Mouse Model Of Acute Lung Injury Research Articles

Salecan ameliorates LPS-induced acute lung injury through regulating Keap1-Nrf2/HO-1 pathway in mice

Acute lung injury (ALI) is a severe clinical condition with high mortality, characterized by rapid onset and limited treatment options. The pathogenesis of ALI involves inflammation and oxidative stress. The polysaccharide salecan, a water-soluble β-(1,3)-D-glucan, has been found to possess numerous pharmaceutical effects, including anti-inflammatory properties, inhibition of oxidative stress, and anti-fatigue effects. This study aims to investigate the protective effect and underlying mechanism of salecan against LPS-induced ALI in mice. Using an in vivo LPS-induced ALI mouse model and an in vitro RAW264.7 cell system, we investigated the role of salecan in ALI with various experimental approaches, including histological staining, quantitative real-time PCR, flow cytometry, western blot analysis, and other relevant assays. Pre-treatment with salecan effectively attenuated LPS-induced ALI in vivo, reducing the severity of pulmonary edema, inflammation, and oxidative stress. NMR-based metabolomic profiling analysis revealed that salecan attenuated LPS-induced metabolic imbalances associated with ALI. Furthermore, salecan downregulated Keap1 and upregulated Nrf2 and HO-1 protein levels, indicating its modulation of the Keap1-Nrf2/HO-1 signaling pathway as a potential mechanism underlying its protective effects against ALI. In vitro studies on RAW264.7 cells revealed that salecan exhibited binding affinity towards macrophages, thereby alleviating LPS-induced apoptosis and inflammation, which underpin its therapeutic potential against ALI. Our study suggests that salecan can alleviate LPS-induced ALI by modulating oxidative stress, inflammatory response, and apoptosis through the activation of the Keap1-Nrf2/HO-1 pathway. These findings provide novel insights into the potential therapeutic use of salecan for the treatment of ALI.

Two new and effective food-extracted immunomodulatory agents exhibit anti-inflammatory response activity in the hACE2 acute lung injury murine model of COVID-19.

The coronavirus disease 2019 (COVID-19) spread rapidly and claimed millions of lives worldwide. Acute respiratory distress syndrome (ARDS) is the major cause of COVID-19-associated deaths. Due to the limitations of current drugs, developing effective therapeutic options that can be used rapidly and safely in clinics for treating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections is necessary. This study aims to investigate the effects of two food-extracted immunomodulatory agents, ajoene-enriched garlic extract (AGE) and cruciferous vegetables-extracted sulforaphane (SFN), on anti-inflammatory and immune responses in a SARS-CoV-2 acute lung injury mouse model. In this study, we established a mouse model to mimic the SARS-CoV-2 infection acute lung injury model via intratracheal injection of polyinosinic:polycytidylic acid (poly[I:C]) and SARS-CoV-2 recombinant spike protein (SP). After the different agents treatment, lung sections, bronchoalveolar lavage fluid (BALF) and fresh faeces were harvested. Then, H&E staining was used to examine symptoms of interstitial pneumonia. Flow cytometry was used to examine the change of immune cell populations. Multiplex cytokines assay was used to examine the inflammatory cytokines.16S rDNA high-throughput sequencing was used to examine the change of gut microbiome. Our results showed that AGE and SFN significantly suppressed the symptoms of interstitial pneumonia, effectively inhibited the production of inflammatory cytokines, decreased the percentage of inflammatory cell populations, and elevated T cell populations in the mouse model. Furthermore, we also observed that the gut microbiome of genus Paramuribaculum were enriched in the AGE-treated group. Here, for the first time, we observed that these two novel, safe, and relatively inexpensive immunomodulatory agents exhibited the same effects on anti-inflammatory and immune responses as neutralizing monoclonal antibodies (mAbs) against interleukin 6 receptor (IL-6R), which have been suggested for treating COVID-19 patients. Our results revealed the therapeutic ability of these two immunomodulatory agents in a mouse model of SARS-CoV-2 acute lung injury by promoting anti-inflammatory and immune responses. These results suggest that AGE and SFN are promising candidates for the COVID-19 treatment.

ICAM-1 targeted and ROS-responsive nanoparticles for the treatment of acute lung injury.

Acute lung injury (ALI) is an inflammatory disease caused by multiple factors such as infection, trauma, and chemicals. Without effective intervention during the early stages, it usually quickly progresses to acute respiratory distress syndrome (ARDS). Since ordinary pharmaceutical preparations cannot precisely target the lungs, their clinical application is limited. In response, we constructed a γ3 peptide-decorated and ROS-responsive nanoparticle system encapsulating therapeutic dexamethasone (Dex/PSB-γ3 NPs). In vitro, Dex/PSB-γ3 NPs had rapid H2O2 responsiveness, low cytotoxicity, and strong intracellular ROS removal capacity. In a mouse model of ALI, Dex/PSB-γ3 NPs accumulated at the injured lung rapidly, alleviating pulmonary edema and cytokine levels significantly. The modification of NPs by γ3 peptide achieved highly specific positioning of NPs in the inflammatory area. The ROS-responsive release mechanism ensured the rapid release of therapeutic dexamethasone at the inflammatory site. This combined approach improves treatment accuracy, and drug bioavailability, and effectively inhibits inflammation progression. Our study could effectively reduce the risk of ALI progressing to ARDS and hold potential for the early treatment of ALI.

Mechanism of isorhynchophylline in lipopolysaccharide-induced acute lung injury based on proteomic technology.

Isorhynchophylline (IRN), a tetracyclic indole alkaloid, has anti-inflammatory and antioxidant activities against cardiovascular diseases and central nervous system disorders. Acute lung injury (ALI) is a manifestation of inflammation concentrated in the lungs and has a high incidence rate and mortality The purpose of this study is to explain the mechanism of IRN in the treatment of acute lung injury and to provide a new scheme for clinical treatment. The experimental mice were divided into three groups: CTRL, LPS, LPS+IRN. The mouse model of ALI was established by inhaling LPS solution through nose. After continuous administration of IRN solution for 7 days, the mice in LPS+IRN group were killed and the lung tissue was collected for detection. Proteomic (Data are available via ProteomeXchange with identifier PXD050432) results showed that 5727 proteins were detected in mouse lung tissues, and 16 proteins were screened out. IRN could reverse the trend of these differential proteins. In addition, IRN can act on integrin αM to reduce neutrophil recruitment and thereby produce anti-inflammatory effects and may suppress neutrophil migration through the leukocyte transendothelial migration pathway. TUNEL and RT-PCR experiments revealed that LPS-induced ALI in mice increases the apoptosis of lung tissues, damage to alveolar epithelial cells and levels of inflammatory factors. Treatment with IRN can repair tissues, improve lung tissue pathology and reduce lung inflammation.

Upregulation of PGC-1α expression by pioglitazone mediates prevention of sepsis-induced acute lung injury.

The imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages plays a critical role in the pathogenesis of sepsis-induced acute lung injury (ALI). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may modulate macrophage polarization toward the M2 phenotype by altering mitochondrial activity. This study aimed to investigate the role of the PGC-1α agonist pioglitazone (PGZ) in modulating sepsis-induced ALI. A mouse model of sepsis-induced ALI was established using cecal ligation and puncture (CLP). An in vitro model was created by stimulating MH-S cells with lipopolysaccharide (LPS). qRT-PCR was used to measure mRNA levels of M1 markers iNOS and MHC-II and M2 markers Arg1 and CD206 to evaluate macrophage polarization. Western blotting detected expression of peroxisome proliferator-activated receptor gamma (PPARγ) PGC-1α, and mitochondrial biogenesis proteins NRF1, NRF2, and mtTFA. To assess mitochondrial content and function, reactive oxygen species levels were detected by dihydroethidium staining, and mitochondrial DNA copy number was measured by qRT-PCR. In the CLP-induced ALI mouse model, lung tissues exhibited reduced PGC-1α expression. PGZ treatment rescued PGC-1α expression and alleviated lung injury, as evidenced by decreased lung wet-to-dry weight ratio, pro-inflammatory cytokine secretion (tumor necrosis factor-α, interleukin-1β, interleukin-6), and enhanced M2 macrophage polarization. Mechanistic investigations revealed that PGZ activated the PPARγ/PGC-1α/mitochondrial protection pathway to prevent sepsis-induced ALI by inhibiting M1 macrophage polarization. These results may provide new insights and evidence for developing PGZ as a potential ALI therapy.

The Therapeutic Potential of (+)-Afzelechin for Alleviating Sepsis-Associated Pulmonary Injury.

Sepsis-induced acute lung injury (ALI) poses a common and formidable challenge in clinical practice, currently lacking efficacious therapeutic approaches. This study delves into the evaluation of (+)-afzelechin (AZC), a natural compound derived from Bergenia ligulata with a diverse array of properties, encompassing antioxidant, anticancer, antimicrobial, and cardiovascular effects to ascertain its effectiveness and underlying mechanisms in mitigating sepsis-induced ALI through animal experimentation. An ALI mouse model induced by sepsis was established through lipopolysaccharide (LPS) administration, and various analytical techniques, including quantitative real-time polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay were employed to gauge inflammatory cytokine levels, lung injury, and associated signaling pathways. The animal experiments revealed that AZC offered safeguards against lung injury induced by LPS while reducing inflammatory cytokine levels in both blood serum and lung tissue. Western blotting experiments revealed AZC's downregulation of the toll-like receptor (TLR)4/NF-κB pathway and the upregulation of PI3K/Akt, coupled with inhibition of the Hippo and Rho signaling pathways. These findings underscore AZC's efficacy in ameliorating sepsis-induced ALI by modulating cytokine storms and curtailing inflammation via the regulation of TLR4/NF-κB, PI3K/Akt, Hippo, and Rho signaling pathways. This work serves as a foundation for additional exploration into AZC's mechanisms and its potential as a therapy for sepsis-induced ALI. Animals in accordance with Kyungpook National University (IRB No. KNU 2022-174).

Magnetically controlled nanorobots induced oriented and rapid clearance of the cytokine storm for acute lung injury therapy

Cytokine storms characterized by excessive secretion of circulating cytokines and immune-cell hyperactivation are life-threatening systemic inflammatory syndromes. The new strategy is in great demand to inhibit the cytokine storm. Here, we designed a type of magnetically controlled nanorobots (MAGICIAN) by fusing neutrophil membranes onto Fe3O4 nanoparticles (Fe3O4NPs). In our study, the receptors of neutrophil membranes were successfully coated to the surface of Fe3O4NPs. The associated membrane functions of neutrophils were highly preserved. MAGICIAN could in vitro neutralize the inflammatory cytokines including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ). Interestingly, MAGICIAN could be navigated to the liver sites under magnetic control and accelerated the cytokine clearance by the liver. Administration of MAGICIAN could efficiently relieve the inflammation in the acute lung injury mouse model. In addition, MAGICIAN displayed good biosafety in systemic administration. The present study provides a safe and convenient approach for the clearance of cytokine storms, indicating the potential for clinical application in acute lung injury therapy.

Intratracheal administration of programmable DNA nanostructures combats acute lung injury by targeting microRNA-155

Acute lung injury (ALI) is an acute inflammatory process that can result in life-threatening consequences. Programmable DNA nanostructures have emerged as excellent nanoplatforms for microRNA-based therapeutics, offering potential nanomedicines for ALI treatment. Nonetheless, the traditional systematic administration of nanomedicines is constrained by low delivery efficiency, poor pharmacokinetics, and nonspecific side effects. Here, we identify macrophage microRNA-155 as a novel therapeutic target using the magnetic bead sorting technique. We further construct a DNA nanotubular nucleic acid drug antagonizing microRNA-155 (NT-155) for ALI treatment through intratracheal administration. Flow cytometry results demonstrate that NT-155, when inhaled, is taken up much more effectively by macrophages and dendritic cells in the bronchoalveolar lavage fluid of ALI mice. Furthermore, NT-155 effectively silences the overexpressed microRNA-155 in macrophages and exerts excellent inflammation inhibition effects in vitro and ALI mouse models. Mechanistically, NT-155 suppresses microRNA-155 expression and activates its target gene SOCS1, inhibiting the p-P65 signaling pathway and suppressing proinflammatory cytokine secretion. The current study suggests that deliberately designed nucleic acid drugs are promising nanomedicines for ALI treatment and the local administration may open up new practical applications of DNA in the future.

The Protective Role of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Protein Sigma-1 Receptor in Regulating Endothelial Inflammation and Permeability Associated with Acute Lung Injury.

Earlier studies from our lab identified endoplasmic reticulum (ER) chaperone BiP/GRP78, an important component of MAM, to be a novel determinant of endothelial cell (EC) dysfunction associated with acute lung injury (ALI). Sigma1R (Sig1R) is another unique ER receptor chaperone that has been identified to associate with BiP/GRP78 at the MAM and is known to be a pluripotent modulator of cellular homeostasis. However, it is unclear if Sig1R also plays a role in regulating the EC inflammation and permeability associated with ALI. Our data using human pulmonary artery endothelial cells (HPAECs) showed that siRNA-mediated knockdown of Sig1R potentiated LPS-induced the expression of proinflammatory molecules ICAM-1, VCAM-1 and IL-8. Consistent with this, Sig1R agonist, PRE-084, known to activate Sig1R by inducing its dissociation from BiP/GRP78, blunted the above response. Notably, PRE-084 failed to blunt LPS-induced inflammatory responses in Sig1R-depleted cells, confirming that the effect of PRE-084 is driven by Sig1R. Furthermore, Sig1R antagonist, NE-100, known to inactivate Sig1R by blocking its dissociation from BiP/GRP78, failed to block LPS-induced inflammatory responses, establishing that dissociation from BiP/GRP78 is required for Sig1R to exert its anti-inflammatory action. Unlike Sig1R, the siRNA-mediated knockdown or Subtilase AB-mediated inactivation of BiP/GRP78 protected against LPS-induced EC inflammation. Interestingly, the protective effect of BiP/GRP78 knockdown or inactivation was abolished in cells that were depleted of Sig1R, confirming that BiP/GRP78 knockdown/inactivation-mediated suppression of EC inflammation is mediated via Sig1R. In view of these findings, we determined the in vivo relevance of Sig1R in a mouse model of sepsis-induced ALI. The intraperitoneal injection of PRE-084 mitigated sepsis-induced ALI, as evidenced by a decrease in ICAM-1, IL-6 levels, lung PMN infiltration, and lung vascular leakage. Together, these data evidence a protective role of Sig1R against endothelial dysfunction associated with ALI and identify it as a viable target in terms of controlling ALI in sepsis.

Regulator of G protein signaling protein 6 alleviates acute lung injury by inhibiting inflammation and promoting cell self-renewal in mice

BackgroundAcute respiratory distress syndrome (ARDS) is a disease with high mortality and morbidity. Regulator of G protein signaling protein 6 (RGS6), identified as a tumor suppressor gene, has received increasing attention owing to its close relationship with oxidative stress and inflammation. However, the association between ARDS and RGS6 has not been reported.MethodsCongruously regulated G protein-coupled receptor (GPCR)-related genes and differentially expressed genes (DEGs) in an acute lung injury (ALI) model were identified, and functional enrichment analysis was conducted. In an in vivo study, the effects of RGS6 knockout were studied in a mouse model of ALI induced by lipopolysaccharide (LPS). HE staining, ELISA, and immunohistochemistry were used to evaluate pathological changes and the degree of inflammation. In vitro, qRT‒PCR, immunofluorescence staining, and western blotting were used to determine the dynamic changes in RGS6 expression in cells. The RGS6 overexpression plasmid was constructed for transfection. qRT‒PCR was used to assess proinflammatory factors transcription. Western blotting and flow cytometry were used to evaluate apoptosis and reactive oxygen species (ROS) production. Organoid culture was used to assess the stemness and self-renewal capacity of alveolar epithelial type II cells (AEC2s).ResultsA total of 110 congruously regulated genes (61 congruously upregulated and 49 congruously downregulated genes) were identified among GPCR-related genes and DEGs in the ALI model. RGS6 was downregulated in vivo and in vitro in the ALI model. RGS6 was expressed in the cytoplasm and accumulated in the nucleus after LPS stimulation. Compared with the control group, we found higher mortality, more pronounced body weight changes, more serious pulmonary edema and pathological damage, and more neutrophil infiltration in the RGS6 knockout group upon LPS stimulation in vivo. Moreover, AEC2s loss was significantly increased upon RGS6 knockout. Organoid culture assays showed slower alveolar organoid formation, fewer alveolar organoids, and impaired development of new structures after passaging upon RGS6 knockout. In addition, RGS6 overexpression decreased ROS production as well as proinflammatory factor transcription in macrophages and decreased apoptosis in epithelial cells.ConclusionsRGS6 plays a protective role in ALI not only in early inflammatory responses but also in endogenous lung stem cell regeneration.Graphical

Circular RNA circVAPA modulates macrophage pyroptosis in sepsis-induced acute lung injury through targeting miR-212-3p/Sirt1/Nrf2/NLRP3 axis.

Sepsis-induced acute lung injury (ALI) is an inflammatory condition involving the pyroptosis of macrophages. This study investigated the role of circular RNA hsa_circ_0006990 (circVAPA) in regulating macrophage pyroptosis in ALI and the underlying mechanisms. The expression pattern of circVAPA was examined in the mouse model of ALI and in the LPS-treated RAW264.7 macrophage cell line. Lung tissue damage was evaluated by haematoxylin and eosin staining, immunohistochemistry and a myeloperoxidase activity assay. The molecular mechanisms were investigated by luciferase reporter assay, western blot, RT-qPCR and ELISA. circVAPA was down-regulated in the lung tissues of ALI mice and LPS-induced RAW264.7 cells. circVAPA over-expression alleviated lung tissue injury and dampened LPS-induced pyroptosis and Th17-associated inflammatory responses. miR-212-3p was identified as a target of circVAPA, and miR-212-3p negatively regulated the expression of Sirt1. Sirt1 knockdown largely abolished the effect of circVAPA over-expression on pyroptosis. CircVAPA/miR-212-3p/Sirt1 axis also regulates Nrf2 and NLRP3 expression upon LPS challenge. By targeting miR-212-3p, circVAPA over-expression negatively regulates the expression of Sirt1 and pyroptosis-related factors (Nrf2 and NLRP3), which alleviates the inflammatory damages in sepsis-induced ALI.

Synergistic Enhancement of Isoforskolin and Dexamethasone Against Sepsis and Acute Lung Injury Mouse Models

Synergistic Enhancement of Isoforskolin and Dexamethasone Against Sepsis and Acute Lung Injury Mouse Models

Network Pharmacology and Experimental Validation of the Anti-Inflammatory Effect of Tingli Dazao Xiefei Decoction in Acute Lung Injury Treatment.

Tingli Dazao Xiefei Decoction (TDXD) is a Traditional Chinese Medicine (TCM) formula used to treat acute lung injury (ALI). However, the precise mechanism of TDXD in treating ALI remains unclear. We investigated the therapeutic mechanism of TDXD against ALI using a complementary approach combining network pharmacology, molecular docking, and in vitro and in vivo experiments. Potential drug targets of TDXD and relevant target genes associated with ALI were retrieved from Chinese medicines and disease genes databases. Bioinformatics technology was employed to screen potential active ingredients and core targets. Validation experiments were conducted using a lipopolysaccharide (LPS)-induced ALI mouse (C57BL/6J) model, LPS-induced inflammatory RAW264.7 cells, and molecular docking between active compounds of TDXD and potential targets. Network pharmacology suggested that the mechanism of TDXD against ALI involved phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) / phosphatase and tensin homolog (PTEN) and Janus kinase 2 (JAK2) / signal transducer and activator of transcription 3 (STAT3) pathways. Quercetin, β-sitosterol, kaempferol, isorhamnetin, and L-stepholidine were identified as the main active compounds of TDXD that exerted anti-ALI effects. Molecular docking indicated that these compounds exhibited good binding capabilities (≤ -5kcal/mol) to key targets in PI3K/AKT/PTEN and JAK2/STAT3 signaling pathways. In the animal model, TDXD alleviated injuries and inflammatory responses in lung tissues, accompanied by inhibition of expression of tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), STAT3, and Suppressor of Cytokine Signaling 3 (SOCS3) mRNA, and key proteins in PI3K/AKT/PTEN and JAK2/STAT3 pathways (all P values < 0.05). Cell based experiments showed that TDXD dose-dependently inhibited the expression of essential proteins in PI3K/AKT/PTEN and JAK2/STAT3 pathways (P < 0.05). This study revealed that the mechanism of TDXD in ALI treatment might involve simultaneous regulation of PI3K/AKT/PTEN and JAK2/STAT3 pathways.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair.

To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.

Imaging peroxynitrite in endoplasmic reticulum stress and acute lung injury with a near-infrared fluorescent probe

BackgroundThe cellular endoplasmic reticulum (ER) is responsible for various functions, including protein synthesis, folding, distribution, and calcium ion storage. Studies have linked ER stress with acute lung injury (ALI), which can result in oxidative stress and even cell death. Peroxynitrite (ONOO−) is a well-known reactive oxygen species (ROS) that contributes to various physiological and pathological processes in oxidative stress diseases. To understand the role of ER ONOO− in ALI, it is crucial to accurately measure its level in the ER. Unfortunately, there is currently no probe available to detect ER ONOO− in an ALI model. ResultsTo address this, we developed three near-infrared (NIR) fluorescent probes (DCM-F-ONOO, DCM-Cl-ONOO, and DCM-Br-ONOO) for the detection of ONOO− using pentafluorobenzenesulfonate (PFBS) moieties as fluorescence quenchers. Through comprehensive testing, we selected DCM-Br-ONOO as the best NIR fluorescent probe due to its rapid response (within 3 min), high selectivity, good sensitivity (LOD = 2.3 nM), and approximately 66-fold enhanced response to ONOO− in fluorescence intensity. The probe was successfully applied to detect changes in ONOO− levels induced by different drugs in the ER of living cells. Importantly, a significant increase in the level of ONOO− was observed in the ER of an ALI cell model (4.5-fold) and an ALI mouse model (2.5-fold) using the probe, which is essential for understanding the role of ONOO− in ER-associated diseases. SignificanceUsing DCM-Br-ONOO as a probe, present work further validated that the elevated levels of ONOO− secretion were accompanied by the ALI progressed. These findings may provide valuable results for figuring out the biological roles that ONOO− played in ALI.

INOS inhibitor S-methylisothiourea alleviates smoke inhalation-induced acute lung injury by suppressing inflammation and macrophage infiltration

ObjectiveWe investigated the effects of the inducible NO synthase (iNOS) inhibitor, S-methylisothiourea (SMT), in a mouse model of smoke inhalation-induced acute lung injury (ALI) and explored the underlying molecular mechanism. Methods and analysisA mouse model of smoke inhalation-induced ALI was established. RNA-sequencing (seq) analysis was conducted to identify the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed for functional annotation of DEGs. Moreover, an immunofluorescence assay using macrophage marker F4/80 was performed to assess macrophage infiltration. A hypoxia-induced HUVEC model was used to mimic smoke inhalation-induced injury in endothelial cells. Finally, a transwell assay was used to analyze the chemoattractive effects of endothelial cells on macrophages. ResultsSMT markedly alleviated the pulmonary pathological symptoms, edema, and inflammatory response in the mouse smoke inhalation-induced ALI model. RNA-seq analysis revealed that SMT may diminish lung injury by regulating the levels of genes associated with inflammatory responses, cell chemokines, and adhesion. In vivo data revealed that the protective effects of SMT against smoke inhalation-induced ALI were partly achieved by inhibiting the production of adhesion molecules and infiltration of macrophages. Furthermore, in vitro data from the hypoxia-induced HUVEC model revealed that SMT reduced macrophage chemotaxis by inhibiting the production of chemokines and adhesion molecules in endothelial cells. ConclusioniNOS inhibitor SMT protects the lungs from smoke inhalation-induced ALI by reducing the production of pro-inflammatory cytokines, adhesion molecules, and chemokines in endothelial cells, thereby inhibiting inflammation and macrophage infiltration.

Lianhua Qingwen protects LPS-induced acute lung injury by promoting M2 macrophage infiltration

Ethnopharmacological relevanceTraditional Chinese medicine Lianhua Qingwen (LHQW) was used to treat regular seasonal influenza. In recent years, LHQW exerts significant therapeutic effects in treating influenza and Coronavirus Disease 2019 (COVID-19). However, the potential mechanisms are not yet understood and need further study. Aim of studyThis study aims to look into the influence of LHQW on lung inflammation and macrophage phenotype, and to clarify the connection between macrophage plasticity and LHQW. MethodsThe cell viability, marker expression, response to LPS stimulation, and phagocytosis of Raw264.7 were detected after LHQW treatment. In an LPS-induced acute lung injury (ALI) mouse model, the alleviating effect of LHQW on lung injury was investigated. The total macrophages and M2 macrophages in mice lungs and the peripheral blood monocytes after LHQW treatment were detected. The cell viability and polarization of peripheral blood macrophages treated with LHQW were detected. ResultsHere, we demonstrate that LHQW protects LPS-induced ALI by promoting M2 macrophage infiltration. LHQW treatment inhibited the inflammatory response and pro-inflammatory phenotype of Raw264.7 macrophages. High concentrations of LHQW promoted the phagocytic capacity of Raw264.7 macrophages. In an ALI mouse model, LHQW alleviated lung injury and no significant hepatotoxicity was observed. By Immunohistochemistry (IHC) analysis, LHQW increased the infiltration of macrophages, mainly M2 macrophages. Consistent with Raw264.7, LHQW also decreased the expression of M1 markers in peripheral blood macrophages. In addition, LHQW blood plasma promoted the M2-type polarization of peripheral blood macrophages. ConclusionsTaken together, our data demonstrate that LHQW reduces the inflammatory response and ameliorates acute lung injury by promoting anti-inflammatory polarization of macrophages.

Songorine inhibits oxidative stress-related inflammation through PI3K/AKT/NRF2 signaling pathway to alleviate lipopolysaccharide-induced septic acute lung injury

Objective The present study aimed to investigate the protective action and mechanism of songorine on sepsis-induced acute lung injury (ALI). Methods The sepsis-induced ALI mouse and cell models were established by lipopolysaccharide (LPS) induction. Lung injury was assayed by hematoxylin and eosin staining, lung injury score, and lung wet-to-dry (W/D) weight ratio. Apoptosis in lung tissues was evaluated by TUNEL assay, and the expression of apoptosis-related markers (Bcl2, Bax, and caspase-3) was measured by western blotting. Levels of pro-inflammatory factors and oxidative stress markers in the bronchoalveolar lavage fluid (BALF) of mice were measured by ELISA and RT-qPCR. The expression of PI3K/AKT/NRF2 pathway-related proteins was analyzed by western blotting. Results Songorine treatment at 40 mg/kg mitigated sepsis-induced ALI, characterized by improved histopathology, lung injury score, and lung W/D weight ratio (p < 0.05). Moreover, songorine markedly attenuated sepsis-induced apoptosis in lung tissues; this was evidenced by an increase in Bcl2 levels and a decrease in Bax and caspase-3 levels (p < 0.01). Also, songorine reduced levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β and MPO) and oxidative stress regulators (SOD and GSH) in the BALF of LPS-induced sepsis mice and RAW264.7 cells (p < 0.05). In addition, songorine upregulated the PI3K/AKT/NRF2 pathway-related proteins in LPS-induced sepsis mice and RAW264.7 cells (p < 0.05). Furthermore, LY294002 (a PI3K inhibitor) treatment reversed the protective effect of songorine on sepsis-induced ALI. Conclusion Songorine inhibits oxidative stress-related inflammation in sepsis-induced ALI via the activation of the PI3K/AKT/NRF2 signaling pathway.

Mesenchymal Stromal Cells Primed by Toll-like Receptors 3 and 4 Enhanced Anti-Inflammatory Effects against LPS-Induced Macrophages via Extracellular Vesicles.

Although it has been suggested that toll-like receptor (TLR) 3 and TLR4 activation alters mesenchymal stromal cells (MSCs)' immunoregulatory function as anti- or pro-inflammatory phenotypes, we have previously confirmed that TLR4-primed hUCB-MSCs alleviate lung inflammation and tissue injury in an E. coli-induced acute lung injury (ALI) mouse model. Therefore, we hypothesized that strong stimulation of TLR3 or TLR4 prompts hUCB-MSCs to exhibit an anti-inflammatory phenotype mediated by extracellular vesicles (EVs). In this study, we compared the anti-inflammatory effect of TLR3-primed and TLR4-primed hUCB-MSCs against an LPS-induced ALI in vitro model by treating MSCs, MSC-derived conditioned medium (CM), and MSC-derived extracellular vesicles (EVs). LPS-induced rat primary alveolar macrophage and RAW 264.7 cells were treated with naïve, TLR3-, and TLR4-primed MSCs and their derived CM and EVs. Flow cytometry and ELISA were used to evaluate M1-M2 polarization of macrophages and pro-inflammatory cytokine levels, respectively. LPS-stimulated macrophages showed significantly increased pro-inflammatory cytokines compared to those of the normal control, and the percentage of M2 macrophage phenotype was predominantly low. In reducing the inflammatory cytokines and enhancing M2 polarization, TLR3- and TLR4-primed MSCs were significantly more effective than the naïve MSCs, and this finding was also observed with the treatment of MSC-derived CMs and EVs. No significant difference between the efficacy of TLR3- and TLR-primed MSCs was observed. Strong stimulation of TLR3- and TLR4-stimulated hUCB-MSCs significantly reduced pro-inflammatory cytokine secretion from LPS-induced macrophages and significantly enhanced the M2 polarization of macrophages. We further confirmed that TLR-primed MSC-derived EVs can exert anti-inflammatory and immunosuppressive effects alone comparable to MSC treatment. We hereby suggest that in the LPS-induced macrophage in vitro model, EVs derived from both TLR3 and TLR4-primed MSCs can be a therapeutic candidate by promoting the M2 phenotype.

GLUT1 contributes to impaired epithelial tight junction in the late phase of acute lung injury

Dysfunction of epithelial barrier is crucial for the development of acute lung injury (ALI). This study was aimed to evaluate the role of glucose transporter 1 (GLUT1) in dysregulation of epithelial tight junction in ALI. GLUT1 was inhibited with specific antagonists WZB117 or BAY876 to see the effects on epithelial tight junction in a well-established LPS-induced mouse ALI model as well as in vitro cultured epithelial cells. Pharmacological inhibition of GLUT1 with WZB117 at either a low or high dose had no effects on lung injury and inflammation 24 h after LPS challenge, but significantly decreased the pulmonary inflammatory responses induced by LPS at 72 h with a high dose, which was verified by treatment with BAY876. WZB117 or BAY876 also recovered the expression of epithelial tight junction proteins ZO-1 and occludin. In cultured BEAS-2B and A549 cells, LPS induced increased GLUT1 expression, accompanied by decreased expression of tight junction protein ZO-1 and occludin. Blockade of GLUT1 restored LPS-induced disruption of ZO-1 and occludin in BEAS-2B rather than A549. Taken together, our results showed that GLUT1 is responsible for dysfunction of epithelial tight junctions in the late phase of LPS-induced ALI.