间充质干细胞外泌体通过抑制氧化应激与炎症减轻放射性肺损伤OA

OBJECTIVE To investigate the protective effect of mesenchymal stem cell-derived exosomes(MSC-Exo)against radiation-induced lung injury(RILI)and the underlying mechanisms.METHODS ①MSC-Exo were isolated from the culture supernatant of human umbilical cord mesenchymal stem cells via ultracentrifugation and characterized by transmission electron microscopy and nanoparticle tracking analysis.Western blotting was used to detect the expressions of exosomal markers(heat shock protein 70,tumor susceptibility gene 101,and cluster of differentiation 9 and Calnexin).② In vivo:Male C57BL/6J mice were randomly divided into control,irradiation(IR),and IR+MSC-Exo groups.Mice in the IR and IR+MSC-Exo groups received a single dose of 20 Gy γ-ray whole-thorax irradiation.The IR+MSC-Exo group was administrated with MSC-Exo(1×1012 particles·kg-1)via tail vein injection on 0,3,7,10,14 days after irradiation.Lung tissues were collected on 7,28,and 140 days post-irradiation for histopatho-logical examination by hematoxylin-eosin and Masson's trichrome staining.Bronchoalveolar lavage fluid(BALF)was collected on 7 and 28 days to measure the levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),IL-18,and interferon-γ(IFN-γ)by ELISA.On 7 days post irradiation,F4/80 and IL-1β were detected by immunofluorescence staining while the superoxide dismutase(SOD)activity,reduced glutathione(GSH)content,and malondialdehyde(MDA)level were measured using commercial kits.mRNA expressions of TNF-α,IL-1β,IL-4 and IL-10 were determined by RT-qPCR while the phosphory-lation of nuclear factor-κB p65(NF-κB p65)was analyzed by Western blotting.On 140 days post-irradi-ation,E-cadherint and Vimentin were detected by immunofluorescence staining,mRNA expressions of α smooth muscle actin(α-SMA)and collagen typeⅠalpha 1 chain(Col1a1)were detected by RT-qPCR.③ In vitro:Mouse lung epithelial(MLE-12)cells were divided into control,IR,and IR+MSC Exo groups.Cells in the IR and IR+MSC Exo groups were exposed to a single dose of 10 Gy γ-ray irradiation.The IR+MSC Exo group was treated with MSC Exos(5×1012 particles·L-1)after irradiation.At 24 h after treatment,the SOD activity,GSH content,and MDA level were measured.Cell viability was assessed by CCK-8 assay,mRNA levels of TNF-α,IL-1β,IL-4 and IL-10 were detected by RT-qPCR,and NF-κB p65 phosphorylation was analyzed by Western blotting.RESULTS ① The isolated MSC-Exo appeared round or cup-shaped with a diameter of 70-150 nm,expressed HSP70,TSG101 and CD9,and were negative for calnexin.② In vivo:Compared with the control group,pathological injury and collagen deposition were pronounced in lung tissues of irradiated mice,BALF levels of IFN-γ,TNF-α,IL-18 and IL-1β increased,the SOD activity and GSH content decreased,but the MDA level elevated.The mRNA expressions of TNF-α and IL-1β,the level of F4/80+and IL-1β were upregulated.The level of E-cadherint was decreased,the level of Vimentin was increased,the level of NF-κB p65 phosphoryla-tion,and the expressions of α-SMA and Col1a1 were upregulated.Compared with the IR group,MSC-Exo treatment significantly ameliorated pulmonary pathological changes,suppressed collagen deposition,reduced BALF levels of IFN-γ,TNF-α,IL-18 and IL-1β,restored the SOD activity and GSH content,low-ered MDA levels,downregulated TNF-α and IL-1β,alleviated F4/80 and IL-1β level,increased E-cad-herint level,decreased Vimentin level,inhibited NF-κB p65 phosphorylation and EMT,and decreased α-SMA and Col1a1 expressions.③ In vitro:Compared with the control group,the IR group showed decreased cell viability,reduced SOD activity and GSH content,an elevated MDA level,upregulated TNF-α and IL-1β mRNA,and increased NF-κB p65 phosphorylation.MSC-Exo treatment significantly restored cell viability,elevated SOD activity and GSH content,reduced the MDA level,suppressed TNF-α and IL-1β mRNA expressions,and inhibited NF-κB p65 phosphorylation.CONCLUSION MSC-Exo may exert protective effects against RILI by regulating NF-κB p65 phosphorylation,preventing exces-sive activation of the NF-κB pathway,inhibiting oxidative stress and inflammation and alleviating fibrosis.