β‑Ecdysterone promotes autophagy and inhibits apoptosis in osteoporotic rats

Abstract

Osteoporosis is an aging process of skeletal tissues with characteristics of reductions in bone mass and microarchitectural deterioration of bone tissue. The present study aimed to investigate the effects of glucocorticoid‑induced osteoporosis on osteoblasts and to examine the roles of β‑ecdysterone (β‑Ecd) involved. In the present study, an in vivo model of osteoporosis was established through the subcutaneous implantation of prednisolone (PRED) into Sprague‑Dawley rats, with or without a subcutaneous injection of β‑Ecd (5 or 10 mg/kg body weight). Expression of Beclin‑1 and microtubule‑associated protein 1A/1B‑light chain 3I/II and apoptosis in lumbar vertebrae tissues was measured by immunofluorescence and TUNEL assays, respectively. Serum concentration of calcium and phosphorus, and the activity of tartrate‑resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were measured by biochemical assay. Reverse transcription‑quantitative polymerase chain reaction and western blotting was used for detect the expression of related genes and proteins. PRED treatment inhibited bone formation by decreasing bone mineral density, and suppressing the expression of Runt‑related transcription factor 2 and bone morphogenetic protein 2, while enhancing the activity of alkaline phosphatase, upregulating the expression of receptor activator of nuclear factor-κB ligand, and increasing the serum content of calcium, phosphorus and tartrate‑resistant acid phosphatase in rats. Additionally, PRED was revealed to inhibit autophagy through the downregulation of Beclin‑1, autophagy protein 5 and microtubule‑associated protein 1A/1B‑light chain 3I/II expression, whereas it induced the apoptosis, through the activation of caspase‑3 and the suppression of apoptosis regulator BCL2 expression. Notably, the PRED‑induced alterations in bone formation, autophagy and apoptosis were revealed to be attenuated by β‑Ecd administration. In conclusion, the findings of the present study suggested that β‑Ecd may be a promising candidate for the development of therapeutic strategies for the treatment of osteoporosis, through the induction of autophagy and the inhibition of apoptosis in vivo.

Figure 1.

3D-reconstructed micro-CT images of the 5th lumbar vertebrae isolated from rats in the various treatment groups. Control, untreated rats; PRED, rats received PRED for 4 weeks; PRED + ALN, rats received PRED and ALN for 4 weeks; PRED + β-Ecd, rats received PRED and β-Ecd (5 or 10 mg/kg) for 4 weeks. CT, computed tomography; PRED, prednisolone; ALN, alendronate; β-Ecd, β-ecdysterone.

Figure 2.

Biochemical parameters in rats from various experimental groups. The serum activity or expression of (A) ALP, (B) Ca, (C) TRAP and (D) P were detected using a biochemical analyzer. Control, untreated rats; PRED, rats received PRED for 4 weeks; PRED + ALN, rats received PRED and ALN for 4 weeks; PRED + β-Ecd, rats received PRED and β-Ecd (5 or 10 mg/kg) for 4 weeks. Data are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 vs. control; ^#P<0.05, ^##P<0.01 vs. PRED. ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase; Ca, calcium; P, phosphorus; PRED, prednisolone; ALN, alendronate; β-Ecd, β-ecdysterone.

Figure 3.

Immunofluorescence and TUNEL staining in rats from various experimental groups. The protein expression of Beclin-1 and LC3I/II was detected using immunofluorescence staining. Apoptosis was assessed using TUNEL staining. Magnification, ×200. Control, untreated rats; PRED, rats received PRED for 4 weeks; PRED + ALN, rats received PRED and ALN for 4 weeks; PRED + β-Ecd, rats received PRED and β-Ecd (5 or 10 mg/kg) for 4 weeks. TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling; LC3I/II, microtubule-associated protein 1A/1B-light chain 3I/II; PRED, prednisolone; ALN, alendronate; β-Ecd, β-ecdysterone.

Figure 4.

Expression of RUNX2, RANKL and BMP2 in rats from the various experimental groups. The mRNA expression levels of (A) RUNX2, (B) RANKL and (C) BMP2 were measured using the reverse transcription-quantitative polymerase chain reaction. (D) The protein expression levels of RUNX2, RANKL and BMP2 were detected using western blot analysis. Control, untreated rats; PRED, rats received PRED for 4 weeks; PRED + ALN, rats received PRED and ALN for 4 weeks; PRED + β-Ecd, rats received PRED and β-Ecd (5 or 10 mg/kg) for 4 weeks. Data are expressed as the mean ± standard deviation. **P<0.01 vs. control; ^##P<0.01 vs. PRED. RUNX, Runt-related transcription factor; RANKL, receptor activator of nuclear factor-κB ligand; BMP, bone morphogenetic protein; PRED, prednisolone; ALN, alendronate; β-Ecd, β-ecdysterone.

Figure 5.

Expression of Beclin-1, ATG5, LC3I/II, Bcl-2 and caspase-3 in rats from various experimental groups. The mRNA expression levels of (A) Beclin-1 and (B) ATG5 were measured using reverse transcription-quantitative polymerase chain reaction. (C) The protein expression levels of Beclin-1, ATG5, LC3I/II, Bcl-2 and caspase-3 were detected using western blot analysis. Control, untreated rats; PRED, rats received PRED for 4 weeks; PRED + ALN, rats received PRED and ALN for 4 weeks; PRED + β-Ecd, rats received PRED and β-Ecd (5 or 10 mg/kg) for 4 weeks. Data are expressed as the mean ± standard deviation. **P<0.01 vs. control; ^##P<0.01 vs. PRED. ATG, autophagy protein; LC3I/II, microtubule-associated protein 1A/1B-light chain 3I/II; Bcl, B-cell lymphoma; PRED, prednisolone; ALN, alendronate; β-Ecd, β-ecdysterone.