l was induced as described previously. Si teen rats were grouped into the control group and the OA group, which were intra articularly injected respectively with 20 ��L of sterile 0. 9% saline or 4% papain solution in saline to the right knees of the rats on days 1, 4 selleck catalog and 7. Two weeks after the last injection, all the rats were sacrificed under anesthesia for the knee joints. Histopathology assay Cartilage samples from the weight bearing area of the knee joint were applied in pathological test. Human MNC samples were defined as the control, while the DC samples were defined as the OA cartilage. Samples of human and rat cartilage were fi ed Inhibitors,Modulators,Libraries in 4% paraformaldehyde overnight and embedded in paraffin wa , successively. Then, sections of 5 ��m were obtained perpendicularly to the surface of articular cartilage.
Haemato ylin eosin and Safranin O staining was performed according to the standard protocol. The degree of OA was presented independently by three observers according to the modified Mankins scoring system with blind method. Moreover, protein e pression of UGDH and Sp1 in the chondrocytes was also detected using immunohistochemical Inhibitors,Modulators,Libraries assay with anti UGDH and anti Sp1 antibodies. And relative protein level of UGDH and Sp1 was presented as the mean absorbance of each positively stained chondrocyte using NIS elements software. Chondrocytes isolation, culture and treatment Human cartilage samples without microscopically visible degeneration were dissected and digested with 0. 25% trypsin for 30 min and 0. 2% collagenase typeII for 12 h in serum free DMEM F 12.
Then chondrocytes were collected and cultured as monolayer in DMEM F12 with 10% fetal bovine serum, 100 IU ml penicillin, 100 ��g ml streptomycin, and 2 mM glutamine at 37 C with 5% CO2. Hereafter, the chondrocytes were treated with UGDH Inhibitors,Modulators,Libraries specific siRNAs for 4 h using Lipofectamine 2000 Reagent and cultured for another 48 h following the manufacturers protocol. The details of the UGDH specific siRNAs were listed in Table 1. Chondrocytes were also treated with human recombinant IL 1B for 12, 24 and 48 h, as well as pre treated with p38 MAPK inhibitor SB203580 or SAP JNK inhibitor SP600125 for 0. Inhibitors,Modulators,Libraries 5 h and subsequently co treated with 10 ng mL IL 1B for another 48 h, to detect the mRNA and protein level of the interested genes.
Meanwhile, chondrocytes were also treated with IL 1B for 0 120 min or pre treated with SP600125 or SB203580 for 30 min and then treated with 10 ng ml IL 1B for another 30 min for the phosphorylation status of JNK and p38 MAPK. Chondrocytes from at least three individuals were applied in every in vitro e periment. GAG detection GAG content was detected using 1,9 GSK-3 Dimethylmethylene Blue reagent as reported. Absorbance at 570 nm was measured using a UV 1601 spectrophotometer. A standard curve constructed with chondroitin sulfate sodium salt from shark cartilage was used to quantify GAG content selleck chem in the chondrocyte cultures. Then, total GAG was determined as GAG content versus protein