Endogenous n-3 fatty acids protect ovariectomy induced bone loss by attenuating osteoclastogenesis

Abstract

Beneficial effects of n-3 fatty acids (FA) on bone mineral density (BMD) have been reported in mice, rats and human beings, but the precise mechanisms involved have not been described. This study used the Fat-1 mouse, a transgenic model that synthesizes n-3 FA from n-6 FA to directly determine if outcome of bone health were correlated with n-3 FA. Ovariectomized (Ovx) and sham operated wild-type (WT) and Fat-1 mice were fed an AIN-93M diet containing 10% corn oil for 24 weeks. BMD was analysed by dual energy x-ray absorptiometry. Fat-1 Ovx mice exhibited significantly lower level of osteotropic factors like receptor activator of NF-kappaB ligand and tartrate-resistant acid phosphatase (TRAP)5b in serum and higher BMD in distal femoral metaphysis, proximal tibial metaphysis, femoral diaphysis and lumbar vertebra as compared to WT Ovx mice. LPS-stimulated bone marrow (BM) cells from Fat-1 Ovx mice produced significantly lower level of pro-inflammatory cytokines like tumour necrosis factor-alpha, interleukin (IL)-1-beta, IL-6 and higher level of anti-inflammatory cytokines like IL-10, IFN-gamma and higher level of nitric oxide as compared to BM cells from WT Ovx mice. LPS-stimulated COX-II activity as well as NF-kappaB activation in BM cells from Fat-1 Ovx mice was significantly less as compared to BM cells from WT Ovx mice. Furthermore, Fat-1 BM cells generated significantly less number of TRAP osteoclast-like cells as compared to WT BM cells. In conclusion, we offer further insight into the mechanisms involved in preventing the BMD loss in Ovx mice by n-3 FA using a Fat-1 transgenic mouse model.

Keywords: bone mineral density; inflammation; n-3 fatty acids; osteoclasts; osteoporosis.

Figure 1

Endogenous n‐3 fatty acids reduce serum RANKL and TRAP5b. After 20 weeks on experimental diet serum was separated from blood collected retro‐orbitally from WT and Fat‐1 transgenic sham and Ovx mice (seven mice/groups). Serum was analysed for (A) free serum RANKL and (B) TRAP5b levels using standard ELISA kits. Each bar represents the mean ± S.E.M. of seven duplicate samples. Value with different superscripts are significantly different at P < 0.05 by Newman‐Keuls’ one way ANOVA with multiple comparison test.

Figure 2

Endogenous n‐3 fatty acids modulate LPS‐stimulated cytokines production. Bone marrow cells from WT and Fat‐1 transgenic mice were cultured in the presence of LPS (5 μg/ml). After 24 hrs, culture media were collected and analysed for TNF‐α,, IL‐1β, IL‐6, IL‐10 and IFN‐γ by standard ELISA techniques. Each value represents the mean ± S.E.M. of two independent triplicate cultures. P‐value <0.05 was considered significant by Student’s t‐test.

Figure 3

Endogenous n‐3 fatty acids increase LPS‐stimulated nitric oxide production. Bone marrow cells from WT and Fat‐1 transgenic mice were cultured in the presence of LPS (5 μg/ml). After 24 hrs, culture media were collected and analysed for nitric oxide using quantichrome nitric oxide assay kit. Each value represents the mean ± S.E.M. of two independent triplicate cultures. P‐value <0.05 was considered significant by Student’s t‐test.

Figure 4

Endogenous n‐3 fatty acids decrease LPS‐stimulated COX‐II production. Bone marrow cells from WT and Fat‐1 transgenic mice were cultured in the presence of LPS (5 μg/ml). After 24 hrs, cells were collected and cytosolic proteins were prepared. 100 μg of cytosolic proteins were analysed for COX‐II activity using COX Activity Assay Kit. Each value represents the mean ± S.E.M. of two independent triplicate cultures. P‐value <0.05 was considered significant by Student’s t‐test.

Figure 5

Endogenous n‐3 fatty acids decrease LPS‐stimulated NF‐κB activation. Bone marrow cells from WT and Fat‐1 transgenic mice were cultured in the presence of LPS (5 μg/ml). After 24 hrs, cells were collected and cytosolicand nuclear proteins were prepared. (A) 30 μg of cytosolic proteins were analysed for phosphorylated I‐κB‐α and total I‐κB‐α level by western blot. Relative expression of I‐κB‐α, pI‐κB‐α and pI‐κB‐α/total I‐κB‐α is shown. The intensity of the bands was determined by densitometry. (B) 10 μg of nuclear proteins were analysed for p65 NF‐κB and p50 NF‐κB‐DNA binding activity using TransAM Transcription Factor Assay kit. Each value represents the mean ± S.E.M. of two independent triplicate cultures. P‐value <0.05 was considered significant by Student’s t‐test.

Figure 6

Endogenous n‐3 fatty acids suppress osteoclast differentiation in bone marrow (BM) cell culture. BM cells (1 × 10⁶) from WT and Fat‐1 mice were cultured in the presence of sRANKL and macrophage colony‐stimulating factor (M‐CSF). (A) Formation of TRAP + multinucleated cells in cultures of BM cells isolated from WT and Fat‐1 transgenic mice in the presence of sRANKL and M‐CSF. (B) TRAP⁺ multinucleated cells count in cultures of BM cells isolated from WT and Fat‐1 mice.. *Significantly different from WT control at P < 0.001 by Student’s t‐test.