High Cholesterol Induces Apoptosis And Autophagy Through The ROS-activated AKT/FOXO1 Pathway In Tendon-derived Stem Cells

BackgroundHigh cholesterol may inhibit the tenogenic differentiation in tendon derived stem cells(TDSCs),but the effect of cholesterol on the viability and properties of TDSCs was not illuminated.There are studies indicating that apoptosis and autophagy participate in the development of tendinopathy.Our aim is to investigate whether apoptosis and autophagy are involved in highcholesterol related tendinopathy and reveal the possible mechanisms and signaling pathways involved.Methods1.High cholesterol inhibits the proliferation of TDSCs and then induces apoptosis and autophagy of TDSCsThe OD values of TDSCs stimulated by different concentrations of cholesterol were detected by CCK-8 assay at day 1,day 3,day 5 and day7,and the optimal concentration of cholesterol was selected according to the results.The proliferation rate of TDSCs was detected by Ki67 staining,and the cell cycle after cholesterol stimulation was detected by flow cytometry.The cell migration was tested by a wound healing assay.After cholesterol stimulation,the apoptotic cells were detecte and the apoptotic TDSCs were counted under a fluorescence microscope.Finally,the expression levels of apoptotic proteins such as cleaved-caspase 3,BAX,Bcl-XL and PARP were detected by western blotting.LC3-?/LC3-1 and p62 were detected by western blotting.Subsequently,TDSCs were transfected with mRFP-GFP-LC3 adenovirus vector.Then,observe the autophagosomes and autolysosomes under a fluorescence microscope.2.Inhibition the autophagy of TDSCs enhances apoptosis,while repression of apoptosis diminishes autophagyTDSCs were mixed with 3-MA(an inhibitor of autophagy)and treated with cholesterol.Then detected the protein expression of cleaved-caspase 3,Bax,Bcl-XL and PARP.Then counted apoptotic cells by a tunel assay and detected the viability of TDSCs with a cck-8 assay kit.Similarly,TDSCs were mixed with Z-VAD-FMK(an inhibitor of apoptosis)and treated with cholesterol.Then detected the protein expression of LC3-?/LC3-1 and p62 by western blotting.3.High cholesterol-induced ROS initiate apoptosis and autophagy in TDSCsAfter different concentrations and times of cholesterol stimulation,the ROS levels were detected by a DCFH-DA probe.Then a ROS scavenger was added in TDSCs before cholesterol stimulation.The number of autophagosomes and autolysosomes were counted and the proportion of apoptotic TDSCs were calculated.At last,western blotting was carried out to detect the protein expression of apoptosis and autophagy like cleaved-caspase 3,Bax,Bcl-XL,PARP,LC3-?/LC3-1 and p62.4.High cholesterol activated ROS and up-regulated the AKT/FOXO1 pathway in TDSCs,leading to apoptosis and autophagyWestern blotting was carried out to detect the total and phosphorylated AKT and FOXO1 proteins.In addition,we detected alterations in apoptosis and autophagy-related proteins such as LC3-?,LC3-1,p62,cleaved-caspase 3,Bax,Bcl-XL and PARP in the presence of the FOXO1 inhibitor AS 1842856 using western blotting.Next,changes of AKT/FOXO1 signaling pathway in TDSCs treated by NAC was also detected by western blot.5.High cholesterol induces apoptosis and autophagy in vivoThe 10-month-old ApoE knockout mice were selected as the high cholesterol group and the 10-month-old C57BL/6 mice as the control group.HE staining was used to stain the histological sections of Achiles tendons.Bonar score was used to quantify the morphology.At last,immunohistochemical staining of LC3-?,LC3-1,p62,cleaved-caspase 3,Bax,Bcl-XL,PARP and FOXO1 were tested.Results1.High cholesterol inhibits the viability of TDSCs and induces apoptosis of autophagy in TDSCslOmg/dL and 100mg/dL cholesterol stimulation had the significant inhibitory effect on TDSCs,so we select 10mg/dL as the optimal concentration and the proportion of ki67-positive cells decreased significantly under this concentration.Wound healing assay indicated that the migration of TDSCs decreased markedly 24h after the stimulation of 10mg/dL cholesterol.In addition,cholesterol can block the cell cycle in the G0/G1 phase and reduce the number of cells in the G2/M and S phases.Flow cytometry analysis showed that 1Omg/dL cholesterol significantly increased the number of apoptotic cells,and TUNEL staining also indicated that apoptotic TDSCs were increased.Treatment with cholesterol for 12h and 24h upregulated cleaved caspase 3 and proapoptotic protein BAX,downregulated PARP and antiapoptotic proteins Bcl-xl.TDSCs were transfected with mRFP-GFP-LC3 adenovirus for 24h to detect autophagy flux.10mg/dL cholesterol was used to stimulate TDSCs after the transfection.Western blotting method showed that LC3-?/LC3-1 were upregulated while p62,which is an adaptor protein mediating the ubiquitination and degradation of LC3 was downregulated.Moreover,the number of yellow and red puncta both increased markedly in the merged pictures,indicating that cholesterol increase autophagosomes and autolysosomes in TDSCs.All in all,cholesterol induces apoptosis and autophagy in TDSCs respectively.2.Inhibition the autophagy of TDSCs enhances apoptosis,while repression of apoptosis diminishes autophagy3-MA(an autophagy inhibitor)and cholesterol produced fewer autophagosomes and autolysosomes compared with cholesterol alone,and the difference between groups was statisticaly significant.After mixed with 3-MA,LC3-?/LC3-1,Bcl-xl,PARP were down-regulated while p62,cleaved-caspase 3 and BAX were up-regulated.In addition,3-MA increased the proportion of tunel-positive TDSCs and inhibited its proliferation.Furthermore,Z-VAD-FMK,an apoptosis inhibitor,down-regulated LC3-?/LC3-1 while up-regulated p62.These results indicated that inhibition of autophagy enhances apoptosis in TDSCs,while repression of apoptosis diminishes autophagy.3.High cholesterol-induced ROS initiate apoptosis and autophagy in TDSCsCholesterol affects the ROS level in TDSCs in a dose and time dependent manner.ROS level after cholesterol stimulation was up-regulated with the concentration increased from 0,1,10 to 100mg/dL,and also up-regulated increased after 3h,6h,12h and 24h gradually.ROS scavenger NAC markedly eliminated ROS level of TDSCs and autophagosomes and autolysosomes were reduced under the fluorescence microscope after transfected with mRFP-GFP-LC3 adenovirus.NAC also reduced the number of apoptotic cells but thed difference was not significant compared with the control group.In addition,NAC down-regulated caspase 3,Bax,LC3-?/LC3-1 and up-regulated Bcl-xl,PARP and p62 proteins.Flow cytometry technique showed that apoptotic cells were relatively less after NAC treatment,but did not return to normal level.Western blotting indicated that NAC dramatically decreased the protein expression such as cleaved caspase 3,Bax,LC3-?/LC3-1 and increased Bcl-xl,PARP and p62.These results suggest that the activated ROS is an activator of cholesterol induced apoptosis and autophagy.4.High cholesterol activated ROS and up-regulated the AKT/FOXO1 signaling pathway in TDSCs,leading to apoptosis and autophagyAfter the treatment of cholesterol,FOXO1 was up-regulated in a time-dependent manner,while the phosphorylated AKT and phosphorylated FOXO1 were down-regulated in TDSCs.AS 1842856,an inhibitor of FOXO1,when treated with cholesterol significantly inhibited apoptosis and autophagy in TDSCs compared to cholesterol alone.NAC up-regulated phosphorylated AKT and phosphorylated FOXO1 proteins and down-regulated FOXO1 proteins.These results suggest that reactive oxygen species(ROS)may be the upstream of AKT/FOXO1 signaling pathway and mediate cholesterol-induced apoptosis and autophagy of TDSCs.5.High cholesterol induces apoptosis and autophagy in vivoAchilles tendons in the hypercholesterolemic group showed significant histopathological abnormalities and higher Bonar scores.Immunohistochemical analysis confirmed the increased expression of cleaved-caspase 3,BAX,LC3-? and FOXO1 and decreased expression of p62in the hypercholesterolemic group.These results showed that apoptosis and autophagy were both occred when the mice fed with cholesterol.ConclusionTaken together,the results in this study suggeste that high cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells,which indicate one of the pathogenesis of highcholesterol induced tendonopathy.The application of stains,apoptosis,autophagy and FOXO1 inhibitors may be helpful in the treatment of hypercholesterolemia tendonosis and improve the prognosis and quality of patients.