Study On The Effect Of Gubishu Tablet On Histomorphology,oxgen Free Radical And Coll-Ⅱ,mmp-1,mmp-13 Of Knee Osteoarthritis

Objective:1. To observe the effect of Gubishu on histomorphology and oxgen free radical of experimental knee osteoarthritis.2. To investigate the effects of Gubishu containing serum on the proliferation of rabbit articular chondrocytes cultured in vitro and the gene,protein expression of Coll-Ⅱ,MMP-1,MMP-13 of rabbit articular chondrocytes stimulated by interleukin-1beta.3. To explore the mechanism of action of Gubishu tablet in preventing and treating osteoarthritis on the tissue,cellular,protein and genetic level.Method:1.40 SD rats were randomly divided into 4 groups, blank group, model group, GuBishu group, kanggu Zengsheng group. Knee OA models were made by the modified Hulth method. Every group received a daily administration by the way of ig for 6 weeks, and then the articular cartilage of medial epicondyle femur were harvested to observe the pathological changes microscopically through the hematoxylin-eosin,Masson and Safranin-o-fast green-iron hematoxylin staining, and scoring based on the Mankin's. And the level of the serum SOD,MDA,NO were detected at the same time.2. Articular chondrocytes were obtained from the cartilage of 1-month rabbit and cultured in vitro. They were randomly divided into 8 groups, blank and Gubishu groups in different concentrations (5%,10%,15%, 20%),MTT assay method was adopted to observe the influence of Gubishu containing serum with different concentrations to the proliferation of chondrocytes after incubated 1,3,5,7 and 9days.3. Articular chondrocytes were obtained from the cartilage of 1-month rabbit and cultured in vitro. The passage I chondrocytes were used. They were divided into 4 groups, control groups,model groups,Gubishu groups and Kangguzengsheng groups. After 4 days cultured, IL-1β10ng/ml were added in the control groups,Gubishu groups and Kangguzengsheng groups. RT-PCR assay method was adopted to observe the influence of every different groups on the expression of Coll-Ⅱ,MMP-1 and MMP-13 mRNA of chondrocytes stimulated by interleukin-1beta after incubated 24h,48h and 72h.4. Articular chondrocytes were obtained from the cartilage of 1-month rabbit and cultured in vitro. The passage I chondrocytes were used. They were divided into 4 groups, control groups,model groupa,Gubishu groups and Kangguzengsheng groups. After 4 days cultured, IL-1β10ng/ml were added in the control groups,Gubishu groups and Kangguzengsheng groups. In-Cell Western assay method was adopted to observe the influence of every different groups on the expression of Coll-Ⅱ,MMP-1 and MMP-13 of chondrocytes stimulated by interleukin-lbeta after incubated 24h,48h and 72h.Result:1. The Mankin's score and the level of the serum SCD,MDA,NO was significant difference between GuBishu group and model group(P<0.05).2. The proliferation of chondrocytes were dependent on the concentration in Gubishu groups. At same time point, there was significant value between every groups,20% concentration was greatest(P<0.05); There was significant differences between 5%,10% and 20% concentration of the blank groups at same time point (P<0.05), and was not between 15% and 20% concentration at the 1,3,5 and 7days (P>0.05),20% concentration of the blank group was greatest. 20% concentrations of Gubishu containing serum was significantly greater than 20% concentrations of blank group at the 1,3,5 and 7days (P<0.05).3. The expression of Coll-ⅡmRNA of Gubishu groups were increased but lower of MMP-land MMP-13 mRNA than model groups at all the time points.4. The expression of Coll-Ⅱ,MMP-land MMP-13 of chondrocytes were significant differences between control groups and model groups at same time point (P<0.05); The expression of Coll-Ⅱof chondrocytes were significant differences between Gubishu groups and model groups at 48h,72h (P<0.05); There was significant differences between Gubishu groups and model groups in expression of MMP-land MMP-13 at same time point (P<0.05).Conclusion:1. Gubishu troche is effective to postpone degeneration of articular cartilage via inhibiting the oxygen free radical and NO and promoting the biological effect of SOD.2.20% concentrations of Gubishu containing serum can significantly increase the proliferation of chondrocytes, and bring the logarithmic growth period forward to the 3day.3. Gubishu can increase the expression of Coll-ⅡmRNA and decrease MMP-1 and MMP-13 mRNA.4. Gubishu containing serum can significantly inhibit the degradation and destroy of Coll-Ⅱcaused by IL-1β, also inhibit the IL-1β-induced MMP-1,MMP-13.