Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis

Abstract

Background: Alamandine is a newly characterized peptide of renin angiotensin system. Our study aims to investigate the osteo-preservative effects of alamandine, explore underlying mechanism and bring a potential preventive strategy for postmenopausal osteoporosis in the future.

Methods: An ovariectomy (OVX)-induced rat osteoporosis model was established for in vivo experiments. Micro-computed tomography and three-point bending test were used to evaluate bone strength. Histological femur slices were processed for immunohistochemistry (IHC). Bone turnover markers and nitric oxide (NO) concentrations in serum were determined with enzyme-linked immunosorbent assay (ELISA). The mouse embryo osteoblast precursor (MC3T3-E1) cells were used for in vitro experiments. The cell viability was analysed with a Cell Counting Kit‑8. We performed Alizarin Red S staining and alkaline phosphatase (ALP) activity assay to observe the differentiation status of osteoblasts. Western blotting was adopted to detect the expression of osteogenesis related proteins and AMP-activated protein kinase/endothelial nitric oxide synthase (AMPK/eNOS) in osteoblasts. DAF-FM diacetate was used for semi-quantitation of intracellular NO.

Results: In OVX rats, alamandine alleviated osteoporosis and maintained bone strength. The IHC showed alamandine increased osteocalcin and collagen type I α1 (COL1A1) expression. The ELISA revealed alamandine decreased bone turnover markers and restored NO level in serum. In MC3T3-E1 cells, alamandine promoted osteogenic differentiation. Western blotting demonstrated that alamandine upregulated the expression of osteopontin, Runt-related transcription factor 2 and COL1A1. The intracellular NO was also raised by alamandine. Additionally, the activation of AMPK/eNOS axis mediated the effects of alamandine on MC3T3-E1 cells and bone tissue. PD123319 and dorsomorphin could repress the regulating effect of alamandine on bone metabolism.

Conclusion: Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.

Keywords: AMPK/eNOS axis; Alamandine; MrgD; Ovariectomy-induced osteoporosis; Renin angiotensin system.

Fig. 1

Animal protocol. Experimental grouping: Sham group, OVX group, OVX + alamandine group and OVX + alamandine + PD123319 group (a). Experimental timeline and the schematic diagram of tissue collection and detection (b)

Fig. 2

Representative Micro-CT three-dimensional images of trabecular bone microarchitecture with quantitative results of Micro-CT and three-point bending test. Sagittal and transverse planes of the femur were visualized. A region of interest (ROI) with 1.5 mm height was chosen starting 0.4 mm from the lowest end of the growth plate to the proximal end of the femur. Three-dimensional images of the right side distal femurs (a) and the trabecular bone microarchitecture (b). The scale bars represent 1 mm. Quantitative results of Micro-CT analysis expressed as BMD (c), BV/TV (d), Tb.N (e), Tb.Th (f), Tb.Sp (g) and SMI (h). Sample size n = 6 specimens/group. Quantitative results of the mechanical properties (i–n). The histograms are ultimate load (i), ultimate displacement (j), energy to failure (k), stiffness (l), bending stress (m) and bending strain (n) of the femur diaphysis (cortical bone). Sample size n = 4 specimens/group. ***: P < 0.001, **: P < 0.01, *: P < 0.05

Fig. 3

Effects of different treatment on the bone tissue and bone turnover markers of model rats. OCN (a) and COL1A1 (b) expression were detected with the sections of proximal femur (femoral head). The magnifications are × 1.5 (left parts) and × 10 (right parts), scale bars represent 1 mm and 100 μm respectively. Positive area (%) fold of Sham was semi-quantified by Image J software (c, d). Sample size n = 5 specimens /group. Serum concentrations of OCN (e) and CTX-I (f) were detected. Sample size n = 4 specimens/group. ***: P < 0.001, **: P < 0.01, *: P < 0.05

Fig. 4

Effects of diverse intervention on cell viability and osteogenic differentiation of MC3T3-E1 cells. Cell viability under different concentrations of alamandine (a), PD123319 and compound C with 100 nM alamandine respectively (b, c). Sample size n = 6 wells/group. ARS staining after osteogenic induction for 21 days (d, e). ALP staining after osteogenic induction for 14 days (f, g). The magnification is × 10 and the scale bars represent 100 μm (e, g). Mineralized area (%) and ALP staining positive area (%) were used to semi-quantify the osteogenic mineralization capacity and ALP activity respectively (h, i). Sample size n = 5 images/group. ***: P < 0.001, **: P < 0.01, ns: P > 0.05

Fig. 5

Effects of various interference on protein expression of MrgD, osteogenesis and AMPK/eNOS, and intracellular NO generation in MC3T3-E1 cells. The expression of MrgD (a, b). Representative images of osteogenic proteins (RUNX2, OPN and COL1A1) expression (a, c–e). The expression of pho-eNOS and eNOS (a, f). The expression of AMPKα and its phosphorylated form (a, g). The gels were cropped, the samples derive from the same experiment and the gels/blots were processed in parallel. The images of phosphorylated form and total amount of AMPKα and eNOS were obtained from the same membranes which were stripped and re-probed respectively. Semi-quantitation of intracellular NO was performed by DAF-FM diacetate (h, i). Sample size n = 3 images/group. The magnification is × 40 and the scale bars represent 10 μm. ***: P < 0.001, **: P < 0.01, *: P < 0.05, ns: P > 0.05

Fig. 6

Effects of different treatment on the bone tissue and serum NO of model rats. pho-AMPKα (a) and pho-eNOS (b) expression were detected with the sections of proximal femur (femoral head). The magnifications are × 1.5 (left parts) and × 10 (right parts), scale bars represent 1 mm and 100 μm respectively. Positive area (%) fold of Sham was semi-quantified by Image J software (c, d). Sample size n = 5 specimens /group. Serum concentration of NO (e) was detected. Sample size n = 4 specimens/group. ***: P < 0.001, *: P < 0.05

Fig. 7

Classical vs. protective arm of the renin-angiotensin system (a). Alamandine combined with its receptor MrgD, attenuates OVX-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis (b)