Molecular imaging of chemokine-like receptor 1 (CMKLR1) in experimental acute lung injury.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

GLUT1 Promotes NLRP3 Inflammasome Activation of Airway Epithelium in Lipopolysaccharide-Induced Acute Lung Injury.

Acute lung injury (ALI) is a devastating clinical syndrome caused by different factors, with high morbidity and mortality. Lung injury and inflammation caused by lipopolysaccharide (LPS) can be modulated by NLRP3 inflammasome activation, yet its exact function within the airway epithelium is still unknown. Meanwhile, glucose transporter protein 1 (GLUT1) contributes to a number of inflammatory illnesses, including ALI. The present study aimed to assess GLUT1's function in NLRP3 inflammasome activation of airway epithelium in LPS-induced acute lung injury. BALB/c mice and BEAS-2B cells were exposed to LPS (5 mg/kg and 200 µg/mL, respectively), with or without GLUT1 antagonists (WZB117 or BAY876). LPS up-regulated pulmonary expression of NLRP3 and GLUT1 in mice, which could be blocked by WZB117 or BAY876. Pharmacological inhibition of GLUT1 in vivo significantly attenuated lung tissue damage, neutrophil accumulation, and proinflammatory factors release (TNF-α, IL-6, and IL-1ß) in LPS-exposed mice. Meanwhile, the activation markers of NLRP3 inflammasome (ASC, caspase-1, IL-1ß, and IL-18) induced by LPS were also suppressed. In cultured BEAS-2B cells, LPS induced an increase in GLUT1 expression and triggered activation of the NLRP3 inflammasome, both of which were inhibited by GLUT1 antagonists. These results illustrate that GLUT1 participates in LPS-induced ALI and promotes the activation of the NLRP3 inflammasome in airway epithelial cells.

VNS-mediated α7nAChR signaling promotes SPM synthesis via regulation of netrin-1 expression during LPS-induced ALI.

The α7 nicotinic acetylcholine receptor (α7nAChR) plays a crucial role in the cholinergic anti-inflammatory pathway (CAP) during sepsis-associated acute lung injury (ALI). Increasing evidence suggests that specialized pro-resolving mediators (SPMs) are important in resolving α7nAChR-mediated ALI resolution. Our study aims to elucidate the pivotal role of α7nAChR in the CAP during LPS-associated acute lung injury (ALI). By employing vagus nerve stimulation (VNS), we identified α7nAChR as the key CAP subunit in ALI mice, effectively reducing lung permeability and the release of inflammatory cytokines. We further investigated the alterations in SPMs regulated by α7nAChR, revealing a predominant synthesis of lipoxin A4 (LXA4). The significance of α7nAChR-netrin-1 pathway in governing SPM synthesis was confirmed through the use of netrin-1 knockout mice and siRNA-transfected macrophages. Additionally, our evaluation identified a synchronous alteration of LXA4 synthesis in the α7nAChR-netrin-1 pathway accompanied by 5-lipoxygenase (5-LOX), thereby confirming an ameliorative effect of LXA4 on lung injury and macrophage inflammatory response. Concurrently, inhibiting the function of LXA4 annulled the lung-protective effect of VNS. As a result, our findings reveal a novel anti-inflammatory pathway wherein VNS modulates netrin-1 expression via α7nAChR, ultimately leading to LXA4 synthesis and subsequent lung protection.

Characterization of Chemotaxis-Associated Gene Dysregulation in Myeloid Cell Populations in the Lungs during Lipopolysaccharide-Mediated Acute Lung Injury.

During endotoxin-induced acute lung injury (ALI), immune cell recruitment resulting from chemotaxis is mediated by CXC and CC chemokines and their receptors. In this study, we investigated the role of chemokines and their receptors in the regulation of myeloid cell populations in the circulation and the lungs of C57BL/6J mice exhibiting LPS-mediated ALI using single-cell RNA sequencing. During ALI, there was an increase in the myeloid cells, M1 macrophages, monocytes, neutrophils, and other granulocytes, whereas there was a decrease in the residential alveolar macrophages and M2 macrophages. Interestingly, LPS triggered the upregulation of CCL3, CCL4, CXCL2/3, and CXCL10 genes associated with cellular migration of various subsets of macrophages, neutrophils, and granulocytes. Furthermore, there was an increase in the frequency of myeloid cells expressing CCR1, CCR3, CCR5, and CXCR2 receptors during ALI. MicroRNA sequencing studies of vehicle versus LPS groups identified several dysregulated microRNAs targeting the upregulated chemokine genes. This study suggests that chemokine ligand-receptors interactions are responsible for myeloid cell heterogenicity and cellular recruitment to the lungs during ALI. The single-cell transcriptomics allowed for an in-depth assessment and characterization of myeloid cells involved in immune cell trafficking during ALI.

In vivo tracking of mesenchymal stem cell dynamics and therapeutics in LPS-induced acute lung injury models.

Mesenchymal stem cells (MSCs) have been widely used to treat various inflammatory and immune-related diseases in preclinical and clinical settings. Intravital microscopy (IVM) is considered the gold standard for investigating pathophysiological conditions in living animals. However, the potential for real-time monitoring of MSCs in the pulmonary microenvironment remains underexplored. In this study, we first constructed a lung window and captured changes in the lung at the cellular level under both inflammatory and noninflammatory conditions with a microscope. We further investigated the dynamics and effects of MSCs under two different conditions. Meanwhile, we assessed the alterations in the adhesive capacity of vascular endothelial cells in vitro to investigate the underlying mechanisms of MSC retention in an inflammatory environment. This study emphasizes the importance of the "lung window" for live imaging of the cellular behavior of MSCs by vein injection. Moreover, our results revealed that the upregulation of vascular cell adhesion molecule 1 (VCAM1) in endothelial cells post-inflammatory injury could enhance MSC retention in the lung, further ameliorating acute lung injury. In summary, intravital microscopy imaging provides a practical method to investigate the therapeutic effects of MSCs in acute lung injury.

STING mediates LPS-induced acute lung injury by regulating ferroptosis.

The pathogenesis of acute lung injury is not fully understood. Stimulator of interferon genes (STING) and ferroptosis have been implicated in various pathological and physiological processes, including acute lung injury (ALI). However, the relationship between STING and ferroptosis in lipopolysaccharide (LPS)-induced ALI is unclear. We found that LPS stimulation activated STING and ferroptosis. Furthermore, STING knockout and ferroptosis inhibitor alleviated lung inflammation and epithelial cell damage. Also, STING knockout reduced inflammation injury and ferroptosis. Notably, the ferroptosis inducer reversed the alleviation of inflammation caused by STING knockout. These results show that STING participates in the inflammation injury of ALI by regulating ferroptosis. Results also showed that p-STAT3 levels increased after STING knockout, suggesting that STING negatively regulates STAT3 activation. Besides, STAT3 inhibitor aggravated ferroptosis after STING knockout, indicating that STING regulates ferroptosis through STAT3 signaling. In conclusion, STING mediates LPS-induced ALI by regulating ferroptosis, indicating that STING and ferroptosis may be new targets for ALI treatment.

MSCs alleviate LPS-induced acute lung injury by inhibiting the proinflammatory function of macrophages in mouse lung organoid-macrophage model.

Acute lung injury (ALI) is an inflammatory disease associated with alveolar injury, subsequent macrophage activation, inflammatory cell infiltration, and cytokine production. Mesenchymal stem cells (MSCs) are beneficial for application in the treatment of inflammatory diseases due to their immunomodulatory effects. However, the mechanisms of regulatory effects by MSCs on macrophages in ALI need more in-depth study. Lung tissues were collected from mice for mouse lung organoid construction. Alveolar macrophages (AMs) derived from bronchoalveolar lavage and interstitial macrophages (IMs) derived from lung tissue were co-cultured, with novel matrigel-spreading lung organoids to construct an in vitro model of lung organoids-immune cells. Mouse compact bone-derived MSCs were co-cultured with organoids-macrophages to confirm their therapeutic effect on acute lung injury. Changes in transcriptome expression profile were analyzed by RNA sequencing. Well-established lung organoids expressed various lung cell type-specific markers. Lung organoids grown on spreading matrigel had the property of functional cells growing outside the lumen. Lipopolysaccharide (LPS)-induced injury promoted macrophage chemotaxis toward lung organoids and enhanced the expression of inflammation-associated genes in inflammation-injured lung organoids-macrophages compared with controls. Treatment with MSCs inhibited the injury progress and reduced the levels of inflammatory components. Furthermore, through the nuclear factor-κB pathway, MSC treatment inhibited inflammatory and phenotypic transformation of AMs and modulated the antigen-presenting function of IMs, thereby affecting the inflammatory phenotype of lung organoids. Lung organoids grown by spreading matrigel facilitate the reception of external stimuli and the construction of in vitro models containing immune cells, which is a potential novel model for disease research. MSCs exert protective effects against lung injury by regulating different functions of AMs and IMs in the lung, indicating a potential mechanism for therapeutic intervention.

PEBP4 deficiency aggravates LPS-induced acute lung injury and alveolar fluid clearance impairment via modulating PI3K/AKT signaling pathway.

Acute lung injury (ALI) is a common clinical syndrome, which often results in pulmonary edema and respiratory distress. It has been recently reported that phosphatidylethanolamine binding protein 4 (PEBP4), a basic cytoplasmic protein, has anti-inflammatory and hepatoprotective effects, but its relationship with ALI remains undefined so far. In this study, we generated PEBP4 knockout (KO) mice to investigate the potential function of PEBP4, as well as to evaluate the capacity of alveolar fluid clearance (AFC) and the activity of phosphatidylinositide 3-kinases (PI3K)/serine-theronine protein kinase B (PKB, also known as AKT) signaling pathway in lipopolysaccharide (LPS)-induced ALI mice models. We found that PEBP4 deficiency exacerbated lung pathological damage and edema, and increased the wet/dry weight ratio and total protein concentration of bronchoalveolar lavage fluid (BALF) in LPS-treated mice. Meanwhile, PEBP4 KO promoted an LPS-induced rise in the pulmonary myeloperoxidase (MPO) activity, serum interleuin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels, and pulmonary cyclooxygenase-2 (COX-2) expression. Mechanically, PEBP4 deletion further reduced the protein expression of Na+ transport markers, including epithelial sodium channel (ENaC)-α, ENaC-γ, Na,K-ATPase α1, and Na,K-ATPase ß1, and strengthened the inhibition of PI3K/AKT signaling in LPS-challenged mice. Furthermore, we demonstrated that selective activation of PI3K/AKT with 740YP or SC79 partially reversed all of the above effects caused by PEBP4 KO in LPS-treated mice. Altogether, our results indicated the PEBP4 deletion has a deterioration effect on LPS-induced ALI by impairing the capacity of AFC, which may be achieved through modulating the PI3K/AKT pathway.

Tropomodulin1 exacerbates inflammatory response in macrophages by negatively regulating LPS-induced TLR4 endocytosis.

The excessive inflammation caused by the prolonged activation of Toll-like receptor 4 (TLR4) and its downstream signaling pathways leads to sepsis. CD14-mediated endocytosis of TLR4 is the key step to control the amount of TLR4 on cell membrane and the activity of downstream pathways. The actin cytoskeleton is necessary for receptor-mediated endocytosis, but its role in TLR4 endocytosis remains elusive. Here we show that Tropomodulin 1 (Tmod1), an actin capping protein, inhibited lipopolysaccharide (LPS)-induced TLR4 endocytosis and intracellular trafficking in macrophages. Thus it resulted in increased surface TLR4 and the upregulation of myeloid differentiation factor 88 (MyD88)-dependent pathway and the downregulation of TIR domain-containing adaptor-inducing interferon-ß (TRIF)-dependent pathway, leading to the enhanced secretion of inflammatory cytokines, such as TNF-α and IL-6, and the reduced secretion of cytokines, such as IFN-ß. Macrophages deficient with Tmod1 relieved the inflammatory response in LPS-induced acute lung injury mouse model. Mechanistically, Tmod1 negatively regulated LPS-induced TLR4 endocytosis and inflammatory response through modulating the activity of CD14/Syk/PLCγ2/IP3/Ca2+ signaling pathway, the reorganization of actin cytoskeleton, and the membrane tension. Therefore, Tmod1 is a key regulator of inflammatory response and immune functions in macrophages and may be a potential target for the treatment of excessive inflammation and sepsis.

Antimicrobial Drug Penetration Is Enhanced by Lung Tissue Inflammation and Injury.

Rationale: Pneumonia is a frequent and feared complication in intubated critically ill patients. Tissue concentrations of antimicrobial drugs need to be sufficiently high to treat the infection and also prevent development of bacterial resistance. It is uncertain whether pulmonary inflammation and injury affect antimicrobial drug penetration into lung tissue.Objectives: To determine and compare tissue and BAL fluid concentrations of ceftaroline fosamil and linezolid in a model of unilateral acute lung injury in pigs and to evaluate whether dose adjustment is necessary to reach sufficient antimicrobial concentrations in injured lung tissue.Methods: After induction of unilateral acute lung injury, ceftaroline fosamil and linezolid were administered intravenously. Drug concentrations were measured in lung tissue through microdialysis and in blood and BAL fluid samples during the following 8 hours. The primary endpoint was the tissue concentration area under the concentration curve in the first 8 hours (AUC0-8 h) of the two antimicrobial drugs.Measurements and Main Results: In 10 pigs, antimicrobial drug concentrations were higher in inflamed and injured lung tissue compared with those in uninflamed and uninjured lung tissue (median ceftaroline fosamil AUC0-8 h [and interquartile range] = 26.7 mg ⋅ h ⋅ L-1 [19.7-39.0] vs. 16.0 mg ⋅ h ⋅ L-1 [13.6-19.9], P = 0.02; median linezolid AUC0-8 h 76.0 mg ⋅ h ⋅ L-1 [68.1-96.0] vs. 54.6 mg ⋅ h ⋅ L-1 [42.7-60.9], P = 0.01), resulting in a longer time above the minimal inhibitory concentration and in higher peak concentrations and dialysate/plasma ratios. Penetration into BAL fluid was excellent for both antimicrobials, but without left-to-right differences (ceftaroline fosamil, P = 0.78; linezolid, P = 1.00).Conclusions: Tissue penetration of two commonly used antimicrobial drugs for pneumonia is enhanced by early lung tissue inflammation and injury, resulting in longer times above the minimal inhibitory concentration. Thus, lung tissue inflammation ameliorates antimicrobial drug penetration during the acute phase.

RNA-Seq profiling of circular RNAs in mice with lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) is a serious illness that develops suddenly, progresses rapidly, has a poor treatment response and a high mortality rate. Studies have found that circular RNAs (circRNA) play a critical role in several diseases, but their role in ALI remains unclear. The aim of this study was to identify circRNAs that are associated with ALI and investigate their potential molecular mechanisms. A comparison of lung circRNA and microRNA expression profiles in mice with ALI and controls was performed by RNA-sequencing. A bioinformatic analysis was conducted to identify differentially expressed (DE) RNAs, to construct competitive endogenous RNA (ceRNA) networks, and to analyze their function and pathways. Then, a protein-protein interaction (PPI) network was generated by the Search Tool for the Retrieval of Interacting Genes database, and hub genes were identified using Cytoscape. Furthermore, a key ceRNA subnetwork was constructed based on these hub genes. Overall, we found 239 DE circRNAs and 42 DE microRNAs in ALI mice compared to controls. Additionally, the molecular mechanism of ALI was further understood by building ceRNA networks based on these DE genes. ALI-induced circRNAs are mostly function in the inflammatory response and metabolic processes. Moreover, DE circRNAs are primarily involved in the nuclear factor (NF)-kappa B and PI3K-Akt signaling pathways. Seven hub genes were derived from the PPI network of 191 genes, followed by the construction of circRNA-miRNA-hub gene subnetworks. In this study, circRNA profiles are remarkably changed in mice with LPS-triggered ALI, and their potential contribution to the disease is revealed.

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.

FTO Deficiency Alleviates LPS-induced Acute Lung Injury by TXNIP/NLRP3-mediated Alveolar Epithelial Cell Pyroptosis.

N6-methyladenosine (m6A) plays a role in various diseases, but it has rarely been reported in acute lung injury (ALI). The FTO (fat mass and obesity-associated) protein can regulate mRNA metabolism by removing m6A residues. The aim of this study was to examine the role and mechanism of the m6A demethylase FTO in LPS-induced ALI. Lung epithelial FTO-knockout mice and FTO-knockdown/overexpression human alveolar epithelial (A549) cell lines were constructed to evaluate the effects of FTO on ALI. Bioinformatics analysis and a series of in vivo and in vitro assays were used to examine the mechanism of FTO regulation. Rescue assays were conducted to examine whether the impact of FTO on ALI depended on the TXNIP/NLRP3 pathway. In LPS-induced ALI, RNA m6A modification amounts were upregulated, and FTO expression was downregulated. In vivo, lung epithelial FTO knockout alleviated alveolar structure disorder, tissue edema, and pulmonary inflammation and improved the survival of ALI mice. In vitro, FTO knockdown reduced A549 cell damage and death induced by LPS, whereas FTO overexpression exacerbated cell damage and death. Mechanistically, bioinformatics analysis revealed that TXNIP was a downstream target of FTO. FTO deficiency mitigated pyroptosis in LPS-induced ALI via the TXNIP/NLRP3 pathway. Rescue assays confirmed that the impact of FTO on the TXNIP/NLRP3 pathway was significantly reversed by the TXNIP inhibitor SRI-37330. Deficiency of FTO alleviates LPS-induced ALI via TXNIP/NLRP3 pathway-mediated alveolar epithelial cell pyroptosis, which might be a novel therapeutic strategy for combating ALI.

Indole-3-Acetic Acid Protects Against Lipopolysaccharide-induced Endothelial Cell Dysfunction and Lung Injury through the Activation of USP40.

Lung microvascular endothelial cell (EC) dysfunction is the pathological hallmark of acute respiratory distress syndrome. Heat shock protein 90 (HSP90) is a key regulator in control of endothelial barrier disruption and inflammation. Our recent study has demonstrated that ubiquitin-specific peptidase 40 (USP40) preserves endothelial integrity by targeting HSP90ß for its deubiquitination and inactivation. Indole-3-acetic acid (IAA), a plant hormone of the auxin class, can also be catabolized from dietary tryptophan by the intestinal microbiota. Accumulating evidence suggests that IAA reduces oxidative stress and inflammation and promotes intestinal barrier function. However, little is known about the role of IAA in endothelial cells and acute lung injury. In this study, we investigated the role of IAA in lung endothelial cell function in the context of acute lung injury. IAA exhibited EC barrier protection against LPS-induced reduction in transendothelial electrical resistance and inflammatory responses. The underlying mechanism of IAA on EC protective effects was investigated by examining the influence of IAA on degrees of HSP90 ubiquitination and USP40 activity. We identified that IAA, acting as a potential activator of USP40, reduces HSP90 ubiquitination, thereby protecting against LPS-induced inflammation in human lung microvascular endothelial cells as well as alleviating experimental lung injury. Furthermore, the EC protective effects of IAA against LPS-induced EC dysfunction and lung injury were abolished in USP40-deficient human lung microvascular endothelial cell and lungs of USP40 EC-specific knockout (USP40cdh5-ECKO) mice. Taken together, this study reveals that IAA protects against LPS-induced EC dysfunction and lung injury through the activation of USP40.

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.

Plumbagin ameliorates LPS-induced acute lung injury by regulating PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling pathways.

Acute lung injury (ALI) is a major pathophysiological problem characterized by severe inflammation, resulting in high morbidity and mortality. Plumbagin (PL), a major bioactive constituent extracted from the traditional Chinese herb Plumbago zeylanica, has been shown to possess anti-inflammatory and antioxidant pharmacological activities. However, its protective effect on ALI has not been extensively studied. The objective of this study was to investigate the protective effect of PL against ALI induced by LPS and to elucidate its possible mechanisms both in vivo and in vitro. PL treatment significantly inhibited pathological injury, MPO activity, and the wet/dry ratio in lung tissues, and decreased the levels of inflammatory cells and inflammatory cytokines TNF-α, IL-1ß, IL-6 in BALF induced by LPS. In addition, PL inhibited the activation of the PI3K/AKT/mTOR signalling pathway, increased the activity of antioxidant enzymes CAT, SOD, GSH and activated the Keap1/Nrf2/HO-1 signalling pathway during ALI induced by LPS. To further assess the association between the inhibitory effects of PL on ALI and the PI3K/AKT/mTOR and Keap1/Nrf2/HO-1 signalling, we pretreated RAW264.7 cells with 740Y-P and ML385. The results showed that the activation of PI3K/AKT/mTOR signalling reversed the protective effect of PL on inflammatory response induced by LPS. Moreover, the inhibitory effects of PL on the production of inflammatory cytokines induced by LPS also inhibited by downregulating Keap1/Nrf2/HO-1 signalling. In conclusion, the results indicate that the PL ameliorate LPS-induced ALI by regulating the PI3K/AKT/mTOR and Keap1-Nrf2/HO-1 signalling, which may provide a novel therapeutic perspective for PL in inhibiting ALI.

Aqueous extract of Descuraniae Semen attenuates lipopolysaccharide-induced inflammation by inhibiting ER stress and WNK4-SPAK-NKCC1 pathway.

Sepsis causes systemic inflammatory responses and acute lung injury (ALI). Despite modern treatments, sepsis-related ALI mortality remains high. Aqueous extract of Descuraniae Semen (AEDS) exerts anti-endoplasmic reticulum (ER) stress, antioxidant and anti-inflammatory effects. AEDS alleviates inflammation and oedema in ALI. Sodium-potassium-chloride co-transporter isoform 1 (NKCC1) is essential for regulating alveolar fluid and is important in ALI. The NKCC1 activity is regulated by upstream with-no-lysine kinase-4 (WNK4) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). This study aimed to investigate the effects of AEDS on lipopolysaccharide (LPS)-induced ALI model in A549 cells, considering the regulation of ER stress, WNK4-SPAK-NKCC1 cascades, inflammation and apoptosis. Cell viability was investigated by the CCK-8 assay. The expressions of the proteins were assessed by immunoblotting analysis assays. The levels of pro-inflammatory cytokines were determined by ELISA. The expression of cytoplasmic Ca2+ in A549 cells was determined using Fluo-4 AM. AEDS attenuates LPS-induced inflammation, which is associated with increased pro-inflammatory cytokine expression and activation of the WNK4-SPAK-NKCC1 pathway. AEDS inhibits the WNK4-SPAK-NKCC1 pathway by regulating of Bcl-2, IP3R and intracellular Ca2+. WNK4 expression levels are significantly higher in the WNK4-overexpressed transfected A549 cells and significantly decrease after AEDS treatment. AEDS attenuates LPS-induced inflammation by inhibiting the WNK4-SPAK-NKCC1 cascade. Therefore, AEDS is regarded as a potential therapeutic agent for ALI.

CGRP is essential for protection against alveolar epithelial cell necroptosis by activating the AMPK/L-OPA1 signaling pathway during acute lung injury.

Alveolar epithelial cell (AEC) necroptosis is critical to disrupt the alveolar barrier and provoke acute lung injury (ALI). Here, we define calcitonin gene-related peptide (CGRP), the most abundant endogenous neuropeptide in the lung, as a novel modulator of AEC necroptosis in lipopolysaccharide (LPS)-induced ALI. Upon LPS-induced ALI, overexpression of Cgrp significantly mitigates the inflammatory response, alleviates lung tissue damage, and decreases AEC necroptosis. Similarly, CGRP alleviated AEC necroptosis under the LPS challenge in vitro. Previously, we identified that long optic atrophy 1 (L-OPA1) deficiency mediates mitochondrial fragmentation, leading to AEC necroptosis. In this study, we discovered that CGRP positively regulated mitochondrial fusion through stabilizing L-OPA1. Mechanistically, we elucidate that CGRP activates AMP-activated protein kinase (AMPK). Furthermore, the blockade of AMPK compromised the protective effect of CGRP against AEC necroptosis following the LPS challenge. Our study suggests that CRGP-mediated activation of the AMPK/L-OPA1 axis may have potent therapeutic benefits for patients with ALI or other diseases with necroptosis.

Umbilical cord MSC-derived exosomes improve alveolar macrophage function and reduce LPS-induced acute lung injury.

Acute lung injury (ALI) is a severe condition that can progress to acute respiratory distress syndrome (ARDS), with a high mortality rate. Currently, no specific and compelling drug treatment plan exists. Mesenchymal stem cells (MSCs) have shown promising results in preclinical and clinical studies as a potential treatment for ALI and other lung-related conditions due to their immunomodulatory properties and ability to regenerate various cell types. The present study focuses on analyzing the role of umbilical cord MSC (UC-MSC))-derived exosomes in reducing lipopolysaccharide-induced ALI and investigating the mechanism involved. The study demonstrates that UC-MSC-derived exosomes effectively improved the metabolic function of alveolar macrophages and promoted their shift to an anti-inflammatory phenotype, leading to a reduction in ALI. The findings also suggest that creating three-dimensional microspheres from the MSCs first can enhance the effectiveness of the exosomes. Further research is needed to fully understand the mechanism of action and optimize the therapeutic potential of MSCs and their secretome in ALI and other lung-related conditions.