The Anti-aging Function And Mechanisms Of Extracellular Vesicles Derived From Stem Cells On Aged Tissue

BACKGROUND AND OBJECTIVE:Cellular senescence has attracted more and more attention in recent years.Senescent cells are characterized by their cell cycle arrest,functional impairment,and metabolic change.Recent research has found that the pathologic progression of variousage-related diseases are closely related to cellular senescence.After the accumulation of senescent cells in the body,on the one hand,the proliferation and function of senescent cells decrease obviously,which affects the repair and function of tissues.On the other hand,senescent cells themselves secrete a variety of inflammatory factors,which are called senescent secretory phenotypes.These inflammatory factors not only affect surrounding normal cells,but also recruit a large number of inflammatory cells,affecting tissue repair and damaging tissue homeostasis.In recent years,several studies have shown that removing senescent cells or reversing cellular senescence can significantly delay the pathological progress of age-related diseases and increases healthylifespan.These results suggest that interfering with the process of cellular senescence may be a new approach to preventing and treating age-related diseases.In the past,great achievements have been made in the use of stem cells to treat age-related diseases.Recent studies have found that the small Extracelluar vesicls(s EVs)secreted by stem cells are considered to be an important medium for the function of stem cells.A large number of studies have suggested that the extracellular vesicles derived from stem cells have the effect of improving the aging phenotype and delaying the pathological progress in a variety of aged-related disease models,with almost no tumor risk and immune response,and are easy to store and transport.Urine derived stem cells(USCs)are a subpopulation of mesenchymal stem cells that can be obtained from urine noninvastively and relatively indefinitely.Compared with other tissue-derived mesenchymal stem cells,they have great clinical application prospect.In addition to adult mesenchymal stem cells,the study also found that Embryonic stem cells(ESCs)also showed a strong potential of tissue repair.However,due to the totipotency of ESCs,ESCs transplantation carries a risk of teratoma formation in vivo and cannot be used directly for disease treatment.Extracelluar vesicls secreted by embryonic stem cells(ESC-s EVs)possess parental cell-like therapeutic effects and almost have no risk of teratoma formation.Given that ESCs can maintain the undifferentiated state in vitro and can proliferate indefinitely,it can continuously produce ESC-s EVs with stable performance,which is convenient for industrial production.It is expected to become a new tool for the treatment of age-related diseases.Currently,few studies have been conducted on the role of urine stem cells-derived extracellular vesicles(USC-s EVs)and ESCs derivedextracellular vesicles(ESC-s EVs)in age-related diseases.In this study,skin pressure ulcer and osteoarthritis models of aged mice were constructed,and the roles and related mechanisms of USC-s EVs and ESC-s EVs in aged tissues were investigated.METHODS:1.Skin pressure ulcer in aged mice induced by D-galactose was constructed,and the effect of USC-s EVs loaded in human acellular amniotic membrane(HAAM)on skin wound healing of aging mice and the effect of USC-s EVs loaded in HAAM on senescence cells and SASP level at wound site were observed.In vitro experiments were conducted to evaluate the role of USC-s EVs in improving the aging phenotype of D-galactose-induced senescent human dermal fibroblasts(HDFs)and its related molecular mechanisms.2.ACLT was used to establish OA model in C57BL/6 mice.The therapeutic effect of intra-articular local injection of ESC-s EVs on OA model and the effect on aged chondrocytes were evaluated.The in vitro model of OA was constructed by treating chondrocytes with IL-1?,the therapeutic effects of ESC-s EVs on chondrocytes were observed and the effect of ESC on apoptosis of senescent chondrocytes was assessed to investigate whether the anti-aging function of ESC-s EVs is related to inducing apoptosis of senescent cells.Since the binding of highly expressed FOXO4 protein to phosphorylated P53 in senescence cells is the key factor in senescence cell resistance to apoptosis,the effect of ESC-s EVs on the expression of FOXO4 protein in senescence chondrocytes was further evaluated,and the related micro RNAs that regulate the expression of FOXO4 protein in ESC-s EVs were identified.RESULTS:1.The HAAM loaded with USC-s EVs promotes the healing of skin pressure ulcer in aged mice by ameliorating cellular senescence andsecretion of SASP.1)The release curve showed that HAAM could controlled-release USC-s EVs in vitro.2)Gross observation,HE,and Masson staining showed that USC-s EVs could accelerate skin pressure ulcer healing in aged mice.3)Immunohistochemical results of P16 and SASP in wound site showed that USCs EVs could ameliorate cellular senescence and SASP secretion in wound site.4)In vitro experiments showed that USC-s EVs could reduce the number of senescent cells,down-regulate the expression of age-related P16 and P21 proteins,restore the proliferation ability of HDFs,and reduce the secretion of age-related SASP.5)In vivo experiments showed that USC-s EVs alleviated cellular senescence and recovered age-related dysfunction by activating Sirt1 signaling.2.ESC-s EVs attenuate the development of osteoarthritis in mice by inducing senescent chondrocyte apoptosis.1)Safranin O and methyl green staining,IHC for collagen ?and P16 showed that ESC-s EVs could attenuate the development of osteoarthritis in mice byreducing the number of senescent chondrocytes.IF co-staining for P16 and Tunel showed that ESC-s EVs could induce apoptosis of senescent chondrocytes.2)In vitro experiments showed that ESC-s EVs could alleviate age-related phenotype of chondrocyte induced by IL-1?.3)Western blot,IF co-staining and co-immunoprecipitation showed that ESC-s EVs reduced the binding of FOXO4 to P-P53 by down-regulating the expression of FOXO4 protein in senescent chondrocytes,which was conducive to the function of P-P53 in promoting the apoptosis of senescent cells.4)The overexpression of FOXO4 in chondrocytes transfected with adenovirus blocked the effect of ESC-s EVs on ameliorating age-related phenotype of chondrocytes treated with IL-1? and the effect of ESC-s EVs on inducing apoptosis of senescent chondrocytes.5)qPCR assay showed that mi R-664a-3p,mi R-23b-3p,mi R-532-3p,mi R-150-5p and mi R-421 that down-regulated FOXO4 were rich in ESC-s EVs,and the specific inhibitors of the above mi RNAs significantly block the effect of ESC-s EVs on inducing apoptosis of senescent chondrocytes.CONCLUTIONS:1.USC-s EVs promoted wound regeneration in aged mice by alleviating cellular senescence and the secretion of SASP.2.USC-s EVs could alleviate the aging phenotype and the secretion of SASP in senescent HDFs,and restore the proliferation and collagen secretion capacity of HDFs by activating the Sirt1 pathway.3.ESC-s EVs could significantly delay the pathological progression of degenerative knee OA in mice and maintain the normal function of articular cartilage.4.ESC-s EVs could induce apoptosis of senescent cells and ameliorate cellular senescence by down-regulating the expression of FOXO4 protein in senescent chondrocytes.5.USC-s EVs and ESC-s EVs may be a promising tool for treating age-related diseases.