Hexamerization: explaining the original sin of IgG-mediated complement activation in acute lung injury.

Although antibody-mediated lung damage is a major factor in transfusion-related acute lung injury (ALI), autoimmune lung disease (for example, coatomer subunit α [COPA] syndrome), and primary graft dysfunction following lung transplantation, the mechanism by which antigen-antibody complexes activate complement to induce lung damage remains unclear. In this issue of the JCI, Cleary and colleagues utilized several approaches to demonstrate that IgG forms hexamers with MHC class I alloantibodies. This hexamerization served as a key pathophysiological mechanism in alloimmune lung injury models and was mediated through the classical pathway of complement activation. Additionally, the authors provided avenues for exploring therapeutics for this currently hard-to-treat clinical entity that has several etiologies but a potentially focused mechanism.

Effects of CDDO-EA in sepsis-induced acute lung injury: mouse model of endotoxaemia.

OBJECTIVE: Aim: The aim of this research is to clarify the potential effect of CDDO-EA against experimentally sepsis induced lung injury in mice. PATIENTS AND METHODS: Materials and Methods: Mice have divided into four groups: Sham group CLP group, Vehicle-treatment group, CDDO-EA-treated group: mice in this group received CDDO-EA 2mg/kg intraperitoneally, 1hr before CLP, then the animals were sacrificed 24hr after CLP. After exsAngpuinations, tissue samples of lung were collected, followed by markers measurement including, TNF-α, IL-1ß, VEGF, MPO, caspase11, Angp-1and Angp-2 by ELISA, gene expression of TIE2 and VE-cadherin by qRT-PCR, in addition to histopathological study. RESULTS: Results: A significant elevation (p<0.05) in TNF-α, IL-1ß, MPO, ANGP-2, VEGF, CASPASE 11 in CLP and vehicle groups when compared with sham group. CDDO-EA group showed significantly lower levels p<0.05, level of ANGP-1 was significantly lower p<0.05 in the CLP and vehicle groups as compared with the sham group. Quantitative real-time PCR demonstrated a significant decrement in mRNA expression of TIE2&ve-cadherin genes p<0.05 in sepsis & vehicle. CONCLUSION: Conclusions: CDDO-EA has lung protective effects due to its anti-inflammatory and antiAngpiogenic activity, additionally, CDDO-EA showes a lung protective effect as they affect tissue mRNA expression of TIE2 and cadherin gene. Furthermore, CDDO-EA attenuate the histopathological changes that occur during polymicrobial sepsis thereby lung protection effect.

Mexenone protects mice from LPS-induced sepsis by EC barrier stabilization.

Blood vessels permit the selective passage of molecules and immune cells between tissues and circulation. Uncontrolled inflammatory responses from an infection can increase vascular permeability and edema, which can occasionally lead to fatal organ failure. We identified mexenone as a vascular permeability blocker by testing 2,910 compounds in the Clinically Applied Compound Library using the lipopolysaccharide (LPS)-induced vascular permeability assay. Mexenone suppressed the LPS-induced downregulation of junctional proteins and phosphorylation of VE-cadherin in Bovine Aortic Endothelial Cells (BAECs). The injection of mexenone 1 hr before LPS administration completely blocked LPS-induced lung vascular permeability and acute lung injury in mice after 18hr. Our results suggest that mexenone-induced endothelial cell (EC) barrier stabilization could be effective in treating sepsis patients.

Paeoniflorin attenuates particulate matter-induced acute lung injury by inhibiting oxidative stress and NLRP3 inflammasome-mediated pyroptosis through activation of the Nrf2 signaling pathway.

Particulate matter (PM), the main component of air pollutants, emerges as a research hotspot, especially in the area of respiratory diseases. Paeoniflorin (PAE), known as anti-inflammatory and immunomodulatory effects, has been reported to alleviate acute lung injury (ALI). However, the effect of PAE on PM-induced ALI and the underlying mechanisms are still unclear yet. In this study, we established the PM-induced ALI model using C57BL/6J mice and BEAS-2B cells to explore the function of PAE. In vivo, mice were intraperitoneally injected with PAE (100 mg/kg) or saline 1 h before instilled with 4 mg/kg PM intratracheally and were euthanized on the third day. For lung tissues, HE staining and TUNEL staining were used to evaluate the degree of lung injury, ELISA assay was used to assess inflammatory mediators and oxidative stress level, Immunofluorescence staining and western blotting were applied to explore the role of pyroptosis and Nrf2 signaling pathway. In vitro, BEAS-2B cells were pretreated with 100 µM PAE before exposure to 200 µg/ml PM and were collected after 24h for the subsequent experiments. TUNEL staining, ROS staining, and western blotting were conducted to explore the underlying mechanisms of PAE on PM-induced ALI. According to the results, PAE can attenuate the degree of PM-induced ALI in mice and reduce PM-induced cytotoxicity in BEAS-2B cells. PAE can relieve PM-induced excessive oxidative stress and NLRP3 inflammasome-mediated pyroptosis. Additionally, PAE can also activate Nrf2 signaling pathway and inhibition of Nrf2 signaling pathway can impair the protective effect of PAE by aggravating oxidative stress and pyroptosis. Our findings demonstrate that PAE can attenuate PM-induced ALI by inhibiting oxidative stress and NLRP3 inflammasome-mediated pyroptosis, which is mediated by Nrf2 signaling pathway.

Cholesterol-Modified Anti-Il6 siRNA Reduces the Severity of Acute Lung Injury in Mice.

Small interfering RNA (siRNA) holds significant therapeutic potential by silencing target genes through RNA interference. Current clinical applications of siRNA have been primarily limited to liver diseases, while achievements in delivery methods are expanding their applications to various organs, including the lungs. Cholesterol-conjugated siRNA emerges as a promising delivery approach due to its low toxicity and high efficiency. This study focuses on developing a cholesterol-conjugated anti-Il6 siRNA and the evaluation of its potency for the potential treatment of inflammatory diseases using the example of acute lung injury (ALI). The biological activities of different Il6-targeted siRNAs containing chemical modifications were evaluated in J774 cells in vitro. The lead cholesterol-conjugated anti-Il6 siRNA after intranasal instillation demonstrated dose-dependent therapeutic effects in a mouse model of ALI induced by lipopolysaccharide (LPS). The treatment significantly reduced Il6 mRNA levels, inflammatory cell infiltration, and the severity of lung inflammation. IL6 silencing by cholesterol-conjugated siRNA proves to be a promising strategy for treating inflammatory diseases, with potential applications beyond the lungs.

ANGPTL2 knockdown induces autophagy to relieve alveolar macrophage pyroptosis by reducing LILRB2-mediated inhibition of TREM2.

Acute lung injury (ALI) is featured with a robust inflammatory response. Angiopoietin-like protein 2 (ANGPTL2), a pro-inflammatory protein, is complicated with various disorders. However, the role of ANGPTL2 in ALI remains to be further explored. The mice and MH-S cells were administrated with lipopolysaccharide (LPS) to evoke the lung injury in vivo and in vitro. The role and mechanism of ANGPTL was investigated by haematoxylin-eosin, measurement of wet/dry ratio, cell count, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling, reverse transcription quantitative polymerase chain reaction, immunofluorescence, enzyme-linked immunosorbent assay, detection of autophagic flux and western blot assays. The level of ANGPTL2 was upregulated in lung injury. Knockout of ANGPTL2 alleviated LPS-induced pathological symptoms, reduced pulmonary wet/dry weight ratio, the numbers of total cells and neutrophils in BALF, apoptosis rate and the release of pro-inflammatory mediators, and modulated polarization of alveolar macrophages in mice. Knockdown of ANGPTL2 downregulated the level of pyroptosis indicators, and elevated the level of autophagy in LPS-induced MH-S cells. Besides, downregulation of ANGPTL2 reversed the LPS-induced the expression of leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and triggering receptor expressed on myeloid cells 2 (TREM2), which was reversed by the overexpression of LILRB2. Importantly, knockdown of TREM2 reversed the levels of autophagy- and pyroptosis-involved proteins, and the contents of pro-inflammatory factors in LPS-induced MH-S cells transfected with si ANGPTL2, which was further inverted with the treatment of rapamycin. Therefore, ANGPTL2 silencing enhanced autophagy to alleviate alveolar macrophage pyroptosis via reducing LILRB2-mediated inhibition of TREM2.

MiR-21-5p modulates LPS-induced acute injury in alveolar epithelial cells by targeting SLC16A10.

Sepsis is a systemic inflammatory response syndrome resulting from the invasion of the human body by bacteria and other pathogenic microorganisms. One of its most prevalent complications is acute lung injury, which places a significant medical burden on numerous countries and regions due to its high morbidity and mortality rates. MicroRNA (miRNA) plays a critical role in the body's inflammatory response and immune regulation. Recent studies have focused on miR-21-5p in the context of acute lung injury, but its role appears to vary in different models of this condition. In the LPS-induced acute injury model of A549 cells, there is differential expression, but the specific mechanism remains unclear. Therefore, our aim is to investigate the changes in the expression of miR-21-5p and SLC16A10 in a type II alveolar epithelial cell injury model induced by LPS and explore the therapeutic effects of their targeted regulation. A549 cells were directly stimulated with 10 µg/ml of LPS to construct a model of LPS-induced cell injury. Cells were collected at different time points and the expression of interleukin 1 beta (IL-1ß), tumor necrosis factor-α (TNF-α) and miR-21-5p were measured by RT-qPCR and western blot. Then miR-21-5p mimic transfection was used to up-regulate the expression of miR-21-5p in A549 cells and the expression of IL-1ß and TNF-α in each group of cells was measured by RT-qPCR and western blot. The miRDB, TargetScan, miRWalk, Starbase, Tarbase and miR Tarbase databases were used to predict the miR-21-5p target genes and simultaneously, the DisGeNet database was used to search the sepsis-related gene groups. The intersection of the two groups was taken as the core gene. Luciferase reporter assay further verified SLC16A10 as the core gene with miR-21-5p. The expression of miR-21-5p and SLC16A10 were regulated by transfection or inhibitors in A549 cells with or without LPS stimulation. And then the expression of IL-1ß and TNF-α in A549 cells was tested by RT-qPCR and western blot in different groups, clarifying the role of miR-21-5p-SLC16A10 axis in LPS-induced inflammatory injury in A549 cells. (1) IL-1ß and TNF-α mRNA and protein expression significantly increased at 6, 12, and 24 h after LPS stimulation as well as the miR-21-5p expression compared with the control group (P < 0.05). (2) After overexpression of miR-21-5p in A549 cells, the expression of IL-1ß and TNF-α was significantly reduced after LPS stimulation, suggesting that miR-21-5p has a protection against LPS-induced injury. (3) The core gene set, comprising 51 target genes of miR-21-5p intersecting with the 1448 sepsis-related genes, was identified. This set includes SLC16A10, TNPO1, STAT3, PIK3R1, and FASLG. Following a literature review, SLC16A10 was selected as the ultimate target gene. Dual luciferase assay results confirmed that SLC16A10 is indeed a target gene of miR-21-5p. (4) Knocking down SLC16A10 expression by siRNA significantly reduced the expression of IL-1ß and TNF-α in A549 cells after LPS treatment (P < 0.05). (5) miR-21-5p inhibitor increased the expression levels of IL-1ß and TNF-α in A549 cells after LPS stimulation (P < 0.05). In comparison to cells solely transfected with miR-21-5p inhibitor, co-transfection of miR-21-5p inhibitor and si-SLC6A10 significantly reduced the expression of IL-1ß and TNF-α (P < 0.05). MiR-21-5p plays a protective role in LPS-induced acute inflammatory injury of A549 cells. By targeting SLC16A10, it effectively mitigates the inflammatory response in A549 cells induced by LPS. Furthermore, SLC16A10 holds promise as a potential target for the treatment of acute lung injury.

Inhaled Macrophage Apoptotic Bodies-Engineered Microparticle Enabling Construction of Pro-Regenerative Microenvironment to Fight Hypoxic Lung Injury in Mice.

Oxygen therapy cannot rescue local lung hypoxia in patients with severe respiratory failure. Here, an inhalable platform is reported for overcoming the aberrant hypoxia-induced immune changes and alveolar damage using camouflaged poly(lactic-co-glycolic) acid (PLGA) microparticles with macrophage apoptotic body membrane (cMAB). cMABs are preloaded with mitochondria-targeting superoxide dismutase/catalase nanocomplexes (NCs) and modified with pathology-responsive macrophage growth factor colony-stimulating factor (CSF) chains, which form a core-shell platform called C-cMAB/NC with efficient deposition in deeper alveoli and high affinity to alveolar epithelial cells (AECs) after CSF chains are cleaved by matrix metalloproteinase 9. Therefore, NCs can be effectively transported into mitochondria to inhibit inflammasome-mediated AECs damage in mouse models of hypoxic acute lung injury. Additionally, the at-site CSF release is sufficient to rescue circulating monocytes and macrophages and alter their phenotypes, maximizing synergetic effects of NCs on creating a pro-regenerative microenvironment that enables resolution of lung injury and inflammation. This inhalable platform may have applications to numerous inflammatory lung diseases.

PTPRO inhibits LPS-induced apoptosis in alveolar epithelial cells.

Acute lung injury (ALI) and its severe manifestation, acute respiratory distress syndrome (ARDS), represent critical clinical syndromes with multifactorial origins, notably stemming from sepsis within intensive care units (ICUs). Despite their high mortality rates, no selective cure is available beside ventilation support. Apoptosis plays a complex and pivotal role in the pathophysiology of acute lung injury. Excessive apoptosis of alveolar epithelial and microvascular endothelial cells can lead to disruption of lung epithelial barrier integrity, impairing the body's ability to exchange blood and gas. At the same time, apoptosis of damaged or dysfunctional cells, including endothelial and epithelial cells, can help maintain tissue integrity and accelerate recovery from organ pro-inflammatory stress. The balance between pro-survival and pro-apoptotic signals in lung injury determines patient outcomes, making the modulation of apoptosis an area of intense research in the quest for more effective therapies. Here we found that protein tyrosine phosphatase receptor type O (PTPRO), a poorly understood receptor-like protein tyrosine phosphatase, is consistently upregulated in multiple tissue types of mice under septic conditions and in the lung alveolar epithelial cells. PTPRO reduction by its selective short-interfering RNA (siRNA) leads to excessive apoptosis in lung alveolar epithelial cells without affecting cell proliferation. Consistently PTPRO overexpression by a DNA construct attenuates apoptotic signaling induced by LPS. These effects of PTPTO on cellular apoptosis are dependent on an ErbB2/PI3K/Akt/NFκB signaling pathway. Here we revealed a novel regulatory pathway of cellular apoptosis by PTPRO in lung alveolar epithelial cells during sepsis.

Nebulized Inhalation of Peptide-Modified DNA Origami To Alleviate Acute Lung Injury.

Acute lung injury (ALI) is a severe inflammatory lung disease, with high mortality rates. Early intervention by reactive oxygen species (ROS) scavengers could reduce ROS accumulation, break the inflammation expansion chain in alveolar macrophages (AMs), and avoid irreversible damage to alveolar epithelial and endothelial cells. Here, we reported cell-penetrating R9 peptide-modified triangular DNA origami nanostructures (tDONs-R9) as a novel nebulizable drug that could reach the deep alveolar regions and exhibit an enhanced uptake preference of macrophages. tDONs-R9 suppressed the expression of pro-inflammatory cytokines and drove polarization toward the anti-inflammatory M2 phenotype in macrophages. In the LPS-induced ALI mouse model, treatment with nebulized tDONs-R9 alleviated the overwhelming ROS, pro-inflammatory cytokines, and neutrophil infiltration in the lungs. Our study demonstrates that tDONs-R9 has the potential for ALI treatment, and the programmable DNA origami nanostructures provide a new drug delivery platform for pulmonary disease treatment with high delivery efficiency and biosecurity.

PLD2 deficiency alleviates endothelial glycocalyx degradation in LPS-induced ARDS/ALI.

- Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a life-threatening condition marked by severe lung inflammation and increased lung endothelial barrier permeability. Endothelial glycocalyx deterioration is the primary factor of vascular permeability changes in ARDS/ALI. Although previous studies have shown that phospholipase D2 (PLD2) is closely related to the onset and progression of ARDS/ALI, its role and mechanism in the damage of endothelial cell glycocalyx remains unclear. We used LPS-induced ARDS/ALI mice (in vivo) and LPS-stimulated injury models of EA.hy926 endothelial cells (in vitro). We employed C57BL/6 mice, including wild-type and PLD2 knockout (PLD2-/-) mice, to establish the ARDS/ALI model. We applied immunofluorescence and ELISA to examine changes in syndecan-1 (SDC-1), matrix metalloproteinase-9 (MMP9), inflammatory cytokines (TNF-α, IL-6, and IL-1ß) levels and the effect of external factors, such as phosphatidic acid (PA), 1-butanol (a PLD inhibitor), on SDC-1 and MMP9 expression levels. We found that PLD2 deficiency inhibits SDC-1 degradation and MMP9 expression in LPS-induced ARDS/ALI. Externally added PA decreases SDC-1 levels and increases MMP9 in endothelial cells, hence underlining PA's role in SDC-1 degradation. Additionally, PLD2 deficiency decreases the production of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in LPS-induced ARDS/ALI. In summary, these findings suggest that PLD2 deficiency plays a role in inhibiting the inflammatory process and protecting against endothelial glycocalyx injury in LPS-induced ARDS/ALI.

Erratum - LncRNA gadd7 promotes mitochondrial membrane potential decrease and apoptosis of alveolar type II epithelial cells by positively regulating MFN1 in an in vitro model of hyperoxia-induced acute lung injury.

This corrects the article published in European Journal of Histochemistry 2023;67:3535. doi: 10.4081/ejh.2023.3535.

Deficiency of interleukin-19 exacerbates acute lung injury induced by intratracheal treatment of hydrochloric acid.

Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.

Exploring immune-related pathogenesis in lung injury: Providing new insights Into ALI/ARDS.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a significant global burden of morbidity and mortality, with lung injury being the primary cause of death in affected patients. The pathogenesis of lung injury, however, remains a complex issue. In recent years, the role of the immune system in lung injury has attracted extensive attention worldwide. Despite advancements in our understanding of various lung injury subtypes, significant limitations persist in both prevention and treatment. This review investigates the immunopathogenesis of ALI/ARDS, aiming to elucidate the pathological processes of lung injury mediated by dendritic cells (DCs), natural killer (NK) cells, phagocytes, and neutrophils. Furthermore, the article expounds on the critical contributions of gut microbiota, inflammatory pathways, and cytokine storms in the development of ALI/ARDS.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

The protective effect of Lonicera japonica Thunb. against lipopolysaccharide-induced acute lung injury in mice: Modulation of inflammation, oxidative stress, and ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Various components of Lonicera japonica Thunb. (LJT) exhibit pharmacological activities, including anti-inflammatory and antioxidant effects. Nevertheless, the relationship between LJT and ferroptosis remains largely unexplored. AIM OF THE STUDY: The purpose of this research was to look into the role of LJT in regulating LPS-induced ferroptosis in ALI and to compare the effects of different parts of LJT. MATERIALS AND METHODS: We established a mice ALI model by treating with LPS. Administered mice with different doses of Lonicerae Japonicae Flos (LJF), Lonicera Japonica Leaves (LJL) and Lonicerae Caulis (LRC) extracts, respectively. The levels of IL-6, IL-1ß, TNF-α, IL-4, IL-10, and PGE2 in bronchoalveolar lavage fluid (BALF) were measured using enzyme-linked immunosorbent assay. Furthermore, the concentrations of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS), and total ferrous ions (Fe2+) in lung tissues were evaluated. Hematoxylin and eosin staining was conducted to examine the morphological structure of lung tissues. Transmission electron microscopy was used to investigate the ultrastructural morphology of mitochondria. Furthermore, the effects of LJT were evaluated via immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction analyses. Finally, employing molecular docking and molecular dynamics research techniques, we aimed to identify crucial components in LJT that might inhibit ferroptosis by targeting nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4). RESULTS: We observed that pretreatment with LJT significantly mitigated LPS-induced lung injury and suppressed ferroptosis. This was supported by reduced accumulation of pro-inflammatory cytokines, ROS, MDA, and Fe2+, along with increased levels of anti-inflammatory cytokines, SOD, GSH, Nrf2, and GPX4 in the lung tissues of ALI mice. Luteolin-7-O-rutinoside, apigenin-7-O-rutinoside, and amentoflavone in LJT exhibit excellent docking effects with key targets of ferroptosis, Nrf2 and GPX4. CONCLUSIONS: Pretreatment with LJT may alleviate LPS-induced ALI, possibly by suppressing ferroptosis. Our initial results indicate that LJT activates the Nrf2/GPX4 axis, providing protection against ferroptosis in ALI. This finding offers a promising therapeutic candidate for ALI treatment.

miRNA-206-3p alleviates LPS-induced acute lung injury via inhibiting inflammation and pyroptosis through modulating TLR4/NF-κB/NLRP3 pathway.

Acute lung injury (ALI) is life-threatening. MicroRNAs (miRNAs) are often abnormally expressed in inflammatory diseases and are closely associated with ALI. This study investigates whether miRNA-206-3p attenuates pyroptosis in ALI and elucidates the underlying molecular mechanisms. ALI mouse and cell models were established through lipopolysaccharide (LPS) treatment for 24 h. Subsequently, the models were evaluated based on ultrasonography, the lung tissue wet/dry (W/D) ratio, pathological section assessment, electron microscopy, and western blotting. Pyroptosis in RAW264.7 cells was then assessed via electron microscopy, immunofluorescence, and western blotting. Additionally, the regulatory relationship between miRNA-206-3p and the Toll-like receptor (TLR)4/nuclear factor (NF)-κB/Nod-like receptor protein-3 (NLRP3) pathway was verified. Finally, luciferase reporter gene and RNA pull-down assays were used to verify the targeting relationship between miRNA-206-3p and TLR4. miRNA206-3p levels are significantly decreased in the LPS-induced ALI model. Overexpression of miRNA-206-3p improves ALI, manifested as improved lung ultrasound, improved pathological changes of lung tissue, reduced W/D ratio of lung tissue, release of inflammatory factors in lung tissue, and reduced pyroptosis. Furthermore, overexpression of miRNA-206-3p contributed to reversing the ALI-promoting effect of LPS by hindering TLR4, myeloid differentiation primary response 88 (MyD88), NF-κB, and NLRP3 expression. In fact, miRNA-206-3p binds directly to TLR4. In conclusion, miRNA-206-3p alleviates LPS-induced ALI by inhibiting inflammation and pyroptosis via TLR4/NF-κB/NLRP3 pathway modulation.

Vaporization of perfluorocarbon attenuates sea-water-drowning-induced acute lung injury by deactivating the NLRP3 inflammasomes in canines.

Seawater-drowning-induced acute lung injury (SD-ALI) is a life-threatening disorder characterized by increased alveolar-capillary permeability, an excessive inflammatory response, and refractory hypoxemia. Perfluorocarbons (PFCs) are biocompatible compounds that are chemically and biologically inert and lack toxicity as oxygen carriers, which could reduce lung injury in vitro and in vivo. The aim of our study was to explore whether the vaporization of PFCs could reduce the severity of SD-ALI in canines and investigate the underlying mechanisms. Eighteen beagle dogs were randomly divided into three groups: the seawater drowning (SW), perfluorocarbon (PFC), and control groups. The dogs in the SW group were intratracheally administered seawater to establish the animal model. The dogs in the PFC group were treated with vaporized PFCs. Probe-based confocal laser endomicroscopy (pCLE) was performed at 3 h. The blood gas, volume air index (VAI), pathological changes, and wet-to-dry (W/D) lung tissue ratios were assessed. The expression of heme oxygenase-1 (HO-1), nuclear respiratory factor-1 (NRF1), and NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasomes was determined by means of quantitative real-time polymerase chain reaction (qRT-PCR) and immunological histological chemistry. The SW group showed higher lung injury scores and W/D ratios, and lower VAI compared to the control group, and treatment with PFCs could reverse the change of lung injury score, W/D ratio and VAI. PFCs deactivated NLRP3 inflammasomes and reduced the release of caspase-1, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) by enhancing the expression of HO-1 and NRF1. Our results suggest that the vaporization of PFCs could attenuate SD-ALI by deactivating NLRP3 inflammasomes via the HO-1/NRF1 pathway.

Echinatin attenuates acute lung injury and inflammatory responses via TAK1-MAPK/NF-κB and Keap1-Nrf2-HO-1 signaling pathways in macrophages.

Echinatin is an active ingredient in licorice, a traditional Chinese medicine used in the treatment of inflammatory disorders. However, the protective effect and underlying mechanism of echinatin against acute lung injury (ALI) is still unclear. Herein, we aimed to explore echinatin-mediated anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated ALI and its molecular mechanisms in macrophages. In vitro, echinatin markedly decreased the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated murine MH-S alveolar macrophages and RAW264.7 macrophages by suppressing inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) expression. Furthermore, echinatin reduced LPS-induced mRNA expression and release of interleukin-1ß (IL-1ß) and IL-6 in RAW264.7 cells. Western blotting and CETSA showed that echinatin repressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways through targeting transforming growth factor-beta-activated kinase 1 (TAK1). Furthermore, echinatin directly interacted with Kelch-like ECH-associated protein 1 (Keap1) and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to enhance heme oxygenase-1 (HO-1) expression. In vivo, echinatin ameliorated LPS-induced lung inflammatory injury, and reduced production of IL-1ß and IL-6. These findings demonstrated that echinatin exerted anti-inflammatory effects in vitro and in vivo, via blocking the TAK1-MAPK/NF-κB pathway and activating the Keap1-Nrf2-HO-1 pathway.

[Heme oxygenase-1 (HO-1) gene knockout affects the balance of lung immune cell composition and aggravates inflammatory injury in lung tissues of LPS-induced acute lung injury (ALI) mice].

Objective To evaluate the effects of heme oxygenase-1 (HO-1) gene deletion on immune cell composition and inflammatory injury in lung tissues of mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods C57BL/6 wild-type (WT) mice and HO-1 conditional-knockout (HO-1-/-) mice on the same background were randomly divided into four groups (n=5 in every group): WT control group, LPS-treated WT group, HO-1-/- control group and LPS-treated HO-1-/- group. LPS-treated WT and HO-1-/- groups were injected with LPS (15 mg/kg) through the tail vein to establish ALI model, while WT control group and HO-1-/- control group were injected with an equivalent volume of normal saline through the tail vein, respectively. Twelve hours later, the mice were sacrificed and lung tissues from each group were collected for analysis. Histopathological alterations of lung tissues were assessed by HE staining. The levels of mRNA expression of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 were determined by PCR. The percentages of neutrophils (CD45+CD11b+Ly6G+Ly6C-), total monocytes (CD45+CD11b+Ly6Chi), pro-inflammatory monocyte subsets (CD45+CD11b+Ly6ChiCCR2hi) and total macrophages (CD45+CD11b+F4/80+), M1 macrophage (CD45+CD11b+F4/80+CD86+), M2 macrophage (CD45+CD11b+F4/80+CD206+), total T cells (CD45+CD3+), CD3+CD4+ T cells, CD3+CD8+ T cells and myeloid suppressor cells (MDSCs, CD45+CD11b+Gr1+) were detected by flow cytometry. Results Compared with the corresponding control groups, HE staining exhibited increased inflammation in the lung tissues of both LPS-treated WT and HO-1-/- model mice; mRNA expression levels of TNF-α, IL-1ß and IL-6 were up-regulated; the proportions of neutrophils, total monocytes, pro-inflammatory monocyte subsets, MDSCs and total macrophages increased significantly. The percentage of CD3+, CD3+CD4+ and CD3+CD8+ T cells decreased significantly. Under resting-state, compared with WT control mice, the proportion of neutrophils, monocytes and pro-inflammatory monocyte subset increased in lung tissues of HO-1-/- control mice, while the proportion of CD3+ and CD3+CD8+ T cells decreased. Compared with LPS-treated WT mice, the mRNA expression levels of TNF-α and IL-1ß were up-regulated in lung tissues of LPS-treated HO-1-/- mice; the proportion of total monocytes, pro-inflammatory monocyte subsets, M1 macrophages and M1/M2 ratio increased greatly; the percentage of CD3+CD8+ T cells decreased significantly. Conclusion The deletion of HO-1 affects the function of the lung immune system and aggravates the inflammatory injury after LPS stimulation in ALI mice.