Investigation of the potential beneficial effects on bone from chrysin, a bioactive phytochemical

The experimental design had three phases: neonatal intervention, no intervention, and adult intervention. During the neonatal period (PND 6-20) the suckling rat pups were randomly assigned into groups, these groups determined what experimental treatments were administered to the rat pups. During the neonatal phase of the experiments, the treatments (10 mL/kg) were given orally every day in the morning (08:00-10:00). All data was reported as mean ± SEM. The data analysis was conducted using GraphPad Prism 9 software (GraphPad Software Inc., California, USA). A one-way ANOVA was employed to compare any differences between means, followed up with the Bonferroni post hoc test for multiple comparisons. Descriptive statistics (means and standard deviations) were performed on all assays.Western blotting was carried out to assess the impact of chrysin on the RANK signalling pathways. Immunofluorescence was conducted to determine how chrysin effects the translocation of nuclear factor kappa B (NFκB) to the nucleus. Micro-computed tomography (microCT) was utilised to assess the tibia bones in Sprague-Dawley rats. The parameters evaluated of the whole bone, length, volume, surface area, bone volume fraction, segmented surface density, and the thickness, number, and spacing of trabecular bone. The parameters evaluated for the midpoint of the bone, volume, area, bone volume fraction, segmented surface density, thickest and thinnest part of the bone midpoint.The IC50 (14µM) was determined in a pilot study performed previously. Western blotting revealed no significant changes in the phosphorylation of mitogen-activated protein kinases (MAPKs), specifically ERK, JNK, and P38, and NFκB signally pathways when treated to chrysin. Immunofluorescence investigation showed a significant inhibition in the amount of NFκB that translocated to the nucleus from the cytoplasm when treated with chrysin, when compared to the positive RANK ligand (RANKL) control. Micro-CT analysis of tibial bones indicated no significant differences in most of the parameters analysed except when an unpaired T-test was performed comparing the control group and the chrysin-treated group. Chrysin significantly increased the area, volume, and segmented bone density of the midpoint of the bones.