AKI血清预处理BMSCs外泌体减轻HK-2细胞缺氧/复氧损伤的体外研究OA

Objective The aim of this study was to investigate the protective effect of exosomes derived from acute kidney injury(AKI)serum-preconditioned bone marrow mesenchymal stem cells(AKI-Exos)on hypoxia/reoxygenation(H/R)-induced injury in Human Kidney-2(HK-2)cells,and to determine whether this protection is mediated through inhibition of pyroptosis.Methods HK-2 cells were treated with different concentrations of cobalt chloride(CoCl₂).The CCK-8 assay was employed to screen for the appropri-ate concentration and duration to establish an in vitro hypoxia/reoxygenation(H/R)injury model.The experiment included the follow-ing groups:a blank control group,an H/R model group,an H/R+normal exosomes(Nor-Exo)group,and an H/R+AKI serum-preconditioned exosomes(AKI-Exo)group.Bone marrow mesenchymal stem cells were preconditioned with either normal fetal bovine serum or rat AKI serum,and Nor-Exos and AKI-Exos were isolated by ultrafiltration.The morphology and phenotype of exosomes were characterized by transmission electron microscopy,nanoparticle tracking analysis,and Western blotting for exosomal markers Alix,CD63 and TSG101.Uptake of exosomes by HK-2 cells was observed using fluorescent labeling.ELISA was employed to detect IL-1β and IL-18 levels in the cell culture supernatant of each group.Western blotting was used to assess the expression of pyroptosis-related proteins NLRP3,GSDMD,and IL-1β.Results Nor-Exos and AKI-Exos were successfully prepared and identified.Both exhibited typical bilayer membrane vesicular morphology,with particle sizes mainly within the exosomal range,expressed exosomal markers Alix,CD63,and TSG101,and could be internalized by HK-2 cells.Compared with the blank control group,H/R treatment markedly reduced HK-2 cell viability,significantly increased IL-1β and IL-18 levels in the supernatant,and upregulated the expression of pyroptosis-related proteins NLRP3,GSDMD,and IL-1β.Compared with the H/R group,both the H/R+Nor-Exo group and H/R+AKI-Exo group showed restored cell viability,reduced IL-1β and IL-18 levels,and decreased expression of NLRP3,GSDMD,and IL-1β to varying degrees,with a more pronounced improvement observed in the AKI-Exo group.Conclusion Exosomes derived from AKI serum-preconditioned bone marrow mesenchymal stem cells can attenuate H/R-induced injury and inflammatory responses in HK-2 cells by suppressing pyroptosis.These findings suggest that AKI-Exos may represent a potential therapeutic strategy for ischemia-reperfusion-related acute kidney injury.