Olive oil and vitamin D synergistically prevent bone loss in mice

Abstract

As the Mediterranean diet (and particularly olive oil) has been associated with bone health, we investigated the impact of extra virgin oil as a source of polyphenols on bone metabolism. In that purpose sham-operated (SH) or ovariectomized (OVX) mice were subjected to refined or virgin olive oil. Two supplementary OVX groups were given either refined or virgin olive oil fortified with vitamin D3, to assess the possible synergistic effects with another liposoluble nutrient. After 30 days of exposure, bone mineral density and gene expression were evaluated. Consistent with previous data, ovariectomy was associated with increased bone turnover and led to impaired bone mass and micro-architecture. The expression of oxidative stress markers were enhanced as well. Virgin olive oil fortified with vitamin D3 prevented such changes in terms of both bone remodeling and bone mineral density. The expression of inflammation and oxidative stress mRNA was also lower in this group. Overall, our data suggest a protective impact of virgin olive oil as a source of polyphenols in addition to vitamin D3 on bone metabolism through improvement of oxidative stress and inflammation.

Figure 1. Mice body weight and composition.

Body weight and composition were assessed at the end of the experiment. Values are means ± SEM. *p<0.05 vs RO-SH, ^‡p<0.05 vs VO-SH based on ANOVA analysis with Tukey’s post hoc test. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy. Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3.

Figure 2. Bone parameters in mice fed with olive oil enriched or not with vitamin D3.

Femoral cortical, primary and secondary spongiosa bone mineral density (A) and serum levels of CTX1 and PINP (B) in mice fed with olive oil enriched or not with vitamin D3.Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice received refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed. *p<0.05 vs RO-SH, ^‡p<0.05 vs VO-SH, ^$p<0.05 vs VO-OVX, ^£p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; BMD, bone mineral density; CTX-1, collagen type 1 cross-linked C-telopeptide; PINP, N-terminal propeptide of type I procollagen.

Figure 3. Transcript levels of osteoblast metabolism markers in mice femurs.

Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ^‡p<0.05 vs VO-SH, ^#p<0.05 vs RO-OVX, ^$p<0.05 vs VO-OVX, ^£p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; ALP, alkaline phosphatase; OCN, osteocalcin; OPN, osteopontin; Col1a1, type I collagen; Lrp5, low density lipoprotein receptor-related protein 5; Sfrp1, secreted frizzled related sequence protein 1; Sost1, sclerostin; Esr1, estrogen receptor 1.

Figure 4. Transcript levels of osteoclast metabolism markers in mice femurs.

Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ^‡p<0.05 vs VO-SH, ^#p<0.05 vs RO-OVX, ^$p<0.05 vs VO-OVX, ^£p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; TRAP, tartrate-resistant acid phosphatase; Ctsk, catepsin K; Itg-β3, β3-integrin; MMP-2, matrix metalloproteinase 2; RANK, Receptor activator of nuclear factor-kappaB (NF-kappaB); RANKL, RANK ligand; OPG, osteoprotegerin.

Figure 5. Transcript levels of oxidative stress and inflammation markers in mice femurs.

Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ^‡p<0.05 vs VO-SH, ^#p<0.05 vs RO-OVX, ^$p<0.05 vs VO-OVX, ^£p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; IL-1β, interleukin-1β; IL-6, interleukin-6; CCl2, chemokine (C-C motif) ligand 2; Tlr2, toll like receptor 2; Tlr4, toll like receptor 4; Nos2, nitric oxide synthase.

Figure 6. Schematic representation of the bone sparing effect of virgin olive oil enriched with vitamin D3.

The effect of ovariectomy is represented by dotted boxes, the arrow (before the gene name), showing the modification of gene expression. The effect of virgin olive oil and vitamin D3 is represented by the grey boxes, the arrow (after the gene name) showing the impact on gene expression. The arrows between the genes outline regulation pathways. ALP, alkaline phosphatase; CCl2, chemokine (C-C motif) ligand 2; Col1a1, type I collagen; Ctsk, catepsin K; Esr1, estrogen receptor 1; IL-1β, interleukin-1β; IL-6, interleukin-6; Itg-β3, β3-integrin; Lrp5, low density lipoprotein receptor-related protein 5; MMP-2, matrix metalloproteinase 2; Nos2, nitric oxide synthase; OCN, osteocalcin; OPN, osteopontin; Sost1, sclerostin; Tlr2, toll like receptor 2; Tlr4, toll like receptor 4; TRAP, tartrate-resistant acid phosphatase.