Fig. 5: ALE down-regulates AB-EVs release and attenuates bone-fat imbalance and VD3-induced vascular calcification in aged OVX mice.

a Total protein contents of EVs isolated from the conditioned media of osteoclasts treated with solvent (OC-CM), ALE (OC^ALE-CM), AB (OC^AB-CM), or AB + ALE (OC^AB+ALE-CM). n = 5 biologically independent samples per group. b–d, Quantification of the percentages of ARS⁺ (b) and ORO⁺ (c) areas in BMSCs with different treatments under osteogenic or adipogenic induction, or ARS⁺ areas (d) in VSMCs with different treatments under osteogenic induction. n = 5 biologically independent cells per group. e–f μCT-reconstructed images of femurs from 16-month-old Sham or OVX mice in different groups (e) and quantification of Tb. BV/TV, Tb. N, Tb. Th, and Tb. Sp (f). Scale bars: 1 mm. n = 5 biologically independent animals per group. g–h PLIN immunofluorescence staining images of femur sections (g) and quantification of the number of PLIN⁺ (red) adipocytes in bone marrow (h). Scale bar: 100 μm. n = 5 biologically independent animals per group. i qRT-PCR for Pparγ expression in femurs. n = 5 biologically independent animals per group. j–l OCN immunostaining images (j), quantification of the number of OCN-stained (brown) osteoblasts on BS (k), and ELISA for OCN (l). Scale bar: 50 μm. n = 5 biologically independent animals per group. m–n ARS staining images (m) and quantification of the percentage of ARS⁺ areas (red; n). Scale bar: 200 μm. n = 5 biologically independent animals per group. o Vascular calcium content measurement. n = 5 biologically independent animals per group. p–q, RUNX2 immunostaining images (p) and quantification of the percentage of RUNX2⁺ (red) areas (q). Scale bar: 200 μm. n = 5 biologically independent animals per group. r qRT-PCR for Alpl expression. n = 5 biologically independent animals per group. Experiments in b–d were repeated independently three times with similar results. The illustrated results represented one of the three independent experiments. The other experiments were performed with at least five biological replicates per group without independent repetition. Data were presented as mean ± SD. Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test. Source data are provided as a Source Data file.