Exosomal miR-451a Derived from Bone Marrow Mesenchymal Stem Cells Inhibits Macrophage M1 Polarization Through the MIF/CD74 Signaling Pathway to Alleviate Acute Lung Injury

ZBP1 aggravates acute lung injury in mice by promoting the macrophage inflammatory phenotype.

Macrophages play a key role in the development of sepsis-induced acute respiratory distress syndrome (ARDS). Recent evidence has proved that glycolysis plays an important role in regulating macrophage polarization through metabolic reprogramming. Bone marrow mesenchymal stem cells (BMSCs) can alleviate sepsis-induced lung injury and possess potent immunomodulatory and immunosuppressive properties via secreting exosomes. However, it is unknown whether BMSCs-derived exosomes exert their therapeutic effect against sepsis-induced lung injury by inhibiting glycolysis in macrophages. Therefore, the present study aimed to evaluate the anti-inflammatory effects of exosomes released from BMSCs on acute lung injury induced by lipopolysaccharide (LPS) in mice and explored the possible underlying mechanisms in vitro and in vivo. We found that BMSCs inhibited M1 polarization and promoted M2 polarization in MH-S cells (a murine alveolar macrophage cell line) by releasing exosomes. Further experiments showed that exosomes secreted by BMSCs modulated LPS-treated MH-S cells polarization by inhibiting cellular glycolysis. Moreover, our results showed that BMSCs-derived exosomes down-regulated the expression of several essential proteins of glycolysis via inhibition of hypoxia-inducible factor 1 (HIF-1)α. Finally, a model of LPS-induced ARDS in mice was established, we found that BMSCs-derived exosomes ameliorated the LPS-induced inflammation and lung pathological damage. Meanwhile, we found that intratracheal delivery of BMSCs-derived exosomes effectively down-regulated LPS-induced glycolysis in mice lung tissue. These findings reveal new mechanisms of BMSCs-derived exosomes in regulating macrophage polarization which may provide novel strategies for the prevention and treatment of LPS-induced ARDS.

Numerous studies have shown that the M2 polarization of alveolar macrophages (AM) plays a protective role in acute lung injury (ALI). Mesenchymal stem cells (MSCs) secreted exosomes have been reported to be involved in inflammatory diseases by the effects of polarized M1/M2 macrophage populations. However, whether bone marrow mesenchymal stem cells (BMMSCs) derived exosomes could protect from ALI and its mechanisms are still unclear. Here, we explored the role of exosomes from BMMSC in rat AM polarization and the lipopolysaccharide- (LPS-) induced ALI rat model. Furthermore, the levels of exosomal miR-223 in BMMSCs were measured by RT-qPCR. Additionally, miR-223 mimics and its inhibitors were used to verify the vital role of miR-223 of BMMSCs-derived exosomes in the polarization of M2 macrophages. The results showed that BMMSCs-derived exosomes were taken up by the AM. Exosomes derived from BMMSCs promoted M2 polarization of AM in vitro. BMMSCsexosomes effectively mitigated pathological injuries, lung edema, and the inflammation of rats from LPS-induced ALI, accompanied by an increase of M2 polarization of AM in lung tissue. Interestingly, we also found that miR-223 was enriched in BMMSCs-derived exosomes, and overexpression of miR-223 in BMMSCs-derived exosomes promoted M2 polarization of AM while depressing miR-223 showed opposite effects in AM. The present study demonstrated that BMMSCs-derived exosomes triggered alveolar M2 polarization to improve inflammation by transferring miR-223, which may provide new therapeutic strategies in ALI.

USP18 improves mitochondrial homeostasis by stabilizing PKM2 and promoting M2 polarization in macrophages to relieve acute lung injury.

The present study aimed to investigate the therapeutic effect of monoammonium glycyrrhizinate (MAG) on lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice and possible mechanism. Acute lung injury was induced in BALB/c mice by intratracheal instillation of LPS, and MAG was injected intraperitoneally 1 h prior to LPS administration. After ALI, the histopathology of lungs, lung wet/dry weight ratio, protein concentration, and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were determined. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the BALF were measured by ELISA. The activation of NF-κB p65 and IκB-α of lung homogenate was detected by Western blot. Pretreatment with MAG attenuated lung histopathological damage induced by LPS and decreased lung wet/dry weight ratio and the concentrations of protein in BALF. At the same time, MAG reduced the number of inflammatory cells in lung and inhibited the production of TNF-α and IL-1β in BALF. Furthermore, we demonstrated that MAG suppressed activation of NF-κB signaling pathway induced by LPS in lung. The results suggested that the therapeutic mechanism of MAG on ALI may be attributed to the inhibition of NF-κB signaling pathway. Monoammonium glycyrrhizinate may be a potential therapeutic reagent for ALI.

Objective To evaluate the role of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in bone marrow mesenchymal stem cells (MSCs)-induced reduction of endotoxin-induced acute lung injury (ALI) in rats. Methods Thirty-two clean-grade healthy male Sprague-Dawley rats, weighing 180-250 g, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), ALI group, MSCs group and brusatol plus MSCs group (group B+ MSCs). Lipopolysaccharide 20 mg/kg was intravenously infused to establish the model of acute lung injury. Phosphate buffered saline(PBS)1 ml was intravenously infused at 1 h after establishing the model in group ALI. The equal volume of sterile saline and PBS was given instead in group C. PBS (1 ml) containing MSCs 1×106 cells was intravenously infused at 1 h after establishing the model in group MSCs. Brusatol 0.4 mg/kg was intraperitoneally injected every other day during 10 days before establishing the model, and MSCs were given at 1 h after establishing the model in group B+ MSCs. Bronchoalveolar lavage fluid (BALF) was collected and lung tissues were removed at 6 h after establishing the model. The protein concentration and neutrophil count in BALF were determined, and the wet/dry weight ratio (W/D ratio) were calculated. The pathological changes of lung tissues were observed by hematoxylin-eosin staining. The expression of Nrf2 and HO-1 by Western blot, activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) and content of malondialdehyde (MDA) were determined . Results Compared with group C, the W/D ratio and total cell count and protein concentration in BALF were significantly increased, the MPO activity was enhanced, the MDA content was increased, the SOD activity was weakened, and the expression of Nrf2 was up-regulated (P<0.05), and the pathological changes were accentuated in group ALI. Compared with group ALI, the W/D ratio and total cell count and protein concentration in BALF were significantly decreased, the MPO activity was weakened, the MDA content was decreased, the SOD activity was enhanced, and the expression of Nrf2 and HO-1 was up-regulated (P<0.05), and the pathological changes were significantly attenuated in group MSCs. Compared with group MSCs, the total cell count was significantly increased, the MPO activity was enhanced, the MDA content was increased, the expression of Nrf2 and HO-1 was down-regulated (P<0.05), and the pathological changes were accentuated in group B+ MSCs. Conclusion Nrf2/HO-1 signaling pathway is involved in bone marrow MSCs-induced reduction of endotoxin-induced acute lung injury in rats. Key words: NF-E2 related factor 2; Heme oxygenase-1; Mesenchymal stromal cells; Acute lung injury; Endotoxemia

Zanubrutinib ameliorates lipopolysaccharide-induced acute lung injury via regulating macrophage polarization

MicroRNA (miR)-9-5p has been shown to affect lung cancer progression and lung fibrosis, but the efficacy of miR-9-5p in acute lung injury (ALI) remained indefinite. The study was performed to probe the modulating mechanism of miR-9-5p in ALI via regulating macrophage polarization. The ALI mouse model was established and blood samples of ALI patients were obtained. MiR-9-5p levels in ALI mice and ALI patients were detected. Mouse pulmonary macrophages were extracted from bronchoalveolar lavage fluid and polarized into M1 and M2 macrophages. Intervention of miR-9-5p expression was performed to observe the effects on M1 polarization and M2 polarization in lung macrophages, inflammatory factors in BALF, wet/dry weight ratio (W/D) in lung tissues, myeloperoxidase (MPO) activity in lung tissues, and lung tissue lesion condition. MiR-9-5p levels were elevated in the lung tissues of ALI mice and ALI patients. MiR-9-5p silencing could repress lung macrophages in ALI mice polarized toward the M1 phenotype and promoted the polarization toward the M2 phenotype, reduced the lung lesions, the lung water content, and the secretion levels of the pro-inflammatory factors TNF-α, IL-6, and IL-1β in BALF, increased the secretion of the anti-inflammatory factor IL-10, as well as impeded the MPO activity in the lung tissues of ALI mice. MiR-9-5p deletion ameliorates LPS-induced inflammatory infiltration in lung tissues via inhibiting the polarization of mouse lung macrophages to the M1 phenotype and promoting the polarization to the M2 phenotype.

To determine the role of matrix metalloproteinase-8 (MMP-8) in acute lung injury (ALI), we delivered LPS or bleomycin by the intratracheal route to MMP-8(-/-) mice versus wild-type (WT) mice or subjected the mice to hyperoxia (95% O(2)) and measured lung inflammation and injury at intervals. MMP-8(-/-) mice with ALI had greater increases in lung polymorphonuclear neutrophils (PMNs) and macrophage counts, measures of alveolar capillary barrier injury, lung elastance, and mortality than WT mice with ALI. Bronchoalveolar lavage fluid (BALF) from LPS-treated MMP-8(-/-) mice had more MIP-1alpha than BALF from LPS-treated WT mice, but similar levels of other pro- and anti-inflammatory mediators. MIP-1alpha(-/-) mice with ALI had less acute lung inflammation and injury than WT mice with ALI, confirming that MIP-1alpha promotes acute lung inflammation and injury in mice. Genetically deleting MIP-1alpha in MMP-8(-/-) mice reduced the increased lung inflammation and injury and mortality in MMP-8(-/-) mice with ALI. Soluble MMP-8 cleaved and inactivated MIP-1alpha in vitro, but membrane-bound MMP-8 on activated PMNs had greater MIP-1alpha-degrading activity than soluble MMP-8. High levels of membrane-bound MMP-8 were detected on lung PMNs from LPS-treated WT mice, but soluble, active MMP-8 was not detected in BALF samples. Thus, MMP-8 has novel roles in restraining lung inflammation and in limiting alveolar capillary barrier injury during ALI in mice by inactivating MIP-1alpha. In addition, membrane-bound MMP-8 on activated lung PMNs is likely to be the key bioactive form of the enzyme that limits lung inflammation and alveolar capillary barrier injury during ALI.

Pre‐treatment with the heat shock protein 90 (hsp90) inhibitor, 17‐ AAG, reduces the magnitude of LPS‐induced ALI in mice. We now tested the hypothesis that post‐treatment with 17‐AAG would also reduce the magnitude of LPS‐induced ALI. 24h after the intratracheal instillation of vehicle or LPS (1.5mg/kg), mice received either vehicle or 17‐AAG (i.p. 10mg/kg). 48h later bronchoalveolar lavage fluid (BALF) was collected, and the number of cells and protein concentration were estimated as indicators of inflammation and permeability, respectively. In separate groups, lung capillary permeability was estimated by the Evans’ Blue Dye (EBD) method. LPS alone increased BALF cellularity (452±50 ×103cells/ml vs 33±2 ×103cells/ml in vehicle) and BALF protein concentration (204±5% from vehicle). 17‐AAG post‐treatment reduced the LPS‐induced BALF cellularity (140±32 ×103cells/ml) and protein concentration (119±18% from vehicle). Furthermore, LPS produced a 193±18% increase in EBD extravasation which was also significantly reduced to 135±27% in mice post‐treated with 17‐AAG. In additional mice, pulmonary function tests were performed. LPS caused a nearly 100% increase in airway resistance that was completely prevented by post‐treatment with 17‐AAG. These data strongly suggest important reparative actions of hsp90 inhibitors when administered after the LPS insult.

ObjectiveEmerging evidence has revealed that exosomal microRNAs (miRNAs) are implicated in human diseases. However, role of exosomal miR-125b-5p in sepsis-induced acute lung injury (ALI) remains further explored. We focused on the effect of exosomal miR-125b-5p on ALI progression via targeting topoisomerase II alpha (TOP2A).MethodsThe ALI mouse models were established by cecal ligation and perforation, which were then treated with miR-125b-5p agomir or overexpressed TOP2A. Next, the pathological structure of ALI mouse lung tissues were observed, miR-125b-5p, TOP2A and vascular endothelial growth factor (VEGF) expression was determined, and the lung water content, inflammatory response, protein content in bronchoalveolar lavage fluid (BALF) and cell apoptosis in ALI mouse lung tissues were assessed. Exosomes were extracted from endothelial cells (ECs) and identified, which were then injected into the modeled mice to observe their roles in ALI. The targeting relationship between miR-125b-5p and TOP2A was confirmed.ResultsMiR-125b-5p was downregulated while TOP2A was upregulated in ALI mice. MiR-125b-5p elevation or ECs-derived exosomes promoted VEGF expression, improved pathological changes and restrained lung water content, inflammatory response, protein content in BALF and cell apoptosis in lung tissues ALI mice. TOP2A overexpression reversed the repressive role of miR-125b-5p upregulation in ALI, while downregulated miR-125b-5p abrogated the effect of ECs-derived exosomes on ALI. TOP2A was confirmed as a direct target gene of miR-125b-5p.ConclusionOur study indicates that ECs-derived exosomes overexpressed miR-125b-5p to protect from sepsis-induced ALI by inhibiting TOP2A, which may contribute to ALI therapeutic strategies.

Human Umbilical Cord Mesenchymal Stem Cells Reduce Fibrosis of Bleomycin-Induced Lung Injury

Uncontrolled inflammation and excessive M1 macrophage polarization are key drivers of acute lung injury (ALI). Scutellarin (SCU), a natural flavonoid compound, possesses anti-inflammatory activity, but its precise mechanism remains unclear. This study aimed to investigate whether SCU alleviates ALI by targeting guanine nucleotide-binding protein 2 (GBP2) and regulating alveolar macrophage polarization. A lipopolysaccharide (LPS)-induced ALI mouse model was used to evaluate the therapeutic effects of SCU. Macrophage polarization and lung injury severity were assessed histologically and by cytokine analysis. Transcriptomic profiling (RNA-seq) identified GBP2 as a candidate target. GBP2 was knocked down or overexpressed in MH-S cells to evaluate its role in LPS-induced polarization. Co-immunoprecipitation, molecular docking, and immunofluorescence were performed to confirm the interaction between GBP2 and STAT3. SCU pre-treatment significantly alleviated lung injury, reduced inflammatory cytokine levels, and improved the wet-to-dry lung weight ratio. It modulated macrophage polarization by downregulating LPS-induced M1 polarization in alveolar macrophages. Mechanistically, SCU downregulated GBP2 expression and suppressed activation of the JAK2/STAT3 signaling pathway in LPS-stimulated models. SCU ameliorates LPS-induced ALI by modulating alveolar macrophage polarization through inhibition of the GBP2/JAK2/STAT3 pathway. These findings suggest that SCU may serve as a potential therapeutic agent for ALI.

Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are respiratory failures caused by excessive alveolar inflammation with high mortality. In this study, we investigated the effects of bone marrow mesenchymal stem cells (BMSCs) on lung injury of lipopolysaccharide (LPS)-induced ALI and explored the associated mechanisms. BMSCs were isolated, cultured, identified by staining with CD34 and CD44 surface markers. LPS-induced ALI mouse model was generated by injecting with LPS and divided into ALI group and ALI+BMSCs group. Mice treated without any reagents were assigned as Control, mice transplanted with BMSCs were assigned as BMSCs group. Regulatory T (Treg) and Th17 percentages were evaluated using flow cytometry. Proresolving mediators (resolvin E1 (RvE1), protectin D1 (ProD1)) in lung tissue and cytokines (interleukin-6 (IL-6) and IL-17) in serum were analyzed by ELISA. Myeloperoxidase (MPO) activity was determined. Cultured cells demonstrated typical characteristics of BMSCs. BMSCs transplantation (ALI+BMSCs) obviously alleviated LPS-induced ALI in mice. BMSCs transplantation significantly decreased MPO activity in LPS-induced ALI in mice compared to the Control group (p < 0.05). BMSCs transplantation markedly increased Treg percentages and decreased dendritic cells (DCs) and Th17 cells percentages compared to those of the Control group (p < 0.05). BMSCs transplantation remarkably enhanced RvE1 and ProD1 levels in LPS-induced ALI (ALI+BMSCs) compared to the ALI group (p < 0.05). BMSCs transplantation significantly attenuated IL-6 and IL-17 levels in serum of mice treated with LPS (ALI+BMSCs) compared to those of the ALI group (p < 0.05). In conclusion, BMSCs transplantation effectively attenuated LPS-induced pathological injury of ALI in mice, at least partly through promoting proresolving mediators RvE1 and ProD1 and modulating the balance of Treg/Th17.

Nrf2 activation protects against intratracheal LPS induced mouse/murine acute respiratory distress syndrome by regulating macrophage polarization