Cepharanthine Prevents Estrogen Deficiency-Induced Bone Loss by Inhibiting Bone Resorption

Abstract

Osteoporosis is a common health problem worldwide caused by an imbalance of bone formation vs. bone resorption. However, current therapeutic approaches aimed at enhancing bone formation or suppressing bone resorption still have some limitations. In this study, we demonstrated for the first time that cepharanthine (CEP, derived from Stephania cepharantha Hayata) exerted a protective effect on estrogen deficiency-induced bone loss. This protective effect was confirmed to be achieved through inhibition of bone resorption in vivo, rather than through enhancement of bone formation in vivo. Furthermore, the in vitro study revealed that CEP attenuated receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast formation, and suppressed bone resorption by impairing the c-Jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathways. The inhibitory effect of CEP could be partly reversed by treatment with anisomycin (a JNK and p38 agonist) and/or SC79 (an AKT agonist) in vitro. Our results thus indicated that CEP could prevent estrogen deficiency-induced bone loss by inhibiting osteoclastogenesis. Hence, CEP might be a novel therapeutic agent for anti-osteoporosis therapy.

Keywords: Akt; JNK; cepharanthine; osteoclasts; osteoporosis.

Figure 1

CEP prevented OVX-induced bone loss in vivo. (A) Representative 3D micro-CT images of trabecular bone of proximal tibias from each group were reconstructed. Scale bar = 1 mm and 500 μm (B–G) The BV/TV, Conn.D, SMI, Tb.N, Tb.Th, and Tb.Sp values of micro-CT data from each sample were obtained and analyzed (H). H&E staining was performed for the histomorphological analysis. Scale bar = 200 μm (I–L) The BV/TV, Tb.N, Tb.Th, and Tb.Sp values of histomorphological sections were measured and quantified. LD and HD represent the dose of 5 and 20 mg/kg CEP, respectively. Values are expressed as mean ± SD, n = 10; ^*P < 0.05, ^**P < 0.01 vs. the control group.

Figure 2

CEP impaired OVX-induced osteoclastogenesis in vivo, but does not affecting osteogenesis. (A) TRAP staining was performed on decalcified sections of distal femurs. Representative images of osteoclasts (red arrows) were measured. Scale bar = 100 μm (B) Representative images of calcein (green) and andalizarin red (red) labels were visualized. Scale bar = 200 μm (C,D) The N.Ob/BS and ObS/BS were measured with Toluidine Blue-stained sections. (E,F) The N.Oc/BS and OcS/BS were measured with TRAP-stained sections. (G,H) The MAR and BFR/BS were measured with undecalcified sections. (I,J) The serum levels of P1NP and CTX-1 were analyzed with ELISA. Values are expressed as mean ± SD, n = 10; ^*P < 0.05, ^**P < 0.01 vs. the control group.

Figure 3

CEP attenuated RANKL-induced osteoclast formation in vitro. (A) BMMs were seeded at a density of 2 × 10⁴ cells/well and treated with the indicated concentrations of CEP in the presence of 30 ng/mL M-CSF for 48 or 96 h. The cell viability was quantified using the CCK8 assay. (B) TRAP staining was performed on BMMs treated with different doses of CEP in osteoclastogenic medium for 4 days. Scale bar = 200 μm. (C,D) The number and area of TRAP positive osteoclasts were analyzed. (E–J) The mRNA expression levels of Cathepsin K, TRAP, CTR, V-ATPase a3, V-ATPase d2, and DC-STAMP in BMMs treated with the indicated concentrations of CEP for 4 days were quantified. K BMMs were treated with 0.5 μM CEP for day 0–day 2 (Early-stage), day 2–day 4 (Late-stage), or day 0–day 4 (Early + Late stage) in osteoclastogenic medium. Scale bar = 200 μm. (L,M) The number and the area of osteoclasts were measured. All experiments were repeated independently for three times. Values are expressed as mean ± SD; ^*P < 0.05, ^**P < 0.01 vs. the control group.

Figure 4

CEP inhibited F-actin ring formation and bone resorption in vitro. (A) BMMs were cultured with osteoclastogenic medium for 4 days and subsequently seeded on bone slices in the presence of indicated dilutions of CEP for another 48 h. Cells were fixed and stained to detect F-actin rings. Scale bar = 50 μm. (B) The area of the F-actin rings was quantified. (C) Cells were removed from bone slices and bone resorption pits were observed using SEM. Scale bar = 200 μm. (D) The area of bone resorption pits was measured. Values are expressed as mean ± SD; ^*P < 0.05, ^**P < 0.01 vs. the control group.

Figure 5

CEP suppressed RANKL-induced activation of the JNK and AKT pathways in vitro. (A) BMMs were cultured in osteoclastogenic medium with or without CEP treatment for 0, 1, 2, and 3 days. Cell lysates of samples from day 0 to day 3 were collected and analyzed by Western blotting using specific antibodies against NFATc1, c-Fos, and CatK. (B) The gray levels of NFATc1, c-Fos, and CatK were analyzed by normalization to α-tubulin. (C) After culture for 3 days, the cells were collected and the mRNA expression levels of NFATc1 and c-Fos were analyzed. (D) BMMs were pre-treated with or without CEP for 2 h and were subsequently treated with RANKL for 0, 5, 10, 20, 30, and 60 min. Cells were collected and lysates were analyzed by Western blotting using primary antibodies specific to p-p65, p65, p-ERK1/2, ERK, p-JNK1/2, JNK1/2, p-p38, p38, p-IκBα, IκBα, p-AKT, AKT, p-PI3K, PI3K, and α-tubulin. (E,F) The gray levels of p-JNK1/2, p-AKT, and p-PI3K were analyzed by normalization to total JNK1/2, AKT, and PI3K. Values are expressed as mean ± SD; ^*P < 0.05, ^**P < 0.01 vs. the control group.

Figure 6

ANI and SC79 partly reversed the inhibitory effects of CEP on RANKL-induced osteoclastogenesis. (A) BMMs were cultured in osteoclastogenic medium containing CEP for 4 days in the presence of DMSO, ANI, and/or SC79. Cells were fixed and TRAP staining was performed. Scale bar = 200 μm. (B) The number and size of TRAP positive osteoclasts were quantified. (C) BMMs were pre-treated with CEP for 2 h and subsequently stimulated with RANKL in the presence of DMSO, ANI, and/or SC79 for 20 min. Cell lysates were collected and analyzed by Western blotting using primary antibodies specific to p-AKT, AKT, p-JNK1/2, and JNK. (D) The gray levels of p-JNK1/2 and p-AKT were analyzed by normalization to total JNK1/2 and AKT. (E) BMMs were cultured with osteoclastogenic medium for 4 days, and subsequently seeded on bone slices with CEP treatment in the presence of DMSO, ANI and/or SC79 for another 48 h. Bone resorption pits were observed under SEM. Scale bar = 200 μm (F) The area of the bone resorption pits was measured. Values are expressed as mean ± SD; ^*P < 0.05, ^**P < 0.01 vs. the control group.