The Study On The Mechanism Of IGF-1/Akt Pathway In Promoting Aging Of Chondrocytes In Rats

Inhibition of chondrocyte senescence is an urgent problem to be solved in vitro culture of chondrocytes and osteoarthrosis.Therefore,this study will explore the regulation of Insulin-like growth factors-1/protein kinase B(IGF-1/Akt)signal on the senescence of rat chondrocytes in vitro,in order to inhibit cell senescence,improve the culture of chondrocytes in vitro,maintain the normal physiological activity of chondrocytes,and provide a theoretical basis for the treatment of cartilage defects.In this experiment,IGF-1 and IGF-1 downstream signal factor Akt were added to the rat chondrocytes in vitro,and the cell level,gene level and protein level were detected.The third and fifth generation chondrocytes in vitro were divided into 5 groups: blank control group,IGF-1 group(100ng/mL IGF-1),MK+IGF-1 group(5ng/mL+100ng/mL IGF-1),MK group(5ng/mL MK2206),and negative control group(Dimethyl sulfoxide,DMSO).When cells were cultured to 4d,7d,10 d,15d and 20 d,24h was added to the drug,and then the chondrocyte senescence related beta galactosidase staining was performed.After the cells were cultured to the fusion state,the drugs were treated with 24 h.The mRNA expression level of Akt,p53 and p21 in chondrocytes was detected by RT-PCR.The protein expression levels of Akt,p-Akt,p53,p-p53 and p21 were identified by Western-blot.The results of this experiment showed that the positive rate of senescence-associated ?-galactosidase staining increased gradually with the increase of culture time in the two generation of cell.When cultured 15 d and 20 d,the positive rate of cell staining increased significantly,and the results were significantly different from those of culture 4,7 and 10d(P<0.05).Compared with the control group,the positive rate of cell staining was the highest in group IGF-1,and the lowest in group MK.On the whole,the positive rate of fifth generation chondrocytes was higher than that of third generation chondrocytes.The effect of IGF-1 on Akt in cartilage cells,from the gene level,the mRNA level of Akt in group IGF-1 was the highest(P<0.01),the Akt expression of IGF-1+MK group decreased,and mRNA expression level of Akt in negative control group was significantly different from that of IGF-1 group.The expression trend of total protein and phosphorylated protein in Akt was consistent with that of mRNA.On the effect of p53,the mRNA level of p53 in group IGF-1 was the highest(P<0.01),and p53 expression in IGF-1+MK group decreased significantly(P<0.01),and p53 expression in negative control group was not significantly different from that of control group(P>0.05).From the protein level,the trend is basically the same as that of the gene level.The expression level of p21 mRNA in group IGF-1 was the highest,which was significantly different from the control group(P<0.05),and the mRNA expression level of p21 in IGF-1+MK group decreased,and the difference was very significant compared with the IGF-1 group(P<0.01),and the expression level of p21 in the negative control group was significantly different from that of the control group(P<0.05).Although the level of protein expression of p21 is not as significant as that of the gene level,the change trend is consistent.In addition,the expression level of related protein genes in the fifth generation of chondrocytes was higher than that in the third generation of chondrocytes in general.The results of this experiment show that: IGF-1 promoted the expression and activation of Akt,andsenescence-associated ?-galactosidase,and the expression of senescence mediating factors p53 and p21 in chondrocytes,thus promoting chondrocyte senescence.Inhibiting the activity of Akt in the IGF-1 signaling pathway can reverse the above effects of IGF-1,which suggests that the effect of iIGF-1 on the aging of chondrocytes may be mediated partly by Akt.