| ObjectiveTo observe the effective of Wubisanyao pill (WBSY) in ovariectomized osteoporosis rats (OVX), bone histomorphometry is used for evaluation, then from the viewpoint of bone formation negative regulator, aiming at osteopontin and adrenergic receptor β-2protein, to show its mechanism, and offer further scientific evidences for Chinese medicine in treating osteoporosis.Methods77female SD rats were randomly divided into three groups:blank control group (n=12), the sham group (n=12), OVX group (n=53). Model rats were anesthetized with sodium pentobarbital, then both ovaries were removed, sham rats only removed only a few fat tissue around ovaries.15days after operation,53OVX rats were further randomly divded into four groups:model group (n=13), raloxifene group (n=13), WBSY in high dose (n=14), WBSY in low dose (n=13). Then, drugs were administrated in o.p, raloxifene group,5mg/kg raloxifene in body weight; WBSY in high dose (n=14), with a dose of20g/kg WBSY crude drug; WBSY in low dose (n=14), with a dose of10g/kg WBSY crude drug. Drugs were administrated for6days in every week, and with1day no administration, the total duration was3months. Other model rat, sham rats, and control rats were administrated with pure water in the same volume.Respectively,15days and3days before the rats were sacrificed,30mg/kg tetracycline hydrochloride were intraperitoneal injected twice, to make fluorescent label in the bone. After last time of drug administration, intraperitoneal injection of45mg/kg sodium pentobarbital for anesthesia, the rats were decapitated, then the right tibia were dissected, and fixed with4%paraformaldehyde solution, then undecalcified bone slice were made for bone morphometry detection, TBV%TRS%, TFS, MAR, mAR and OSW and other index were recorded. Used left tibia to make frozen section, for analysis of osteopontin and adrenergic receptor β-2expression were detected in bone marrow.Statistical Methods:The obtained experimental data were expressed as mean±standard deviation (x±s), ANOVA analysis was applied, and LSD method was used to compare differences between groups.Results1The effect of WBSY on bone histomorphometry in ovariectomized rat tibia1.1The effect of WBSY on tibia TBV%in OVX ratsIn contrast with sham rats, OVX rats showed significant decrease in tibia TBV%. Compared with model rats, WBSY in high dose and low dose showed significant higher level in TBV%, there isn’t obvious difference in WBSY high dose and low dose. Raloxifene treated OVX rats showed much greater TBV%than model rats.1.2The effect of WBSY on tibia TRS%in OVX ratsCompared with sham rats, OVX rats showed obvious down-regulation in tibia TRS%; this indicated that bone resorption was enhanced in OVX rats. Compared with model rats, WBSY in high dose and low dose, as well as raloxifene treated rats showed significant decrease in TRS%.1.3The effect of WBSY on tibia TFS%in OVX ratsIn comparison with sham rats, OVX rats showed great increase in tibia TFS%. Compared with model rats, WBSY in high dose and low dose, as well as raloxifene treated rats showed decrease in TFS%.1.4The effect of WBSY on tibia MAR in OVX ratsOVX rats showed significant increase in tibia MAR than that in sham rats. Compared with model rats, WBSY in high dose and low dose, as well as raloxifene treated rats showed decrease in MAR.1.5The effect of WBSY on tibia mAR in OVX ratsIn contrast with sham rats, OVX rats showed obvious suppression in tibia mAR. Compared with model rats, WBSY in high dose showed decrease in mAR, while no obvious difference showed between WBSY in low dose and model rats. mAR in raloxifene was significant low than in model rats, but not obvious with sham rats.1.6The effect of WBSY on tibia OSW in OVX ratsIn comparison with sham rats, OVX rats showed significant increase in tibia OSW WBSY in high dose and low dose showed no obvious difference compared with model rats. OSW in raloxifene was significant low than in model rats, but not obvious with sham rats.2WBSY on osteopontin expression in bone marrow of OVX ratsOPN expression positive area in bone marrow of OVX rats was significant high than that of sham rats. Compared with OVX rats, OPN area of WBSY in high dose, in low dose and raloxifene were obvious low. Compared with sham rats, OPN area of WBSY in high dose, in low dose showed obvious increase and raloxifene show no obvious difference. OPN area in high dose of WBSY was great low than that in low dose.3WBSY on adrenergic receptor β-2protein expression in bone marrow of OVX ratsARP2expression positive area in bone marrow of OVX rats was significant high than that of sham rats. Compared with OVX rats, ARβ2area of WBSY in high dose, in low dose and raloxifene were obvious low. Compared with sham rats, ARβ2area of WBSY in low dose showed obvious increase; High dose of WBSY and raloxifene show no obvious difference.In all, compared with normal control, sham rats showed no difference in all these index, this eliminated the effect of ovariectomy operation Conclusion1. Three months after ovariectomy in rats, TBV%, a main parameter presented for bone volume, showed great reduce, while TRS%, parameter for bone formation, and TFS%, MAR, mAR as well as OSW, parameters for bone resorption, showed greatly increase, these indicated that ovariectomy in female rats could produce a type of osteoporosis characterized in high transformation rate with bone resorption greater than bone formation. Except high and low dose of WBSY in OSW and low dose in mAR, this formulae could make other listed indexes reversion in some extent. These showed WBSY could inhibit bone great rate of transformation, then produce some prevention in ovariectomical osteoporosis.2. Three months after ovariectomy, OPN and ARβ2protein expression in bone marrow of tibia showed obvious up-regulation, these indicated OPN and ARβ2took great role in osteoporosis development; And WBSY could inhibit this over-expression. This implied OPN and ARβ2might be possible target for WBSY to prevent osteoporosis development. |
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