The Role And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell Derived Exosomes In Acute Skin Photo-damage

Objective:With the ozone layer becoming more and more invasive,people exposed to ultraviolet?UV?increased significantly,long-term exposure to excess UV can cause skin sunburn,photoaging and tumorigenesis.Human umbilical cord mesenchymal stem cells?hucMSC?conditioned medium are reported to protect UV damage and delay skin aging.Exosome is a key component secreted by hucMSC,which is abundant in the conditioned medium.However,the role of hucMSC-exosome?hucMSC-ex?in skin UV damage has not been reported.This study aimed to investigate whether hucMSC-ex can prevent UV-induced skin damage and elucidate its mechanism of action.Methods:HucMSC were isolated by tissue adherent method,the differentiation potential of hucMSC was identified by adipogenic and osteogenic induction experiments.30%sucrose density gradient ultrafiltration centrifugation was used to separate huc MSC-ex.The surface markers were analyzed by western blot,the morphology and size were observed by transmission electron microscopy,the particle size distribution and concentration were detected by Nanosight.HucMSC-ex pretreatment exposed skin,UVA and UVB irradiation were used to make SD rat acute photodamage model,the protective effect by observing macroscopic observation,HE and immunohistochemical staining.Western blot was used to detect the expression of PCNA,p-NF-?B and TNF-?in tissue proteins,the expression of 8-OHDG and?H₂AX were detected by immunohistochemistry and immunofluorescence.Confocal microscopy was used to detect the internalization of PKH-67-labeled hucMSC-ex by HaCaT cells.UVB and H₂O₂ were used to establish the oxidative damage cell model of HaCaT.Immunofluorescence was used to analyze the effect of hucMSC-ex on the expression of reactive oxygen species?ROS?.Cellular immunofluorescence was used to detect the expression of PCNA and Caspase3.The proliferation,apoptosis and inflammation of HaCaT cells were detected by western blot.The expression of SIRT1 and TNF-?in HaCaT cells was analyzed by qRT-PCR.The expression of 14-3-3?and SIRT1 in the exosome of the overexpression 14-3-3?group was detected by western blot.Ad-14-3-3?-ex and H₂O₂ co-treated HaCaT cells.The expression of SIRT1 was detected by immunofluorescence,western blot and qRT-PCR.HaCaT cells were transfected with overexpressing 14-3-3?adenovirus,SIRT1 expression was detected by immunofluorescence and western blot.Results:Alizarin oil red O stain positive.Exosome expresses CD63and CD9,under the electron microscope as cup structure,nanosight analysis of the diameter of about 100nm.The skin of the control group was smooth and delicate,and the condition was healthy.The skin of the model group was markedly reddened,desquamated,and rough.The results of HE showed that the skin of the control group was clear and there was no pathological change.However,in the model group,the skin epidermis cells in the model group had large necrosis,and some of them still adhered and the stratified structure was disordered.A large number of inflammatory cells infiltrated the dermis,and the reticulated fibers were swollen and degenerated.In the hucMSC-ex group,the epidermis was red and the desquamation was significantly reduced.The cell necrosis and shedding were significantly reduced,and the inflammatory cell infiltration was less.The expression of DNA damage products was significantly reduced,p-NF-kB levels were significantly down-regulated,and PCNA was significantly up-regulated.HucMSC-ex was enriched in cytoplasm of HaCaT,ROS and 8-OHDG were significantly decreased,the expression of Bax,p-NF-?B,TNF-?were significantly down-regulated,the expression of PCNA and Bcl2 were significantly up-regulated.The expression of SIRT1 was down-regulated in a time-and dose-dependent manner in HaCaT cells under oxidative stress,SIRT1was highly expressed in hucMSC-ex-treated group.Ad-14-3-3?-ex significantly up-regulated the expression of SIRT1 in HaCaT cells,promoted autophagy activation,decreased DNA damage and apoptosis.Overexpression 14-3-3?adenovirus transfected HaCaT cells significantly promoted SIRT1 expression.Conclusion:HucMSC-ex reduced ROS production,DNA damage,and promoted autophagy activation to exert cytoprotection.HucMSC-ex up-regulated SIRT1 expression in HaCaT cells by carrying 14-3-3?to relieve UV and H₂O₂-induced oxidative stress damage.