| Objective: To investigate the effects of extract from rabbit skin inflamed by vaccinia virus(neurotropin/NTP) combined with sodium hyaluronate(HA) for injection on the traumatic arthritis of knee joint in rabbits, and to provide the animal experimental data for clinical drug treatment of traumatic arthritis.Method: Healthy adult New Zealand rabbits were used to establish the animal models of traumatic arthritis of knee joint. The animal models were established through disconnecting the anterior and posterior cruciate ligaments of right knee joints of the rabbits and removing their right medial meniscus completely. Complete disconnection of the cruciate ligaments was confirmed by Drawer test. After establishment of the traumatic arthritis animal models, the model animals, disregarding female and male,were divided into four groups, 10 rabbits per group. Animals in Group NTP+HA were treated with injection of neurotropin combined with sodium hyaluronate into the traumatic osteoarthritis knees. Rabbits in Group NTP or HA received neurotropin or sodium hyaluronate in the same manner, respectively. Animals that served as controls were injected with normal saline into the traumatic arthritis knees. All model animals were injected with drugs or saline twice a week from fifth week on after the operation for establishment of the animal model for 5 weeks. Three days before or 2, 4, 6, 8 and 10 weeks after the operation, the joint fluid was collected form right knee joints of the animals, and IL – 1, TNF-α and MMP – 3 were quantitated by ELISA method. All experimental animals were sacrificed after two weeks of withdrawal of injection, and arthrodial cartilage of the keen jiont was for morphological analysis or histopathological examination.Results: The results of 38 rabbits were actually involved in the analysis because one rabbit died in Group control and Group HA, respectively.(1) Three days before or 2 and4 weeks after the operation, the values of joint fluid IL-1 of rabbits from groups NTP+HA, NTP, HA and Control were 42.892 ± 4.54, 45.497 ± 4.43, 44.281 ± 4.54,43.319±1.72 or 112.660±5.49, 112.294±4.54, 112.087±6.77, 109.151±3.07 and97.118±4.54, 97.231±4.17, 100.479±3.61, 98.220±2.67, respectively; the values of joint fluid TNF-α were 71.650±4.53, 72.767±4.54, 75.150±3.92, 74.680±4.54 or121.435 ± 4.54, 123.274± 5.15, 124.680 ±4.58, 125.372 ± 5.39 and 172.963 ±6.33,168.837±4.44, 169.362±4.54, 172.889±3.88, respectively; and the joint fluid MMP –3 levels of rabbits from groups NTP+HA, NTP, HA and Control were 0.409 ± 0.100,0.388±0.153, 0.381±0.100, 0.309±0.100 or 1.734±0.099, 1.754±0.092, 1.665±0.100, 1.659±0.155 and 3.891±0.100, 3.958±0.094, 3.987±0.098, 3.901±0.147,respectively. The joint fluid IL-1, TNF-α and MMP-3 levels in all experimental animals at 2 or 4 weeks after the surgery were significantly higher than those at 3 days before the surgery(p<0.05). No significant differences in rabbits joint fluid IL-1, TNF-α and MMP-3 levels were observed among groups NTP+HA, NTP, HA and Control at 3 days before or 2 and 4 weeks after the operation(p>0.05). These results indicated that the animal models of traumatic arthritis knee joint had been established successfully in rabbits.(2) At 6 or 8, and 10 weeks after the surgery, the joint fluid IL-1 levels of in animals from groups NTP+HA, NTP, HA and Control were 66.814 ± 4.54,82.614 ± 3.82,78.365±4.54,88.743±3.60 or 52.382±4.33, 72.113±4.54, 64.210±4.54,81.517±2.54,and 44.336±4.54、64.704±5.39、59.894±4.54、76.550±3.90, respectively; the TNF-α levels were 124.810 ± 4.54,144.614 ± 4.54,133.136 ± 7.92, 64.780 ± 4.54 or108.651±4.54,126.882±4.54,117.630±4.19,158.621±4.54,and 86.552±4.54,110.673±4.54,97.812±4.54,147.423±4.55, respectively; the MMP-3 levels were 2.191±0.100,3.023±0.100, 2.675±0.092, 3.480±0.100 or 1.680±0.100, 2.650±0.100, 2.073±0.100, 2.973±0.100, and 1.248±0.100, 2.008±0.103, 1.730±0.100, 2.522±0.100,respectively. After 4 or 6 weeks of treatment with drugs by injecting into the knee joints(namely, 8 or 10 weeks after the surgery), the joint fluid IL-1, TNF-α and MMP-3 values in the rabbits from groups NTP+HA, NTP or HA remarkably decreased entirely when compared with the values in controls(p<0.05), and then, the values described above in animals from group NTP+HA were significantly lower than those in rabbits of groups NTP or HA(p<0.05), and the values in animals of group HA were slightly lower than those in group NTP rabbits. In addition, the joint fluid IL-1, TNF-α levels in animals from groups NTP+HA, NTP or HA markedly decreased after 4 or 6 weeks of treatment with drugs in comparation with those at 2 or 4 weeks after the surgery, although slightly increased compared with those at 3 days before the surgery. Moreover, although the the joint fluid MMP-3 values in rabbits from groups NTP+HA, NTP or HA slightly increased significantly after 4 or 6 weeks of treatment with drugs when compared with those values at 3 days before the surgery, dramatically decreased in comparation with the values at 4weeks after the surgery. However, the joint fluid IL-1, TNF-α and MMP-3 values in controls after 4 or 6 weeks of the treatment were much more significantly higher than the values at 3 days before the surgery(p<0.01), even though slightly lower significantly than the values at at 4 weeks after the surgery(p<0.05).(3) The morphological observations after 2-week withdrawal of treatmentin were described below: in group control, the gross examination showing: synovium hyperplased nodularly, less and turbid synovial fluid,the surface had severe deep cracks and a large area ulcer, osteophyte formation and serious exposure of subchondral bone. Histopathological examination showing: cartilage injury seriously, a large area of cartilage fibrosis, the number of the cartilage cell was decreased, With karyolysis or nuclear pyknosis, Tide line was not obvious or disappear,some tissue was no staining in the HE hematoxylin; in group HA,the gross examination showing: articular cartilage hyperplasia slightly, less and yellow synovial fluid, the surface had severe cracks and ulcer,no osteophyte formation and no exposure of subchondral bone. Histopathological examination showing:cartilage injury moderately, the surface of cartilage fibrosis,the deep of cracks was the middle layer,a large number of the cartilage cells gathered, Tide line was irregular or break and With a little karyolysis or nuclear pyknosis, staining in the HE hematoxylin was inhomogeneous;in Group NTP, the gross examination showing: articular cartilage hyperplasia moderately or seriously, synovial fluid was yellow and melicera, the surface of cartilage had severe cracks and the depth was deep to the bone layer,some part had obvious ulcer, some osteophyte formation and exposure of subchondral bone. Histopathological examination showing: cartilage injury moderately or seriously, the surface of cartilage fibrosis, more and deep cracks, appropriate amount of the cartilage cells gathered, Tide line was not obvious or disappear, With karyolysis or nuclear pyknosis, some deep tissue was no staining in the HE hematoxylin; in group NTP+HA, the gross examination showing:synovial hyperemia and hyperplasia longitudinally, increased synovial fluid effusion,cartilage surface roughness, the cracks were unconspicuous, some cartilage was soften, no osteophyte formation. Histopathological examination showing: cartilage injury moderately longitudinally, no cracks, cartilage surface roughness,the number of cartilage cells gathered was increased, Tide line was holonomic, matrix stained tinge. All the results show that extract from rabbit skin inflamed by vaccinia virus(neurotropin/NTP)could exert certain protective effect on articular cartilage, and the combination of NTP and sodium hyaluronate(HA) could enhance the effects of protection and repair on articular cartilage of knee joint.Conclusion:(1) The extract from rabbit skin inflamed by vaccinia virus(neurotropin) could exert a certain protective effect on articular cartilage, but the effect was poorer than Sodium hyaluronate.(2) Administration of neurotropin combined with sodium hyaluronate injected into traumatic osteoarthritis knees could synergistically enhance the effects of protection and repair on the articular cartilage in rabbits. |
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