| ObjectiveUsing bone resorption as the breakthrough point in animal experiments, the paper aims at observing the effect of Qing’e Fang on ovariectomized rats’OPN, BSP and their receptors, integrin alpha v beta 3.The mechanism of Qing’e Fang on effective prevention and treatment of postmenopausal osteoporosis was discussd from the standpoint of bone resorption,providing the experimental basis for the clinical application.Methods1. Selecting、Grouping、Modeling、Treatment and Drawn: We selected 120 healthy female SD rats,all three-month-old and in SPF level.They were randomized into 2 groups: 90 in the OVX group and 30 in the Sham group.The ovaries of rats in the OVX group were removed while those of the Sham group were kept. The OVX group was randomly divided into 3 sub-groups:Qing’e Fang-treat-group, Gushukang-treat-group, Saline-treat-group.Three days after operation, the rats of the Qing’e Fang-treat-group were treated with Qing’e Fang 3.6g/kg/d, the rats of Sham-operation-group and Saline-treat-group took NS 2ml/d, and the rats of Gushukang-treat-group took Gushukang 1.8g/kg/d. In each group,10 rats were randomly chosen, to be killed on 4,8 and 12weeks after treatment. Sample of 24h-urine collected a day before were centrifuged to remove sediment and were stored in a -20℃ refrigerator for testing later.Ventral aortic blood was collectd after the anesthesia took effect,and was centrifuged to collect serum to be stored in a -20℃ refrigerator.The shin、femur and lumbar were separated and stored in a -80℃ refrigerator for testing.2.Indexes Detection:Bone mineral densities (BMD) were measured by dual-energy X-ray absorptiometry (DXA) for the Lumbar vertebra. The bone tissues from the proximal shin were measured by Motic Med 6.0 digital medical image analysis system. Bone biological force of the femur were measured by IG-A1000N universal material testing machine.Concentrations of serum MMP-9、Cath-K、 TRACP5b and urine DPD were measured by ELISA.Detection of urine creatinine content of biochemical method.The expressions of OPN、BSP、Itgαv、Itgβ3 p38mRNA in bone tissue were measured by quantitative PCR (Q-PCR).The content of OPN、BSP、Itgαv、Itgβ3、p38 protein in bone tissue were measured by Western Bloting.Results1. Results of BMD:Some time after treatment,the BMD of the Saline-treat-group were significantly lower compared with the Sham-treat-group in week 8 and week 12(P<0.01). The BMD of Qing’e Fang-treat-group and Gushukang-treat-Group were higher than that of Saline-treat-group after testing for 12 weeks(P<0.01).2.Bone tissue form analysis:The trabecular bone of the saline-group changed obviously after operating for 12 weeks:trabecular bone became thinner and showed discontinuous, poor integrity and a lots of bone lacuna fomation. Sham-treat-group showed uniform distribution of trabecular bone, regular arrangement, and normal trabecular gap. The number of bone trabecula reduced in the two Traditional Chinese Medicine group and trabecular bone became thinner and showed micro fracture.Compared with the saline-group, the number of trabecular bone of the two treatment groups reduced in obviously with regular arrangement and better structural integrity.After treatment,compared with the Saline-treat-group, the area percentage of trabecular of the Qing,e Fang-treat-group was significantly increased after weekt 8 and week 12(P<0.01 or P<0.05).The area percentage of trabecular of the Qing,e Fang-treat-group was lower than the Sham-group and the only statistical significance appeared after treating 12 weeks(P<0.05).3. Results of bone biological force:Compared with the Sham-group, bone biological force of the saline-group was significantly reduced for three time point (P<0.01 or P< 0.05).After treatment, compared with the Saline-treat-group, bone biological force of the Qing’e Fang-treat-group were significantly increased after week 12(P<0.01)and compared with the Sham-treat-group, bone biological force of the Qing’e Fang-treat-group was significantly reduced in week 8(P<0.01). Compared with the Qing’e Fang-treat-group, the bone biological force of the Gushukang-treat-group showed no statistical significance (P> 0.05).4. Results of MMP-9、Cath-K、TRACP5b:Concentrations of serum MMP-9、 Cath-K、TRACP5b of the Saline-treat-group were increased at first and then decreased.Compared with the Sham-group,greater increase were shown after treating for 4,8 and 12 weeks(P<0.01 or P<0.05). Numerically, the serum of Qing’e Fang-treat-group was significantly reducd compared with the Saline-group, and they had statistical significance except the MMP-9 at 4 weeks and TRACP5b at 12 weeks (P<0.01 or P< 0.05).5. Results of DPD/Cr:After operation, the DPD/Cr of the Saline-treat-group were gradually increased.The two treatment group were increased at first and then decreased. Numerically, the two treatment group were lower than the Saline group after treatment in 4,8 and 12 weeks(P<0.01 or P<0.05), There was no statistical significance between the two groups(P>0.05).6. Results of OPN、BSP、Itgαv、Itgβ3 mRNA and protein in bone tissue:Twelve weeks after operation, compared with the Sham-group,the results of the Saline-group were significantly increased(P<0.01). After treatment, the results of two treatment groups were significantly lower than the Saline-treat-group(P<0.01).The results of two treatment groups were significantly higer than the Sham-group(P<0.01).7. Results of p38mRNA and protein in bone tissue:Ovariectomy, three sampling time point of the saline group, bone tissue p38mRNA and protein content were significantly higher than that in sham group (P<0.01),the two drug group bone tissue p38mRNA and protein content decreased after treatment, p38mRNA in bone tissue of the Qing’e Fang-treat-group compared with the saline-treat-group were significantly increased after treating for 8 and 12weeks(P<0.01),while p38 protein in bone tissue of the Qing’e Fang-treat-group compared with the saline-treat-group were significantly increased after treament 12 weeks(P<0.01). The two treatment group had no statistical significance(P> 0.05).Conclusion:1.Ovariectomy promotes the expression of OPN、BSP、Itgαv,Itgβ3 and interaction and enhance the function of osteoclast, which leads to the occurrence of osteoporosis2. Qing’e Fang can inhibit osteoclastic bone resorption by reducing the expression of bone tissue OPN, BSP and its receptor Itgαv,Itgβ3.Qing’e Fang plays a role in the prevention and treatment of postmenopausalosteoporosis. |
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