| BackgroundsSkin aging is a continuous dynamic process.Clinically,skin aging is mainly manifested as wrinkle formation,loss of elasticity,and pigmentation.Histologically,skin aging is characterized by thinning of epidermis,atrophy of dermis,flattening of the dermoepidermal junction,accumulation of abnormal elastic tissue in the dermis,and reduced amounts of subcutaneous white adipose tissue(s WAT).Although the pathological mechanism of skin aging is complex,previous studies found that the pathogenesis mainly included gene mutations,oxidative stress,matrix metalloproteinases(MMPs),increased chronic inflammation,and cell senescence.Recent study has revealed that injection of adipose-derived mesenchymal stem cells(ADMSCs)can prevent skin aging phenotype but the underlying mechanism is still unknown.Nuclear Receptor Interacting Protein 1(NRIP1),also known as RIP 140(Receptor Interacting Protein of 140 kDa),regulates gene expression by interacting with nuclear receptors,thereby participating in the process of metabolism and aging.Our previous studies found that deletion of Nrip1 extended longevity in female mice and delayed cell senescence in adipose tissue.Therefore,we hypothesized that NRIP1 may play an important part in delaying skin aging by regulating the function of ADMSCs.ObjectivesThe aim of this study is to investigate the role of NRIP1 on the skin aging phenotypes,to explore the impact of NRIP1 on the proliferation,senescence,differentiation and quiescence of ADMSCs cells,to reveal how NRIP1 regulates IGF1/mTOR/NF?B pathway in delaying skin aging,and to provide a potential therapeutic target for skin aging.Methods1.The mouse genotype was analyzed by agarose gel electrophoresis.Skin tissues were collected from C57BL/6J(B6)mice of different ages,Nrip1-/-and Nrip1+/+ mice of the same age.Skin aging phenotypes were detected by HE staining and SA-?-Gal staining.2.ADMSCs were isolated from young and old B6 mice.MTT assay was applied to evaluate cell proliferation.Annexin-V staining was applied to evaluate cell apoptosis.The colony-forming capability of ADMSCs was determined by the CFU assay.After establishing the model of cell replicative senescence,SA-?-Gal staining was applied to detect cell senescence.After the induction of adipogenic differentiation in ADMSCs,Oil red O staining was applied to detect adipogenesis and qRT-PCR was performed to detect the expressions of adipogenic differentiation-related genes.3.ADMSCs were isolated from Nrip1-/-and Nrip1+/+ mice.MTT assay was applied to evaluate cell proliferation.Annexin-V staining was applied to evaluate cell apoptosis.The colony-forming capability of ADMSCs was determined by the CFU assay.After establishing the model of cell replicative senescence,SA-?-Gal staining was applied to detect cell senescence.After the induction of adipogenic differentiation in ADMSCs,Oil red O staining was applied to detect adipogenesis and qRT-PCR was performed to detect the expressions of adipogenic differentiation-related genes.PI staining was used to detect cell cycle.To identify the quiescent cell population,immunocytochemistry was performed to stain cells with Ki67.4.Prepare Nrip1-/-ADMSCs and siRNA mediated Nrip1 knockdown ADMSC s.qRT-PCR was performed to detect the expressions of senescence-related genes(p16,p21 and p53).senescence-associated secretory phenotype(SASP)and NF?B pathway-related genes in Nrip1 knockout and knockdown ADMSCs.Western Blotting was performed to detect the levels of p65,p-p65,mTOR and p-mTOR in siNrip1 treated ADMSCs.Immunohistochemistry was used to detect p-p65 levels in young and old B6 skin samples,as well as in Nrip1-/-and Nrip1-/-skin samples.Results1.Compared with young B6 mice,the thickness of dermis and subcutaneous white adipose tissue(sWAT)from old mice was significantly reduced,and the percentage of SA-?-Gal positive cells in the sWAT was significantly increased.Compared with Nrip1-/-mice,the thickness of dermis and sWAT in Nrip1-/-mice was significantly increased,and the percentage of SA-?-Gal positive cells in sWAT was significantly reduced.2.Compared with the ADMSCs from young B6 mice,the cell proliferation of ADMSCs from old B6 mice was significantly reduced,the percentage of apoptotic cells was significantly increased,the ability of colony formation was significantly reduced,the percentage of SA-?-Gal positive cells was significantly increased in replicative senescence,adipogeneisis was significantly reduced,and the expression of genes related to adipogenesis(Ppary,Fabp4 and Adiponectin)was significantly reduced.3.Compared with the ADMSCs from Nrip1+/+ mice,the cell proliferation of ADMSCs from Nrip1-/-mice was significantly reduced,the percentage of apoptotic cells was significantly reduced,the ability of colony formation was significantly reduced,the percentage of SA-?-Gal positive cells was significantly decreased in replicative senescence,adipogeneisis was significantly reduced,and the expression of genes related to adipogenesis(Ppar?,Fabp4 and Adiponectin)was significantly reduced.The percentage of quiescence cells in Nrip1-/-ADMSCs was significantly higher compared to Nrip1+/+ADMSCs.4.The expression of senescence-associated genes(p21 and p53),SASP(IL6 and IL1a)and NF?B pathway-related genes(p65,mTOR and Igf1)decreased significantly in both Nrip1-/-and siNrip1 ADMSCs compared to Nrip1+/+ and siCon ADMSCs.The expression of p16 increased in Nrip1-/-ADMSCs compared to Nrip1+/+ ADMSCs.The western blot results showed that at protein level,mTOR,p65,and p-p65 were significantly lower in siNrip1 ADMSCs than siCon ADMSCs,while p-mTOR showed suggestively lower in siNRIP1 ADMSCs.Increased p-p65 levels were seen in the sWAT of skin at old B6 mice.Moreover,p-p65 levels Were reduced in Nrip1-/-mice compared with Nrip1+/+ mice.Conclusions1.The skin aging phenotypes were verified in B6 mice,including thinner dermal and s WAT and increased accumulation of senescent cells in the sWAT.2.The deletion of Nripl can increase the thickness of the dermis and sWAT,and reduce the accumulation of senescent cells in sWAT,thereby delaying skin aging.3.Based on the data of B6 mice,we found that the function of ADMSCs gradually decreased with aging,which includes the increased number of senescent cells,increased apoptosis,and decreased proliferation capability,self-renewal ability,and adipogenic differentiation potential.4.The deletion of Nrip1 can reduce cell senescence,inhibit apoptosis,and maintain ADMSCs quiescence,thereby preserving self-renewal ability,proliferation,and adipogenic differentiation potential.5.The deletion of Nrip1 can delay cell senescence of ADMSCs by inhibiting p53,p21 and SASP(IL-1a and IL-6)and maintain cell quiescence by down-regulating mTOR expression.NRIP1 may delay skin aging through the IGF1/mTOR/NF?B pathway. |
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