Epimedin B protects against bone loss and inflammation in diabetic osteoporosis rats by regulating OPG/RANKL pathway

Abstract

Background: Diabetes is a common disease contributing to osteoporosis. Epimedin B (EB), a major ingredient of Herba Epimedii, has been found to be effective in preventing osteoporosis in mice. However, the potential of EB to ameliorate diabetic osteoporosis (DOP) remains elusive. In this study, our goal is to investigate the functions and underlying mechanisms of EB in the progression of DOP.

Methods: A DOP rat model was established via a high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). DOP rats were daily administered with EB or vehicle via intragastric administration for 8 weeks. Body weights and blood glucose levels were measured every 4 weeks during the drug administration period. Blood samples and femoral tissues were collected for further analysis. Bone parameters and bone histopathological changes were detected. Bone formation and resorption markers as well as inflammatory factors were detected using enzyme-linked immunosorbent assay kits. Reverse-transcription quantitative polymerase chain reaction and western blotting were conducted to measure the expression of osteoprotegerin (OPG) and Rev-Erbα, receptor activator of NF-κB ligand (RANKL).

Results: EB improved weight loss and lowered blood glucose of DOP rats. EB promoted the formation of bone trabeculae and altered several bone microstructure parameters in DOP rats. EB ameliorated improved bone structure, restored histological abnormalities of femoral bone, and reduced the number of bone marrow adipocytes in DOP rats. EB inhibited excessive bone resorption and inflammation and increased bone formation in DOP rats. EB regulated the OPG/RANKL axis in DOP rats.

Conclusion: EB attenuates STZ-induced DOP in rats by maintaining the balance between bone formation and resorption and inhibiting inflammation through regulating the OPG/RANKL axis.

Keywords: Bone formation; Bone resorption; Diabetes; Epimedin B; Inflammation; OPG/RANKL axis; Osteoporosis.

Fig. 1

EP improves weight loss and blood glucose of DOP rats. (A) Chemical structure of Epimedin B (EB). (B) Change in body weights. (C) Change in blood glucose concentrations. ^*p < 0.05, ^***p < 0.001 vs. Control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. DOP group

Fig. 2

EB prevents bone loss in DOP rats. (A) Three-dimensional images of bone trabecula. (B) Bone mineral density (BMD). (C) Relative bone volume over total volume (BV/TV). (D) Trabecular separation (Tb.Sp). (E) Trabecular number (Tb.N). (F) Maximum load for the proximal femur. ^***p < 0.001 vs. Control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. DOP group

Fig. 3

EB ameliorates histological abnormalities of femoral bone in DOP rats. (A) Representative hematoxylin-eosin staining of femur sections. (B) Representative hematoxylin-eosin staining of bone marrow adipocytes. (C) Quantification of the number of adipocytes per square millimeter. ^***p < 0.001 vs. Control group; ^###p < 0.001 vs. DOP group

Fig. 4

EB inhibits bone turnover and inflammation in DOP rats. (A) ELISA of osteocalcin (OCN) level in the serum. (B) ELISA of bone alkaline phosphatase level in the serum. (C) ELISA of tartrate-resistant acid phosphatase-5b (TRACP-5b) level in the serum. (D) ELISA of IL-6 level in the serum. (E) ELISA of TNF-α level in the serum. (F) ELISA of MCP-1 level in the serum. ^**p < 0.01, ^***p < 0.001 vs. Control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. DOP group

Fig. 5

EB targets the RANKL/OPG axis in DOP rats. (A) RT-qPCR of OPG mRNA level in rat femur. (B) RT-qPCR of RANKL mRNA level in rat femur. (C) Representative images of OPG and RANKL protein bands. (D) Relative protein levels of OPG and RANKL normalized to β-actin. ^***p < 0.001 vs. Control group; ^##p < 0.01, ^###p < 0.001 vs. DOP group