Structural and biomechanical analysis of femurs from mice treated with diclofenac, miR-15b and miR-365

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Abstract
Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat pain, but they have side effects, including the inhibition of bone healing. Diclofenac (DF), a member of the NSAID group, affects bone health adversely. One potential approach to protect bones from the effects of NSAIDs involves the administration of short nucleic acids, such as microRNAs (miRNAs). This study aimed to determine whether two specific miRNAs, miR-15b and miR-365, could mitigate the effects of DF on bone.Methods: We used the C57BL/6J mouse strain and the MC3T3-E1 pre-osteoblast cell line derived from this mouse strain. Female C57BL/6J mice were treated with DF and miR-15b or miR-365 mimics. After euthanising the mice, we analysed their femurs using micro-computed tomography (μCT) and dynamic mechanical analysis (DMA). In addition, we performed experiments in cultured MC3T3-E1 cells, which were transfected with either miR-15b or miR-365. We assessed the relative mRNA levels of osteoblast differentiation markers using real-time PCR.Results: Our findings indicated that miR-15b and miR-365 were effective in reversing the detrimental effects of DF on bone mineral density. DF decreased the bone’s storage modulus (E’), while miR-15b and miR-365 ameliorated this effect. In the preosteoblast MC3T3-E1 cells, DF did not significantly regulate marker genes; however, the administration of miR-15b and miR-365 reduced the gene expression of Runx2, Alp, and Satb2.Conclusions: In summary, the impact of DF on the structural and mechanical properties of bone was not mediated by gene regulation in osteoblasts. However, osteoblasts were responsive to the administration of miR-15b and miR-365.