Assessment of BMC and BMD on whole body, femora and LV in OVX-induced osteoporotic mice
BMC and BMD were measured using DXA to determine value of bone minerals on experimental mice in total body and 2 specific regions including femora and L4-L6 vertebrae. In terms of total body BMC and BMD, OVX-induced osteoporotic mice showed significant reduction compared to sham-operated mice. The body BMC values in OVX group (0.8001 ± 0.0250 g) was 0.156 g lower than sham group (0.956 ± 0.0250 g), while E2 (0.882 ± 0.0817 g) and SOS 0.01, 0.1 and 1 µM groups (0.887 ± 0.0871 g, 0.888 ± 0.0880 g and 0.889 ± 0.0891 g, respectively) were significantly higher than OVX group (Fig. 1A). The value of body BMD in sham group (0.030 ± 0.0006 g/cm2) was 0.006 g/cm2 higher than OVX group (0.025 ± 0.0008 g/cm2). Administration of E2 increased the body BMD level by 0.031 ± 0.0005 g/cm2. SOS at all concentrations up-regulated the body BMD levels to 0.029 ± 0.0045 g/cm2, 0.030 ± 0.0005 g/cm2 and 0.029 ± 0.0014 g/cm2, respectively, compared to OVX group (Fig. 1A).
In terms of femoral bone mineral level, the femoral BMC were 0.006 g reduced in OVX group (0.040 ± 0.0010 g) compared to sham group (0.046 ± 0.0005 g). Injection of E2 increased the 0.004 g BMC level (0.044 ± 0.0007 g). Administration of SOS with 0.01, 0.1 and 1 µM improved the BMC levels (0.0417 ± 0.0011 g, 0.043 ± 0.0004 g and 0.044 ± 0.0008 g), respectively (Fig. 1B). The levels of femoral BMD in sham group was 0.119 ± 0.0022 g/cm2, while that in OVX group was 0.107 ± 0.0024 g/cm2. E2 group showed recovery of femoral BMD level (0.123 ± 0.0025 g/cm2). In addition, SOS treatment at all concentrations increased the BMD levels (0.111 ± 0.0024 g/cm2, 0.122 ± 0.0019 g/cm2 and 0.121 ± 0.0027 g/cm2), respectively (Fig. 1B).
Additionally, OVX group exhibited a significant reduction of BMC level in L4-L6 vertebrae (0.025 ± 0.0019 g) compared to sham group (0.038 ± 0.0024 g). Injection of E2 significantly increased the BMC level of LV region (0.038 ± 0.0038 g). Administration of SOS at all concentrations markedly improved the value of BMC by 0.031 ± 0.0014 g, 0.033 ± 0.0017g and 0.0292 ± 0.0004 g, respectively (Fig. 1C). The BMD level of LV in sham group was 0.076 ± 0.0009 g/cm2 and that in OVX group was 0.055 ± 0.0008 g/cm2. E2 treatment significantly increased by 49.11% the value of BMD in LV (0.076 ± 0.0057 g/cm2). The BMD levels in L4-L6 vertebrae in SOS-treated groups were 0.058 ± 0.0016 g/cm2, 0.067 ± 0.0009 g/cm2 and 0.067 ± 0.0007 g/cm2, showing 6.17%, 21.05% and 22.45% recoveries, respectively, compared to OVX group (Fig. 1C).
Assessment of histomorphometrical changes in OVX-induced mice
H&E staining revealed well-formed bone marrow with a little bone marrow adiposity in normal femoral bone. In OVX-induced osteoporotic mice, bone marrow adipose tissues were markedly increased at the medullary cavity of femoral shaft. Treatment with 1 µM SOS apparently improved the increase of bone marrow fat in OVX mice (Fig. 2).
Analysis of BMP-2 expression by RT-PCR in OVX-induced mice
BMP-2 attributes to regulation of osteogenic differentiation and bone formation. As illustrated in Fig. 3A, expression mRNA level of BMP-2 was sharply declined in OVX group by 41.65% compared with sham group. On the contrary, E2 treatment slightly increased BMP-2 mRNA level compared to OVX group. mRNA levels of BMP-2 in SOS-treated groups, especially SOS SOS 1 µM group were elevated by 70.68% compared to OVX group (Fig. 3A).
Assessment of RUNX2 expression by RT-PCR in OVX-induced osteoporotic mice
Expression mRNA level of RUNX2, promoted by BMP-2, was decreased in OVX group by 61.15 ± 15.49% compared to sham group. On the other hand, E2 treatment showed a significant increase about 62% on expression mRNA level of RUNX2 compared with OVX group. Also, mRNA level of RUNX2 was recovered from SOS 0.01, 1 µM treatment by 56.70 ± 16.07% and 108.78 ± 11.23% compared to OVX group (Fig. 3B).
Assessment of bone specific matrix genes by RT-PCR in OVX-induced osteoporotic mice
Bone specific matrix genes, ALP, OPN and BSP-1, were known as turnover and maturation of osteo-relative cells 25. Expression mRNA levels of ALP and OPN were promoted by injection of E2 (68.98 ± 6.31% and 49.7 ± 36.69%) contrary to OVX group. Compared with OVX group, particularly SOS 1 µM treatment improved mRNA levels of ALP and OPN (112.03 ± 9.11% and 71.51 ± 4.53%), approaching approximate levels of sham group. In comparison with OVX group, gene expression mRNA levels of BSP-1 were increased in equal levels with sham group in E2 (72.26 ± 19.00%) and SOS-treated group (95.88 ± 14.8%, 98.62 ± 4.63% and 105.78 ± 5.22%, respectively) (Fig. 3C).
Assessment of osteoblastogenesis in AA + β-GP-induced SaOS-2 cells
SaOS-2 cells were differentiated with AA and β-GP and stained with ARS to identify extracellular calcium deposition 26. By contrast with non-treated group, AA + β-GP-induced group were dramatically increased in calcium deposition by 10.5 folds. In addition, SOS co-treated with AA + β-GP group were highly differentiated by 1.44%, 2.57% and 4.11%, respectively in dose-dependent manner compared to single treated with AA + β-GP group (Fig. 4A, 4B). The effect of SOS on SaOS-2 cells viability were determined by MTT assay. After treatment with concentrations of SOS 1, 10, 100 nM for 24h, there was no significant difference in all concentrations of SOS on SaOS-2 cells (Fig. 4C).
Assessment of BMP-2 and RUNX2 expression by RT-PCR in AA + β-GP-induced SaOS-2 cells
To demonstrate the effects of SOS on BMP-2 and RUNX2, which are facilitate osteoblastogenesis through differentiation of osteoblast cells, mRNA expression levels were analyzed by RT-PCR. As showed in Fig. 5A, expression mRNA level of BMP-2 was increased by 56.11% in differentiated with AA + β-GP group compared with non-differentiated group. SOS co-treated with AA + β-GP groups were considerably elevated by 1.6 folds, 1.2 folds and 2 folds, respectively compared to AA + β-GP group. Also, expression mRNA level of RUNX2 was increased by 79.73% in AA + β-GP-treated group compared with non-treated group. Especially, SOS 100 nM treatment was markedly increased mRNA level of RUNX2 about 117% compared to AA + β-GP group (Fig. 5B).
Assessment of bone specific matrix genes by RT-PCR in AA + β-GP-induced SaOS-2 cells
To evaluate the effects of SOS on ALP, OPN and BSP-1, which are key factors of bone turnover and maturation were measured by RT-PCR. Expression mRNA levels of ALP, OPN and BSP-1 were increased by 52.05%, 56.78% and 81.1%, respectively in differentiation of SaOS-2 cells treated with AA + β-GP. The three of bone specific genes, SOS treatment prominently improved expression mRNA levels of ALP, OPN and BSP-1 in dose-dependent manner. In particular, SOS 100 nM has remarkable effects on mRNA levels of ALP, OPN and BSP-1 that were increased by 7.7 folds, 4.3 folds and 3.8 folds, respectively (Fig. 5C).