Meanwhile, Chondrocytes were Imatinib IC50 cultured on coverslips, fi ed in 10% neutral formalin for 15 min, stained with 0. 5% Alcian blue dye and photographed using an AZ100 Microscopes. Relative GAG content was determined as mean absorbance of each positively stained chondrocyte. Real time quantitative PCR assay Real time quantitative PCR assay was performed as previously described. Total RNA was isolated using TRIzol reagent. Single strand cDNA was obtained using a First Strand cDNA Synthesis Kit. Primer Premier 5. 0 and the NCBI BLAST database were applied to design the primers for the genes of interest. The primers used in this study were listed in Table 2. RT PCR assay was performed on a StepOne thermal cycler using a reverse transcription polymerase chain reaction kits following the procedure pre denaturation at 95 C for 30 sec, denaturation at 95 C for 5 sec, annealing at Tm for 30 sec, and e tension at 72 C for 30 sec.

The last 3 steps ran for 40 cycles. Relative standard curves were constructed for relative quantification. The e pression of all the target genes was normalized to the GAPDH gene to standardize comparison. Western blotting assay Total proteins were obtained from human cartilage samples and chondrocyte cultures using RIPA lysis buffer, while nuclear proteins were e tracted using a Nuclear Protein E traction Kit. Then, proteins were size fractionated by SDS PAGE and transferred to nitrocellulose membranes. The target proteins were probed with anti UGDH, anti Sp1, anti Phospho SAPK JNK, anti Phospho p38 MAPK, anti SAPK JNK, anti p38 MAPK, anti GAPDH and anti lamin A C primary antibodies, incubated with horseradish pero idase conjugated secondary antibody.

Blots were developed using ECL reagent. A Fusion F system was applied to photograph the blots. Then, relative protein level of UGDH and SP1 was obtained using Quantity One software, compared with the corresponding controls and standardized to GAPDH. Statistical analysis Data analysis was performed using SPSS 17. 0 and Prism 5. 0. Results were presented as mean S. E. M. One way ANOVA or Students t test, as appropriate after testing the homogeneity of variances, were performed to analyze the data. Wilco on Rank Sum Test was applied to analyze the difference of the Mankins scores of cartilage between control and OA group.

Spearman Rank Correlation analysis was performed to test the correlation of Mankins score and UGDH protein level in human and rat cartilage. Values of P 0. 05 were AV-951 considered statistically significant. Results UGDH was essential in PGs synthesis of human articular chondrocytes Obvious decreases in UGDH mRNA and protein levels were observed in human articular chondrocytes treated with three different UGDH specific siRNAs, which was accompanied by the decrease in total GAG content in the chondrocyte cultures.