Dietary ω-3 Fatty Acid Supplementation Improves Murine Sickle Cell Bone Disease and Reprograms Adipogenesis

Abstract

Sickle cell disease (SCD) is a genetic disorder of hemoglobin, leading to chronic hemolytic anemia and multiple organ damage. Among chronic organ complications, sickle cell bone disease (SBD) has a very high prevalence, resulting in long-term disability, chronic pain and fractures. Here, we evaluated the effects of ω-3 (fish oil-based, FD)-enriched diet vs. ω-6 (soybean oil-based, SD)- supplementation on murine SBD. We exposed SCD mice to recurrent hypoxia/reoxygenation (rec H/R), a consolidated model for SBD. In rec H/R SS mice, FD improves osteoblastogenesis/osteogenic activity by downregulating osteoclast activity via miR205 down-modulation and reduces both systemic and local inflammation. We also evaluated adipogenesis in both AA and SS mice fed with either SD or FD and exposed to rec H/R. FD reduced and reprogramed adipogenesis from white to brown adipocyte tissue (BAT) in bone compartments. This was supported by increased expression of uncoupling protein 1(UCP1), a BAT marker, and up-regulation of miR455, which promotes browning of white adipose tissue. Our findings provide new insights on the mechanism of action of ω-3 fatty acid supplementation on the pathogenesis of SBD and strengthen the rationale for ω-3 fatty acid dietary supplementation in SCD as a complementary therapeutic intervention.

Keywords: adipogenesis; bone histomorphometry; miRNAs; osteogenesis; sickle cell disease.

Figure 1

FD improves bone microarchitecture and reduces bone turnover in humanized sickle cell mice under normoxia. Quantitative histomorphometric analysis of distal femur of healthy (AA) and sickle cell (SS) mice under either SD or FD supplementation under normoxia. (a) Marrow Star Volume (Marrow*V) and increased Node Number (NdN#/TV) in healthy (AA) and sickle cell (SS) mice under either SD or FD supplementation. (b) Osteoclast number (N.Oc/TA), activity (ES/BS) osteoblastic activity (ObS/BS) in healthy (AA) and sickle cell (SS) mice under either SD or FD supplementation. (c) Bone Formation Rate (BFR/BS) and Activation Frequency (AcF), data are shown as mean ± standard deviation (SD). (d) Real-time PCR analysis of bone Runx2, Sparc, RankL, Rank expression in healthy (AA) and sickle cell (SS) mice under either SD or FD supplementation. (e) Real-time PCR analysis of interleukin- 6 (Il-6) and peroxiredoxin-2 (Prx2) bone expression in healthy (AA) and sickle cell (SS) mice under either SD or FD supplementation. (a–e) Data are shown as mean ± standard deviation (SD) (n = 4; 2 females and 2 males). * p < 0.05 compared to AA; ° p < 0.05 compared to SD.

Figure 2

In SCD mice, FD protects bone from recurrent H/R related damage. Quantitative histomorphometric analysis of distal femur of healthy (AA) and sickle cell (SS) mice under either SD or FD supplementation exposed to recurrent H/R. (a) Left panel. Representative undecalcified section of distal femur stained with Trichoma Goldener’s stain. Microarchitecture was preserved in FD treated mice compared to the SD treated group, expressed as indirect (Marrow Star Volume, M*V) and direct (Node Number, NdN#/TV) parameters (right panel). Scale bar: 1 µM. (b) Bone Volume (BV/TV) and trabecular parameters were better in the FD treated group compared to the FD group. In particular, the Trabecular Number and Thickness were higher and Trabecular Separation was lower in the FD treated group (c) Concerning the cellular activity, the FD group showed reduced osteoclastic number (NOc/TA), increased Osteoblasts Surfaces (OS/BS) and reduced Erosion Surfaces (ES/BS) compared to the SD group. (a–c) Data are shown as mean ± standard deviation (SD) (n = 4; 2 females and 2 males). * p < 0.05 compared to AA; ° p < 0.05 compared to SD.

Figure 3

In SCD mice, FD diet beneficially affects osteoblastogenesis and osteogenic commitment. (a) Real-time PCR analysis of Runx2 and Sparc gene expression in bone from SD/FD AA and FD SS mice exposed to recurrent H/R (rec H/R). (b) Left panel. Osteoprogerin (OPG)/RANKL ratio in SD/FD AA and FD SS mice exposed to recurrent H/R (rec H/R). Right panel. Bone miR205 expression was downregulated in FD SS mice exposed to rec H/R compared to SD SS animals. (c) Real-time PCR analysis of bone interleukin-6 (IL6), matrix metalloproteinase 9 (MMP9) and peroxiredoxin-2 (Prx2) gene expression was reduced in FD SS compared to SD SS mice exposed to recurrent H/R. (d) To assess the number of osteoprogenitors, bone marrow derived cells were cultured in vitro under osteogenic differentiation condition. The CFU-Ob obtained from mesenchymal stem cells of AA and SS mouse groups were stained with alizarin red (upper panel) and quantified (lower panel). By comparing the number of CFU-Ob (colony forming unit-osteoblastic), we observed an increased number of CFU-Ob in FD fed mice, which is in agreement with the increased osteogenic commitment of progenitor cells. (a–c) Data are shown as mean ± standard deviation (SD) (n = 4; 2 females and 2 males). * p < 0.05 compared to AA; ° p < 0.05 compared to SD. (d) Data are shown as mean ± standard deviation (SD); at least six independent experiments have been performed; * p < 0.05 compared to AA; ° p < 0.05 compared to SD.

Figure 4

In SCD mice, FD reduces and re-programs adipogenesis from white to brown adipocyte tissue. (a) Upper panel. Representative images from bone sections of SD/FD AA and SD/FD SS mice exposed to recurrent H/R (rec H/R) stained for Perilipin, a lipid droplet-membrane component. Scale bar: 50 uM. Lower panel. IRS expresses the product of Perilipin staining intensity (between negative = 0 and strong = 3) and the percentage of positive Perilipin stained cells. (b) To assess the number of adipocytic progenitors, bone marrow derived cells were cultured in vitro under adipogenic (A) differentiation condition. The CFU-A obtained from mesenchymal stem cells of AA and SS mouse groups were stained with Oil Red O (ORO) (upper panel) and quantified (lower panel). By comparing the number of CFU-A (colony forming unit-adipogenic), we observed a decreased number of CFU-A in FD SS fed mice, which is in agreement with the decreased adipocyte commitment of progenitor cells. (c) Real-time PCR analysis of peroxisome proliferator-activated receptor2-γ (PPAR2g) in SD/FD AA/SS mice exposed to rec H/R. (d) Upper panel. Representative images of bone sections of SD/FD AA and SD/FD SS mice exposed to recurrent H/R (rec H/R) stained for Uncoupling protein-1 (UCP1, brown). Scale bar: 50 uM. Lower panel. UCP-1 quantification. (e) miR455 expression in bone from SD/FD AA and SD/FD SS mice exposed to recurrent H/R (rec H/R). (a,b,d,e) Data are shown as mean ± standard deviation (SD); at least six independent experiments have been performed; * p < 0.05 compared to AA; ° p < 0.05 compared to SD (c,e) Data are shown as mean ± standard deviation (SD) (n = 4; 2 females and 2 males). * p < 0.05 compared to AA; ° p < 0.05 compared to SD.

Figure 5

Schematic diagram of the multimodal action of FD supplementation that ameliorates sickle cell bone disease. SCD mice display increased osteoclast recruitment/activity and reduced osteoblastogenesis/activity, ending in reduced osteoid formation and bone loss. miR205a negatively affects osteoblast recruitment in favor of adipogenesis, mainly represented by white adipocyte tissue. FD supplementation reduces osteoclastogenesis/osteoclast activity and downregulates miR205a, favoring osteoblastogenesis/activity. Finally, FD re-programing of adipogenesis resulted in the browning of white adipocyte tissue. The multimodal action of FD protects bone from sickle cell related tissue damage. SCD: sickle cell disease; MSC; mesenchymal stem cells.