Oleanolic Acid Exerts Osteoprotective Effects and Modulates Vitamin D Metabolism

Abstract

Oleanolic acid (OA) is a triterpenoid with reported bone anti-resorption activities. The present study aimed to characterize its bone protective effects in vivo and to study its effects on vitamin D metabolism, both in vivo and in vitro. OA significantly increased bone mineral density, improved micro-architectural properties, reduced urinary Ca excretion, increased 1,25(OH)₂D₃ and renal CYP27B1 mRNA expression in mature C57BL/6 ovariectomised (OVX) mice. OA also improved bone properties, Ca balance, and exerted modulatory effects on renal CYP27B1 and CYP24A1 expressions in aged normal female Sprague-Dawley rats. In addition, OA significantly increased renal CYP27B1 mRNA and promoter activity, and suppressed CYP24A1 mRNA and protein expressions in human proximal tubule HKC-8 cells. OA exerted bone protective effects in mature OVX mice and aged female rats. This action on bone might be, at least in part, associated with its effects on Ca and vitamin D metabolism. The present findings suggest that OA is a potential drug candidate for the management of postmenopausal osteoporosis.

Keywords: aging; calcium; oleanolic acid; osteoporosis; ovariectomised; vitamin D.

Figure 1

Chemical structure of oleanolic acid.

Figure 2

The effects of OA on renal and duodenal mRNA and protein expressions in OVX mice. The expression levels of (A) renal CYP27B1 mRNA; (B) renal CYP24A1 mRNA; (C,E) renal CYP27B1 protein; (D,E) renal CYP24A1 protein; (F) renal TRPV5 mRNA; (G) renal CaBP28k mRNA; (H) duodenal TRPV6 mRNA; and (I) duodenal CaBP9k mRNA in OVX mice were studied. Four-month-old ovariectomized (OVX) or sham-operated (Sham) C57BL/6J mice were pair-fed with phytoestrogen-free AIN-93M diet and treated with vehicle (Sham or OVX), E2 (200 μg/kg/day), OA low dose (OAL, 50 mg/kg/day) or OA high dose (OAH, 100 mg/kg/day) for 6 weeks. The mRNA expression level is presented as the ratio of target gene to GAPDH. The protein expression level is shown as the ratio of target protein to β-actin. Data are presented by mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. * p < 0.05 and *** p < 0.001 vs. OVX.

Figure 2

The effects of OA on renal and duodenal mRNA and protein expressions in OVX mice. The expression levels of (A) renal CYP27B1 mRNA; (B) renal CYP24A1 mRNA; (C,E) renal CYP27B1 protein; (D,E) renal CYP24A1 protein; (F) renal TRPV5 mRNA; (G) renal CaBP28k mRNA; (H) duodenal TRPV6 mRNA; and (I) duodenal CaBP9k mRNA in OVX mice were studied. Four-month-old ovariectomized (OVX) or sham-operated (Sham) C57BL/6J mice were pair-fed with phytoestrogen-free AIN-93M diet and treated with vehicle (Sham or OVX), E2 (200 μg/kg/day), OA low dose (OAL, 50 mg/kg/day) or OA high dose (OAH, 100 mg/kg/day) for 6 weeks. The mRNA expression level is presented as the ratio of target gene to GAPDH. The protein expression level is shown as the ratio of target protein to β-actin. Data are presented by mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. * p < 0.05 and *** p < 0.001 vs. OVX.

Figure 3

The effect of OA on renal and duodenal mRNA and protein expressions in aged female rats. The expression levels of (A) renal CYP27B1 mRNA; (B) renal CYP24A1 mRNA; (C,E) renal CYP27B1 protein; (D,E) renal CYP24A1 protein; (F) renal TRPV5 mRNA; (G) renal CaBP28k mRNA; (H) duodenal TRPV6 mRNA; and (I) duodenal CaBP9k mRNA in aged female rats were studied. Thirteen-month-old female rats were fed with a high calcium diet (HCD, 1.2% calcium, 0.65% phosphorous) or a normal calcium diet (NCD, 0.6% calcium, 0.65% phosphorous) and orally administrated with OA (25 mg/kg/day) or its vehicle treatment for 12 weeks. The mRNA expression level is presented as the ratio of the target gene to GAPDH. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. NCD.

Figure 4

Effects of OA on CYP27B1 and CYP24A1 mRNA, protein expressions, and promoter activities in HKC-8 cells. The expression levels of (A) CYP27B1 mRNA; (B) CYP24A1 mRNA; (C) CYP27B1 protein; (D) CYP24A1 protein as well as the promoter activities of (E) CYP27B1 and (F) CYP24A1 in HKC-8 cells were studied. HKC-8 cells were treated with vehicle (0.1% ethanol), 10⁻⁷ M PTH (1–34, human), 10⁻⁵ M Foskolin or 10⁻⁸ M 1,25(OH)₂D₃, and 10⁻⁹ M–10⁻⁵ M OA for 24 h. Cells were harvested by Trizol reagent at indicated time for RT-PCR and real-time PCR analysis (A,B). Relative gene expression was normalized by GAPDH. Total protein was extracted by lysis buffer and separated by SDS-PAGE and immunoblotted with anti-CYP27B1 (C,E), anti-CYP24A1 (D,E) antibody and normalized with β-actin expression. Promoter activities (F,G) were measured by dual luciferase assay and data were normalized against a thymidine kinase (TK) reporter construct. Results are presented as mean ± SEM (n = 3) and analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control.