The Role Of Akt/mTOR Pathway In The Premature Senescence Of Rat Chondrocytes Induced By IGF-1

Chondrocyte is the only cell phenotype in articular cartilage.When the balance of synthesis and decomposition of extracellular matrix in chondrocytes is destroyed,degenerative diseases such as osteoarthritis?OA?occur in cartilage.The senescent chondrocytes gradually accumulate with age,which leads to the changes of the internal micro-balance of articular cartilage,and then triggering this degenerative disease.It is considered that aging play a role in the development of OA.Insulin-like growth factor-1?IGF-1?is a classical anabolic hormone and growth factor,which can enhance the proliferation of multiple types of cells.Studies have found that IGF-1 also accelerates aging.For example,IGF-1 enhanced cellular senescnece in mouse and human fibroblasts,and rat vascular smooth muscle cells.Recent studies have found that Akt/m TOR is a major pathway for cell senescence.But the effects of IGF-1 on chondrocyte cellular senescence in rats and the molecular mechanisms are still poorly understood.Therefore,our study investigated that the effects of IGF-1 on chondrocyte senescence and the regulation of Serine-threonine kinase/mammalian target of rapamycin?Akt/m TOR?pathway in aging.In order to provide some theoretical support for the culturing chondrocytes in vitro,the maintenance of physiological activity of chondrocyte,the inhibition of cellular senescence and the mechanism of senescence,and the prevention of cartilage disease.In this experiment,the rat articular chondrocytes were randomly divided into 6 groups: control group,IGF-1 group?100 ng/m L IGF-1?,MK2206+IGF-1 group?10 n M MK2206+100 ng/m L IGF-1?and MK2206 group?10 n M MK2206?,Rapamycin+IGF-1 group?10 ?M Rapamycin+100 ng/m L IGF-1?and Rapamycin group?10 ?M Rapamycin?.Chondrocytes were cultured for 24 h at 37? in a 5% CO₂ incubator after treatment.The expression of Akt,p-Akt,m TOR,p-m TOR,p70S6 K,p-p70S6 K,p53 and p21 were identified by Western blot.The expression of senescence mediated factors p53 and p21 were identified by immunofluorescence.Senescence-associated ?-galactosidase?SA-?-gal?staining kit was used to investigate the changes of chondrocyte senescence.The levels of senescence-associated secretory phenotype Interleukin-6?IL-6?and Interleukin-8?IL-8?were detected by ELISA kits.The results showed that the cells volume,the activity of ?-galactosidase,and the SA-?-gal-positive cells were significantly increased in IGF-1-treated chondrocytes.Compared with control group,the protein levels of p53 and p21 were significantly increased in IGF-1-treated chondrocytes?P<0.05?.The effect of Akt pathway on IGF-1-induced cellular senescence.IGF-1 treatment significantly increased Akt phosphorylation compared with control group,while MK2206 significantly inhibited this effect.The level of p-Akt in MK2206+IGF-1 group was significantly decreased.Next,from the results of many senescence markers,we found that the positive rate of SA-?-gal in IGF-1 group was the highest.After chondrocytes were treated with MK2206,the positive rate of SA-?-gal in MK2206+IGF-1 group was significantly lower than that in IGF-1 group.The protein levels of p53 and p21 in IGF-1 group were the highest.MK2206 significantly decreased the elevation of p53 and p21 protein levels induced by IGF-1.The levels of IL-6 and IL-8 in IGF-1-treated chondrocytes were significantly higher than that in control group.MK2206 significantly decreased the elevation of IL-6 and IL-8 secretion induced by IGF-1.The effect of m TOR pathway on IGF-1-induced chondrocyte senescence.IGF-1 treatment significantly increased m TOR phosphorylation compared with control group,while MK2206 significantly inhibited this effect.The level of p-m TOR in MK2206+IGF-1 group was significantly decreased.Subsequently,Rapamycin pretreatment decreased the level of p-m TOR in IGF-1-treated chondrocytes?P<0.05?.From the results of many senescence markers,we found that the positive rate of SA-?-gal in Rapamycin+IGF-1 group significantly lower than that in IGF-1 group?P<0.01?.The levels of p53 and p21 protein in IGF-1 group were the highest.Rapamycin significantly decreased the elevation of p53 and p21 protein levels induced by IGF-1.Compared with IGF-1 group,the expression of p53 and p21 protein in Rapamycin+IGF-1 group decreased?P<0.05,P<0.05?.The levels of IL-6 and IL-8 in IGF-1-treated chondrocytes were significantly higher than that in control group.Rapamycin significantly decreased the elevation of IL-6 and IL-8 secretion induced by IGF-1.Compared with IGF-1 group,the expression of IL-6 and IL-8 in Rapamycin+IGF-1 group decreased?P<0.05,P<0.01?.In conclusion,IGF-1 promotes the activation of Akt and m TOR in Akt/m TOR pathway,and increases the ?-galactosidase activity,the expression of senescence mediated factors p53 and p21,and the secretion of senescence-associated secretory phenotype IL-6 and IL-8 in chondrocytes,thus promoting chondrocyte premature senescence.The inhibition of Akt and m TOR pathways can reverse the above effects of IGF-1.It was suggested that Akt/m TOR pathway is involved in IGF-1 induced chondrocyte premature senescence.