Apelin Regulates The Senescence Of Mesenchymal Stem Cells Through AMPK/Autophagy Pathway

Myocardial infarction(MI)is characterized by high prevalence and high mortality,especially in the elderly.In recent years,a large number of animal experiments and clinical trials have confirmed that Mesenchymal stem cells(MSCs)transplantation has a good therapeutic effect on MI,and it has gradually become a research hotspot in the medical field.MSCs treatment of MI is mainly autologous transplantation.However,for MI,an age-related disease,the overall quality of MSCs ages with the patient’s own age,leading to a decline in function.Therefore,it is of great significance to modify bonemarrow-derived MSCs(BMSCs)in vitro to enhance their functions.Objective:1.Comparison of the functional difference between young mesenchymal stem cells(YMSCs)and aging mesenchymal stem cells(AMSCs).2.To determine the role of Apelin rejuvenate the senescence of MSCs via AMPK/Autophagy pathway.Methods:1.CCK8 assay,Ki67 immunofluorescence staining and cumulative population multiplication assay were used to detect the proliferation ability of MSCs cells.2.SA-β-gal assay was used to detect the senescence of MSCs.3.The m RNA expression of Apelin in MSCs was detected by RT-PCR.4.The concentration of Apelin in Cd M derived from YMSCs and AMSCs were detected by ELISA Kit.5.The expression levels of Apelin and autophagy-related protein p62,Beclin,LC3ΙΙ/Ι in YMSCs and AMSCs were detected by Western blot.6.The number of autophagosomes in YMSCs and AMSCs cells was evaluated by transmission electron microscopy.7.si RNA-plasmids were used to knock down Apelin in YMSCs;SA-β-gal assaing kit was used to detect MSC senescence;Western blot was used to detect Apelin,aging-related proteins p16,p21,autophagy-related protein p62,Beclin,LC3ΙΙ /Ι.The number of autophagosomes in cells was detected by transmission electron microscope.8.Western blot was used to detect the level of Apelin in Apelin-overexpressed AMSCs infected by lentivirus.After autophagy inhibitor(3-MA)and AMPK inhibitor(Compound C)were given respectively,the aging degree was detected by SA-β-gal kit,Western blot was used to detect AMPK,p-AMPK,autophagy-related protein p62,Beclin,LC3ΙΙ/Ι.The number of autophagosomes in cells was detected by transmission electron microscope.Results:1.The proliferative ability of AMSCs was decreased.The results of CCK8 assay and Ki67 immunofluorescence showed that the proliferation of AMSCs was significantly lower than that of YMSCs(P< 0.001,P<0.01).2.AMSCs exhibited cellular senescence phenotype.The positive rate of SA-β-gal in AMSCs was significantly higher than that in YMSCs(P<0.01).AMSCs stopped growing in P7 generation,while YMSCs could continued to expand until about P11 generation.With the increase of genaration,the proliferation rate of AMSCs gradually slowed down and was much lower than that of YMSCs.3.The expression level of Apelin in AMSCs was low and autophagy is impaired.The results of Western blot,m RNA,ELISA kits showed that the levels of Apelin protein in AMSCs,Apelin in Cd M,Apelin-m RNA were significantly lower than those in YMSCs(P<0.05).Compared with YMSCs,the expression of aging-related protein p16,p21,autophagy-related protein p62 was greatly increased,but the expression of Beclin,LC3ΙΙ/Ι was significantly decreased in AMSCs(P<0.05).Furthermore,the results of transmission electron microscopy showed that the number of autophagosomes in AMSCs was significantly less than that in YMSCs(P<0.01).4.Knockdown of Apelin induced YMSC senescence to via regulating Autophagy.The results of Western blot showed that the expression of Apelin in Apelin-si RNAtreated YMSCs was decreased(P<0.001).The expression of aging-related protein p16,p21 and autophagy-related protein p62 were significantly increased(P<0.001),whereas the expression of Beclin,LC3ΙΙ/Ι was decreased in Apelin-si RNA-treated YMSCs(P<0.001).Furthermore,the positive rate of SA-β-gal was also significantly increased in Apelin-si RNA-treated YMSCs(P<0.01).5.Overexpression of Apelin in AMSCs rejuvenates cells through AMPK/Autophagy pathway.Compared with AMSCs,the results of Western blot showed that the expression of Apelin in AMSCs was increased(P<0.01),the positive rate of SA-β-gal was decreased(P<0.01),the number of autophagosomes was increased(P<0.01),the expression of aging-related protein p16 and p21 was decreased(P<0.01),autophagy-related protein p62 was decreased,but Beclin,LC3ΙΙ/Ι were increased in Apelin-overexpressed AMSCs(P<0.001).However,3-MA weakened this effect.Furthermore,the expression of p-AMPK was increased(P<0.001),the positive rate of SA-β-gal was decreased after overexpression of Apelin(P<0.01).The number of autophagosomes in cells was increased(P<0.001),but the above effects were decreased by AMPK inhibitor Compound C.Conclusion:1.Compared with YMSCs,the proliferation ability of AMSCs was significantly decreased.2.Compared with YMSCs,Apelin,Apelin-m RNA and the concentration of Apelin in Cd M in AMSCs were greatly decreased,whereas the expression of aging-related protein p16 and p21 was greatly increased.3.Knocking down Apelin induced YMSCs senescence through regulating autophagy.4.Overexpression of Apelin rejuvenated AMSCs through activating AMPK/Autophagy pathway.