The Mechanism Of BMSCs-derived Exosomes Alleviate Hyperoxia-induced Lung Injury Via The Manipulation Of Mir-425

Background:Hyperxia-induced lung injury(HILI)is a type of acute lung injury caused by exposure to high oxygen environment,which may further develop into acute respiratory distress syndrome(ARDS).Hyperoxia can lead to proliferation inhibition and apoptosis increase of type II alveolar epithelial cells,resulting in pulmonary histopathological changes and pulmonary edema.Despite the relentless efforts spent in the study about HILI,an explicit understanding of the mechanism of HILI remains elusive.There are currently no effective therapeutic interventions to address this problem,so mechanisms and therapeutic targets of HILI are in extremely urgent need.Previous studies have shown that bone marrow mesenchymal stem cells are a type of pluripotent stem cells that can differentiate into various types of cells to participate in tissue repair.New researchs have reported that bone marrow mesenchymal stem cells-derived exosomes(BMSCs-Exos)played an important role in regulating immunity and tissue repair.MicroRNAs(miRs)are an important type of exosomal content that can be carried by exosomes to participate in the regulation of protein expression and affect cell functions.It has been reported that miR-425 was reduced in the ARDS patient exosomes.miR-425 reduction in lung fibroblasts contributes to the fibrosis through upregulating KDM6A and then activates the TGF-? signaling pathway.However,the role and mechanism of BMSCs-Exos and miR-425 in hyperoxia-induced lung injury remains unclear.Objective:This study explores the role and mechanism of BMSCs-Exos and miR-425 in HILIMethods:1.Take 8-week SD rat bone marrow to isolate bone marrow mesenchymal stem cells(BMSCs),and extract BMSCs-Exos by high-speed centrifugation.The exosomes were extracted from BMSCs treated with exosome inhibitor GW4689 as BMSCs-NC.BMSCs were identified by osteoblast and adipocyte differentiation test and flow cytometry.BMSCs-Exos were identified surface marker protein expression by Western blot,morphology by transmission electron microscopy detection and diameter detection by nano particle size tracking analysis respectively.The total protein volume of BMSCs-Exos and BMSCs-NC isolated by the same amount of BMSCs was assayed by BCA method.After co-culturing exosomes labeled by PKH26 with RLE-6TN of rats ? alveolar epithelial cell line,the uptake of BMSCs-Exos by RLE-6TN cells was observed under confocal microscopy.2.Newborn SD rats were randomly divided into four groups.Normoxia group:reared in a normal environment;Hyperoxia group:reared in a 90%high-oxygen environment for 7 days;BMSCs-NC group:Each tail vein of rats were injected with BMSCs-NC extracted from the same amount of BMSCs,they were kept in a 90%hyperoxic environment for 7 days;BMSCs-Exos group:Each tail veins of rats were injected with BMSCs-Exos extracted from the same amount of BMSCs,and then they were kept in a 90%high oxygen environment for 7 day.HE staining,ratio of the wet lung to the dry lung and Tunel staining were used to evaluate histopathological change,lung tissue edema and cell apoptosis of each SD rats group respectively.The expression level of miR-425 in lung tissues of each SD rats group was evaluated by Q-PCR.Western blot was used to evaluate PI3K and AKT protein phosphorylation expression levels in lung tissues of each SD rats group.3.RLE-6TN was induced by H2O2 to construct a cell model of oxidative stressinjury.RLE-6TN was treated with BMSCs-NC and BMSCs-Exos respectively.Proliferation activity and apoptosis of RLE-6TN cells in each group was evaluated by MTT,flow cytometry and Western blot.The expression level of miR-425 in RLE-6TN cells of each group was evaluated by Q-PCR.4.BMSCs-Exos,BMSCs-Exos-NC(transfection inhibition control group)and BMSCs-Exos-in-miR-425(transfection inhibition miR-425)were treated with H2O2-induced RLE-6TN cells respectively.The expression level of miR-425 in RLE-6TN cells of each group was evaluated by Q-PCR;Proliferation and apoptosis of RLE-6TN cells in each group was evaluated by MTT,flow cytometry and Western blot respectively.5.The target relationship between miR-425 and PTEN(3 '-UTR)was speculated by bioinformatics website analysis.Luciferase reporter assay was used to confirm the target interaction between miR-425 and 3'-untranslated region(3'-UTR)of PTEN gene.BMSCs-NC,BMSCs-Exos,BMSCs-Exos-NC and BMSCs-Exos-in-miR-425 were treated with H2O2-induced RLE-6TN cells,respectively.The expression of PTEN mRNA and protein in RLE-6TN cells of each group was evaluated by Q-PCR and Western blot.6.RLE-6TN cells were transfected with overexpressed PTEN plasmid(oe-PTEN)and its control plasmid(oe-NC)respectively,and then which were treated with H2O2 induction after co-culturing them with BMSCs-Exos.Thus,BMSCs-Exos+oe-PTEN group and BMSCs-Exos+oe-NC group were formed.Proliferation,apoptosis,PI3K and AKT protein phosphorylation expression levels of RLE-6TN cells of the two groups was evaluated by MTT,flow cytometry and Western blot respectively.7.BMSCs-NC,BMSCs-Exos,BMSCs-Exos-NC and BMSCs-Exos-in-miR-425 was treated with H2O2-induced RLE-6TN cells respectively.The phosphorylation levels of PI3K and AKT of RLE-6TN cells in each group was evaluated by Western blot.Results:1.Isolated BMSCs were spindle-shaped,which expressed stem cell tag proteins CD90 and CD 105 and differentiated into osteoblasts and adipocytes in differentiation mediums respectively.The diameter of spherical vesicles secreted by BMSCs was 50-120 nm,which expressesed exosomal-specific proteins CD63 and TSG101.The total of proteins volume from BMSCs-Exos extracted from the same amount of BMSCs were obviously higher than those from BMSCs-NC extracted from the same amount of BMSCs(P<0.05).RLE-6TN cells may uptake BMSCs-Exos.2.Compared with BMSCs-NC group and Hyperoxia group respectively,BMSCs-Exos group significantly reduced the pathological change of hyperoxic lung tissue,pulmonary edema and cell apoptosis in neonatal SD rats;BMSCs significantly increased miR-425 expression levels and PI3K and AKT protein phosphorylation expression levels in lung tissues of SD rats(P<0.05).3.Compared with BMSCs-NC group and H2O2 group respectively,BMSCs-Exos significantly promoted cells proliferation and decreased apoptosis of H2O2-induced RLE-6TN cells;BMSCs-Exos significantly increased miR-425 expression levels of H2O2-induced RLE-6TN cells(P<0.05).4.Compared with BMSCs-Exos and BMSCs-Exos-NC(transfection inhibition control group)respectively,BMSCs-Exos-in-miR-425 group significantly not only increased apoptosis of H2O2-induced RLE-6TN cells,but also inhibited cells proliferation and miR-425 expression level(P<0.05).5.PTEN(3'-UTR)was a direct target of miR-425.Compared with BMSCs-Exos-NC group and BMSCs-Exos group respectively,BMSCs-Exos-in-miR-425 group significantly increased PTEN mRNA and protein expression levels of H2O2-induced RLE-6TN cells(P<0.05).6.Compared with BMSCs-Exos+oe-NC group,BMSCs-Exos+oe-PTEN group not only significantly increased apoptosis of H2O2-induced RLE-6TN cells,but also inhibited cells proliferation and PI3K and AKT phosphorylation expression levels(P<0.05).7.Compared with BMSCs-Exos group and BMSCs-Exos-NC group respectively,BMSCs-Exos-in-miR-425 group significantly decreased PI3K and AKT phosphorylation expression levels of H2O2-induced RLE-6TN cells(P<0.05).Conclusion:BMSCs-Exos delivers miR-425 into type II alveolar epithelial cells and lung tissues to activate the PI3K/AKT signaling pathway by inhibiting PTEN expression,to reduce the apoptosis of type II alveolar epithelial cells and increase cells proliferation,which may be the mechanism that BMSCs-Exos alleviates hyperoxia-induced lung injury.