| BackgroundAging has become a serious social problem in our country.According to the data released by the China’s National Committee on Aging,to date,those older than 60 years account for 17.3%of the total population,and it is estimated that by 2050 there will be 487 million elderly representing 34.7%of China’s population.Aging can result in a substantial increase of cardiovascular disease,cerebrovascular disease and tumor,leading to higher mortality in the elderly.Meanwhile,aging-related type 2 diabetes,osteoarthritis and vascular dementia markedly compromise the quality of life in the aged population,imposing an enormous burden on the society.A better understanding of how aging occurs will promote the development of strategies to slow down the aging process,which will have a significant positive impact on the society.Currently,aging mechanisms remain largely unresolved and measures to delay aging are limited,which warrant further study.Aging is a complex process involving cellular senescence.It is believed that cellular senescence results in aging.Unlike apoptotic cells,senescent cells can survive for long periods of time with phenotypical changes.In 2008,Coppe et al.coined for the first time the term "senescence-associated secretory phenotype,SASP".SASP refers to the phenomenon that senescent cells express a variety of secreted proteins which include inflammatory factors,chemoattractants,matrix metalloproteinases,etc.These proteins exert various pathophysiological activities and can have deleterious effects on the tissue microenvironment.With the increase of age,senescent cells accumulate and the SASP persists,which can induce chronic inflammation responsible for dysfunction of glycolipid metabolism,coronary artery atherosclerosis,vascular dementia,or cancer progression.Therefore,interventions targeting chronic inflammation caused by aging may be effective options for delaying the aging process,which can be applied for the prevention and treatment of aging-related diseases.Adipose tissue is an important endocrine organ which can secrete adipokines and inflammatory factors to regulate energy metabolism and chronic inflammation.Adipose tissue plays an important role in aging.Metabolic dysregulation and abnormal cytokine secretion in adipose tissue are the main reasons for the rising incidence of type 2 diabetes and organ dysfunction seen in the elderly.Senescent adipocytes can produce and secret adipokines,inflammatory cytokines and matrix metalloproteinases,leading to the acquisition of SASP in adipose tissue that can persist resulting eventually in chronic inflammation and functional impairment.Dysfunction of adipose tissue affects insulin metabolism,inducing insulin resistance and metabolic syndrome.Meanwhile,senescent adipocytes can recruit immune cells,especially CD3+,CD4+ T cells and regulatory T cells(Treg)into adipose tissue.These cells together with the SASP enhance chronic inflammatory response in adipose tissue.Therefore,effective inhibition of the SASP and inflammation is key to improving adipose tissue function and reducing the incidence of aging-related metabolic disorders.It has been shown that the JNK/p53 signaling pathway is a key regulator of SASP.JNK is a member of the mitogen-activated protein kinase family that governs cell development,proliferation,differentiation and apoptosis,thereby playing an important role in a wide range of biological processes.p53 is a downstream molecule of the JNK signaling pathway,which regulates cell apoptosis and senescence.Dysregulation of the JNK/p53 signaling pathway can lead to abnormal activation of SASP,which is closely associated with chronic inflammation,diabetes and cancer onset and progression.Activation of JNK up-regulates p53 expression,which mediates cell cycle arrest,resulting in cell senescence and the SASP.Cell cycle arrest in return can induce JNK activation,thus forming a positive feedback loop to amplify SASP effects.To date,there is still a lack of interfering strategies to control the JNK/p53 signaling pathway.L-carnitine is a ubiquitous natural substance in mammals and plays an essential role in lipid metabolism.Studies have shown that L-carnitine inhibits the activities of both JNK and p53,thus reducing the JNK/p53 pathway-induced inflammatory response and improving inflammation conditions.Based on these findings,we speculate that administration of L-carnitine can suppress the JNK/p53 signaling pathway in adipose tissue,thereby leading to the control of SASP and the reduction of chronic inflammation and insulin resistance.In this study,we used Wistar rats that underwent a natural aging process to examine the effects of L-carnitine on the aging of rat adipose tissue.Furthermore,we explored if L-carnitine was able to improve the chronic inflammatory conditions in adipose tissue through the regulation of SASP.Objectives1.To determine if L-carnitine modifies the SASP therefore resulting in the improvement of inflammatory conditions and dysfunction in adipose tissue;2.To uncover if the inhibition of adipose tissue SASP by L-carnitine is through the regulation of the JNK/p53 signaling pathway.Methods1.Animal protocolThree-month and 18-month old male Wistar rats were randomly divided into 4 groups:young animal group(3-month rat,n=4),young animal+L-carnitine group(3-month rat,n=4),old animal group(18-month rat,n=4),and old animal+L-carnitine group(18-month rat,n=4).All animals were maintained in a clean,pathogen-and stress-free environment.Free access to water and separate cages were provided.A regular activity-rest pattern was followed and animals were fed a basic diet.Gavage administration of L-carnitine(300 mg/kg/day)was conducted for 4 weeks.Afterwards,animals were anesthetized,and subcutaneous adipose tissue and epididymal adipose tissue were harvested for analysis.2.Immunohistochemical analysisImmunohistochemical analysis was performed to measure the expression of CD3,CD4,Foxp3 and CD68,and assess the infiltration of T cells,CD4 T cells,Treg cells and macrophages in adipose tissue.3.Western blottingTotal protein was extracted from rat adipose tissue and Western blotting was performed to detect the expression of p21,p16,IL-6,IL-1β,TGF-1β,TNF-a,p-Ser IRS1,IRS1,p-AKT,AKT,p-JNK,JNK,p53 and β-actin.4.Quantitative RT-PCRTotal RNA was extracted from adipose tissue using the Trizol method.Quantitative RT-PCR was performed to detect the mRNA level of adiponectin,leptin,p21,p16,IGF,MCP-1 and MMP-3.Results1.L-carnitine delayed the aging process of adipose tissueThe levels of aging markers p21 and p16 were significantly higher in both subcutaneous and epididymal white adipose tissues from the old rats compared with those of the young rats(p<0.05).Treatment with L-carnitine in young rats did not cause remarkable changes of p16 and p21 compared with non-treated young rats.However,L-carnitine administration resulted in a significant reduction of p16 and p21 in old rats compared with non-treated old rats(p<0.05).These results indicate that L-carnitine delays the aging process of adipose tissue in rats.2.L-carnitine repressed SASP in adipose tissueRT-qPCR showed that mRNA levels of adiponectin,leptin,IGF,MCP-1 and MMP-3 in both subcutaneous and epididymal white adipose tissues from the old rats were significantly higher than those of young rats.L-carnitine did not affect mRNA levels of adiponectin,leptin,IGF,MCP-1 and MMP-3 in young rats.In contrast,L-carnitine significantly reduced mRNA levels of adiponectin,leptin,IGF,MCP-1 and MMP-3 in both subcutaneous and epididymal adipose tissues in old rats(p<0.05).These findings indicate that L-carnitine represses SASP in adipose tissue.3.L-carnitine inhibited inflammatory response and improved inflammatory conditionsTo observe the improvement of L-carnitine on chronic inflammation of aging adipose tissue,we detected the infiltration of immune cells and the expression of inflammatory factors in subcutaneous and epididymal white adipose tissue of rats.3.1 L-carnitine improved Immune cell infiltration in aging adipose tissueCD3,CD4,Foxp3 and CD68 expression was increased in both subcutaneous and epididymal white adipose tissues in old rats compared with that of young rats,indicating that there was more T cell,CD4+ T cell,Treg cell and macrophage infiltration in old rats.L-carnitine significantly suppressed CD3,CD4,Foxp3 and CD68 expression in old rats(p<0.05),indicating that L-carnitine inhibits infiltration of T cells,CD4+ T cells,Treg cells and macrophages into adipose tissues in old rats.3.2 L-carnitine inhibited the expression of Inflammatory cytokines in aging adipose tissueOld rats had significantly higher levels of IL-6,IL-1β,TGF-1β and TNF-a in both subcutaneous and epididymal white adipose tissues than young rats(p<0.05).L-carnitine intervention did not affect these cytokines in young rats but significantly decreased the levels of above cytokines in old rats(p<0.05).These data indicate that aging results in chronic inflammation in adipose tissue and such condition can be mitigated by L-carnitine.4.L-carnitine improved the function of PI3K/AKT insulin signaling pathway Compared with young rats,old rats had reduced IRS-1,p-AKT/AKT and elevated p-Ser IRS 1/IRS 1 in both subcutaneous and epididymal white adipose tissues,and such reduction and increase were statistically significant.L-carnitine did not regulate IRS-1,p-AKT/AKT or p-Ser IRS1/IRS1 in young rats but markedly enhanced IRS-1,p-AKT/AKT expression and reduced p-Ser IRS1/IRS1 levels in old rats(p<0.05).These results indicate that the function of insulin signaling pathway is diminished in aging adipose tissue of rats,and L-carnitine can improve the activation of insulin signaling pathway in aging adipose tissue.5.L-carnitine inhibited the JNK/p53 signaling pathwayCompared with young rats,old rats had increased p-JNK/JNK and p53 in both subcutaneous and epididymal white adipose tissues(p<0.05).L-carnitine did not affect p-JNK/JNK and p53 in young rats but significantly decreased p-JNK/JNK and p53 in old rats(p<0.05).These findings indicate that JNK and p53 expression is enhanced in the aging adipose tissue,the JNK/p53 signaling pathway is involved in aging and L-carnitine can inhibit the activation of the JNK/p53 signaling pathway.Conclusions1.The aging adipose tissue presents chronic inflammation condition with the increased expression of inflammatory cytokines,adipokines,matrix metal lo-proteinases and the infiltration of large numbers of immune cells.2.L-carnitine can improve the chronic inflammation of aging adipose tissue by reducing the infiltration of immune cells and inhibiting the secretion of SASP factors.3.Through the repression of the JNK/p53 signaling pathway,it is possible L-carnitine improves chronic inflammatory response and dysfunction in aging adipose tissue. |
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