Abstract:

Objective To discuss the improvement effect of exosomes （Exo） derived from human adipose-derived stem cells （hADSCs） and human dermal fibroblasts （HDFs） on ultraviolet-induced skin wrinkles in photoaged nude mice， and to clarify its effect. Methods The Exo were isolated from hADSCs and HDFs， respectively， and identified by Western blotting method， designated as hADSCs-Exo and HDFs-Exo. Twenty-eight nude mice were randomly divided into control group， model group ［injected with Hank’s balanced salt solution （HBSS）］， hADSCs-Exo group （injected with hADSCs-Exo）， and HDFs-Exo group （injected with HDFs-Exo）， and there were 7 mice in each group. Except for control group， the other three groups were used to establish a photoaging model on the dorsal skin. After 4 weeks， the gross morphological changes of dorsal skin of the nude mice in various groups were observed， and the wrinkle severity scores were evaluated； HE staining was used to observe the pathomorphology of skin tissue of the nude mice in various groups； ELISA was used to detect the levels of interleukin （IL）-1β， IL-6， and tumor necrosis factor α （TNF-α） in skin tissue of the nude mice in various groups. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting method were used to detect the expression levels of collagen Ⅰ （Col Ⅰ）， matrix metalloproteinase （MMP）-1， MMP-2， MMP-3， MMP-9， and MMP-13 mRNA and protein in skin tissue of the nude mice in various groups； immunohistochemical staining was used to observe the protein expression of Col Ⅰ， tropoelastin， and fibrillin-1 in skin tissue of the nude mice in various groups. Results The particles isolated from hADSCs and HDFs exhibited typical vesicle-like structures with diameters of 50-100 nm， and highly expressed CD81， CD63， heat shock protein 70 （HSP70）， and tumor susceptibility gene 101 protein （TSG101）， indicating successful isolation of hADSCs-Exo and HDFs-Exo. The dorsal skin of the nude mice in control group was smooth without looseness or wrinkles， while severe wrinkles， rough epidermis， dryness， and pigmentation were observed in model group. Compared with model group， the dorsal skin of the nude mice in hADSCs-Exo group showed fewer deep wrinkles and mild looseness， whereas the wrinkles in HDFs-Exo group were significantly alleviated compared with control group， the wrinkle severity score in model group was significantly increased （P<0.05）， compared with model group， the wrinkle severity socres of the mice in hADSCs-Exo and HDFs-Exo groups were significantly decreased （P<0.05）. Compared with hADSCs-Exo group， the wrinkle severity score of the mice in HDFs-Exo group was decreased （P<0.05）. The HE staining results showed that clear skin tissue stratification and thin epidermis in control group， while disordered structure， loose arrangement， and thickened epidermis were observed in model group. Compared with model group， the skin lesions in hADSCs-Exo and HDFs-Exo groups were alleviated， with thinner epidermis， clearer stratification， and normalized structure in HDFs-Exo group. The ELISA results showed that compared with control group， the levels of IL-1β， IL-6， and TNF-α in skin tissue of the mice in model group were significantly increased （P<0.05）， and compared with model group， the levels of I6-1β， IL-6， and TNF-α in skin tissue of the mice in hADSCs-Exo and HDFs-Exo groups were significantly decreased （P<0.05）. Compared with hADSCs-Exo group， the levels of IL-1β， IL-6， and TNF-α in skin tissue of the mice in HDFs-Exo group were decreased （P<0.05）. The RT-qPCR and Western blotting results showed that compared with control group， the expression levels of Col Ⅰ mRNA and protein in skin tissue of the mice in model group were significantly decreased （P<0.05）， while the levels of MMP-1， MMP-2， MMP-3， MMP-9， and MMP-13 were significantly increased （P<0.05）. Compared with model group， the expression levels of Col Ⅰ in skin tissue of the mice in hADSCs-Exo and HDFs-Exo groups were significantly increased （P<0.05）， while the levels of MMP-1， MMP-2， MMP-3， MMP-9， and MMP-13 were significantly decreased （P<0.05）. Compared with hADSCs-Exo group， the expression levels of Col Ⅰ in skin tissue of the mice in HDFs-Exo group were increased （P<0.05）， while the levels of MMP-1， MMP-2， MMP-3， MMP-9， and MMP-13 were further decreased （P<0.05）. The immunohistochemical staining results showed that compared with control group the staining intensities of tropoelastin， fibrillin-1， and Col Ⅰ in skin tissue of the mice in model group were significantly weakened， and compared with model group， the staining intensities of tropoelastin， fibrillin-1， and Col Ⅰ in hADSCs-Exo and HDFs-Exo groups were enhanced. Compared with hADSCs-Exo group， the staining intensities in HDFs-Exo group were stronger. Conclusion The Exo derived from hADSCs and HDFs significantly improve ultraviolet-induced skin wrinkles in the photoaged nude mice， with HDFs-Exo exhibiting superior effects compared with hADSCs-Exo.

Key words: Human adipose-derived stem cells, Human dermal fibroblasts, Exosomes, Photoaging, Skin wrinkle