Effects Of Panax Notoginseng Saponins And Exosomes Released From Keratinocytes On Human Epidermal Melanocytes

Objective: To observe effects of Panax Notoginseng saponins(PNS)on proliferation,tyrosinase activity,melanogenesis and autophagy of human epidermal melanocytes.(2)the high-throughput sequencing technology was conducted to detect the expression profile of miRNA of exosomes released by keratinocytes and fibroblasts.(3)The exosomes respectively released by keratinocytes and fibroblasts were used to intervene melanocytes to observe the internalization of exosomes and effects on the proliferation,tyrosinase activity and melanogenesis of melanocytes.Methods: The human epidermal melanocyte suspension was obtained from the human foreskin.Many keratinocytes and fibroblasts were mixed in primary melanocytes.To prepare relatively pure melanocytes,primary cells were treated with 0.05% trypsin-EDTA when cells at 80-90% confluence.The melanocytes were treated with PNS at concentrations of 0,10,30,60,90,and 120?g/ml,respectively.The growth and proliferation of melanocytes were observed under the optical microscope.In addition,tyrosinase activity,melanin content and cell proliferation rate were detected after treating for 24 or 48 hours.In our study,the autophagy in melanocytes treated with PNS(60?g/ml)was measured with the help of a Autophagy assay kit.(2)Human foreskin melanocytes,keratinocytes and fibroblasts were prepared.The culture medium of keratinocytes and fibroblasts was collected by enrichment.Then,we used an exo Easy Maxi kit to extract exosomes in the conditioned medium.we used the high-throughput sequencing instrument Illumina Hi Seq to exam the miRNA expression profiles.We aimed to show the differences of miRNA expression profiles between exosomes released by keratinocytes and fibroblasts.The differentially expressed micro RNAs were screened,and their target genes were predicted.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis of their target genes were performed.(3)The collected medium was used to extract exosomes with the ultracentrifugation.The the extracted exosomes were labelled with PKH67.Human melanocytes were co-cultured with pre-labeled exosomes released by keratinocytes and fibroblasts.After 48 h of incubation,the proliferation of melanocytes and the internalization process of exosomes were observed by optical microscope or confocal microscope.The tyrosinase activity and melanin content were also detected.Results:(1)Differential trypsinization was used to prepare relatively pure melanocytes.Purified melanocytes were treated with PNS at different concentrations and we found that PNS can promote the proliferation,tyrosinase activity,and melanin synthesis of melanocytes in a concentration-dependent manner.The promotion effect reached the peak when the concentration was 60?g/ml.In addition,our study also suggested that PNS can increase the level of autophagy in melanocytes.(2)High-throughput sequencing analysis showed that among the two sources of exosomes,267 differentially expressed miRNAs were detected,of which 119 were up-regulated and 148 were down-regulated.GO analysis showed that the target genes of these differentially expressed miRNA were mainly enriched in protein binding in the molecular function and were mainly concentrated in the cytoplasm,nucleus,plasma membrane,and cytosol in the cellular components,which were closely related to cell growth and metabolism.KEGG pathway analysis showed that target genes regulated by differentially expressed miRNA were mainly involved in MAPK signaling pathway,Ras signaling pathway,Rap1 signaling pathway,c AMP signaling pathway,endocytosis,axon guidance,Wnt signaling pathway,and regulation of actin cytoskeleton,neurotrophin signaling pathway,pathways in cancer,etc.(3)Exosomes derived from keratinocytes and fibroblasts were pre-labeled with PKH67 and co-cultured with melanocytes for 48 hours.The former showed bright green fluorescent particles in melanocytes,whereas the latter was not observed.Exosomes released by keratinocytes could promote the proliferation,tyrosinase activity,and melanin synthesis of melanocytes,and exosomes released by fibroblasts did not have this promoting effect.Conclusions:(1)NS promotes the proliferation,tyrosinase activity,and melanin synthesis of melanocytes in a concentration-dependent manner.This promoting effect peaks at a concentration of 60?g/ml.In addition,PNS can also increase the level of autophagy in melanocytes.(2)High-throughput sequencing analysis showed that there were 267 differentially expressed miRNA in both exosomes,of which 119 were up-regulated and 148 were down-regulated.(3)These differentially expressed miRNAs may have an effect on melanocyte biology.Exosomes released by keratinocytes can be transported to melanocytes and have an effect on cell proliferation,tyrosinase activity,and melanin synthesis.Exosomes released from fibroblasts cannot be internalized by melanocytes and there is no significant effect on the cell proliferation,tyrosinase activity,and melanin synthesis.