Abstract
Spinal cord injury (SCI) causes a significant amount of bone loss, which results in osteoporosis (OP). The neuropeptide substance P (SP) and SP receptors may play important roles in the pathogenesis of OP after SCI. To identify the roles of SP in the bone marrow mesenchymal stem cell derived osteoblasts (BMSC-OB) in SCI rats, we investigated the expression of neurokinin-1 receptors (NK1R) in BMSC-OB and the effects of SP on bone formation by development of BMSC-OB cultures. Sixty young male Sprague-Dawley rats were randomized into two groups: SHAM and SCI. The expression of NK1R protein in BMSC-OB was observed using immunohistochemistry and Western blot analysis. The dose- and time-dependent effects of SP on the proliferation, differentiation and mineralization of BMSC-OB and the expression of osteoblastic markers by in vitro experiments. The expression of NK1R in BMSC-OB was observed on plasma membranes and in cytoplasm. One week after osteogenic differentiation, the expression of NK1R was significantly increased after SCI at mRNA and protein levels. However, this difference was gradually attenuated at 2 or 3 weeks later. SP have the function to enhance cell proliferation, inhibite cell differentiation and mineralization at a proper concentration and incubation time, and this effect would be inhibited by adding SP or NK1R antagonist. The expression of RANKL/OPG was significantly increased in tibiae after SCI. Similarly, the RANKL/OPG expression in SCI rats was significantly increased when treating with 10−8 M SP. SP plays a very important role in the pathogenesis of OP after SCI. The direct effect of SP may lead to increased bone resorption through the RANKL/OPG axis after SCI. In addition, high expression of SP also results in the suppression of osteogenesis in SCI rats. Then, the balance between bone resorption and bone formation was broken and finally osteoporosis occurred.
Highlights
Osteoporosis (OP), one of common complications caused by spinal cord injury (SCI), manifests rapid bone loss [1,2,3], bone-ultrastructural degeneration [4], decline of bone-biomechanical property and increases fracture risk [5, 6]
To confirm our findings regarding neurokinin-1 receptors (NK1R) expression, we performed Western blot analysis using the antibody against NK1R and PCR experiment by specific primers, the results showed that the expression of both the protein and mRNA levels were in consistent with the immunoreactivity at 1 week, but this discrepancy disappeared gradually at 2 weeks and 3 weeks after osteogenic differentiation (Fig 1D and 1E)
As a widely distributed neurotransmitter and neuromodulator, substance P (SP) may plays an important role in bone formation of osteoblasts which needs to be further discussed [24], especially whether SP receptor is present in osteoblasts remains considerably controversial
Summary
Osteoporosis (OP), one of common complications caused by spinal cord injury (SCI), manifests rapid bone loss [1,2,3], bone-ultrastructural degeneration [4], decline of bone-biomechanical property and increases fracture risk [5, 6]. The mechanism of SCI-induced OP is poorly elucidated. Immobilization was generally considered to be the main cause of SCIinduced bone loss [7,8,9], but current studies suggested that the mechanisms of OP induced by SCI are more complicated and neural lesion itself may be involved in the bone loss [10, 11]. Compared to disuse OP, the levels of bone resorption markers are significantly increased in SCI-induced OP. The sites and rate of bone loss in SCI-induced OP are different from that in disuse OP [12, 13]. Understanding the pathogenesis of SCI-induced OP will be benefit in the consideration of new treatment strategies [15, 16]
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