| Purpose To study the expression of vertebral Sclerostin, Wnt/β-catenin pathway and the bone mass in rats after ovariectomized and estrogen or PTH treatment; to investigate the intervertebral disc degeneration in rats after ovariectomized and estrogen or PTH treatment, as well as the Wnt/β-catenin pathway changes during the process. To comprehensive evaluate the changes of Wnt/β-catenin pathway in spine after ovariectomized and estrogen or PTH treatment.Methods The first part: Forty three-month-old female SD rats were ovariectomized and divided into four groups. Another 10 age-matched rats received sham operations as controls. Eight weeks later, the rats were administered the estrogen or PTH intervention for 12 weeks. After the lumbar BMD were measured, the rats were sacrificed and lumbar spine and blood were collected for q PCR, immunohistochemistry, Western blot and other tests. The second part: Thirty three-month-old female SD rats were ovariectomized and divided into three groups. Another ten age-matched rats received sham operations as controls. Eight weeks later, the rats were administered the estrogen or PTH intervention for 12 weeks. Then the rats were sacrificed, and discs and vertebras were collected for q PCR, Western blot, immunohistochemistry, HE staining and other tests.Results Part I:(1) After ovariectomized, the vertebral MEF2 s and SOST m RNA expression in OVX group increased, and estrogen treatment can decrease their expression(p <0.05). The expression of MEF2 s and SOST m RNA were lower in OVX+E2+PTH group than OVX+PTH group(p <0.05).(2) After ovariectomized, numbers of Sclerostin-positive osteocytes in vertebrae increased, and estrogen treatment can decrease the numbers of Sclerostin-positive osteocytes. Numbers of Sclerostin-positive osteocytes in OVX+E2+PTH group were similar with OVX+PTH group. The Western blot results of Sclerostin were consistent with PCR and IHC results.(3) The vertebral osteocyte density of Sham group was 426.83±68.14 #/mm2, and in OVX group it decreased to 293.53±53.24 #/mm2, after estrogen treatment the osteocyte density increased to 377.30±48.22 #/mm2(p <0.05). In OVX+E2+PTH group, the osteocyte density was 451.44±55.57 #/mm2, slightly lower than theOVX+PTH group(463.81±64.92 #/mm2), but the difference was not statistically significant.(4) After ovariectomized, numbers of β-catenin-positive osteoblasts in vertebrae decreased, and estrogen treatment can increase the numbers of β-catenin-positive osteoblasts. Numbers of β-catenin-positive osteoblasts in OVX+E2+PTH group were slightly higher than OVX+PTH group. The Western blot results of nuclear β-catenin were consistent with PCR and IHC results, and β-catenin concentration of OVX+E2+PTH group was slightly higher than the OVX+PTH group.(5) Compared with sham controls, OVX rats had lower vertebral BV/TV, Tb.Th, Tb.N and Conn.Dn, and higher BS/BV and Tb.Sp(p < 0.05). The estrogen treatment restored the BS/BV, Tb.Th, Tb.Sp and Conn.Dn to the level of sham controls(p > 0.05). The differences between OVX+E2+PTH group and OVX+PTH group were not statistically significant(p > 0.05).(6) Compared with Sham group, the biomechanical properties of rat vertebrae in OVX group reduced for about 30%(p < 0.05), estrogen treatment improved the biomechanical properties, but did not return to the Sham group level. Compared with the OVX+PTH group, vertebral biomechanical properties increased in OVX+E2+PTH group, but the difference was not statistically significant.(7) Compared with the Sham group(0.1748±0.01056 g/cm2), lumbar BMD of OVX rats decreased to 0.1419±0.01068 g/cm2(p < 0.05), and BMD of OVX+E2 group increased to 0.1596±0.01396 g/cm2(p < 0.05). The lumbar BMD of OVX+E2+PTH group was a little higher than OVX+PTH group, but the difference was not statistically significant.(8) Compared with Sham group, OVX group has significantly higher serum P1 NP and CTX-1(p < 0.05), and estrogen treatment may decrease them, but the difference between OVX group and OVX+E2 group was not statistically significant. Part II:(1) in Sham group, cells in nucleus pulposus were mostly the notochord cells, while in OVX group the notochord cells were gradually replaced by chondrocyte-like cells and fibrocartilaginous tissue in annulus fibrosus proliferated. Estrogen or PTH treatment can partly recover the number of notochord cells and decrease chondrocyte-like cells, as well as improve the station of annulus fibrosus and finally improve the disc degeneration.(2) After ovariectomized, the rats’ endplate got roughened and irregularities emergence in the margin, as well as the endplate porosity decreased. After the estrogen or PTH treatment, smoothness ofendplate got recovered and endplate porosity increased.(3) Compared with the Sham group, expression of Aggrecan and Col2a1 m RNA in OVX group decreased, while Col1a1, MMP-3 and MMP-9 m RNA expression increased. Estrogen or PTH treatment can increase the Aggrecan, Col2a1 m RNA expression and decrease Col1a1, MMP-3 and MMP-9 m RNA expression. The IHC staining results of Aggrecan, type II collagen and MMP-3 were consistent with PCR results.(4) Compared with Sham group, the expression of β-catenin, c-myc and cyclin D1 m RNA in disc of OVX group reduced, indicating that the Wnt/β-catenin pathway down-regulated after rats were ovariectomized, and either estrogen or PTH treatment can increase their expression(p < 0.05). The immunohistochemical staining results showed that β-catenin staining could be observed in nucleus pulposus cells in Sham group, while the staining intensity of β-catenin was significantly lower in OVX group. Compared with the OVX group, staining intensity of β-catenin in OVX+E2 group and OVX+PTH group both increased. Western-Blot results of nucleus β-catenin were consistent with IHC staining results.Conclusions(1) Estrogen appears to be a regulator of Sclerostin, and the effect may involve suppressing MEF2 s. Estrogen and PTH can increase vertebral Wnt/β-catenin pathway activity, and estrogen in combination with PTH has a stronger effect on Wnt/β-catenin pathway than PTH applied alone. Combined treatment with PTH and estrogen is not more beneficial for vertebral bone mass and strength than PTH applied alone in ovariectomized rats.(2) After the rats were ovariectomized, the notochord cells decreased and the chondrocyte-like cells emerged, the endplate porosity decreased, the Aggrecan and type II collagen content decreased, with the disc degeneration occurred and Wnt/β-catenin pathway activity decreased. Estrogen and PTH treatment can increase notochord cells and endplate porosity, improve Aggrecan and type II collagen content, thus improving the disc degeneration, and the Wnt/β-catenin pathway activity increased in the process. |
PDF Full Text Request