| Objective: Aging refers to the inevitable functional decline of the physiological organs,internal environment unstable and anti-stress ability decrease with the increase of age.The irreversible degenerative changes of body structure and components happened which finally tend to death.Aging plays an important role in the normal development of the body,maintaining tissue homeostasis,and inhibiting tumor cell proliferation.Aging is also one of the most important risk factors for human diseases such as cancer,cardiovascular disease,diabetes,and neurodegenerative diseases.Therefore,understanding how aging works,how it affects organ function,and how to prevent and slow down aging becomes crucial.Mesenchymal stem cells,also known as Mesenchymal stromal cells(MSC),are therapeutic candidates for a variety of human diseases.Their therapeutic effect of immune regulation and tissue repair can be achieved through multidirectional differentiation and multiple cytokines secretion when they migrate into the injury region.In recent years,different types of mesenchymal stem cells have shown strong application prospects in the clinical treatment and laboratory research of various aging-related diseases,and they have also shown their unique improving effects in aging frailty symptoms and decreased body functions.However,the molecular mechanism of the MSC anti-aging effect is unclear.There are still many problems in quality control,standard setting,risk control in clinical application,etc.,which has affected the in-depth development of MSC applications.Therefore,the specific mechanism of its role in delaying aging is a problem that needs to be solved urgently.Adipose-derived mesenchymal stem cell(ADSC)is a type of adult pluripotent cells extracted from adipose tissue with multi-differentiation potential(adipogenic,osteogenic and chondrogenic differentiation,etc.)and self-renewal ability.ADSC has the advantages of large internal storage,easy access(through liposuction),easy in vitro culture and expansion.It has become a "popular cell source" for clinical stem cell therapy.ADSC has been used in many clinical treatments around the world in which demonstrated powerful effect in tissue repair,anti-inflammatory and immune regulations.At the same time,our research team found that ADSCs conditioned medium can promote skin collagen formation and resist photoaging of fibroblasts.However,there are scientific reports on the effects of ADSC in delaying cell senescence and animal aging.In order to confirm whether stem cells can delay cell senescence and animal body aging,this study uses mouse embryonic fibroblasts(MEF)as a cell replicative senescence model and POLG knockin mice as an animal model for progeria.Adipose-derived mesenchymal stem cell is used for cell therapy and to verify its anti-aging effect,as well as explore the specific molecular mechanism of its anti-aging effect.This research may provide a theoretical basis for the clinical application and quality control of stem cells.Method:1.The mouse embryonic fibroblasts(MEF)and adipose stem cells(ADCSs)of C57 mice were extracted,and Transwell cell co-culture system was used for MEF and ADSC co-culture;2.In order to detect the anti-aging effect of ADSC on MEF.Cell senescence β-gal kit was used to detect MEF cell senescence,Immunofluorescence staining to detect DNA damage of MEF cells,Western blot to detect senescence-related protein expression in MEF,and CCK8 to detect MEF cell viability and proliferation;3.Seahorse Cell Energy detector was used for the changes of ECAR and OCR of MEF cells after co-culture;4.The differential metabolites of MEF cells before and after co-cultivation and between different generations was detected by metabolomics technology,and relevant bioinformatics analysis on the obtained data were conducted.Then use STITCH online database for further analysis of the differential metabolites;5.Western blot was used to detect the expression of mitochondrial-related biomarker proteins and autophagy-related proteins in MEF cells before and after ADSC co-culture;6.Mito SOX detects the changes in mitochondrial ROS levels of MEF cells before and after co-culture,flow cytometry detects changes of ROS levels in MEF cells,JC-1staining detects changes in mitochondrial membrane potential,and cellular immunofluorescence co-localization detects the activation of mitochondrial autophagy in MEF cells;7.Breeding POLG knockin mice and subjecting them to regular injections of ADSCs treatment to observe their general morphological changes;8.Take mouse heart and brain tissue for Immunohistochemistry staining and senescence β-gal staining which related to autophagy and aging,then extract tissue protein for detection of aging related protein.Result:1.ADSCs can effectively delay the replicative senescence of MEF cells with passage.Under the effect of ADSCs,the proportion of β-gal-blue-stained cells in MEF was significantly reduced,and γH2AX which indicated the level of DNA damage was also reduced.Western blot was used to detect senescence-related proteins p16,p21,and p53.The content is significantly reduced,and the results are statistically significant;2.ADSCs affect the vitality and metabolic balance of MEF cells.Through CCK8 cell viability detection,the cell viability and proliferation rate of MEF were significantly accelerated after co-cultivated with ADSCs.At the same time,the OCR and ECAR of MEF cells showed a downward trend,and the basic level of cell respiration and ATP production decreased which were detected by Seahorse cell energy metabolism measurement instrument.This trend is the same as the trend of cell senescence;3.Metabolomics testing suggests that ADSCs may affect the mitochondrial function of MEF cells.Through the detection and analysis of MEF cell metabolites,it was found that the co-MEF-P4 and MEF-P4 groups,and the MEF-P3 and MEF-P4 groups had familiar differential metabolites.Using STITCH online to analysis these familiar differential metabolites,we found which were closely related to cell proliferation,biomacromolecule synthesis and mitochondrial function.4.ADSCs can promote MEF mitophagy and maintain the homeostasis of MEF cells.Through Western blot detection,it was found that the content of mitochondrial marker proteins Tom20,Tim23,HSP60 decreased.And mitochondrial ROS,as well as total cell ROS content decreased,and mitochondrial membrane potential JC-1 increased significantly.These suggested that those damaged mitochondria were effectively removed and mitochondrial quality was improved.At the same time,the autophagy marker protein P62 decreased,the marker LC3-I decreased,and the immunofluorescence co-localization test showed that the fusion of LAMP1 and HSP60 was enhanced,indicating that the mitophagy process of cells was activated.ADSC promotes the activation of mitophagy,promptly damaged mitochondria clearance in MEF,improves the quality control of mitochondria,and regulates the homeostasis of MEF cells.5.ADSCs can postpone the progeria mice aging caused by mitochondrial dysfunction: by regularly injecting ADSCs into POLG knockin mice,after 3 treatments,the weight of the mice is significantly higher than that of the non-treatment group.There is also a significant improvement in the phenomenon of alopecia and kyphosis.Western blot detection of P62 and Tom20 of mice heart and brain tissues,combined with SA-β-gal staining and LC3 immunohistochemical detection,verified that it can promote mitophagy and delay the aging process.Conclusion:1.ADSCs can delay the replicative senescence of MEF cells,enhance their cell viability,and promote cell proliferation;2.Under the effect of ADSC,the internal environment of MEF tends to be homeostasis,the synthesis of macromolecules is enhanced,and the metabolic balance is transformed from catabolism to anabolism.This change is highly familiar with the natural youthful state of MEF;3.ADSC activate MEF mitophagy and improve the quality of cell mitochondria as well as the homeostasis of the intracellular environment.Thereby achieving the effects of regulating metabolic balance and delaying aging;4.Allogeneic ADSC transplantation treatment can promote autophagy activation in progeria mice with mitochondrial dysfunction,and improve the phenotype of progeria. |
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