[Adipose tissue-derived extracellular vesicles ameliorate zoledronic acid-induced functional impairment of bone marrow mesenchymal stem cells: an in vitro study].

Objective: To investigate the effects of adipose tissue-derived extracellular vesicles (AT-EVs) on zoledronic acid (ZA)-induced impairment of proliferation, migration, osteogenic differentiation and apoptosis of bone marrow mesenchymal stem cells (BMSCs), and to preliminarily explore the underlying molecular mechanisms. Methods: AT-EVs were isolated from subcutaneous adipose tissue of 4-week-old male SPF SD rats, and characterized by transmission electron microscopy, nanoparticle tracking analysis and Western blotting; the uptake of AT-EVs by BMSCs was observed. BMSCs derived from the femurs and tibias of rats of the same batch were isolated and cultured, and identified by flow cytometry and tri-lineage differentiation. The working concentration of ZA was screened by cell counting kit-8 (CCK-8). BMSCs were divided into control, ZA (5 μmol/L), and ZA+AT-EVs (5 μmol/L ZA+50 μg/ml AT-EVs) groups; proliferation was evaluated by CCK-8 combined with 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, and migration was evaluated by wound healing and Transwell assays; for the apoptosis assay, 10 μmol/L ZA was applied and detected by flow cytometry after Annexin V/PI staining. The osteogenic differentiation experiment included five groups (Control, Osteogenic induction, Osteogenic induction+AT-EVs, ZA, and ZA+AT-EVs), in which alkaline phosphatase (ALP) staining and Alizarin red S staining were performed, mineralized nodule formation was quantified by the cetylpyridinium chloride decolorization method, and the mRNA expression of Runx2, ALP and OCN was detected by real-time quantitative PCR. Cells from the ZA and ZA+AT-EVs groups after 7 days of osteogenic induction were subjected to liquid chromatography-tandem mass spectrometry-based proteomic analysis, followed by principal component analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. Results: AT-EVs exhibited a cup-shaped bilayer membrane structure with a peak diameter of 116.8 nm, were positive for CD9, CD63 and CD81 and negative for Calnexin, and were efficiently taken up by BMSCs. CCK-8 results showed that 5 μmol/L ZA significantly inhibited BMSCs proliferation (P<0.001), and AT-EVs partially restored the proliferation capacity. The EdU incorporation assay further showed that, compared with the Control group, the EdU-positive rate of the ZA group was significantly decreased (P<0.001), while that of the ZA+AT-EVs group was significantly increased compared with the ZA group (P<0.01), consistent with the CCK-8 results. Compared with the ZA group, the ZA+AT-EVs group showed enhanced BMSCs migration (all P<0.05) and a significantly reduced total apoptosis rate (P<0.001). Western blotting further showed that AT-EVs down-regulated the ZA-induced elevation of cleaved caspase-3 (P<0.001) and BAX (P<0.01) and up-regulated Bcl-2 expression. AT-EVs significantly ameliorated the ZA-induced suppression of BMSCs ALP expression and mineralized nodule formation (P<0.001). qPCR showed that AT-EVs up-regulated the ZA-suppressed expression of Runx2, ALP and OCN (P<0.05), whereas no significant difference was found between the Osteogenic induction+AT-EVs group and the Osteogenic induction group. Proteomic analysis identified 483 differentially expressed proteins (344 up-regulated and 139 down-regulated), which were mainly enriched in steroid biosynthesis, the cGMP-PKG signaling pathway and cytoskeleton regulation pathways. Conclusions: AT-EVs can effectively ameliorate the ZA-induced impairment of proliferation, migration and osteogenic differentiation of BMSCs and reduce apoptosis in vitro, and their effects may be related to the overall down-regulation of steroid biosynthesis pathway-related proteins and the predominant up-regulation of cGMP-PKG signaling pathway-related proteins in the ZA+AT-EVs group.