Dendrobium officinale Orchid Extract Prevents Ovariectomy-Induced Osteoporosis in Vivo and Inhibits RANKL-Induced Osteoclast Differentiation in Vitro

Abstract

Background:Dendrobium officinale, a traditional Chinese medical herb with high value that is widely used in Asia, possesses many positive effects on human health, including anti-chronic inflammation, anti-obesity, and immune modulation properties; however, whether D. officinale has inhibitory effects on postmenopausal osteoporosis remains unknown. Objective: We investigated the effects of D. officinale extract (DOE) on ovariectomy-induced bone loss in vivo and on osteoclastogenesis in vitro. Methods:In vivo, female rats were divided into a sham-operated (sham) group and five ovariectomized (OVX) subgroups: OVX with vehicle (OVX), OVX with Xian-Ling-Gu-Bao capsule (240 mg/kg body weight/day), and OVX with low-, medium-, and high-dose DOE (150, 300, and 600 mg/kg body weight/day, respectively). Animals in each group were administered their corresponding treatments for 13 weeks. Body weight, serum biochemical parameters, uterine and femoral physical parameters, bone mineral density (BMD), bone biomechanical properties, and bone microarchitecture were obtained. In vitro, the effects of DOE on osteoclastogenesis were examined using RAW264.7 cells. The effects of DOE on osteoclastogenesis and the expression of osteoclast-specific marker genes and proteins were determined. Results: DOE effectively ameliorated serum biochemical parameters, especially alleviated estradiol (E2) deficiency and maintained calcium and phosphorus homeostasis. DOE improved uterine and femoral physical parameters. In addition, DOE improved femoral BMD and biomechanical properties. DOE significantly ameliorated bone microarchitecture. Moreover, DOE inhibited osteoclastogenesis independent of its cytoxicity and suppressed the expression of osteoclast-specific marker genes and proteins. Conclusion: DOE can effectively prevent ovariectomy-induced bone loss in vivo and inhibit osteoclastogenesis in vitro.

Keywords: DOE; bone quality; osteoclastogenesis; ovariectomy; postmenopausal osteoporosis.

FIGURE 1

HPLC chromatogram of reference characteristic of compounds: Vicenin 2 (1); Narigenin (2); Daucosterol (3); and β-Sitosterol (4).

FIGURE 2

DOE treatment ameliorates serum biochemical parameters in OVX rats: (A) TC; (B) TG; (C) HDL-C; (D) LDL-C; (E) Glucose; (F) E2; (G) Calcium; (H) Phosphorus; (I) ALP; (J) BGP; and (K) ACP concentrations. All data are presented as means ± SEM (n = 10). ^∆P < 0.05, ^∆∆P < 0.01, and ^∆∆∆P < 0.001 versus the sham group, and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus the OVX group.

FIGURE 3

DOE treatment improves uterine and femoral physical parameters in OVX rats: (A) uterine weight; (B) organ coefficient; (C) endometrial height; (D) femoral wet weight; (E) organ coefficient; (F) femoral dry weight; (G) organ coefficients; (H) femoral diameter; and (I) femoral length. All data are presented as means ± SEM (n = 10). ^∆∆∆P < 0.001 versus the sham group, and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus the OVX group.

FIGURE 4

DOE treatment improves bone quality in OVX rats: (A) femoral BMD; (B) maximum deflection; (C) maximum load; (D) cortical bone tissue stained with H&E; (E) calculated cortical bone thickness; (F) trabecular bone tissue stained with H&E; and (G) calculated trabecular bone area. Representative images were acquired using a medical image analysis system at an original magnification of ×400. All data are presented as means ± SEM (n = 10). ^∆P < 0.05, ^∆∆P < 0.01, and ^∆∆∆P < 0.001 versus the sham group, and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus the OVX group.

FIGURE 5

DOE treatment inhibits RANKL-induced osteoclastogenesis. (A) RAW264.7 cells were cultured for 5 days with RANKL (50 ng/ml) in the presence of XLGB (10 μg/ml) or the indicated concentrations of DOE and then stained for TRAP. (B) TRAP-positive multinucleated cells with more than five nuclei were considered mature osteoclasts, as observed under a light microscope. (C) The effects of DOE on the viability of RAW264.7 cells as determined by the MTT assay. Representative images were acquired using a light microscope (magnification ×200). Values are means ± SEM for three independent experiments. ^∆∆∆P < 0.001 versus the control, and ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus just RANKL-treated cells.

FIGURE 6

DOE treatment reduces the expression of osteoclast-specific marker genes. Values are means ± SEM of three independent experiments. ^{∆ ∆ ∆}P < 0.001 versus the control, and ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus just RANKL-treated cells.

FIGURE 7

DOE treatment reduces the expression of osteoclast-specific marker proteins. In this experiment, c-Fos, NFATc1, c-Src, cathepsin K, and β-tubulin levels were all detected using western blotting, and results were quantified using the software AlphaView. Representative images are displayed; values are the means ± SEM of three independent experiments.