The Study Of Metoprolol On The Treatment Effect And Mechanism For Postmenopausal Osteoporosis

Both osteoporosis and hypertension are age-related chronic diseases. The rising morbidities of these two diseases have been detected among the postmenopausal women. The severe complications, which are induced by either osteoporosis or hypertension, are proved to pose threats to the health of the postmenopausal women, to result in rather high mortalities and disabilities, and then to create an enormous social and economic burden.Selective β1 adrenergic receptor blockers are recommended as the first-line treatment of hypertension for its advantage on lowering blood pressure with few side-effects. Clinical epidemiological investigations demonstrate that hypertensive patients, who are treated with selective β1 adrenergic receptor blockers, have higher bone mineral density and lower fracture risks. The same effects emerge in the postmenopausal women, and there is no deeply related study on these effects, thus it is of great importance to investigate the effect of metoprolol on the treatment of the postmenopausal osteoporosis.ObjectiveIn order to explore new method for postmenopausal osteoporosis treatment, this study is to invstigate the effects and mechanisms of metoprolol on osteoporosis by treating the osteoblasts and osteoclasts with metoprolol in vitro, as well as OVX-induced osteoporosis in vivo.Methods1. The study of metoprolol on the treatment effect and mechanism for osteoblasts.Cells were obtained from calvaria of new-born SD rats by enzymes digesting and tissue block method. The third generation of osteoblasts was divided into 4 groups, and then incubated with the regular culture medium, which contained different concentrations of metoprolol(0, 0.001, 0.01, and 0.1 μM).(1) Cell proliferation assessment. The MTT assays were used to assess the effect of metoprolol on osteoblast proliferation on the 1st, 4th and 7th days.(2) Cell differentiation assessment. ALP staining and activity assays were used to assess the effect of metoprolol on osteoblast differentiation on the 1st, 4th and 7th days. Alizarin red S staining and quantitative assays of calcium nodules were used to assess the effect of metoprolol on osteoblast mineralization on the 22 nd, 25 th and 28 th days. Moreover, the expression of Collagen 1(Col1a1), ALP, Osteopontin(OPN) and Osteocalcin(OCN) m RNAs were tested by quantitative real-time PCR on the 3rd, 6th and 9th days.2. The study on effect of metoprolol on the formation and TRAP activity of osteoclasts.RAW 264.7(osteoclasts precursor cell line) were induced to osteoclasts by s RANKL. The osteoclasts were then identified by the TRAP staining and bone resorption lacuna. The RAW 264.7 cells were divided into 4 groups, and then incubated with the culture medium, which contained s RANKL and different concentrations of metoprolol(0, 0.001, 0.01, and 0.1 μM).(1) Cell proliferation assessment. The MTT assays were used to assess the effect of metoprolol on the RAW 264.7 cells proliferation on the 3rd, 5th and 7th days.(2) TRAP activity assessment. TRAP activity assay was used to assess the effect of metoprolol on TRAP activity of osteoclasts on the 5th day.3. The study of metoprolol on the treatment effect for OVX rats.32 female SD rats were randomly assigned into sham-operated group(Sham) and three OVX subgroups, i.e. OVX with vehicle(OVX); and OVX with metoprolol of graded doses(150 or 250 mg/kg/day). There were 8 rats in each group. 4 weeks after surgery, daily oral administration started. And this treatment lasted for 12 weeks. Bone mass and bone strength of L4 vertebrae were evaluated by DXA and compression test respectively. Also histological examination of L5 vertebrae by HE staining and Micro-CT scanning of the distal right femur were performed.Results1. The study of metoprolol on the treatment effect and mechanism for osteoblasts.(1) Cell proliferation assessment. The OD values increased with time in each group. At Day 1, the OD value of the group treated with 0.01 μM metoprolol was statistically higher(P < 0.05) than the control group. After culturing for 4 days and 7 days, the OD values of 0.01-μM- and 0.1-μM-groups were significantly higher than that of control group. The results indicated that the proliferation of osteoblasts was promoted by the treatment of metoprolol at the concentrations of 0.01 μM and 0.1 μM.(2) Cell differentiation assessment. The level of cell differentiation was increased over time. At Day 1 and Day 4, ALP staining and activity showed a significantly increased differentiation level in the 0.1-μM- group, and the quantitative increment of ALP activity was statistically significant compared with the control group. These indicators were significantly higher in both of the 0.01-μM- and 0.1-μM-groups than the control group at Day 7. Similar results were detected with Alizarin Red S staining. And at Day 28, calcium deposition of the groups which were treated with metoprolol significantly increased than the control group. Additionally, real-time PCR analysis revealed that the expression levels of osteoblast-specific genes, including Col1a1, ALP, OPN and OCN, were significantly elevated in the 0.01-μM- and 0.1-μM-groups. These results, taken together, suggested a positive effect of metoprolol, at the concentrations of 0.01 μM and 0.1 μM, on the differentiation and mineralization of osteoblasts by a high level of osteoblast-specific genes expression.2. The study on effect of metoprolol on the formation and TRAP activity of osteoclasts.(1) Cell proliferation assessment. In comparation with the control, the OD values were significantly lower by the treatment of 0.1 μM metoprolol on the 3rd, 5th and 7th days(P < 0.05), and by the treatment of 0.01 μM metoprolol on the 5th and 7th days(P < 0.05), which indicated that RAW 264.7 cells proliferation could be inhibited by metoprolol at the concentrations of 0.01 μM and 0.1 μM.(2) TRAP activity assessment. TRAP activity was significantly inhibited by the treatment of metoprolol at the concentrations of 0.01 μM and 0.1 μM at Day 5, which represented a negative effect of metoprolol on osteoclast activity.3. The study of metoprolol on the treatment effect for OVX rats.Metoprolol prevented BMD decrease in the L4 vertebrae induced by OVX(P < 0.05). Higher dosage of metoprolol treatment altered Ultimate load, stiffness and energy absorption(P < 0.05), and significantly alleviated the trabecular bone microarchitecture deterioration of the distal right femur(P < 0.05). And the histological examination of L5 vertebrae which were treated with metoprolol showed a superior microarchitecture of trabecular bone.ConclusionThe present study demonstrated that the oral administration of metoprolol could inhibit the trabecular microarchitecture deterioration and maintain the bone biomechanical competence, thus prevent estrogen deficiency-induced bone loss. These beneficial effects are probably based on promoting the amount and function of osteoblasts by the high expression of osteoblast-specific genes, as well as inhibiting the formation and activity of osteoclasts. Our findings indicate that metoprolol has the potential to develop as an alternative for postmenopausal osteoporosis management of hypertension patients, which may substantially improve current osteoporosis treatment.