The Role Of Alveolar Macrophage Autophagy Regulated By BMSC Exosomes-Mediated MiR-384-5p/Beclin-1 Signaling Pathway In Modulation Of Acute Lung Injury

Background and objective Acute lung injury(ALI)is common critical disease that may develop into highly lethal acute respiratory distress syndrome(ARDS),with the mortality reached as high as 40%.The pathogenesis of ALI is incontrollable inflammation.Alveolar macrophages play a key role in modulation of inflammatory response in ALI.There is a close connection between autophagy and inflammation.Beclin-1,which is one of the most important autophgy-associated proteins,plays a vital role in regulating the connection of autophagy,apoptosis and inflammation.Our previous study found that the protein level of Beclin-1 in alveolar macrophages was increased in a dose-dependent and time-dependent manner after alveolar macrophages were treated with LPS,indicating that Beclin-1 mediated autophagy was involved in the inflammatory response of alveolar macrophages.Exosomes are small membrane vesicles(40-100nm),merging their membrane contents into the recipient cell membrane and delivering effectors including transcription factors,oncogenes,small and large non-coding regulatory RNAs(such as microRNAs(miRNAs)),mRNAs and infectious particles into recipient cells.Recent studies have found that bone marrow mesenchymal stem cell(BMSC)-derived exosomes are expected to treat ALI/ARDS.However,little is known regarding the role of exosomes in alveolar macrophages,and therapeutic mechanisms of exosomes in ALI need further exploration.In this study,we aim to investigate the role of miR-384-5p in the inflammatory response of ALI/ARDS,and confirm the molecular mechanism of alveolar macrophage autophagy regulated by exosomes-mediated miR-384-5p/Beclin-1 signaling pathway,from molecular,cellular,tissue to whole animal level.The presentstudy may lay a foundation for revealing the pathogenesis and progression of ALI/ARDS from the new viewpoint of alveolar macrophage autophagy dysfunction,and may also provide a novel concept for the prevention and treatment of ALI/ARDS.Methods 1.BMSCs were isolated from rat bone marrow using the method of tissue adherent culture,and identified by flow cytometric analysis.A clone was selected after subjected to osteogenic,chondrogenetic,and adipogenic assays to confirm the multidirectional differentiation potential of BMSCs.The ability of proliferation was evaluated by clone formation assay.BMSC-derived exosomes were purified using ultracentrifugation and Total Exosome Isolation Kit,and identified by nanoparticle trafficking analysis(NTA),transmission electron microscopy(TEM)and Western Blot using Alix,CD63 and CD9 as markers.Exosomes were tracked by using Exosome Labeling Kits in vitro and CM-DiI in vivo.2.In vitro,rat alveolar macrophage line NR8383 cells were cultured and divided into four experimental groups as follows:(1)control: alveolar macrophages were cultured under normal condition,(2)LPS: cells were treated with 10μg/mL LPS,(3)LPS+non-Ex: cells were treated with exosome-depleted BMSC-conditioned media in the presence of 10μg/mL LPS,(4)LPS+Ex: cells were co-cultured with BMSC-derived exosomes in the presence of 10μg/mL LPS.Cell viability was determined with Cell Counting Kit-8 assay.Apoptotic ratio was determined with TUNEL staining and Annexin V-FITC/PI double staining.The inflammatory cytokines were measured by ELISA.The levels of miR-384-5p in exosomes and alveolar macrophages were measured by RT-qPCR.The levels of autophagy-associated genes were measured by RT-qPCR,Western Blot and immunofluorescent staining.Autophagy was observed by TEM and assessed by means of the mRFP-GFP-LC3 adenovirus transfection assay.3.In vitro,alveolar macrophages were transfected with miR-384-5p mimic,miR-384-5p inhibitor or the corresponding negative control.The levels of miR-384-5p and Beclin-1 were measured by RT-qPCR and Western Blot.RatBeclin-1 3’-UTR sequences were retrieved from the Entrez Nucleotide database.The potential miRNA binding site in the Beclin-1 3’-UTR was predicted by TargetScan and miRanda.The direct binding effect of miR-384-5p and Beclin-1 3’-UTR was determined with luciferase reporter assay.4.In vivo,adult male Sprague-Dawley rats were used in the experiments.ALI model was induced by the intratracheal(IT)instillation of a nonlethal dose of LPS at 5mg/kg dissolved in 100μL PBS.The treatment groups of ten rats each,which were given simultaneously,were:(1)Sham: 100μL PBS IT,(2)Sham+Ex(V): 4 h later after PBS infusion,50μL exosomes were intravenously(IV)injected through caudal vein,(3)Sham+Ex(T): 4 h later after PBS infusion,50μL exosomes IT,(4)LPS: LPS at 5mg/kg dissolved in 100 μL PBS IT to induce ALI,(5)LPS+Ex(V): 4 h later after LPS treatment,50μL exosomes IV,(6)LPS+Ex(T): 4 h later after LPS treatment,50μL exosomes IT.The lung histological pathology was determined by HE staining.Pulmonary vascular permeability was detected by wet-to-dry ratio and protein concentration in BALF.Inflammatory response was assessed by neutrophil counts and levels of inflammatory cytokines in BALF and serum.Rats were followed up for 7 days to evaluate survival rate.The levels of miR-384-5p in lung tissues were measured by RT-qPCR.The levels of Beclin-1 were measured by RT-qPCR,Western Blot and immunohistochemistry.Results 1.The BMSCs presented as long spindle-shaped fibrocyte-like adherent cells.Flow cytometric analysis demonstrated that BMSCs expressed high levels of CD29,CD44,CD90,and CD105,but were negative for CD11b/c,CD34,and CD45.BMSCs had the ability of proliferation and could differentiate into osteocytes,chondrocyte and adipocytes.BMSC-derived exosomes were obtained from the culture medium of BMSCs.NTA estimated the size of the exosomes was between 63 and 269 nm,and the proper concentration was 1.95×109±0.21×109 particles per mL.TEM showed that the particles were revealed as round-shaped vesicles with double layer membrane structure and diameters about 90 to 100 nm.The protein levels of markers Alix,CD63and CD9 were measured with Western Blot,all of the three markers could be detected in the exosomes.Exosomes were taken up by alveolar macrophages in vitro and assembled in the injured rat lung tissues in vivo.2.Cell Counting Kit-8 assay showed that cell viability decreased prominently after LPS treatment compared with the control group,and improvements in cell viability were observed in the LPS+Ex group.TUNEL and Annexin V-FITC/PI double staining indicated that exosomes could effectively prevent LPS-induced apoptosis.ELISA indicated that exosomes could reduce TNF-a,IL-1β and IL-6 elevation,and promoted IL-10 production.RT-qPCR and Western Blot showed that the exosomes had effects on increasing miR-384-5p levels and decreasing Beclin-1 expression in the alveolar macrophages treated with LPS.TEM showed that exosomes strongly reduced LPS-induced cell autophagy as evidenced by a dramatically decreased number of autophagic vacuoles compared with the LPS group.mRFP-GFP-LC3 assays showed that autophagosome and autolysosome puncta formation were increased in the LPS group compared the control group.Conversely,exosomes attenuated autophagosome and autolysosome puncta formation,indicating that exosomes could stabilize autophagy flux of alveolar macrophages exposed to LPS.3.RT-qPCR and Western Blot showed that miR-384-5p mimic upregulated miR-384-5p expression and reduced Beclin-1 level,whereas the inhibitor downregulated miR-384-5p expression and increased Beclin-1 level.Bioinformatics predicted the 3’-UTR of Beclin-1 mRNA had binding sites for miR-384-5p.Luciferase reporter assay demonstrated that Beciln-1 was one of target genes of miR-384-5p.4.HE staining revealed exosomes attenuated LPS-induced lung injury as shown by histopathology.Exosomes decreased wet-to-dry ratio and protein concentration in BALF.Exosomes attenuated LPS-induced increase in neutrophil counts in BALF.ELISA showed that exosomes reduced TNF-a,IL-1β and IL-6 elevation,and promoted IL-10 production in BALF and serum.Survival analysis indicated that exosomes improved the survival rate of ALI rats within 7 days.RT-qPCR showed that exosomes increased miR-384-5p levels in lung tissues.RT-qPCR,Western Blot andimmunohistochemistry indicated that Beclin-1 mRNA and protein levels were increased in the LPS group,while exosomes could weaken LPS-induced Beclin-1 elevation.In addition,intratracheal injection of exosomes was tended to be more beneficial than intravenous injection on the improvement of ALI.Conclusion 1.BMSCs were specifically isolated and purified by the method of tissue adherent culture.Exosomes were successfully purified from BMSCs conditional medium using ultracentrifugation and Total Exosome Isolation Kit.The properties analysis indicated that BMSC-derived particles collected in our experiments were identified as exosomes.Exosomes were taken up by alveolar macrophages in vitro and assembled in the injured rat lung tissues in vivo.2.In vitro study demonstrated that BMSC-derived exosomes could transit and release miR-384-5p into alveolar macrophages,causing the increase of miR-384-5p in alveolar macrophages.Alveolar macrophage autophagy could be regulated by exosomes-mediated miR-384-5p/Beclin-1 signaling pathway.Therefore,exosomes relieved autophagy stress,alleviated inflammatory response,and then attenuated LPS-induced cell viability loss and cell apoptosis via miR-384-5p/Beclin-1 signaling pathway.3.In vivo study demonstrated that BMSC-derived exosomes could down-regulate Beclin-1 mediated autophagy by increasing miR-384-5p levels in lung tissue of ALI rats.Exosomes stabilized autophagy levels,alleviated inflammatory response,relieved pulmonary vascular permeability,attenuated pathological lung injury,and then improved the prognosis of ALI via modulating miR-384-5p/Beclin-1 signaling pathway.Furthermore,intratracheal injection of exosomes was tended to be more beneficial than intravenous injection on the improvement of ALI.