Experimental Study Of Fuyuan Capsule On Proliferation And Apoptosis Of Chondrcoyte In Experimental Osteoarthritis

Objective: To study of Fuyuan capsule, which is an experimental compound pharmacrutic prescriptions of a traditional Chinese medicine, on articular cartilage histomorphology, proliferation and apoptosis of chondrocytes in experimental osteoarthritis rats. To observe the effects on NO in sera of OA rats and the expression of PCNA,Bax,Bcl-2 and caspase-3 in osteoarthritic rat .To study of the serum containing Fuyuan capsule on the normal rat chondrocyte proliferating and cell cycle progression and synthesis metabolic function in vitro. To study the effects of the serum containing Fuyuan capsule on extracellular signal regulated kinase 1 and 2(ERK 1/2) and p38 signaling pathway in SNP-induced apoptosis of rat chondrocytes in vitro.Methods:1. 150 healthy male Sprague-Dawley(SD) rats were divided into 4 groups randomly: normal group, model contrast group,Zhuanggu guanjiewan group and Fuyuan capsule group. OA models were induced by Hurth's technique. When OA models were successfully induced for different phase, Zhuangguguanjiewan group and Fuyuan capsule group were respectively fed with Zhuangguguanjiewan and Fuyuan capsule, and normal group and model contrast group were fed with 0.9 N.S at the same time. The effects of Fuyuan capsule on histomorphology of cartilage in knee OA rats were evaluated according to macroscopic grading score, X-ray radiography score and Mankin,s score. Ultrastructure and apoptotic indexs of chondrocytes was determined by transmission electron microscopy and TUNEL, NO concentration in sera of rats was delected by nitrare reductase method,Bax and Bcl-2 were determined by immunochistochemistry technology and the expression of PCNA,p53 and caspase-3 mRNA were evaluated by RT-PCR technology.2. Separating and culturing the rat chondrocyte in vitro with SD rats aged 21- 28 d, healthy adult male SD rats were selected for preparation of serum containing Chinese herbs.The second subculture chondrocytes were divided into different groups and were treated with different concentration serum containing Fuyuan capsule. The cytoactive and proliferation were detected by methyl thiazolyl tetrazolium(MTT) assay, cell cycle progression were examined by flow cytometry and cellular DNA and matrixâ…¡collagen were determained by 3H-TdR and 3H-Pro.3. The second subculture chondrocytes were treated with Sodium nitroprusside(SNP) to induce apoptosis of rat chondrocytes.U0126,a special inhibitor of ERK1/2, and SB203580, a special inhibitor of p38 were used to block ERK1/2 and p38 kinase activation. Apoptotic ultramicrostructure was quantified by transmission electron microscopy. Apoptotic rate was quantified by flow cytometric assay,expression of p-ERK/2,p-p38 and p53 were evaluated by western blot analysis ,NO in the culture supernatants was delected by nitrare reductase method and activation of caspase-3 was determined by colorimetric assay . Results:1.The effects of Fuyuan capsule on histomorphology of articular cartilage in knee OA ratsBy Hulth's technique articular cartilage showed obvious osteoarthritic histomorphology changes at early phase and these osteoarthritic histomorph- ology changes became more obvious and severe at the metaphase and the advanced phase. Compared with the model group , histomorphology changes ,macroscopic grading score, X-ray photograph score and Mankin,s score in Fuyuan capsule groups were significantly low.These results suggested that Fuyuan capsule could prevent cartilage from degeneration .2. The effects of Fuyuan capsule on proliferation and apoptosis of chondrocyes of articular cartilage in knee OA ratsApoptotic indexs were significantly increased in model group, but it was decreased in advanced model group. Compared with the model group, Fuyuan capsule could significantly descend apoptotic index. These results suggested that the effects of Fuyuan capsule on preventing cartilage from degeneration may be related with the effect on inhibiting apoptosis. NO concentration in serum of the model group was significantly higher than it in the normal group,and it more and more high in the model group with time delaying. Compared with the model group,Fuyuan could decreased NO concentration and the difference was significant.Expression of PCNA mRNA in normal group was moderate at three time pionts. Expression of PCNA mRNA in model group was significantly increased in the early and metaphase group , but it was significantly decreased in the advanced group.Fuyuan capsule could promote expression of PCNA mRNA, and there were significantly different between Fuyuan capsule and model group. But there were no significantly different between Fuyuan capsule group and Zhuangguguanjiewan group.Compared with normal group, the expession of Bax positive staining cell in model group was significantly raised,but it was descended in the advanced group. Fuyuan capsule could decreased OD of Bax positive staining cell, difference was significant comparing with model group.But there were no significantly difference between Fuyuan capsule group and Zhuangguguan jiewan in early phase group.Compared with normal group ,the expression of Bcl-2 positive staining cell in model group was significantly high, especially in early phase and metaphase model group.Fuyuan capsule could significantly improved the OD of Bcl-2 positive staining cell. But there were no significantly different between Fuyuan capsule group and Zhuangguguanjiewan group. The Bax/Bcl-2 ratio in the model group was significantly higher than it in the normal group. But there was no significantly different among the early phase group, metaphase group and advanced group. At early phase and metaphase, the ratio in Fuyuan capsule and Zhuangguguanjiewan group were significantly lower than it in model group. But there was no significantly different between Fuyuan capsule and Zhuangguguanjiewan group.Expression of p53 mRNA and caspase-3 mRAN in normal group was little and there were no significant change in three phases. Expression of p53 mRNA and caspase-3 mRNA in model group was significantly increased, especially at early phase and metaphase.Fuyuan capsule could descend expression of p53 mRNA and caspase-3 mRNA and there were significantly different between Fuyuan capsule group and model group .But compared with Zhuangguguanjiewan group, Fuyuan capsule could not descend expression of p53 mRNA and caspase-3 mRNA.3.The sera containing Fuyuan capsule could promote proliferation and synthesis metabolic function of chondrocyte in vitroMTT assay showed the sera containing Fuyuan capsule could promote proliferation of chondrocyte .The 10 % and 20 % sera contained Fuyuan capsule group could morely promote proliferation than the 5 % group could, and there was no significantly different between the 10 %and 20 % group. We observed proliferation at 24 h,48h,72 h and 96 h,and we found it was at 48 h that the effects of Fuyuan capsule on promoting proliferation of chondrocytes were most obvious.Flow cytometry results showed that sera contained Fuyuan capsule could promote cell cycle progression. Sera contained Fuyuan capsule could decrease G0/G1 percentages and increase S phase and G2/M percentages and PI, and there were significantly different between Sera contained Fuyuan capsule and blank sera group. The 10% group was best on promoting cell cycle progression.3H-TdR and 3H-Pro results showed that both sera contained Fuyuan cap- sule and zhuangguguanjiewan could improve synthesis metabolic function of chondrocyte in vitro,including cellular DNA and matrixâ…¡collagen. Compar- ed with blank group and blank sera group, there was significantly different .4.The effects of sera contained Fuyuan capsule on extracellular signal regulated kinase 1 and 2(ERK1/2) and p38 signaling pathway with SNP- induced apoptosis of rat chondrocyteCellular typical morphology of apoptosis was observed .Compared with the model group, the serum contained Fuyan capsule could decrease signifi- cantly SNP-induced apoptotic percentages, imported significantly expression of p-ERK1/2 protein and inhibit significantly expression of p-p38 protein and p53 in SNP-induced apoptotic chondrocytes, decrease significantly NO concentration in the culture supernatants and indented significantly caspase-3 activation. When we treat chondrcoyes with U0126, Compared with the U0126 group, the serum contained Fuyan capsule could decrease significantly SNP-induced apoptotic percentages, inhibit significantly expression p53 protein in SNP-induced apoptotic chondrocytes, decrease significantly NO concentration in the culture supernatants and indented significantly caspase-3 activation, but there was no significantly difference on expression of p-ERK 1/2 protein. When we treat chondrcoyes with SB203580, compared with the SB203580 group, the serum contained Fuyan capsule could decrease signifi- cantly SNP-induced apoptotic percentages, inhibit significantly expression p53 protein in SNP-induced apoptotic chondrocytes, decrease significantly NO concentration in the culture supernatants and indented significantly caspase-3 activation, but there was no significantly difference on expression of p38 protein.Conclusions:1. Fuyuan capsule could decrease macroscopic grading score, X-ray photograph score,Mankin,s score and relief ultramicrostructure of articular cartilage in OA knee by Hulth's technique , so it could prevent articular cartilage from OA degeneration.2. Fuyuan capsule could inhibit apoptosis of chondrocyes in OA.3. Fuyuan capsule could decrease NO concentration in serum, inhibit the expression of Bax, promote the expression of Bcl-2 in OA cartilage and adjust the ratio of Bax/Bcl-2.At same time ,it could enhance the expression of PCNA mRNA ,and descend the expression of p53 mRNA and caspase-3 mRNA .4. The sera contained Fuyuan capsule could promote proliferation, increase S phase and G2/M percentages and PI, decrease G0/G1 percentages and improve synthesis metabolic function of chondrocyte in vitro,including cellular DNA and matrixâ…¡collagen.5. The sera contained Fuyuan capsule could inhibit SNP-induced apoptosis of chondrocyte in vitro.6. The sera contained Fuyuan capsule could improve the expression of p-ERK1/2, adjust the expression of p53 and caspase-3 activation, which were downstream target gene of ERK1/2 /MAPK signal pathway, and decrease NO concentration in the culture supernatants ,7. The sera contained Fuyuan capsule could inhibit the expression of p-p38,and adjust the expression of p53 and caspase-3 activation, which were downstream target gene of p38/MAPK signal pathway, and decrease NO concentration in the culture supernatants ,...