FOXO4-DRI Targeting Promotes Rat Degenerated Chondrocyte Apoptosis In Vitro

Objective:To study the effect of FOXO4-DRI on degeneration and aging chondrocytes of rats in vitro,and to explore the mechanism of FOXO4-DRI on the apoptosis-promoting targeting of degeneration and aging chondrocytes in rats.Methods:(1)Take 5-week-old SD rat articular cartilage,digest it with trypsin and type Ⅱ collagenase,centrifuge and put it into an incubator to culture,to obtain SD rat chondrocytes.(2)Identify the obtained cells using Alisin blue,toluidine blue staining and type Ⅱ collagen immunocytochemical staining.(3)Divide the experiment into 2 major groups.Control group(ctrl group): The second generation was taken from chondrocytes of 5-week-old rats.Degenerative senescence group(Sen group): The first generation was taken from chondrocytes of 5-week-old rats,and induced degeneration and senescence by 1L-1β treatment.According to the different reagents added,it is further divided into ctrl + DRI group,Sen + DRI group added with FOXO4-DRI solution,and ctrl + PBS group,Sen + PBS group added with equal volume Phosphate buffered saline,(PBS).(4)Add different concentrations of FOXO4-DRI to the ctrl group and the Sen group.The absorbance at 450 nm is measured by the CCK8 method,and the cell survival rate at different concentrations is calculated to obtain the optimal concentration of FOXO4-DRI.(5)Culture the 4 groups of ctrl + DRI group,ctrl + PBS group,Sen + DRI group Sen + PBS group for 6 days under the effect of optimal FOXO4-DRI concentration and equal volume of PBS.The cell density was monitored and a growth curve was drawn.(6)Under the effect of the optimal FOXO4-DRI concentration and equal volume of PBS,observe the cell morphology of the ctrl + DRI group,ctrl + PBS group,Sen + DRI group,Sen + PBS group for 6 days and observe β Galactosidase aging test.(7)Under the optimal FOXO4-DRI concentration and equal volume of PBS for 3 days,real-time PCR and western blot were used to detect chondrocytes Col2α1,P53,caspase3,p16 mRNA and protein expression levels of ctrl + DRI group,ctrl + PBS group,Sen + DRI group and Sen + PBS group.Results:(1)The chondrocytes taken from 5-week-old rats have a large number of adherent cells within 48 hours.The cells are short fusiform,polygonal,strong refractive,and full in shape.After passage,the cells grow rapidly and occupy about the bottom of the culture flask.It took 3 days.Using 1L-1β to induce degenerative senescent chondrocytes,the cell volume and nucleus were large,and the refractive index became poor.There were finger-like protrusions on the cell surface,which grew in clusters of cell clusters.Over 10 days after passage,they grew up and significantly slowed down.(2)Identification of chondrocytes,toluidine blue stained cytoplasm was blue,nucleus was dark blue,and alixin blue staining showed that the cytoplasm was light blue.Type II collagen immunocytological examination showed that the cytoplasm was brownish yellow.The obtained cells were identified as positive reactions.(3)FOXO4-DRI concentration before 30 umol / L,the cell survival rate of Sen group decreased with the increase of concentration.When the concentration of FOXO4-DRI was 30 umol / L,almost all the cells in the Sen group were apoptotic,while the cell survival rate of the ctrl group was not changed before 30 umol / L,and then there was a downward trend.(4)The proliferation rate of chondrocytes in the ctrl + DRI group and ctrl + PBS group was similar and faster.The chondrocytes in the Sen + PBS group were also proliferating,but they were very slow compared to the ctrl + PBS group.The Sen + DRI group was in negative growth from the beginning,and the number of cells was very small by the 6th day.(5)A large number of chondrocytes were seen in the ctrl + PBS group and ctrl + DRI group,but there was no staining reaction.The chondrocytes in the Sen + PBS group were sparse,but basically all had a dark blue staining reaction,while the cells in the Sen + DRI group were very sparse and most had no obvious chondrocyte morphology,showing an apoptotic morphology in which the cell volume was reduced and rounded.A few non-apoptotic cells showed a dark blue staining reaction.(6)Real Time PCR detection showed that the expression of Col2α1 mRNA in ctrl + DRI group and ctrl + PBS group was significantly higher than that in Sen + DRI group and Sen + PBS group.The mRNA expression of P53,Caspase3 and p16 m RNA in Sen + DRI group was significantly higher than that in ctrl + DRI group.(7)Western blot results showed that the protein expression of Col2α1 in ctrl + PBS group and ctrl + DRI group was significantly higher than that in Sen + PBS group and Sen + DRI group.The expression of apoptosis-related genes P53,Caspase3,p16 in Sen + DRI group was significantly increased.Conclusion:FOXO4-DRI has a targeted pro-apoptotic effect on degenerated and senescent chondrocytes at a certain concentration and will not affect normal chondrocytes.This targeted pro-apoptotic effect is related to the upregulation of P53 expression and the upregulation of Caspase3 and p16 gene expression.