1.The 3D reconstructed human epiderms in vitro was treated with melatonin to observe the effect of melatonin on skin pigmentation;after melatonin was treated with MNT1,a research tool for melanogenesis,the changes in melanin content were detected by Fontana-Masson stain;predicted in the maayanlab online website The expression of melatonin receptor in various skin cells;the co-culture model of MNT1 and human immortalized keratinocyte line(HaCaT)was treated with melatonin,and the changes of melatonin content were observed by Fontana-Masson stain;The supernatant was extracted to treat MNT1,and the content of melanin in MNT1 was observed by ammonia silver staining,and the expression of key genes in melanogenesis was detected by real-time quantitative polymerase chain reaction(q RT-PCR)and Western Blotting.2.After melatonin treatment of HaCaT,the supernatant was extracted to treat MNT1,and then the RNA of MNT1 was extracted for transcriptome sequencing.GO,KEGG,GSEA,and other bioinformatics analyses were performed on the sequencing results,and candidate signaling pathways were verified by Western Blotting.3.After melatonin treatment of HaCaT,the expressions of paracrine factors such as FGF2,ET-1,and PTGS2 were detected by q RT-PCR.Bioinformatics analysis was performed on melatonin-treated primary keratinocytes’ transcriptome GEO sequencing dataset(GSE147588).The interaction of differentially expressed genes was analyzed through the STRING online website to explore the effect of melatonin on keratinocyte paracrine.Possible mechanism of action and validation of candidate signaling pathways by Western Blotting.Results:1.After the 3D epidermis model was treated with melatonin at a concentration of 200 μmol/m L,the color of the epidermis of the model became lighter.The maayanlab website predicts that the melatonin receptor MT1 is relatively highly expressed in keratinocytes.The melatonin content in MNT1 was significantly decreased after melatonin treatment of MNT1 alone and in the co-culture system of MNT1 and HaCaT.In addition,the supernatant extracted from HaCaT treated with melatonin could significantly reduce the melanin content in MNT1 and inhibit the m RNA and protein expression levels of MITF,TYR,TYRP1,and DCT.2.After MNT1 was treated with melatonin-treated HaCaT supernatant,the sequencing results indicated that mitochondrial function and oxidative stress pathway in MNT1 were significantly inhibited.Western Blotting confirmed that the MAPK pathway in MNT1 was significantly inhibited.3.After melatonin treatment of HaCaT,the expressions of paracrine factors such as FGF2,ET-1,and PTGS2 in HaCaT cells were significantly down-regulated.The results of PPI analysis of differentially expressed genes in the GSE147588 dataset suggested that JAK3 and other genes were essential genes for melatonin to affect keratinocyte function.Western Blotting found that melatonin could significantly inhibit the JAK-STAT signaling pathway of HaCaT.Conclusions:1.Melatonin can inhibit skin pigmentation,and melatonin can not only directly inhibit the melanogenesis function of melanocytes but also indirectly inhibit melanogenesis by inhibiting the paracrine function of keratinocytes;2.Melatonin can inhibit the expression of FGF2,ET-1,PTGS2,and other paracrine factors in keratinocytes;3.Melatonin may affect the paracrine effect of keratinocytes by inhibiting the JAK-STAT pathway in keratinocytes.