Abstract
Purpose: To investigate the effect of methylbenzoxime on dexamethasone-induced rat model of osteoporosis.
 Methods: Osteoporosis rat model was prepared by administration of dexamethasone to rats for sixty days. The rats were then divided into five groups of five animals each: normal control, untreated, and 2, 5 and 10 mg/kg treatment groups. All rats were administered dexamethasone for 60 days. Thereafter, rats in the three treatment groups received daily doses of 2, 5 or 10 mg/kg methylbenzoxime for 15 days, while rats in normal control and untreated groups were given equivalent volumes of normal saline in place of methylbenzoxime. After treatment, the rats were sacrificed, and the femur removed for histological assessment of pathological changes using H&E staining. Expressions of Wntn signalling pathway proteins in osteoblasts were assayed using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot assays.
 Results: Methylbenzoxime inhibited osteoblast proliferation, as revealed from 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It increased the expression of osteoprotegerin and downregulated receptor activator for nuclear factor-kappa B ligand. Dexamethasone decreased the expression of Wnt signalling pathway proteins in osteoblasts. However, treatment of the dexamethasone-exposed osteoblasts with methylbenzoxime reversed the inhibition of expressions of Wnt signalling pathway proteins. In vivo studies showed that methylbenzoxime treatment mitigated dexamethasone-induced pathological features in femur. In osteoporotic rats, methylbenzoxime significantly up-regulated the expression of osteocalcin but down-regulated the level of collagen-type I fragments, relative to the untreated group. The effect was significant in the 5 and 10 mg/kg treatment groups, when compared with 2 mg/kg group.
 Conclusion: Methylbenzoxime prevents dexamethasone-induced osteoporosis in vitro and in rats. Therefore, it is a potential therapeutic agent for the management of osteoporosis.
Highlights
Osteoporosis constitutes a medical as well as socio-economic problem characterised by decreased mechanical strength of bones which render them more prone to fracture [1]
Dexamethasone-exposed cells were treated with methylbenzoxime at doses of 5, 10, 15, 20, 25 and 30 μM for 48 h and viability was analysed by MTT assay (Figure 1)
These findings suggest that methylbenzoxime prevents dexamethasone-induced inhibition of primary osteoblast viability
Summary
Osteoporosis constitutes a medical as well as socio-economic problem characterised by decreased mechanical strength of bones which render them more prone to fracture [1]. The growth and development of various tissues (including the bones) are regulated by the activation of Wnt/β-catenin signalling pathway [4]. Up-regulation of Wnt/β-catenin signalling pathway is of immense significance for treatment of osteoporosis. Control group of rats received equal volume of normal saline and those in the treatment groups were injected subcutaneously 0.1 mg/kg dexamethasone and 2, 5 or 10 mg/kg doses of methylbenzoxime daily for 60 days. Analysis of the activity of alkaline phosphatase in rat serum was performed using the commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of manufacturer. Determination of the expression of osteocalcin and collagen type I fragment in rat serum was performed commercially available kits (USCN Life Science, Wuhan, China) in accordance with the instructions of manufacturer.
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