Bone Marrow Mesenchymal Stem Cell Exosomes Regulate Alveolar Macrophage Polarization Via The Glycolytic Pathway To Attenuate Sepsis-Associated ARDS

Objective: To investigate whether bone marrow mesenchymal stem cell-derived exosomes(BMSCs-exo)can alleviate sepsis-related acute respiratory distress syndrome(ARDS)by inhibiting glycolysis to regulate alveolar macrophage polarization and the related mechanism.Methods: In vitro experiments,the changes in M1/M2 polarization markers and glycolysis levels of MH-S cells were observed at different time points after lipopolysaccharide(LPS)stimulation.The BMSCs were pre-treated with the exosome inhibitor GW4869 to determine whether BMSCs could inhibit glycolysis and M1 polarization of MH-S cells by secreting exosomes.BMSCs-exo was extracted by ultracentrifugation combined with the exosome kit method,and PKH26 was used to label BMSCs-exo to observe whether it could enter MH-S cells to perform biological functions.The effects of BMSCs-exo via the glycolysis pathway were determined using the glycolysis inhibitor 2-DG and the glycolysis stimulant glucose.The regulatory activity of HOXA9 on the HK2 and GLUT1 genes was observed through a dual-luciferase reporter gene assay.Finally,the role of HOXA9 in BMSCs-exo inhibition of LPS-stimulated glycolysis in MH-S cells was explored by knocking down the expression of the HOXA9 protein in MH-S cells using small interfering RNA.In vivo experiments,the ARDS model associated with sepsis was induced by intraperitoneal injection of LPS in mice.The BMSCs-exo were traced using small animal live-imaging techniques to observe their ability to enter the lungs of septic mice.The lung damage of septic mice was observed by histopathology.The lung wet-to-dry ratio were measured.The total protein content and inflammatory cell count in bronchoalveolar lavage fluid(BALF),the inflammatory factor levels and lactic acid content in BALF and serum were measured.Finally,we observed whether BMSCs-exo improved the 7-day survival rate of sepsis-associated ARDS mice,and measured the expression levels of glycolysis-related proteins in mouse lung tissue cells and macrophage polarization index.Results: In vitro experiments showed that the m RNA expression of M1 polarization markers IL-1β,IL-6 and TNF-α were significantly increased in MH-S cells after LPS stimulation,while m RNA expression of M2 polarization markers Arg1,Ym1 and CD206 were significantly decreased,protein expression of glycolysis-related genes HK2,PKM2,GLUT1 and LDHA were significantly increased,as well as glucose consumption and lactic acid content,all in a time-dependent manner.Co-cultured MH-S cells with BMSCs at a ratio of 1:2 significantly inhibited the protein expression of MH-S cells glycolysis-related genes HK2,PKM2,GLUT1 and LDHA,while reducing MH-S cells glucose consumption and lactic acid content.Pretreated with GW4869,weakened the inhibitory effect of BMSCs on LPS-stimulated glycolysis in MH-S cells,as well as the inhibition of M1 polarization and the promotion of M2 polarization in MH-S cells,which suggests that BMSCs inhibit LPS-stimulated glycolysis in MH-S cells and regulate their polarization balance by secreting exosomes.The addition of BMSCs-exo to MH-S cells revealed its ability to enter MH-S cells and inhibit LPS-stimulated glycolysis and further modulate the polarization balance in MH-S cells.Furthermore,under the same exposure conditions of LPS stimulation,BMSCs-exo exerted the same effect as the glycolysis inhibitor 2-DG,inhibiting LPS stimulation-induced M1 polarization in MH-S cells,this anti-inflammatory effect of BMSCs-exo was attenuated by the addition of the glycolytic agonist Glucose.The inhibitory effect of BMSCs-exo on LPS-stimulated glycolysis in MH-S cells and the promoting anti-inflammatory effect of BMSCs-exo on M2 polarization were attenuated by transfection with HOXA9 si RNA.In vivo experiments,BMSCs-exo was observed to enter the lungs of sepsis-associated ARDS model mice via the tail vein and significantly increased the 7-day survival rate of septic mice.H&E staining revealed that BMSCs-exo significantly reduced early lung inflammation in mice with sepsis-associated ARDS.In addition,compared with the LPS group,the lung wet-to-dry ratio,the total protein content and the number of inflammatory cells in BALF,and the levels of inflammatory factors such as IL-1β and TNF-α in BALF and serum were significantly reduced in the BMSCs-exo group;the protein expression of HK2,PKM2 and GLUT1 genes related to glycolysis in mouse lung tissue cells were significantly reduced in the BMSCs-exo group,while the protein expression of HOXA9 gene was significantly increased;the lactic acid content in BALF and serum of mice in the BMSCs-exo group was significantly reduced;the expression of M1 polarization markers IL-1β,IL-6 and TNF-α m RNA were significantly decreased and the expression of M2 polarization markers Arg1,Ym1 and CD206 m RNA was significantly increased in the BMSCs-exo group.Conclusion: BMSCs-exo was able to enter LPS-stimulated MH-S cells and significantly inhibit their glycolysis level,promoting MH-S cells to M2 polarization and inhibiting their to M1 polarization,thereby suppressing the inflammatory response.HOXA9 regulated the expression of important rate-limiting enzymes HK2 and GLUT1 of the glycolytic pathway and played an important role in inhibition of LPS-stimulated glycolysis in MH-S cells and promotion of macrophage polarization balance by BMSCs-exo.In addition,BMSCs-exo entered the lungs of sepsis-associated ARDS mice,significantly increased the 7-day survival rate of septic mice,reduced the acute inflammatory symptoms of lung tissue in the early stage of sepsis-associated ARDS,inhibited the glycolysis level regulated the polarization balance of macrophages in lung tissue cells of septic mice,and ultimately alleviated sepsis-associated ARDS.