Prevention of glucocorticoid induced bone changes with beta-ecdysone

Abstract

Beta-ecdysone (βEcd) is a phytoecdysteroid found in the dry roots and seeds of the asteraceae and achyranthes plants, and is reported to increase osteogenesis in vitro. Since glucocorticoid (GC) excess is associated with a decrease in bone formation, the purpose of this study was to determine if treatment with βEcd could prevent GC-induced osteoporosis. Two-month-old male Swiss-Webster mice (n=8-10/group) were randomized to either placebo or slow release prednisolone pellets (3.3mg/kg/day) and treated with vehicle control or βEcd (0.5mg/kg/day) for 21days. GC treatment inhibited age-dependent trabecular gain and cortical bone expansion and this was accompanied by a 30-50% lower bone formation rate (BFR) at both the endosteal and periosteal surfaces. Mice treated with only βEcd significantly increased bone formation on the endosteal and periosteal bone surfaces, and increased cortical bone mass were their controls to compare to GC alone. Concurrent treatment of βEcd and GC completely prevented the GC-induced reduction in BFR, trabecular bone volume and partially prevented cortical bone loss. In vitro studies determined that βEcd prevented the GC increase in autophagy of the bone marrow stromal cells as well as in whole bone. In summary, βEcd prevented GC induced changes in bone formation, bone cell viability and bone mass. Additional studies are warranted of βEcd for the treatment of GC induced bone loss.

Keywords: Autophagy; Beta-ecdysone (βEcd); Bone formation; Glucocorticoid.

Figure 1

Effects of GC and βEcd treatments on the vertebral and femoral trabecular bone microarchitectures, assessed by microCT. Two-month-old mice were treated with βEcd, GC or concurrent treatment of GC + βEcd for 21 days. (A) Lumbar vertebral trabecular bone (LVB) structure measured by micro-CT. (B) Representative trabecular thickness maps were obtained from the LVB by micro-CT where the trabecular thickness is color coded: with blue-green colors indicate thinner trabeculae whereas yellow-red colors fare used for thicker trabeculae. (C) Distal femoral trabecular bone (DFM) structure as measured by microCT. *: p<0.05 between indicated groups.

Figure 2

Effects of βEcd on trabecular bone formation. (A) Surface-based bone formation was measured at the un-decalcified LVB frozen sections. (B) Representative LVB sections from the treatment groups. *: p<0.05 between indicated groups.

Figure 3

Effect of GC and βEcd treatments on cortical bone structure and surface-based bone turnover, assessed by microCT and bone histomorphometry. (A) Cortical bone volume and bone formation were measured at the mid-shaft of the femur. (B) Representative cross-sectional cortical bone sections from the mid-shaft of the femurs. *: p<0.05 between indicated treatment groups.

Figure 4

βEcd increases osteogenic differentiation of bone marrow stromal cells. Bone marrow cells were collected from long bones of male mice, two months of age and maintained in α-MEM with 10% FBS and antibiotics for four days. The adherent cells were collected and cultured in adipogenic or osteogenic media for 21 days. (A) Adipogenic differentiation measured by oil red staining and gene expressions related to adipogenesis (Cebp-α and Ppar-γ). (B) Osteogenesis measured the ratio of CFU-Ob/CFU-F and genes associated with osteoblast differentiation (Runx2 and Bglap1). Data are means ± SD. *: p<0.05 vs. control (PBS). All the studies were performed in triplicate.

Figure 5

Effects of βEcd on osteoblast autophagy in vitro. BMSC cells were obtained from dsRed-LC3 reporter male mice and cultured in osteogenic medium for 10 days before they were treated with PBS, Dexamethasone (Dex, 10^-6 M), βEcd (10^-7M) or combination of Dex + βEcd in serum-starved conditions for eight hours. (A and B) Representative images (A) and quantitation of (B) dsRed-LC dots (autophagosomes, white arrows) in BMSC grown in osteogenic medium for 7 days and after 8 hours serum starvation incubation with Dex or βEcd as indicated. More than 200 cells were analyzed per sample. All the studies were performed in triplicate.

Figure 6

GC decreased while βEcd activated autophagy in bone. (A) RNA was extracted from the tibial shafts of PL, GC or GC+ βEcd treated mice at day 21. The focus RT-PCR gene array for autophagy was performed. RT-PCR data was expressed as fold changes from the PL group. (B) Proteins were extracted from the distal tibiae in animals treated with PL, GC or GC + βEcd. Western blots were performed for Atg7, Atg16L, LC3I/II, total Akt and p-Akt (S473). (C) Relative bend intensity measurements on B (n=3/group). *: p <0.05 vs. PL; #, p<0.05 vs. GC.