Effects of remifemin treatment on bone integrity and remodeling in rats with ovariectomy-induced osteoporosis

Abstract

This study aims to evaluate the effects of Remifemin (isopropanolic extract of Cimicifuga Racemosa) on postmenopausal osteoporosis. 120 female Sprague-Dawley rats were randomly assigned to four groups: sham surgery with vehicle, ovariectomy with vehicle, ovariectomy with estradiol valerate, or ovariectomy with Remifemin. Daily oral administrations of the vehicle, estradiol valerate, or Remifemin began 2 weeks after surgery and lasted to 4, 8, or 12 weeks. Ten rats in each group were sacrificed at each timestep with assessment of bone mineral density, trabecular bone structure, and biomechanical parameters of the femur and lumbar vertebra. Bone turnover markers were evaluated 12 weeks after surgery. Both drugs prevented bone density loss in the distal end of the femur and preserved the trabecular bone structure in both the lumbar vertebra and distal end of the femur following ovariectomy. Both drugs protected bone stiffness at the tested regions and reduced bone reabsorption in ovariectomized rats. The preventive effects of Remifemin against bone-loss can rival those of estradiol valerate if treatment duration is adequately extended. In conclusion, Remifemin may demonstrate equivalent effects to estradiol valerate in terms of preventing postmenopausal osteoporosis.

Figure 1. The BMD of lumbar vertebrae 2–4 (L2-L4) of the four groups.

Comparison of bone mineral density (BMD) of lumbar vertebrae 2–4 (A:L2, B:L3, C:L4) in SHAM, OVX+NS, OVX+E and OVX+iCR groups at 4-, 8- and 12-week postoperation. Data represents the means ± standard deviation (SD ), n = 10 rats in each group. The x-axis represents time after surgery (weeks). Compared with SHAM group, a means p<0.05, b means p<0.01; Compared with OVX+NS group, c means p<0.05, d means p<0.01; Comparison between OVX+iCR and OVX+E group, e means p<0.05, f means p<0.01.

Figure 2. The BMD of femoral proximal and distal ends of the four groups.

Comparison of bone mineral density (BMD) of femoral proximal ends (A) and distal ends (B) in SHAM, OVX+NS, OVX+E and OVX+iCR groups at 4-, 8- and 12-week postoperation. Data represents the means ± standard deviation (SD ), n = 10 rats in each group. The x-axis represents time after surgery (weeks). Compared with SHAM group, a means p<0.05, b means p<0.01; Compared with OVX+NS group, c means p<0.05, d means p<0.01; Comparison between OVX+iCR and OVX+E group, e means p<0.05, f means p<0.01.

Figure 3. Comparisons of the morphometric indicators of L3 among the four groups.

Comparison of bone morphometric indicators (relative bone volume (BV/TV), trabecular number (TbN), trabecular spacing (TbSp), and trabecular thickness (TbTh)) of the third lumbar vertebrae (L3) (A-D) in SHAM, OVX+NS, OVX+E and OVX+iCR groups at 4-, 8- and 12-week postoperation. Data represents the means ± standard deviation (SD), n=6 rats in each group. The x-axis represents time after surgery (weeks). Compared with SHAM group, a means p<0.05, b means p<0.01; Compared with OVX+NS group, c means p<0.05, d means p<0.01; Comparison between OVX+iCR and OVX+E group, e means p<0.05, f means p<0.01.

Figure 4. Comparisons of the morphometric indicators of the femoral distal end among the four groups.

Comparison of bone morphometric indicators (relative bone volume (BV/TV), trabecular number (TbN), trabecular spacing (TbSp), and trabecular thickness (TbTh)) of the femoral distal ends (A-D) in SHAM, OVX+NS, OVX+E and OVX+iCR groups at 4-, 8- and 12-week postoperation. Data represents the means ± standard deviation (SD), n=6 rats in each group. The x-axis represents time after surgery (weeks). Compared with SHAM group, a means p<0.05, b means p<0.01; Compared with OVX+NS group, c means p<0.05, d means p<0.01; Comparison between OVX+iCR and OVX+E group, e means p<0.05, f means p<0.01.

Figure 5. H&E staining of trabecular bone from the left femoral distal ends at 12-week postoperation.

Histological sections (stained with H&E) of trabecular bone from the left femoral distal ends of the four groups at 12 weeks after surgery. The OVX+NS group rats showed sparse and thinning trabeculae, with loss of connectivity and presence of abundant adiposity cells, while estradiol valerate and Remifemin treatment significantly protected trabecular bone from ovariectomy-induced osteopenia.

Figure 6. Changes in biomechanical parameters at L3 and the femoral distal end among the four groups.

Biomechanical changes in bone stiffness and bone strength at L3 (A-B) and the femoral distal end (C-D) in SHAM, OVX+NS, OVX+E and OVX+iCR groups at 4-, 8- and 12-week postoperation. Data represents the means ± standard deviation (SD), n = 6 rats in each group. The x-axis represents time after surgery (weeks). Compared with SHAM group, a means p<0.05, b means p<0.01; Compared with OVX+NS group, c means p<0.05, d means p<0.01.

Figure 7. Comparison of TRACP 5b levels among the four groups at 12-weeks postoperation.

The serum tartrate-resistant acid phosphatase 5b (TRACP 5b) level of SHAM, OVX+NS, OVX+E and OVX+iCR groups at 12-week postoperation were determined using ELISA kit. Data represents the means ± standard deviation (SD), n=10 rats in each group. Compared with SHAM group, a means p<0.05, b means p<0.01; Compared with OVX+NS group, c means p<0.05, d means p<0.01.