The Role And Mechanism Of AMPK-mTOR Signaling Pathway In Tendon Stem/Progenitor Cells Senescence

PartⅠ:Establishment and identification of the rat Achilles tendon natural aging modelObjective:To establish a rat Achilles tendon tissue natural aging model and identify the multi-temporal and spatial changes of Achilles tendon tissue characteristics during the natural aging process,which is used to provide a suitable model for studying the cellular and molecular mechanisms of Achilles tendon aging.Methods:Male SD（Sprague-Dawley）rats aged 2,4,8 and 20 M（Month）were used,2M and 4M were young group,8M was mature group,20M was aged group,2M～8M was the maturation process,and 8M～20M was the aging process.A series of histological stains（H&E,Alizarin red and Alcian blue）were used to observe and evaluate the number and nuclear morphological changes of the cells in the longitudinal section of tendon tissue.WB assay was used to evaluate the expression of senescence markers P16^INK4a and P53 in the tendon tissue.The imaging changes of tendon tissue during the aging process were observed by X-ray and CT.Quantitative reverse transcription polymerase chain reaction（q RT-PCR）and transmission electron microscopy（TEM）were used to evaluate the changes in Achilles tendon tissue composition and collagen fiber diameter,respectively.Results:During the maturation（2M～8M）and aging（8M～20M）process of Achilles tendon tissue,H&E staining found that the cells number is decreased and the nucleus become slender with age.The expression of senescence markers P16^INK4a and P53 was increased,suggesting that cells are gradually senescence.In addition,histological staining observed that heterotopic calcification areas surrounded by osteocyte-chondrocyte-like cells began to appear in the Achilles tendon tissue of the 4M group,and the area of heterotopic ossification gradually was increased with age.On the imaging level,it was also observed that heterotopic ossification began to appear in the4M group.The volume and density of heterotopic ossification showed an upward trend.The composition of Achilles tendon tissue（Col1A1,Col3A1,decorin,biglycan,aggrecan and fibromodulin）showed an upward trend during the maturation period,and a downward trend during the aging period.The diameter of collagen fibers showed an upward trend during the maturation period,and there was no obvious change during the aging period.Conclusion:With the increase of age,the number of cells in Achilles tendon tissue was decreased,the nucleus became slender,and the cells were gradually senescence.At the same time,the volume and density of heterotopic ossification was gradually increased.It was speculated that changes of cell senescence and heterotopic ossification affect each other and promote the aging process of Achilles tendon tissue.At the same time,the afore-mentioned dynamic cell and histological changes might be one of the important initiating factors that affect the composition of Achilles tendon tissue and the diameter of collagen fibers changes.In conclusion,the Achilles tendon tissue of the20M group showed cellular and histological aging changes,indicating that the natural aging of the Achilles tendon tissue being used as an ideal model for tendon aging.The dynamic cellular and histological changes observed during aging also provided important potential directions for better studying the mechanism of age-related changes in Achilles tendon tissue.Part Ⅱ: The role of senescence-related cellular functions and molecular changes of tendon stem/progenitor cells in tendon agingObjective: To explore the role of senescence-related cellular functions and molecular changes of tendon stem/progenitor cells in tendon aging process.Methods: Select 4M rats as the young group and 20 M rats as the aging group,and young TSPCs and aged TSPCs were separated and cultured,respectively.4D Labelfree quantitative proteomics was used to detect the protein expression changes of young and aged TSPCs.q RT-PCR,WB and SA-β-galactosidase staining experiments were used to evaluate and compare the aging status of TSPCs from the two groups.Alamar blue,colony forming ability,and in vitro migration experiments were used to evaluate and compare the changes in the proliferation ability,colony forming ability,and migration ability of TSPCs from the two groups.Osteogenesis,chondrogenic differentiation induction experiments,q RT-PCR,immunofluorescence experiments were used to evaluate and compare the osteogenic,chondrogenic and tenogenic differentiation ability of TSPCs from the two groups.Results: The quantitative proteomics results of young and aged TSPCs showed that 5552 proteins were detected,and 813 proteins changed more than 1.2 times.Among them,there were 386 rising proteins and 427 falling proteins.Gene Ontology（GO）analysis found that differential protein genes are mainly enriched in functions such as proliferation,migration,differentiation and aging.At the same time,compared with young TSPCs,the expression of senescence markers（P16INK4a,P53 and SA-β-gal positive cells）of aged TSPCs was increased,showing a senescent phenotype.Compared with young TSPCs,the in vitro proliferation rate of aged TSPCs was decreased.Compared with young TSPCs,the number of clone-like cell clusters formed by aged TSPCs was decreased.Quantitative analysis showed that the cloning forming ability efficiency of aged TSPCs was also decreased.Compared with young TSPCs,the invitro migration rate of aged TSPCs was also decreased.Compared with young TSPCs,the expression of tenogenic markers Col1A1 and Col3A1 of aged TSPCs was decreased.More Alizarin Red S stained calcium nodules were observed in aged TSPCs,and semi-quantitative results indicated that the signal intensity of aged TSPCs calcium-bound Alizarin Red S was higher.Both groups of TSPCs could form three-dimensional small pellets,but the volume of the small pellet formed by aged TSPCs was larger.Conclusion: TSPCs derived from the tendons of the aging group showed a senescent phenotype,and the proliferation,cloning forming ability,migration,and tenogenic differentiation abilities of aged TSPCs were decreased,and the osteogenic differentiation and chondrogenic differentiation abilities were increased,which were closely associated with the age-related changes in aged tendon,suggesting that the TSPCs cellular and molecular changes during aging process might play a very important role in tendon aging process.Part Ⅲ: The study on the role of AMPK-mTOR signaling pathway in tendon stem/progenitor cells senescenceObjective: To explore the role of AMPK-mTOR signaling pathway in tendon stem/progenitor cells senescence.Methods: Quantitative proteomic difference proteins of young and aged TSPCs were analyzed by KEGG PATHYWAY analysis to evaluate the activity changes of AMPKmTOR signaling pathway.WB experiments were used to further verify the activity changes of AMPK-mTOR signaling pathway in young and aged TSPCs.Using AMPK molecule specific activator AICAR and inhibitor Compound C to interfere with aged TSPCs.Then,WB,q RT-PCR and SA-β-galactosidase staining experiments were used to evaluate the aging status and the changes in AMPK-mTOR signaling pathway activity of TSPCs from different groups.Alamar blue,colony forming ability,in vitro migration and q RT-PCR,cell immunofluorescence experiments were used to evaluate the changes in the proliferation ability,colony forming ability,migration ability and tenogenic differentiation ability of TSPCs from different groups,and to further verify whether TSPCs senescence status and cellular functions changes was regulated through AMPK-mTOR signaling pathway.Results: KEGG PATHYWAY analysis indicated that AMPK-mTOR signaling pathway may be involved in the regulation of TSPCs senescence.At the same time,the WB experimental results further verified that the protein expression of p-AMPK of aged TSPCs was decreased,the protein expression of p-mTOR and p-S6K1 of aged TSPCs was increased,and the AMPK-mTOR signaling pathway activity of aged TSPCs was decreased.At the same time,under the intervention of AICAR,the protein expression of p-AMPK of aged TSPCs was increased,the protein expression of p-mTOR and pS6K1 of aged TSPCs was decreased,and the activity of AMPK-mTOR signaling pathway of aged TSPCs was increased;the expression of senescence markers（P16INK4a,P53 and SA-β-gal positive cells）of aged TSPCs was decreased;the proliferation ability of aged TSPCs was increased;the number of clone-like cell clusters formed by aged TSPCs was increased,quantitative analysis showed that the cloning forming ability efficiency of aged TSPCs was also higher;the in vitro migration rate of aged TSPCs was increased;the expression of tenogenic markers Col1A1 and Col3A1 of aged TSPCs was increased.Under the intervention of Compound C,the protein expression of pAMPK of aged TSPCs was decreased,the protein expression of p-mTOR and p-S6K1 of aged TSPCs was increased,and the activity of AMPK-mTOR signaling pathway of aged TSPCs was decreased;the expression of senescence markers（P16INK4a,P53 and SA-β-gal positive cells）of aged TSPCs was increased;the in vitro proliferation rate of aged TSPCs was decreased;the number of clone-like cell clusters formed by aged TSPCs was decreased,quantitative analysis showed that the cloning forming ability efficiency of aged TSPCs was also lower;the in vitro migration rate of aged TSPCs was decreased;the expression of tenogenic markers Col1A1 and Col3A1 of aged TSPCs was decreased.Conclusion: This study revealed that AMPK-mTOR signaling pathway activity change plays an important role in the TSPCs senescence process,providing a new potential target for the prevention and treatment of age-related tendon diseases.Part Ⅳ: Metformin regulates tendon stem/progenitor cells senescence through AMPK-mTOR signaling pathway to delay tendon agingObjective: To explore whether metformin regulates tendon stem/progenitor cells senescence through AMPK-mTOR signaling pathway to delay tendon aging.Methods: In this experiment,SD rats were fed with a diet containing metformin（0.1%W/W）for six months in vivo.Imaging,H&E staining,immunohistochemistry and WB experiments were used to evaluate the degree of heterotopic ossification of tendon tissue,histological score,the expression of senescence markers and the AMPKmTOR signaling pathway activity.At the same time,q RT-PCR,WB and SA-β-galactosidase staining experiments were used to evaluate the effect of metformin on TSPCs senescence in vitro.WB experiment was used to detect the effect of metformin on the AMPK-mTOR signaling pathway activity in TSPCs.By constructing a lentiviral vector to specifically knock down AMPK molecule,we further verified whether the effect of metformin on TSPCs senescence is mediated by AMPK-mTOR signaling pathway.At the same time,Alamar blue,colony forming ability,in vitro migration and q RT-PCR,cell immunofluorescence experiments were used to evaluate the effects of metformin on TSPCs self-renewal,migration ability and the balance of extracellular matrix synthesis and catabolism,and whether these alterations are mediated by AMPKmTOR signaling pathway.Results: The study showed that rats fed with metformin feed in vivo have a smaller volume of heterotopic ossification in Achilles tendon tissue,a higher histological score,and lower expression of senescence markers P16INK4 a and P53 compared with the control group.At the same time,the protein expression of p-AMPK in the Achilles tendon tissue of the metformin group was increased,the protein expression of p-mTOR and p-S6K1 of the metformin group was decreased,and the AMPK-mTOR signaling pathway of the metformin group was more active.Under the intervention of metformin in vitro,the expression of senescence markers（P16INK4a,P53 and SA-β-gal positive cells）of TSPCs was decreased;the in vitro proliferation rate of TSPCs was increased;the number of clone-like cell clusters formed by TSPCs was increased,quantitative analysis showed that TSPCs the cloning forming ability efficiency was also increased;the in vitro migration rate of TSPCs was increased;the expression of Col1A1,Col3A1 and Decorin of the extracellular matrix synthesis genes of TSPCs was increased,and the expression of MMP-1 of the decomposition gene of TSPCs was decreased.Compared with TSPCs in the metformin intervention group,the expression of senescence markers（P16INK4a,P53 and SA-β-gal positive cells）of TSPCs was increased in the AMPK being specifically knocked down combined metformin group;the in vitro proliferation rate of TSPCs was decreased;the number of clone-like cell clusters formed by TSPCs was decreased,quantitative analysis showed that the cloning forming ability efficiency of TSPCs was also decreased;the in vitro migration ability of TSPCs was decreased;the expression of Col1A1,Col3A1 and Decorin genes of the extracellular matrix of TSPCs was decreased,the expression of MMP-1of the decomposition gene of TSPCs was rose,and the protective effect of metformin on the anti-aging and functional recovery of TSPCs would be partially eliminated when AMPK was specifically knocked down.Conclusion: This study revealed that metformin could delay tendon aging in vivo.At the same time,metformin could regulate TSPCs senescence through the AMPK-mTOR signaling pathway,thereby delaying tendon aging,providing new insights and mechanism exploration for the anti-aging effect of metformin in tendon tissue and an opportunity for a potentially safe and effective treatment for age-related tendon diseases.