| 1.ObjectiveThe main objective of this research was to specify the concern between osteoporosis of ovariectomized SD rats and the OPG/RANKL/RANK system,and discover the mechanism of Er-Zhi-Wan defending the postmenopausal osteoporosis from the perspective of the OPG/RANKL/RANK system.2.Methods2.1 Experimental animal group,building model,model identification,drug intervention and drawing materials The amount of six months old healthy SPF female rats was 170,respectively.After feeding 3 days,the female rats were randomly devided into two groups:the ovariectomized group(n=130)and the sham operation group(n=40).All the experimental rats were anesthetized by inTRACPeritoneal injection of 2%sodium pentobarbital(40mg/kg),were taken the middle abdominal incision.All the experimental rats underwent bilateral ovariectomy,excepte the sham operation group,the rest of steps were same in both groups.After 12 weeks of the surgery,10 rats of each group were randomly selected to do the model identification.The right hind proximal tibia of each rats were done the detection of bone mineral density and the right uterus was taken to do the detection of uterine endometrium thickness after anesthesia to identify the establishment of the model.After success of the model,the ovariectomized group was devided into 4 groups:the model group,the low-dose Er-Zhi-Wan group,the moderate-dose Er-Zhi-Wan group,and the high-dose Er-Zhi-Wan group,with 30 rats in each group.From the 13th week,the low-dose Er-Zhi-Wan group was given 6g/(kg.d)Er-Zhi-Wan lavage,which was dissolved by 2ml normal saline.The moderate-dose Er-Zhi-Wan group was given 9g/(kg.d)Er-Zhi-Wan lavage,which was dissolved by 2ml normal saline.The high-dose Er-Zhi-Wan group was given 12g/(kg.d)Er-Zhi-Wan lavage.which was dissolved by.The model group and the ovariectomized group were both given 2ml normal saline,1 time per day,12 weeks of dose.All the experimental rats were raised and drank water freely in the same conditions.After lavage,In the 16th,20th,24th week,ten rans were selected randomly from each group,the blood was drawn from abdominal aorta and putted into EP tubes respectively and-80 ℃ cryopreserved for measurement.The tibia and the lumbar spine were drawn,meanwhile,and-80℃ cryopreserved for measurement.2.2 Indexes detectionThe purity of OPG、RANKL、TRACP and BGP in the serum was determined by the experimental method of ELISA.The tibia,the lumbar spine and the uterus were made paraffin setions and HE staining,the bone trabecula area of them were measured by Motic Med 6.0 digital medical image analysis system.BMD of the rats was determined by Dual energy X ray bone sonometers.Biomechanical testing of the right tibia was performed through the Three-point bending experiment.The protein and gene expression levels of OPG、RANKL、TRACP and BGP of tibia and the lumbar spine were determined by Western blot and Real time PCR.3.Results3.1 After oophorectomy,compared with the sham operation group,the bone mineral density,the bone morphology and the bone biomechanical indexes of the model group lasted to go down from 16th week and 24th week(p<0.05).The protein and gene expression levels of RANKL、TRACP and BGP in the serum and the lumbar spine are both increasing(p<0.05),but the OPG are decreasing at the same time(p<0.05).As the time gone by,the amplification of the RANKL、TRACP and BGP was slowing down,the decreases in the OPG was also slowing down.3.2 Compared with the model group,the Er-Zhi-Wan could elevate the bone mineral density and the bone trabecula area and the bone biomechanical properties(p<0.05).The protein and gene expression levels of OPG in the serum and the lumbar spine are both increasing(p<0.05),but the RANKL、TRACP and BGP are decreasing at the same time(p<0.05).As the medication time gone by and the increase of drug concentration,the protein and gene expression levels of OPG in the serum and the lumbar spine are both increasing,and the RANKL、TRACP and BGP are decreasing at the same time4.ConclusionDuring the dose range of rats corresponding to the safe dose range of the human:4.1 Er-Zhi-Wan could lift the bone mineral density and the bone trabecula area and the bone biomechanical properties of osteoporosis of ovariectomized model rats.4.2 Er-Zhi-Wan could regulate the abnormal expression of the protein and gene expression levels of OPG、BGP、RANKL and TRACP in the serum and the lumbar spine.Er-Zhi-Wan could promote the protein and gene expression level of OPG in the serum and the lumbar spine,improve the function of osteoblasts and bone formation,bring down the protein and gene expression level of RANKL in the serum and the lumbar spine and the function of osteoclast and the bone absorption,debase the protein and gene expression level of TRACP and BGP in the serum and the lumbar spine and bone metabolic activity.Er-Zhi-Wan can achieve the purpose of prevention and cure of the ovariectomized osteoporosis. |
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