Effects Of High-fat Diet And Sex Hormone Deficiency On Bone Volume And BMMSCs’ Multilineage Differentiation In Mice

Objective:To study the synergistic effects of high-fat diet (HFD) and gonadectomy on osteoporosis and multilineage differentiation potential of bone marrow mesenchymal stem cells (BMMSCs) in mice, so as to investigate the influence of HFD-induced obesity on the development of gonadectomy-induced osteoporosis.Methods:488-week female and male C57mice were given gonadectomy to build osteoporotic models. Half of these mice were then equally divided into4experimental groups according to the diets, sex and treatments, named OVX-HF (ovariectomy-operated mice fed a HF diet), the OVX-LF (ovariectomy-operated mice fed a LF diet), ORX-HF (orchiectomy-operated mice fed a HF diet), ORX-LF (orchiectomy-operated mice fed a LF diet). The other half, which accepted SHAM operation, were divided into4control groups as above. Body weight of each mouse was measured twice a week, and blood glucose concentration was detected once a week. The mice were sacrificed after2week. One femur of each mouse was fixed, decalcified, embedded in paraffin, and prepared for HE staining for trabecular bone area measurement. The other femur, together with tibias and humerus, was applied for culture of BMMSCs. Multilineage differentiation tests were fulfilled with BMMSCs of passage3. BMMSCs were stained for oil red after7-day adipogenic differentiation. RNA and total protein were extracted in the same day, and they were prepared for the measurements of levels of adipogenic genes PPAR-γ and LPL with RT-PCR and Western blot. BMMSCs were stained for ALP and alizarin red after7-day and21-day osteogenic differentiation, respectively. RNA and total protein were extracted in the same day alizarin red staining was executed, and they were prepared for the measurements of levels of osteogenic genes Runx2and Sp7with RT-PCR and Western blot. All the data were tested for normal distribution and homogeneity test of variance, and analyzed with the SPSS20.0software. Results:1. In2months after gonadectomy, the body weights of mice were influenced by the triple effects of sex, operation and diet. The increase of body weight was faster in OVX group than in SHAM group (P<0.05), while lower in ORX group than in SHAM group (P<0.05). The increase of body weight was also faster in HF group than in LF group (P<0.05). The differences above were all significantly.2. In3months after gonadectomy, the blood glucose concentrations of mice were not influenced. No significant differences were detected between each of the gonadectomy and SHAM groups, and HF and LF groups (P>0.05).3. HE staining of femur showed that the trabecular bone area was significantly smaller in gonadectomy groups than in the SHAM groups (P<0.05). No significant differences were detected between HF and LF groups (P>0.05).4. Oil red staining showed that BMMSCs of each group presented some adipogenic differentiation capability with lipid droplets formation after7-day adipogenic induction. The adipogenic differentiation capability was significantly higher in gonadectomy groups than in the SHAM groups (P<0.05). The adipogenic differentiation capability was significantly lower in HF groups than in the LF groups (P<0.05).5. The tests on adipogenic genes showed that after7-day adipogenic induction, the RNA levels of PPAR-γ and LPL were significantly higher in gonadectomy groups than in the SHAM groups (P<0.05), consistently with the results of total protein and adipogenic staining, the RNA levels of PPAR-γ and LPL were significantly lower in HE groups than in the LF groups (P<0.05), consistently with the results of total protein and adipogenic staining.6. ALP staining showed that BMMSCs of each group presented ALP activity after7-day osteogenic induction. The ALP activity was significantly lower in gonadectomy groups than in the SHAM groups (P<0.05). No significant differences were detected between HF and LF groups (P>0.05).7. Alizarin red staining showed that BMMSCs of each group formed mineralized nodules after21days osteogenic induction. The mineralization was significantly lower in gonadectomy groups than in the SHAM groups (P<0.05). No significant differences were detected between HF and LF groups (P>0.05).8. The tests on osteogenic genes showed that after21-day osteogenic induction, the RNA levels of Runx2and Sp7were significantly lower in gonadectomy groups than in the SHAM groups (P<0.05), consistently with the results of total protein and osteogenic staining. No significant differences were detected between HF and LF groups (P>0.05).Conclusion:Sex hormone deficiency can induce bone loss in mice, with decrease of osteogenic differentiation capability and increase of adipogenic differentiation capability of BMMSCs. This might be an important reason in the development of osteoporosis. The obese tendency induced by high-fat diet exerted no significant influences on the sex hormone deficiency-induced osteoporosis and changes of multilineage differentiation potential of BMMSCs in mice.