| 1ObjectiveTo observe the changes of serum OPG, BGP and PINP, and the expressions of VEGF and TGF-β1in femoral head; by watching the research of treating steroid-induced avascular necrosis of femoral head (SANFH) with Wenyang Bushen Prescription in rabbits.To observe the pathologic changes and the ultrastructure of cells in the femoral head, by using an optical microscope and a transmission electron microscope (TEM); so as to explore the mechanism of action of Wenyang Bushen Prescription in prevention and controlling of SANFH.2Methods2.1New Zealand rabbits were randomly divided into normal group and model group, by using the molding method, model group combined the improved horse serum with methylprednisolone, to establish the model of steroid-induced avascular necrosis of femoral head, to evaluate the model by detecting the relevant indicators.2.2After the successfull of modeling, the model group is again divided into four groups:model group, low, middle and high dose treatment group, all treatment groups were underwent intragastric administration with Wenyang Bushen Prescription for4weeks,8weeks,12weeks.2.3After medication intervention for4weeks,8weeks and12weeks, the serum and femoral head were taken for cryopreservation.2.4The OPG, BGP and PINP levels in selected animal serum were detected by enzyme-linked immunosorbent assay.2.5The selected femoral head was given histomorphology observation and detection of percentage of empty lacunae by HE staining.2.6According to the serum OPG, BGP and PINP levels, histomorphology observation and comparison of percentage of empty lacunae, the frozen femoral head tissues were observed under a TEM at the point in time with the most obvious changes.The expressions of VEGF and TGF-β1in the femoral head were detected by Western-blot.3Results3.1Model Identification:After modeling, the rabbits in the model group were loss of body weight, loss of hair, with fewer activities compared to the normal group.The femoral head in the model group showed thinned cartilage layer, sparse and fractured bone trabecula, decreased bone cells, and increased percentage of empty lacunae.Comprehensive observation results showed successful modeling.3.2Results of HE Staining:The model group at4th week,8th week and12th week showed thinned and even fractured bone trabecula, increased empty lacunae, inflated and necrotic fat cells, which were in an increasing trend over time.The percentage of empty lacunae in the model group was increased compared to the normal group (P<0.01). At each point in time, the percentages of empty lacunae in various dose groups of Wenyang Bushen Prescription were significantly reduced compared to the model group (P<0.01).3.3Detection of Serum OPG, BGP and PINP Levels by Enzyme-linked Immunosorbent Assay:The OPG, BGP and PINP levels in the model group were significantly lower than the normal group at4th week,8th week and12th week (P<0.01). At4th week:There were no significant differences in BGP and PINP levels between the low-dose treatment group and the model group (P>0.05), but the OPG level was increased (P<0.05). The OPG, BGP and PINP levels in the medium-and high-dose treatment groups were significantly increased compared to the model group (P<0.01). At8th week:The OPG, BGP and PINP levels in the low-medium-and high-dose treatment groups were significantly increased compared to the model group (P<0.01). At12th week:The OPG, BGP and PINP levels in the low-medium-and high-dose treatment groups were significantly increased compared to the model group (P<0.01). At each point in time, there were differences in the OPG, BGP and PINP levels between the low-and medium-dose treatment groups (P<0.05). However, at each point in time, there were no significant differences in the OPG, BGP and PINP levels between the medium-and high-dose treatment groups (P>0.05), except that at12th week, there were significant difference in OPG level between the medium-and high-dose treatment groups (P<0.01).3.4TEM Observation:the model group compared with normal group, the difference of chondrocytes and osteocyte were in obvious contrast.Compared with the model group, cell structures in various dose groups of Wenyang Bushen Prescription are better.3.5Detection of VEGF and TGF-β1Expressions in Bone Tissue by Western-blot:The expressions of VEGF and TGF-β1in the model group were significantly lower than the normal group (P<0.01).The expressions of VEGF and TGF-β1in each dose treatment groups were significantly higher than the model group (P<0.01).The expressions of VEGF and TGF-(31in the medium-dose treatment group were higher than the low-dose treatment group (P<0.05). However, there were no significant differences between the medium-and high-dose treatment groups (P>0.05).4Conclusion4.1Wenyang Bushen Prescription that serum OPG, BGP, PINP content increased, can improve the activity of osteoblasts, promote bone growth.4.2Wenyang Bushen Prescription inhibit the hypertrophy necrosis of bone marrow fat cell, prevent lipid accumulating in the medullary cavity.4.3Wenyang Bushen Prescription can improve the expression of VEGF in the necrosis of the femoral head organization, promote angiogenesis, thereby increasing the blood supply of the femoral head necrosis area.4.4In Wenyang Bushen Prescription, the effect of middle and high dose groups were better than the low dose group, there was no difference of middle and high dose group in effect. |
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