The Effect And Mechanism Of Metformin On Melanin Synthesis And Melanosome Transfer

Objective:To identify the effects of metformin on melanogenesis and melanosome transfer in zebrafish,human primary melanocytes,and human/mouse melanoma cells,and to explore its related mechanisms.Method:1.Pigment granule in zebrafish was observed at 48hpf,72hpf and 96hpf in 10 mM metformin treatment and control group.The melanin content was determined by sodium hydroxide(NaOH),and tyrosinase activity was determined by levodopa(L-DOPA).The mRNA expression levels of melanogenesis and melanosome transfer-related genes,including MITF,TYR,TYRP1a,DCT,MLPHa,Rab27a,and Myo5a were determined by real-time quantitative PCR(RT-qPCR).2.Four different concentrations of metformin(5 mM,10 mM,20 mM,40 mM)were treated in human primary melanocytes and human/mouse melanoma cells(MNT1 cells,B16F10 cells)for 24h and 48h,relative cell viability was identified by CCK-8,helping to select the appropriate concentrations.Melanin content and tyrosinase activity were measured after the treatment of 5 mM,10 mM metformin,or 1 mM arbutin with or without the stimulating of 1 μM α-melanocyte-stimulating hormone(α-MSH).In primary melanocytes,after the treatment of metformin,the content of cAMP was quantified through an ELISA kit,and the mRNA and protein expression levels of MITF and downstream molecules regulating melanin syntheses,such as TYR,TYRP1,and DCT were detected by RT-qPCR and Western blot.B16F10 cells were treated with 10 mM metformin and/or 20 μM cAMP activator(Forskolin)to detect the changes of melanin content,tyrosinase activity and protein expression of genes related to melanin synthesis.3.Cellular morphology and formation of dendrites were observed under optical microscopy in metformin-treated human primary melanocytes and B16F10 cells,and the number of dendrites in melanocytes was counted and recorded for statistical analysis.The changes in dendrites and filopodia-like structures were investigated under scanning electron microscopy(SEM).In the co-culture system of HaCaT cells and primary melanocytes,the melanosome with gp100 labeling in melanocytes and cytokeratin-positive HaCaT cells were visualized to evaluate the transferred melanosome in keratinocytes through laser confocal microscopy(LCM).The mRNA and protein expression levels of melanosome transfer-related genes,MLPH,Rab27a,and Myo5a,were determined by RT-qPCR and Western blot.The distribution of the cytoskeleton was stained by TRITC-Phalloidin immunofluorescent assay.The protein expression levels of RhoA and Rac1,members of the Rho GTPases which regulate the cytoskeletal change,were verified by Western blot.RhoA inhibitor was used to confirm if the change of dendrites induced by metformin could be reversed.B16F10 cells were treated with 10 mM metformin and/or 20 μM Forskolin,and the protein expression levels of melanosome transfer-related genes and Rho GTPases were detected by Western blot.Result:1.At 48hpf,72hpf,and 96hpf,obvious reduction of the melanin granules was observed in metformin-treated zebrafish and the mean area percentage of melanin granules measured in head regions was significantly decreased(P<0.05).After metformin treatment,melanin content and tyrosinase activity were both remarkably reduced(P<0.05),and the mRNA expression levels of MITF,TYR,TYRP1a,and DCT in melanogenesis and MLPHa,Rab27a,and Myo5a in melanosome transfer were all markedly downregulated(P<0.05).2.The relative cell viability after treatment with 5 mM and 10 mM metformin was all about 90%and above,so these two concentrations were suitable for subsequent research.In primary melanocytes,MNT1 cells,and B16F10 cells,melanin content and tyrosinase activity were obviously reduced after 5 mM,10 mM metformin,or 1 mM arbutin treatment regardless of normal status and α-MSH-induced pigmentation(P<0.05).The level of cAMP and the expression of MITF,TYR,TYRP1,and DCT were declined after metformin treatment.In B16F10 cells,metformin could partially reverse the up-regulation induced by Forskolin of melanin content,tyrosinase activity,TYR and TYRP1 expression.3.After treatment of metformin,the dendrites of human primary melanocytes and B16F10 cells were shortened and reduced,the number of bipolar melanocytes was notably increased and the number of multi-polar melanocytes was prominently decreased(P<0.05),while no obvious change of dendrites was seen after arbutin treatment(P>0.05);under SEM,the number of dendrites and filopodia-like structures was both reduced.In the co-culture system,the melanosomes transferred to keratinocytes were reduced after treatment with metformin and arbutin.The protein expression levels of melanosome transfer-related genes,MLPH,Rab27a,and Myo5a,were significantly down-regulated after metformin treatment.A noticeable difference was detected between the metformin treatment and control group,the F-actin was expressed more intensely and was more inclined to cluster into a retiform bundle in the former.After treatment of metformin,the protein expression levels of RhoA and downstream ROCK1 were upregulated and the Rac1 was downregulated.RhoA inhibitor could partially reverse the inhibitory effect on dendrite formation by metformin.In B16F10 cells,metformin could partially reverse the increase of MLPH,Rab27a,Racl expression and the decrease of RhoA expression induced by Forskolin.Conclusion:1.Metformin could inhibit the melanin content and tyrosinase activity in zebrafish and cells and reduce melanin synthesis;in human primary melanocytes,metformin has a similar effect as arbutin,which is known to inhibit melanin synthesis;the cAMP-MITF pathway may be involved in the inhibitory effect of metformin on melanin synthesis.2.Metformin,unlike arbutin,restrained the formation of dendrites and filopodia-like structures and suppressed melanosome transfer.The cAMP-Rho GTPases pathway may be involved in the inhibitory effect of metformin on dendrite formation and melanosome transfer.3.It obviously revealed that metformin played a negative role in melanin synthesis and melanosome transfer in vivo and in vitro,it may potentially be used as an effective skin-whitening drug to treat refractory pigmentation disorders or prevent hyperpigmentation after laser therapy.