| ObjectiveTreated the ovariectomized osteoporosis model rats and culture osteoclast by Qing’e Fang, to investigate the effect of Qing’e Fang on bone tissueMMP-2、9、13、OPG、RANKL in ovariectomized osteoporosis model rats and inducing osteoclast(OC) formation and bone resorption in vitro. So as to investigate the mechanism of Qing’e Fang on effective prevention and treatment of PMOP from bone resorption and provide the experimental data for clinical medicine.Methods(一) Experiments In Vivo1. Selecting、 Grouping、Modeling、Treatment and Drawn:Selected the3months of healthy and level clean fem-ale SD rats140, randomized into2groups:the OVX group100and the Sham group40. The OVX group was removed the ovary. The Sham group was not removed the ovary.12weeks after operation,10rats were randomly chosen from each group, and were killed.OVX model was proved by Serum E2level, BMD and bone morphologic indicators. The90rats left in model group were randomly divided into3groups:Qing’e Fang-treat-group (30rats), E2-treat-group(30rats), Saline-treat-group(30rats).13weeks after operation, the rats of the Qing’e Fang-treat-group were treated with Qing’e Fang2.15mg/kg/d, the rats of Sham-operation-group and Saline-treat-group took NS2ml/d, and the rats of E2-treat-group took E20.05mg/kg/d.10rats were randomly chosen, in each group, to be killed on4,8,12weeks after treatment. Taking ventral aortic blood after the anesthesia take effect, Seruming-20℃refrigerator; Separating of the shin、femur and lumbar, Seruming-80℃refrigerator.2. Indexes Detection:Bone mineral densities (BMD) were measured by dual-energy X-ray absorptiometry (DXA) for the femur. The bone tissue form of the shin were measured by Motic Med6.0digital medical image analysis system. Bone biological force of the femur were measured by IG-A1000N universal material testing machine; Concentrations of serum E2were measured by ELISA. Vigour of serum TRACP were measured by biochemistry; The expressions of MMP-2、9、13、OPG、RANKLmRNA in bone tissue were measured by quantitative PCR (Q-PCR).The content of MMP-2、9、13、 OPG、RANKL protein in bone tissue were measured by Western Bloting.(二) Experiments In Vitro1. Preparation for Qing’e Fang rat serum:20male SD rats (3-month-old) were randomly divided into2groups:Qing’e Fang-group and NS-group,10rats in each group. The rats of Qing’e Fang-group were perfused with Qing’e Fang2.15g/kg/d, and the NS-group with normal saline2m1/d.The serums containing Qing’e Fang or NS were extracted after7days.2. Indexes Detection2.1The effect of Qing’e Fang rat serum on inducing RAW264.7formation:RAW264.7was induced by RANKL(30ng/ml)and M-CSF(10ng/ml).At the same time, different concentratio-n (10%、15%、20%) Qing’e Fang rat serum and NS rat serum(10%) were added. The form of live cells were observed by light microscope; The number of osteoclast (OC) were counted at2d、4d、6d; Vigour of serum TRACP were measured by biochem istry for cell culture fluid at2d、4d、6d; The exp-ressions of PU.1、Mitf、NFATcl mRNA in cells were measured by Q-PCR at2d、4d、6d.2.2The effect of Qing’e Fang rat serum on osteoclnstic bone resorption in vitro:RAW26-4.7was induced by RANKL(30ng/ml)and M-CSF(10ng/ml) for7days. Osteoclasts were identif-ied by TRACP staining.Then different concentration (10%、15%、20%) Qing’e Fang rat serum and NS rat serum(10%) were added. The form of live cells were observed by light microscope; The number of bone resorption pits on bone slices were counted at24h、48h、72h; Vigour of serum TRACP were measured by biochemistry for cell culture fluid at24h、48h、72h; The expressions of TRACP、RANK、MMP-9mRNA in cells were meas ured by Q-PCR at24h、48h、72h.Results(一) Experiments in Vivo(During the treatment and drawn,the fifteen experimental rats died.)1. OVX model Identification:12weeks after operation, Compared with the sham-group, BMD and Concentrations of serum E2of the rats in the model-group decreased significantly (P<0.01). In the model-group, trabecular of the upper tibia were thinner and fewer than that in sham-group, The difference was obviously (P<0.01).2. Results of BMD:After treatment, compared with the Saline-treat-group, the BMD of the Qing’e Fang-treat-group and the E2-treat-group were significantly increased after treament4、8and12weeks (P<0.01or P<0.05). Compared with the Sham-group, they were reduced after treatment4、8and12weeks, but they were no statistical significance (P>0.05). As time going on, excepting the Saline-treat-group, the BMD of the left groups were increasing.3. Bone tissue form analysis and Results of area percentage of trabecular:The trabecular bone of the sham-group was distributed evenly, arranged regularly, and the three batches was roughly the same. The trabecular bone of the saline-group was sparsely、uneven thickness,the structural integrity was poor after operation12weeks. Compared with the saline-group, the trabecular bone of the two treatment groups were enrich,the number increased, arranged regularly, the structural integrity was good. After treatment, compared with the Saline-treat-group, the area percentage of trabecular of the Qing,e Fang-treat-group and the E2-treat-group were significantly increased after treament4、8and12weeks (P<0.01).Compared with the Sham-group, they were reduced after treament4、8and12weeks. The area percentage of trabecular of the E2-treat-group were higherr than that in the Qing’e Fang-treat-group,and at treament12weeks, they had statistical significance (P<0.05).4. Results of bone biological force:12weeks after operation, compared with the Sham-group, bone biological force of the model-group was significantly reduced (P<0.01). After treatment, compared with the Saline-treat-group, bone biological force of the Qing’e Fang-treat-group and the E2-treat-group were significantly increased after treament4、8and12weeks (P<0.01or P <0.05). Compared with the Sham-group, bone biological force of the Qing’e Fang-treat-group was significantly increased after treament4、8and12weeks (P<0.01or P<0.05). The bone biological force of the E2-treat-group were higherr than that in the Qing’e Fang-treat-group,and at treament12weeks, they had statistical significance (P<0.01).5. Results of E2:After treatment, compared with the Saline-treat-group, Concentrations of serum E2of the Qing,e Fang-treat-group and the E2-treat-group were significantly increased after treament4、8and12weeks (P<0.01or P<0.05). Compared with the Sham-group, they were reduced after treament4、8and12weeks, but they were no statistical significance (P> 0.05). Concentrations of serum E2of the E2-treat-group were higherr than that in the Qing’e Fang-treat-group,and after treament8、12weeks, they had statistical significance (P<0.01).6. Results of TRACP:12weeks after operation, compared with the Sham-group, Vigour of serum TRACP of the model-group was significantly increased (P<0.01). After treatment, compared with the Saline-treat-group, bone biological force of the Qing’e Fang-treat-group and the E2-treat-group were reduced after treament4、8and12weeks. After treament4and8weeks, difference among them had statistical significance (P<0.01or P<0.05). Compared with the Sham-group, they were reduced after treament4、8and12weeks,but they were no statistical significance (P>0.05)7. Results of MMP-2、9、13、RANKLmRNA and protein in bone tissue:12weeks after operation, compared with the Sham-group, them of the model-group was significantly increased (P<0.01). After treatment4weeks, them of two treatment groups were significantly lower than the Saline-treat-group (P<0.01). Them of two treatment groups were significantly higer than the Sham-group (P<0.01). After treatment8weeks, them of two treatment groups were significantly lower than the Saline-treat-group (P<0.01). Them of two treatment groups were higer than the Sham-group. After treatment12weeks, them of two treatment groups were significantly lower than the Saline-treat-group (P<0.01).Except MMP-13mRNA and protein, the left indexes in the two treatment groups were significantly higer than the Sham-group (P<0.01)8. Results of OPGmRNA and protein in bone tissue:12weeks after operation, compared with the Sham-group, OPGmRNA and protein in bone tissue of the model-group was increased (P>0.05). After treatment, compared with the Saline-treat-group, OPGmRNA and protein in bone tissue of the Qing’e Fang-treat-group and the E2-treat-group were significantly increased after treament4、8and12weeks (P<0.01). Compared with the Sham-group, OPGmRNA and protein in bone tissue of the Qing’e Fang-treat-group and the E2-treat-group were significantly reduced after treament4、8and12weeks (P<0.01)(二) Experiments In VitroThe addition of Qing’e Fang rat serum (10%、15%、20%) induced a dose-dependent deer-eased in the number of OC and bone resorption pits, Vigour of serum TRACP of cell cult-ure fluid, PU.1、Mitf、NFATcl、TRACP、RANK、MMP-9mRNA of cells, compared with the NS(10%),the differences had Statistical significance (P<0.01or P<0.05) Conclusion:1. Qing’e Fang could significantly inhibit osteoclast bone absorption.2.The mechanism that Qing’e Fang significantly inhibited osteoclast bone absorption had close relationship to improve ovarian rats serum E2level, decrease bone tissue RANKL/OPG ratio and the expression of MMP-2,9,13, and reduce osteoclasts PU.l, Mitf, NFATcl, TRACP, RANK, MMP-9mRNA expression, reduce the combine of RANKL and RANK. |
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