The Function Of AMPK/SIRT-1 On IL-1β And TNF-α Of Chondrocytes Induced By Advanced Glycation End Products

OBJECTIVE:In order to investigate weather advanced glycation end products(AGEs)increase the level of IL-1β and TNF-α in human articular chondrocytes,and determine the role of AMPK/SIRT-1 and the relationship of AMPK/SIRT-1 and PPARγ in this process.MATERIALS AND METHODS :(1)Human articular chondrocytes treated with different concentrations of AGEs(25μg/ml,50μg/ml,75μg/ml,100μg/ml)for 24 h.Western Blot was used to detect the protein expression levels of intracellular inflammatory factors TNF-α and IL-1β,and q-PCR was used to detect the mRNA levels of TNF-α and IL-1β.(2)Human articular chondrocytes were treated with AGEs(100μg/ml)for 6h,12 h,18h,and 24 h.Western blot was used to detect the protein expression levels of intracellular inflammatory factors TNF-α and IL-1β,and q-PCR was used to detect the mRNA levels of TNF-α and IL-1β.(3)Human articular chondrocytes were treated with different concentrations of AGEs(25μg/ml,50μg/ml,75μg/ml,100μg/ml).Western Blot were used to detect the protein level of p-AMPK,AMPK,SIRT-1.(4)Human articular chondrocytes were treated with AGEs(100μg/ml)for6h,12 h,18h,and 24 h.Western Blot were used to detect the protein level of p-AMPK,AMPK,SIRT-1.(5)Human articular chondrocytes were pretreated with selective AMPK agonist A-769662 and selective AMPK inhibitor Dorsomorphin 2HCl for 1 hour before the treatment of AGEs(100 μg/ml)for 24 hours.Western blot was used to detect the protein expression levels of intracellular inflammatory factors TNF-α and IL-1β,and q-PCR was used to detect the mRNA levels of TNF-α and IL-1β.(6)Human articular chondrocytes were pretreated with selective SIRT-1agonist SRT1720 and selective SIRT-1 inhibitor EX 527 for 1 hour before the treatment of AGEs(100 μg/ml)for 24 hours.Western blot was used to detect the protein expression levels of intracellular inflammatory factors TNF-α and IL-1β,and q-PCR was used to detect the mRNA levels of TNF-α and IL-1β.(7)Human articular chondrocytes pretreated with different concentrations of selective PPARγ agonist pioglitazone(0,10μmol/L,30μmol/L,50μmol/L)for 1 hours,and Western Blot were used to detect the protein levels of p-AMPK,AMPK,SIRT-1.RESULTS :(1)In human articular chondrocytes treated with different concentrations of AGEs for 24 h,the protein expressions and mRNA levels of intracellular inflammatory factors TNF-α and IL-1β was increased(P<0.05)compared with control group(100μg/ml)in a concentration-dependent manner.(2)In human articular chondrocytes treated with AGEs(100μg/ml)for 6h,12 h,18h,24 h the protein expressions and mRNA levels of intracellular inflammatory factors TNF-α and IL-1β was increased(P<0.05)compared with control group(AGEs 100μg/ml 0h)in a time-dependent manner.(3)In human articular chondrocytes treated with different concentrations of AGEs for24 h,the phosphorylation of AMPK and the protein concentration of SIRT-1 was decreased(P<0.05)compared with control group(100μg/ml)in a concentration-dependent manner.(4)In human articular chondrocytes treated with AGEs(100μg/ml)for 6h,12 h,18h,24 h,the phosphorylation of AMPK and the protein concentration of SIRT-1 was decreased(P<0.05)compared with control group(AGEs 100μg/ml 0h)in a time-dependent manner.(5)In human articular chondrocytes pretreated with selective AMPK agonist A-769662 for 1 hour before the treatmentof AGEs(100 μg/ml)for 24 hours,the protein expressions and mRNA levels of intracellular inflammatory factors TNF-α and IL-1β were decreased(P<0.05);In human articular chondrocytes pretreated with selective AMPK inhibitor Dorsomorphin 2HCl for 1 hour before the treatment of AGEs(100 μg/ml)for 24 hours,the protein expressions and mRNA levels of intracellular inflammatory factors TNF-α and IL-1βwere increased(P<0.05).(6)In human articular chondrocytes pretreated with selective SIRT-1 agonist SRT1720 for 1 hour before the treatment of AGEs(100 μg/ml)for 24 hours,the protein expressions and mRNA levels of intracellular inflammatory factors TNF-α and IL-1β were decreased(P<0.05);In human articular chondrocytes pretreated with selective SIRT-1 inhibitor EX 527 for 1 hour before the treatment of AGEs(100 μg/ml)for 24 hours,the protein expressions and mRNA levels of intracellular inflammatory factors TNF-α and IL-1β were increased(P<0.05).(7)In human articular chondrocytes pretreatmented with different concentrations of pioglitazone(10μmol/L,30μmol/L,50μmol/L)for 1h,before treatment with AGEs(100μg/ml)for 24 h,the degree of AMPK phosphorylation and SIRT-1 protein expression were increased(P<0.05)compared with positive control group(AGEs 100μg/ml)in a dose-dependent manner.CONCLUSIONS : AGEs induce the expressions of TNF-α and IL-1β in chondrocytes by reducing the activity of AMPK and downregulating SIRT-1,which could be restored by activation of PPARγ.