Objective To compare the characteristic differences between apoptotic extracellular vesicle (ApoEV) and non-apoptotic cell-derived extracellular vesicle (NCEV) from bone marrow mesenchymal stem cells (BMSCs), and to explore their therapeutic effects on inflammatory macrophages and cisplatin-induced acute kidney injury (AKI) in mice. Methods Staurosporine (STS) was used to induce apoptosis in BMSCs, and the apoptotic model was verified by cell morphology observation, Annexin V/propidium iodide double-staining flow cytometry, and Western blot. NCEV and ApoEV were isolated via differential centrifugation combined with ultracentrifugation, and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. The particle yield and protein yield of extracellular vesicles (EV) per unit donor cell were compared between the two groups. EVs were labeled with DiD, and the uptake of EV by RAW264.7 macrophages was detected using laser scanning confocal microscopy and flow cytometry. Lipopolysaccharide (LPS) was applied to stimulate RAW264.7 cells to establish an in vitro inflammatory cell model. The mRNA expression levels of interleukin 10 (IL-10), arginase 1 (Arg-1), IL-1β, IL-6, IL-18, and tumor necrosis factor α (TNF-α) were determined by real-time fluorescence quantitative PCR. The intracellular reactive oxygen species (ROS) level was measured using 2’, 7’-dichlorodihydrofluorescein diacetate fluorescent probe with laser scanning confocal microscopy and flow cytometry. EVs were labeled with cyanine 7 N-hydroxysuccinimide ester to observe their distribution in major organs and renal tissues of normal mice. A cisplatin-induced AKI mouse model was established, followed by intervention with NCEV or ApoEV. Serum creatinine (Scr), blood urea nitrogen (BUN), renal histopathological injury, kidney injury molecule 1 (Kim-1), cell apoptosis, macrophage infiltration, and DNA damage-related indicators were detected. Based on previously published proteomic data of EV derived from mouse BMSCs, differentially expressed proteins were screened and gene ontology enrichment analysis was performed. Results After STS treatment, BMSCs exhibited typical apoptotic morphology with increased early and late apoptotic rates, alongside upregulated expression of Cleaved Caspase-3 and Bcl-2-associated X protein (Bax). Both NCEV and ApoEV presented spherical or quasi-spherical structures enclosed by a lipid bilayer membrane, with similar particle sizes and Zeta potentials. Bax and tumor susceptibility gene 101 protein were detected in both vesicles, while Cleaved Caspase-3 and Bax were detectable in ApoEV. The particle yield and protein yield of ApoEV were (5.626±0.302)-fold and (2.972±0.124)-fold higher than those of NCEV, respectively (P<0.05). Compared with NCEV, RAW264.7 cells displayed significantly elevated uptake ratio and mean fluorescence intensity for ApoEV (P<0.05). Under LPS stimulation, ApoEV markedly upregulated the mRNA expressions of IL-10 and Arg-1 to levels higher than those in the NCEV treatment group (P<0.05), whereas NCEV significantly suppressed the mRNA expressions of IL-1β and IL-6. Both NCEV and ApoEV reduced intracellular ROS levels, and flow cytometry revealed that the mean 2’, 7’-dichlorofluorescein fluorescence intensity in the ApoEV group was significantly lower than that in the NCEV group (P<0.05). Ex vivo organ imaging demonstrated that the strongest fluorescent signals of both types of EVs appeared in the liver, and obvious signals were also observed in the lung, spleen, and kidney. No significant difference in relative radiation efficiency of each organ was found between the two groups (P>0.05). In cisplatin-induced AKI mice, NCEV significantly reduced serum Scr and BUN levels, while ApoEV significantly decreased serum Scr levels. Both EV preparations alleviated renal tubular histopathological damage and lowered the levels of Kim-1, Bax, TUNEL-positive cells, F4/80-positive macrophages, and serine 139-phosphorylated histone H2AX. No significant intergroup differences in the above renal injury indicators were observed between the two EV intervention groups (P>0.05). Proteomic analysis identified 50 significantly upregulated proteins and 35 significantly downregulated proteins in ApoEV relative to NCEV. The upregulated proteins in ApoEV were mainly enriched in biological processes including extracellular matrix organization, classical complement pathway activation, proteolysis, and angiogenesis. Conclusion ApoEV possess higher production yield, enhanced macrophage uptake capacity and superior anti-inflammatory regulatory activity compared with NCEV. Both ApoEV and NCEV can alleviate renal injury in cisplatin-induced AKI, providing experimental evidence supporting the translational application of ApoEV for AKI treatment.
