Effects Of Advanced Glycation End Products On Proliferation And Autophagy Activity Of Human Chondrocytes

OBJECTIVE:To study the effect of advanced glycation end products(AGEs)on autophagy's activity of human chondrocytes and to explore its mechanism.MATERIALS AND METHODS:(1)Chondrocytes were treated with AGEs at different concentrations and time,the proliferative activity of the cells was detected by CCK-8.(2)Different concentrations of AGEs(50mg/l,100mg/l,200mg/l)were used to treat 6h,12h,24h,48h,and 72h of human chondrocytes respectively,the expression of cell LC3 II protein was also detected by WesternBlot method.(3)After different concentrations of AGEs(50mg/l,100mg/l,200mg/l)treatment of chondrocytes for a certain time,WesternBlot method was used to detect the expression of Beclinl,LC3 II and P62 protein.The autophagic body structure was observed by electronic transmission microscope(TEM),and autophagic double standard adenovirus(mRFP-GFP-LC3)fusion protein was marked to form autophagy.(4)Different concentrations of AGEs(50mg/l,100mg/l,200mg/l)were used to treat chondrocytes 24h,WesternBlot,mTOR,p-mTOR and PPAR gamma proteins were detected by WesternBlot.(5)After the addition of pioglitazone and T0070907 in 2h,the expression of LC3 II,mTOR and p-mTOR protein in the cells was detected by WesternBlot method after 24h was treated with 200mg/l AGEs.RESULTS:(1)CCK-8 results showed that the survival rate of cells in 50mg/LAGEs and 100mg/l treated groups was significantly higher than that in the control group,but the survival rate of cells after 48h was significantly lower than that of the control group.In the 200mg/l AGEs treatment group,the cell survival rate did not change significantly within 6h,and the cell survival rate was lower than that in the control group after 6h(p<0.05).At 24h,the survival rates of the three groups of AGEs treated groups were 1.289±0.011,1.451±0.009 and 0.663±0.014,respectively.(2)After treated with AGEs,the WB results showed that the expression of LC3II protein increased and then decreased.The expression of LC3II protein in the 50 mg/1 and 100 mg/1 AGEs treatment group reached the highest value at 24h,and the expression of LC3II protein gradually decreased with time.The expression of LC3II protein in the 200mg/l AGEs treatment group reached the highest value at 6h,and the expression of LC3II protein gradually decreased with time after 6h.The difference was statistically significant after 12h compared with the control group;the expression of LC3II protein in the three groups of AGEs treatment group after 48h.All were lower than the control group and the difference was statistically significant.(3)Compared with the control group,the expression of LC3 ? and Beclinl protein in the cells increased significantly after 50 mg/l and 100 mg/1 AGEs treatment after 24h,and the expression of P62 decreased significantly.The protein expressions of LC3 II and Beclinl in 100mg/l AGEs treatment group were significantly higher than those in the other three groups,while P62 expression was lower than that in the other three groups.The expression of LC3 ?,Beclinl and P62 in the 200 mg/1 AGEs treatment group was not significantly different from that in the control group.The number of autophagic vesicles and the number of GFP-LC3 were detected by transmission electron microscopy(TEM)and mRFP-GFP-LC3 transfection tracer fluorescence microscopy.The results showed that the number of autophagic corpuscles and the number of GFP-LC3 in cartilage cells after 24 hours of 50 mg/1 AGEs treatment were significantly higher than those in the control and 200mg/l groups.The number of autophagic corpuscles and the number of GFP-LC3 in the 100mg/l treated group were significantly higher than those in the other three groups.However,after 24 hours of AGEs treatment,there was no significant difference in the number of autophagic corpuscles and the number of GFP-LC3 between the 200mg/l group and the control group.(4)After 24 hours of AGEs treatment,the expression of PPAR gamma was significantly lower than that in the control group,and the expression of PPAR gamma decreased with increasing concentration.After 24 hours,the expression of p-mTOR in chondrocytes treated with 100mg/l AGEs was significantly lower than that in the other three groups.The expression of p-mTOR in 200mg/l AGEs treatment group was not significantly different from that in control group.(5)The expression of LC3II in cartilage cells in pioglitazone+AGEs200mg/l group was significantly higher than that in 200mg/l AGEs group,and the expression of p-mTOR was significantly lower than that in 200mg/l AGEs group.The expression of LC3II in chondrocytes of T0070907+ AGEs 200mg/L group was significantly lower than that of 200mg/l AGEs treatment group,and the expression of p-mTOR was significantly higher than that of 200mg/l AGEs group.CONCLUSIONS:1.AGEs can enhance the proliferative activity and autophagic activity of chondrocytes,but decreases with the increase of time and concentration.AGEs affects the proliferation of chondrocytes by affecting cell autophagy level.2.AGEs can affect the autophagic activity of chondrocytes by regulating the mTOR pathway through PPAR gamma.