| Objective: To study the effects of herbal medications—Fuyuan capsule(FYC) on articular cartilage degradation and the expression of matrix metalloproteinase 13 , tissue type inhibitors of metalloproteinases 1, 2 ,interleukin beta 1 and transforming growth factor beta 2 in osteoarthritic rabbits model .To research the proliferation effects of sera contained in FYC on the chondrocyte and p38 signal pathway intervention,we studied the protective effects of FYC on articular cartilage degradation and explored the possible mechanisms of its action.Methods:1. Sixty healthy New Zealand rabbits received the reforming Hulth technique to establish rabbit knee osteoarthritis model.One week after operation the rabbits were randomly divided into 5 groups, named as model group, doxycycline group and FYC low, moderate and high dose groups with 12 rabbits in each group, while the other 6 rabbits were divided into normal group.Treatment groups were perfused with Fuyuan capsule. Samples of joint were collected at the 4th,8th,and 12th week after managements for general, light microscopic and electronmicroscope observation.The distribution and expression of MMP-13,TIMP-2,IL-1βand TGF-β2 of cartilage were documented by immunohistochemistry and mRNA expression of MMP-13,TIMP-1,-2 were detected using reverse transcription-polymerase chain reaction(RT-PCR). From the destructive and protective factors of osteoarthritic cartilage, from the mRNA and protein expression of its, our experiment were designed to fully study the effects of FYC on osteoarthritic cartilage degeneration in knee OA rabbits.2. Articular chondrocytes were obtained from the cartilage of SD rats aged 21-28days adopting method of sequential digestion by 0.2% collagenaseⅡ.After stimulating the third subculture chondrocytes in 24h,48h and 72h respectively with different concentrations of sera contained in FYC (0%,5%,10%,20%), the effects of sera contained in FYC on the proliferation of chondrocytes were assaied by MTT. According to OD, select the best concentration and the suitable time of sera contained in FYC on chondrocytes into later formal experiment.To characterize of chondrocyte,we assessed the expression ofⅡcollagen by immuno- cytochemistry. Application of Western blot to detected the effects of FYC on expression of p-p38 and MMP-13 in chondrocyte. From the signaling pathway, the experiment explored the possible mechanism of prevention of FYC on osteoarthritic cartilage from degeneration .Results:1. The effects of FYC on articular cartilage degradation of knee OA in rabbitsFemoral condylar cartilage in normal group showed normal histology, while femoral condylar cartilage at the 4th week after operation showed obvious pathologic changes that were consistent with those observed in osteoarthritis, such as cartilage fissures and erosions, fibrous osteophyte, swelling or fragmentation of chondrocyte organells, shrinkage of chondrocyte nuclei.The above changes became more obvious and severer at the 8th,12th week after operation,with p<0.05 in macroscopic grading scale and in Mankin's grading scale. FYC moderate and high dose groups were slighter significantly than that in model group at the 12th week after operation, while FYC low dose groups and FYC high and mid-dose groups at the 4th,8th week couldn't perevent cartilage from degeneration.2. Effects of FYC on the protein expression of MMP-13,TIMP-2,IL-1βand TGF-β2 on cartilage of rabbits OAImmunohistochemical test showed that the number of MMP-13 positive staining cell population was large and intensive, while dyeing was yellow or buffy in model group. Model groups were higher significantly than those in normal group at the 4th week(p<0.05),while it were reduced at the 8th and 12th week. The number of MMP-13 positive staining cell population was lower significantly in comparision with that of model group ,there was significant difference(p<0.01).The number of TIMP-2 positive staining cell population was large and intensive and dyeing was yellow or buffy in FYC high dose treatment group at the 4th,8th,12th week .In comparision with that of model group ,there was significant difference(p<0.05).The number of IL-1βpositive staining cell population was large and intensive,dyeing was yellow or buffy in model group at the 4th,8th,12th week. The expression of IL-1βin treatment groups were lower significantly than those in model group(p<0.05),there were not difference between groups. The expression of it in moderate dose groups at the 8th and 12th week were lower significantly in comparision with that of model group(p<0.05).There was positive expression of TGF-β2 in full-thickness chondrocyte cytoplasm of the normal group. Positive chondrocyte population of model groups were reduced at the 4thand 12th week, while there were a statistical significance (P<0.05) between model group and the normal group at the 4th,8th,12th week. Between FYC middle , high dose groups and low dose group there were no significant differences at the 4th week.There were significant difference of TGF-β2 between FYC middle , high dose groups and model groups at the 8th and 12th week (p <0.05).3. Effects of FYC on the expression of MMP-13,TIMP-1,TIMP-2mRNA in rabbits OA cartilageExpression rates of MMP-13 in normal groups were moderate at the 4th,8th,12th week (0.98±0.23). Expression rates of its in model groups were significantly higher at the 4th week (1.32±0.11) and were lower significantly at the 8th and 12th week in comparision with its at the 4th week. Expression rates of its in FYC low,mid and high-dose were high at the 4th and 12th week,compare with its model groups at same stages ,there were no remarkably difference(p>0.05).The rates of its in FYC low and mid-dose group(0.96±0.03,0.94±0.04)at the 8th week was reduced,in comparision with that of model group(1.24±0.06),there was siginificant difference (P<0.05). The rates of its in FYC high-dose group at the 8th and 12th week(0.70±0.01,0.51±0.02)were reduced, in comparision with that of model group,there was siginificant difference (p<0.05). There were no remarkably difference at the 4th week(p>0.05).Expression rates of TIMP-1 and TIMP-2 in normal groups was high at the 4th week, the relative optical density of strap were 2.86±0.65,0.89±0.2. Experimental results showed that TIMP-1 and TIMP-2 changes inconsistent.4. The test results of sera contained in FYC on chondrocyte proliferationThe 48th hour results showed that 5%, 10% and 20% sera contained in FYC could promote proliferation of chondrocyte and there were non-incremental effect among groups. There were remarkably difference (p<0.05) between 10% , 20% sera contained in FYC groups and 5% blank sera group and 5% sera contained in FYC group and 5% blank sera group no significant difference (p>0.05).The 72th hour results showed that 5%,10%,20% sera contained in FYC on chondrocyte proliferation were a promoting role but less than the 48 th hour of its.It was also found that the results of 10% sera contained in FYC at the 48 th and 72th hour were stronger than its of 20% sera contained in FYC. MTT show that in 10% FYC group role, 10ng/ml IL-1βwas strongest on inhibition of proliferation of chondrocyte.5. The effects of sera contained in FYC on chondrocyte on p38 signaling pathwaySera contained in FYC could inhibit p38 protein in chondrocyte activation, Between 10%FYC group and 10% blank sera group there was significant difference (P<0.05). In the IL-1βstimulation, there was a significant difference between 10% blank sera+IL-1 group and 10% FYC +IL-1βgroup (P<0.05).Sera contained in FYC could inhibit MMP-13 in chondrocyte. The expression of MMP-13 in 10% blank sera group and the 10%FYC group were increased significantly, compared with its in the normal groups,while there was a significant difference between 10%FYC group and 10% blank sera group(P<0.01). FYC inhibited the expression of P-P38 of IL-1βsignaling pathway,but inhibit p38 MAPK pathway, MMP-13 expression was not reduced .Conclusion:1. The pathological changes of femoral condyle articular cartilage of reforming Hulth postoperative rabbits coincided with osteoarthritic pathological changes.With the time of model extension cartilage degeneration was severe.It was suitable for further research applications. FYC high dose group could prevent cartilage degeneration at the 12th week. 2. FYC could inhibit the expression of MMP-13 and to enhance the expression of TIMP-1 and TIMP-2 of the degenerative cartilage and to adjust the ratio of MMPs/TIMPs.3. FYC could inhibit the expression of IL-1βand to enhance the expression of TGF-β2 on the cartilage of experimental rabbits OA .It could adjust the balance between MMPs and TIMPs.4. Sera contained in FYC could promote the proliferation of chondrocyte in vitro.5. Sera contained in FYC could inhibit the expression of P-P38 of IL-1 signal transduction pathway, so its promoted the reparation of articular cartilage.6. Sera contained in FYC could inhibit the expression of MMP-13 of IL-1 signal transduction pathway, so its repressed the degradation of extracellular matrix. |
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