关键词: 骨关节炎, 软骨, 骨基质, 骨痹通方, 基质金属蛋白酶类, Wnt信号通路

Abstract: Background　Gubitong Recipe（GR）has a good clinical effect on osteoarthritis（OA）. It also shows a good protective effect against cartilage damage in the OA rat model，but the specific mechanism of action is not yet clear. Objective　To explore the mechanism of protective effect of GR against cartilage matrix degradation in an OA model based on Wnt/β-catenin pathway. Methods　This experiment was carried out from June to September 2020. The viability of SW1353 chondrosarcoma cells was test by MTS method. The OA model of SW1353 chondrosarcoma cells was prepared using IL-1β，and the cells were divided into groups A（blank control，no interventions），B（positive control，intervened with 10 μg/L IL-1β），C（intervened with 10 μg/L IL-1β and 625 mg/L solution of GR），D（intervened with 10 μg/L IL-1β and 1 250 mg/L solution of GR），and E（intervened with 10 μg/L IL-1β and 2 500 mg/L solution of GR）. Relative mRNA expression quantites of MMP-1，MMP-3，and MMP-13 were tested by real-time quantitative polymerase chain reaction，relative protein expression quantites of MMP-1，MMP-3，MMP-13 and β-catenin by Western-blotting，supernatant solution contents of MMP-1，MMP-3，and MMP-13 by ELISA. Results　The concentration of 3 000 mg/L solution of GR partially inhibited the viability of SW1353 chondrosarcoma cells（P<0.05）. Group A had lower relative mRNA expression quantities of MMP-1，MMP-3 and MMP-13 than other four groups（P<0.05）. Group C had lower relative mRNA expression quantities of MMP-1，and MMP-3 than group B（P<0.05）. Group D had lower relative mRNA expression quantities of MMP-1，MMP-3，and MMP-13 than groups B and C（P<0.05），so did group E（P<0.05）. Group E had lower relative mRNA expression quantities of MMP-1，MMP-3 and MMP-13 than group D（P<0.05）. Group A had lower relative protein expression quantities of MMP-1，MMP-3 and MMP-13 than other four groups（P<0.05）. Group B had higher relative protein expression quantities of MMP-1 and MMP-3 than group C，and had higher relative protein expression quantities of MMP-1，MMP-3 and MMP-13 than groups D and E（P<0.05）. Group C had higher relative protein expression quantities of MMP-1 and MMP-13 than group D（P<0.05），and also had higher relative protein expression quantities of MMP-1，MMP-3 and MMP-13 than group E（P<0.05）. The relative protein expression quantities of MMP-1，MMP-3 and MMP-13 in group E were lower than those in group D（P<0.05）. The contents of MMP-1，MMP-3 and MMP-13 in supernatant solution in group A were lower than those of other four groups（P<0.05）. Group B had higher contents of MMP-1，MMP-3 and MMP-13 in supernatant solution than groups C，D，and E（P<0.05）. Group C had higher contents of MMP-1，MMP-3 and MMP-13 in supernatant solution than groups D and E（P<0.05）. The contents of MMP-1，MMP-3 and MMP-13 in supernatant solution in group E were lower than those in group D（P<0.05）. Two more groups，F〔SW1353 chondrosarcoma cells intervened with 10 μg/L IL-1β and 5 μmol/L solution of WAY-262611（an activating agent of Wnt/β-catenin pathway）〕and G（SW1353 chondrosarcoma cells intervened with 10 μg/L IL-1β and 10 μmol/L solution of dexamethasone）were created for further analysis. The relative protein expression quantities of MMP-13 and β-catenin in group A were lower than those of other six groups（P<0.05）. Group F had higher relative protein expression quantities of MMP-13 and β-catenin than groups B，C，D，E and G（P<0.05）. Group D had lower relative protein expression quantity of MMP-13 than groups B and C（P<0.05），so did groups E and G（P<0.05）. Group E had lower relative protein expression quantity of β-catenin than groups B and C（P<0.05），so did group G（P<0.05）. The relative protein expression quantity of MMP-13 in group G was lower than that of group D（P<0.05）. Group D showed higher relative protein expression quantity of β-catenin than groups E and G（P<0.05）. The relative protein expression quantities of MMP-13 and β-catenin in group G were lower than those in group E（P<0.05）. Conclusion　GR may play a partial role in protecting cartilage matrix from degradation，and the protective effect may be increased with the dose of the recipe. The major mechanism may be related to its effect of inhibiting the over-expression of MMPs and abnormal activation of Wnt/β-catenin pathway.

Key words: Osteoarthritis, Cartilage, Bone matrix, Gubitong Recipe, Matrix metalloproteinases, Wnt signaling pathway