| Objective:By intervention of BuShen HuoXue Decoctiontothe rabbit model of steroid-induced avascular necrosis of femoral head, then observed its influences of the main composition content of femoral head and thebone biomechanics,and test the expression of gene and protein of osteogenesis and coed factorn comitant relaton wnt/β-catenin signaling pathway, to explore the mechanism of Bushen Huoxue Decoction to prevent and cure the rabbit model of steroid-induced avascular necrosis of femoral head.Methods:6 months New Zealand white rabbit 104, randomly divided into the blank group 26 and the building group 78. Hayes made mould method was adopted to establish the rabbit model of steroid-induced avascular necrosis of femoral head, used HE to authenticate the model. The 53 successful rabbit model randomly divided into the model group 27 and the treatment group 26. After 1 week of the success of model building,3 groups were given by gavage. The treatment group was given with BuShen HuoXue Decoction of 5.05 g/kg/d, in the morning and evening twice a day at the same time points for 8 weeks, while the model group and the blank group were given by gavage of the same amount of saline. Executed half rabbits respectively after the intervention of 6 and 8 weeks, took the right side of the femoral head to test the biomechanical indexes (elastic modulus, maximum load, strain capacity) and the main component content (calcium and hydroxyproline) of femoral head. Used RT-PCR and Western Blot methods to test the expression of gene and protein of the osteogenesis and concomitant related factor on wnt/(3-catenin signaling pathway on the left side of the femoral head. Compared the changes and relationships of the indicator above on protein between 6 and 8 weeks within the 3 groups.6 and 8 weeks in each group. The changes of gene were compared between the three groups at the 6 groups.Results:1. Light microscope observation:the cartilage layer of the blank group was thick and the number of cartilage cells was normal.The trabecular bone was intact, the arrangement and the shape were regular and normal; while empty lacunae was occasionally. Bone marrow hematopoietic cells were rich, while fat cells were less. The number of cartilage cells in the model group had a substantial reduction, and the cartilage layer became thin or defect. The bone marrow cavity was fillied with a large number of fat cells, and the fat cells volume increased or merged with each other into a bulla.The number of hematopoietic cells in bone marrow decreased, and some of them were nuclear fragmentation and karyolysis. Part of the bone cell was shrinkage or disappearance. There were more empty lacunae. Bone trabeculae became thin, sparse, continuous interrupted, scattered, and part of them necrosis.2. The rate of empty bone lacuna:compared with the model group, the rate of empty bone lacuna of the blank group was lower, was (10.06±0.13)%; while the model group increased significantly, was (29.68±1.23)%. They had significant difference (P<0.01).3. Detection of the main components of the femoral head:3.1 Bone hydroxyproline content:compared 6 weeks with 8 weeks, there was no difference in the blank group, the model group and the treatment group (P>0.05). The trend of 6 and 8 weeks was:the blank group>the treatment group>the model group.Compared 6 weeks to 8 weeks, the blank group and the treatment group were significantly higher, model group increased slightly high. Among them, the blank group and the model group at 6 weeks and 8 weeks had statistical significance (P<0.05); the model group and the treatment group at 6 weeks had statistical significance (P<0.05), was statistically significant (P<0.01)at 8 weeks. Other groups had no difference.3.2 Bone calcium content:there was no difference between the control group and the treatment group (P>0.05) at 6 weeks and 8 weeks. There was significant difference between the model group (P<0.05). The trend of 6 weeks and 8 weeks was not only that the blank group>the treatment group>the model group, but also the blank group and the treatment group were significantly higher, while the model group is significantly lower. Compared with the model groupat 6 weeks (P<0.01) and 8 weeks (P<0.05), the blank group are statistically significant, besides, the treatment group was statistically significant at 6 weeks (P<0.05) and 8 weeks (P<0.01).4. Biomechanics test of femoral head:4.1 Mechanical indexes of bone materials:comparison of elastic modulus The trend of 6 weeks and 8 weeks is that the blank group>the treatment group>the model group, at the same time there were significant differences between the groups (P<0.05). Compared 6 weeks with 8 weeks, the model group is statistically significant (P<0.05). At 6 weeks, compared with the blank group and the model group, compared with the model group and the treatment group, all had statistical significance (P<0.05). At 8weeks, compared with the control group and the model group (P<0.05); compared with model group and treatment group (P<0.01). all have statistical significance.4.2 Mechanical indexes of bone structural:strain capacityAt 6 weeks’ time, the trend was the blank group>the treatment group>the model group, there was significant difference between the groups (P<0.01).The tendency of 8 weeks was that the blank group higher than the treatment group, the treatment group higher than the model group, there was significant difference between the groups (P<0.05). Among them, compared with the blank group and the model group, there was statistically significant between 6 weeks and 8 weeks (P<0.01). Compared with the model group and the treatment group,6 weeks and 8 weeks had statistical significance (P<0.05).Compared 6 weeks to 8 weeks, the blank group was significantly increased, the model group increased slightly, and the treatment group decreased slightly.Maximum load value The trends of 6 weeks and 8 weeks:the treatment group higher than the model group, the blank group higher than the model group, there were significant differences between the groups (P<0.01). The blank group and the treatment group were significantly higher, while the model group was significantly reduced; the model groups were statistically significant (P<0.05). Compared the blank group with the model group, there were significant differences at 6 weeks (P<0.01) and 8 weeks (P<0.05). Compared with the model group with the treatment group, it was statistically significant at 6 weeks (P<0.05) and 8 weeks (P<0.01).5. The relation proteins expression of wnt/β-catenin signaling pathway:(1) compared with the model group, the proteins expression of wntl 11、sFRP2、FZD6、Ror2 was higher, the enhancement effect was on the rise changing over time:while the protein expression of wnt9a was decreased and the enhancement effect was on a downward trend. SFRP3 had the statistical differences between the blank group and the model group only in 8 weeks. SFRP2 had the statistical differences between groups only in 8 weeks. While wnt11 and wnt9a had the statistical differences between groups only in 6 weeks. FZD6 and Ror2 had the statistical differences between groups in 6 and 8 weeks.(2) compared with the model group,the blank group and the treatment group.the expression of DKK-1,sFRP4 and wnt5b declined obviously,which of wntlOb and P-catenin rose. Wnt10b had the statistical differences between groups only in 8 weeks.p-catenin and sFRP4 had the statistical differences between groups only in 6 weeks. DKK-1 and Wnt5b had the statistical differences between groups not only in 6 weeks but also in 8weeks, while the expression of wntl showed no difference.6. The relation gene expression of wnt/β-catenin signaling pathway:(1) compared with the model group in 6 weeks, the expression of wnt11,sFRP2,Ror2 and FZD6 obviously rose, while the expression of wnt9a was decreased obviously; SFRP3 between groups showed no difference. (2)compared with the model group in 6 weeks,the expression of DKK-1,sFRP5,wntl and wnt5b was decreased obviously, while wnt10b and β-catenin increased in the expression. SFRP4 showed no difference.Conclusion:1. BuShen HuoXue Decoction can regulate calcium and phosphorus metabolism; can accelerate matrix synthesis and improve the strength of the femoral head.lt can also strengthen bone strain capacity and maximum load value, improve the elastic modulus of bone and the state of osteoporosis.The bones were reshaped and stabilized in the aspects of structural mechanics and material mechanics.2. The regulation mechanism of BuShen HuoXue Decoction and the differentiation of osteogenesis-into fat within the femoral head between bone marrow mesenchymal stem cells mainly through enhanced the expression of gene and protein of Wnt11,sFRP2,Ror2 and FZD6 in wnt/p-catenin signaling pathway,inhibited the expression of wnt9a;inhibited the expression of DKK-1, wnt5b at the same time.promoted the expression of wntlOb and β-catenin,thus inhibiting the bone marrow mesenchymal stem cells transform into fat cells and promoting the cells transform into osteogenic,accelerating the tissue repair of necrotic bone. |
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