Effects of aldosterone on osteoblast proliferation, differentiation and osteogenic gene expressions in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of aldosterone on cell proliferation, alkaline phosphatase (AKP) activity and osteogenic gene expression in rat osteoblasts and explore the mechanisms.</p><p><b>METHODS</b>Osteoblasts isolated from the skull of neonatal SD rats by enzyme digestion were cultured and treated with different concentrations of aldosterone. The cell proliferation and AKP activity were evaluated using CCK-8 assay kit and AKP assay kit, respectively. The effects of aldosterone on mRNA and protein expressions of the osteogenic genes and epithelial sodium channel (ENaC) gene were investigated using semi-quantitative PCR and Western blotting.</p><p><b>RESULTS</b>Compared with the control cells, the cells treated with 0.01-1.0 µmol/L aldosterone showed obviously enhanced proliferation while lower (1×10µmol/L) or higher (10 µmol/L) concentrations of aldosterone did not significantly affect the cell proliferation. Aldosterone within the concentration range of 1×10to 10 µmol/L did not cause significant changes in AKP activity in the osteoblasts. Treatment with 0.01 to 1.0 µmol/L aldosterone significantly upregulated the expressions of the osteogenic genes and α-ENaC gene at both the mRNA and protein levels.</p><p><b>CONCLUSION</b>Aldosterone within the concentration range of 0.01-1.0 µmol/L stimulates the proliferation and osteogenic gene expressions and enhances α-ENaC gene expression in rat osteoblasts in vitro, suggesting the possibility that ENaC participates in aldosterone-mediated regulation of osteoblast functions.</p>

Optimize aqueous extract protocol for Yugubao formula by orthogonal experiment design / 中国中药杂志

To determine the optimum aqueous extract protocol for Yugubao traditional Chinese medicines formula by using orthogonal experiment design. Through serum pharmacology research, L9(34) orthogonal design with single factor investigation was used to optimize the aqueous extract protocol for Yugubao formula. The effect of water extraction on activity of alkaline phosphatase (ALP) in osteoblast was referred as the evaluation index for investigating four factors: water consumption (A), heating time (B), soaking time (C), and number of decocting (D), analyzing the optimum extraction conditions, and verifying the effectiveness of this process. The optimum aqueous extract protocol for Yugubao was as follows: adding 8 times water into Chinese medical materials, heating for 60 min, soaking for 30 min, and decocting for 1 time. The drug serum of this aqueous extract of Yugubao could significantly up-regulate the osteogenic genes expression. The optimum aqueous extract protocol for Yugubao formula was established in this experiment, providing evidence for the development and utilization of Yugubao traditional Chinese medicines formula.

Role of epithelial sodium channel in rat osteoclast differentiation and bone resorption / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts.</p><p><b>METHODS</b>Multinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts.</p><p><b>RESULTS</b>s Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K.</p><p><b>CONCLUSION</b>s ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.</p>

Effects of sodium on rat osteoblast and the role of epithelial sodium channel / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of sodium on rat osteoblast function and explore the role of epithelial sodium channel (ENaC) in such effects.</p><p><b>METHODS</b>The proliferation and differentiation of rat osteoblasts were evaluated following treatment with 1×10(-4) mol/L to 1 mol/L Na(+). The mRNA expressions of the osteogenic genes and ENaC-α gene in the treated cells were assessed using RT-PCR.</p><p><b>RESULTS</b>Within the concentration of 1×10(-4) mol/L to 1 mol/L, Na(+) showed a two-way effect on the osteoblasts: low-concentration Na(+) (1×10(-4) mol/L) significantly promoted osteoblast differen- tiation, while at higher concentrations (0.5 and 1 mol/L), Na(+) produced an opposite effect. Sodium did not significantly affect osteoblast proliferation. Low-concentration Na(+) significantly increased the transcription of Cbfa1, OPN and OC, while high concentrations of Na(+) decreased their transcription. Low-concentration Na(+) also enhanced the mRNA expression of ENaC-α, but high-concentration Na(+) treatment lowered ENaC-α mRNA expression.</p><p><b>CONCLUSION</b>Na(+) displays a direct dose-related effect on osteoblasts by affecting its differentiation, osteogenic gene expression profile, and ENaC-α gene expression, suggesting the involvement of ENaC in Na(+)-mediated functional modulation of rat osteoblasts.</p>

Piperazinyl estrone prevents bone loss in ovariectomized rats / 药学学报

<p><b>AIM</b>To determine the effect of piperazinyl estrone, a new estrogen derivative, on bone turnover, bone mass and uteri in ovariectomized rats.</p><p><b>METHODS</b>Female Sprague-Dawley rats were ovariectomized (OVX) or sham operated (sham) at the age of 3 months and treated with estrone (E) at 0.75 mg.kg-1.d-1, or with piperazinyl estrone (P-E) at 1 or 10 mg.kg-1.d-1, orally, for 3 months. At the time of death, the uterine weight was measured. Bone histomorphometric analysis of proximal tibial metaphyses (PTM) was performed in undecalcified sections.</p><p><b>RESULTS</b>Bone histomorphometric data showed that the percent trabecular area (% Tb.Ar) of OVX rats with bone high turnover was significantly decreased. The uteri were atrophied. The percent trabecular area (% Tb.Ar) of estrone treated group was increased in decreasing bone turnover manner. But the size and weight of uteri in this group were increased vs OVX group. The bone loss induced by OVX was preserved by P-E treatment, but the mechanism of maintaining bone is different from that of E-treated rats. P-E treatment in low dose did not decrease any bone formation indices, such as percent labeling perimeter, bone formation rate per bone volume (BFR/BV), except bone mineral apposition rate (MAR) compared with E-treated group, and maintained them at OVX level. The uteri were found to be in atrophy compared with the match dose (0.75 mg) of E-treated OVX rats. But rats treated with high dose of P-E showed the same change like E-treated group.</p><p><b>CONCLUSION</b>The finding of this study shows that lower dosage of piperazinyl estrone has effect on preventing the bone losses in OVX rats, while the bone formation and the uterus are not affected, thus supporting the hypothesis that piperazinyl estrone has the potential to prevent postmenopausal bone loss in women with less side effects.</p>

Combination of ginsenosides with low dose estrogen showed synergetic effect on ovariectomy induced osteopenia in rats / 药学学报

<p><b>AIM</b>To determine whether low dose of estrogen in combination with ginsenosides can completely prevent bone loss in ovariectomized rats.</p><p><b>METHODS</b>Four-month-old ovariectomized rats were treated either with 100 and 300 mg.kg-1 of ginsenosides or 30 and 100 micrograms.kg-1 of 17 alpha-ethynylestradiol alone, or ginsenosides 100 mg.kg-1 in combination with 17 alpha-ethynylestradiol 30 micrograms.kg-1 for 10 weeks. Double in vivo fluorochrome labeling was made. The undecalcified longitudinal proximal tibial metaphyseal sections were processed and stained with Goldner's trichrome for histomorphometric analysis of the bone.</p><p><b>RESULTS</b>Body weights and serum cholesterol were increased in ovariectomized (OVX) rats. The rats lost 74% of bone volume and high bone turnover was induced after OVX compared with the sham group. Bone volume increased by 205% in the high dose estrogen treated group while it was increased by 105% in the low dose group. The two doses of estrogen were shown to inhibit osteoclasts surface (by -65% and -55%, P < 0.01) and decrease bone turnover rate (by -85% and -83%, P < 0.01). High dose of estrogen was found to inhibit growth and stimulate uterine weight gain in rats while low dose did not. High dose of ginsenosides increased bone volume by 84% (P < 0.01) and decreased bone turnover rate by -64% (P < 0.05) while lower dose of ginsenosides did not. However, low dose ginsenosides combined with low dose estrogen achieved well preventive effects: increase of 202% in bone volume, decrease of 66% in bone turnover rate and 72% in osteoclasts surface. The combined effect in preventing bone loss equals to that the high dose of estrogen alone did.</p><p><b>CONCLUSION</b>Use of low dose of estrogen plus ginsenosides showed synergistic effect on prevention of osteoporosis induced by ovariectomy.</p>