Inhibition of substance P signaling aggravates the bone loss in ovariectomy-induced osteoporosis

Introduction: Substance P signaling regulates the functions of both osteoblast and osteoclast. Available reports on the effects of substance P on bone mass are contradictory. The objective of this study was to determine the change of substance P expression in the osteoporotic bone of OVX mice. The effects of substance P signaling blockade by using its specific receptor antagonist L-703606 on bone remodeling in sham-operated mice and OVX mice were also investigated. Methods: Forty-eight nine-week-old female C57BL/6J mice were evenly distributed into three groups with sham surgery, OVX or OVX with estrogen replacement. Substance P expression in the bones of each group of mice was evaluated by immunohistochemistry and enzyme immunoassay. Another thirty-two nine-week-old female C57BL/6J mice were divided into a SHAM group (sham surgery followed by vehicle treatment with DMSO), a SHAM + L group (sham surgery followed by 15 mg/kg/d L-703606 repeated intraperitoneal injections), an OVX group (ovariectomy with the same vehicle treatment) and an OVX + L group (ovariectomy with the same L-703606 injections), with 8 mice in each group. Treatment started 3 weeks after surgery and last for 3 weeks. A 2 x 2 factorial experimental design was used to detect the effects of substance P signaling blockade on bone remodeling in sham-operated mice and OVX mice. Techniques including micro-computed tomography, biomechanical testing, histomorphometric analysis, enzyme immunoassay, and real-time PCR were employed. Results: Immunohistochemistry and enzyme immunoassay revealed that substance P expression significantly decreased in the bones of OVX mice both at 3 weeks and 6 weeks after surgery. Micro-CT tomography demonstrated that application of L-703606 led to bone loss in sham-operated mice, and aggravated the micro-structural deterioration of bones in OVX mice. This was shown by reduced BV/TV (Mean bone volume fraction), Tb.N (Mean trabecular number) and Tb.Th (Mean trabecular thickness), and increased Tb.Sp (Mean trabecular separation). Biomechanical analysis demonstrated that blockade of substance P signaling reduced the maximum stress and maximum load of L3 vertebrae and tibiae. Inhibited recruitment of bone mesenchymal stem cells (BMSCs) to bone remodeling sites, which was evidenced by increased number of osteoclasts, decreased number of osteoblasts and increased osteoid volume in the secondary spongiosa, was observed in the mice treated with L-703606. A significant decrease of OPG/RANKL ratio was also found in the bones of mice treated with L-703606. Body weight, uterine weight and serum estradiol level were not significantly different between the mice treated with L-703606 and those treated with vehicle. Conclusion: The results demonstrated that blocking substance P signaling led to bone loss in sham operated mice, and exacerbated the bone loss in OVX mice. Substance P signaling had an important role in the maintenance of bone mass. (C) 2016 Elsevier Ltd. All rights reserved.