Murine Model Of Acute Lung Injury Research Articles

Effects of ozone exposure on lung injury, inflammation, and oxidative stress in a murine model of non-pneumonic endotoxemia.

Recent studies have identified exposure to environmental levels of ozone as a risk factor for the development of acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI) that can develop in humans with sepsis. The aim of this study was to develop a murine model of ALI to mechanistically explore the impact of ozone exposure on ARDS development. Mice were exposed to ozone (0.8 ppm, 3 hr) or air control followed 24 hr later by intravenous administration of 3 mg/kg lipopolysaccharide (LPS) or PBS. Exposure of mice to ozone + LPS caused alveolar hyperplasia; increased BAL levels of albumin, IgM, phospholipids, and proinflammatory mediators including surfactant protein D and soluble receptor for advanced glycation end products were also detected in BAL, along with markers of oxidative and nitrosative stress. Administration of ozone + LPS resulted in an increase in neutrophils and anti-inflammatory macrophages in the lung, with no effects on proinflammatory macrophages. Conversely, numbers of resident alveolar macrophages decreased after ozone + LPS; however, expression of Nos2, Arg1, Cxcl1, Cxcl2, Ccl2 by these cells increased, indicating that they are activated. These findings demonstrate that ozone sensitizes the lung to respond to endotoxin, resulting in ALI, oxidative stress and exacerbated pulmonary inflammation, and provide support for the epidemiologic association between ozone exposure and ARDS incidence.

Impact of lipopolysaccharide-induced acute lung injury in aged mice

Study Aim: As the geriatric population rapidly expands, there has been a concurrent increase in elderly admissions to intensive care units (ICUs). Acute lung injury (ALI) is a prevalent reason for these admissions and carries poorer survival rates for the aged population compared to younger counterparts. The aging lung is subject to physiological, cellular, and immunological changes. However, our understanding of how aging impacts the clinical progression of ALI is limited. This study explored the effect of aging using a murine model of ALI. Methods: Female C57BL/6J mice, aged 7–8 wk (young) and 18 months (aged), were divided into four groups: young controls, aged controls, young with ALI (YL), and aged with ALI (AL). ALI was induced via intratracheal administration of lipopolysaccharide (LPS, 0.5 mg/kg). The animals were euthanized 72 h after LPS exposure. Results: The AL group exhibited a significantly increased wet/dry ratio compared to the other three groups, including the YL group. The bronchoalveolar lavage (BAL) fluid in the AL group had more cells overall, including more neutrophils, than the other groups. Inflammatory cytokines in BAL fluid showed similar trends. Histological analyses demonstrated more severe lung injury and fibrosis in the AL group than in the other groups. Increased transcription of senescence-associated secretory phenotype markers, including PAI-1 and MUC5B, was more prominent in the AL group than in the other groups. This trend was also observed in BAL samples from humans with pneumonia. Conclusions: Aging may amplify lung damage and inflammatory responses in ALI. This suggests that physicians should exercise increased caution in the clinical management of aged patients with ALI.

Modulation of experimental acute lung injury by exosomal miR-7704 from mesenchymal stromal cells acts through M2 macrophage polarization

Acute lung injury (ALI) is a life-threatening condition with limited treatment options. The pathogenesis of ALI involves macrophage-mediated disruption and subsequent repair of the alveolar barriers, which ultimately results in lung damage and regeneration, highlighting the pivotal role of macrophage polarization in ALI. Although exosomes derived from mesenchymal stromal cells have been established as influential modulators of macrophage polarization, the specific role of exosomal microRNAs (miRNAs) remains underexplored. This study aimed to elucidate the role of specific exosomal miRNAs in driving macrophage polarization, thereby providing a reference for developing novel therapeutic interventions for ALI. We found that miR-7704 is the most abundant and efficacious miRNA for promoting the switch to the M2 phenotype in macrophages. Mechanistically, we determined that miR-7704 stimulates M2 polarization by inhibiting the MyD88/STAT1 signaling pathway. Notably, intra-tracheal delivery of miR-7704 alone in a lipopolysaccharide-induced murine ALI model significantly drove M2 polarization in lung macrophages and remarkably restored pulmonary function, thus increasing survival. Our findings highlight miR-7704 as a valuable tool for treating ALI by driving the beneficial M2 polarization of macrophages. Our findings pave the way for deeper exploration into the therapeutic potential of exosomal miRNAs in inflammatory lung diseases.

Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store-operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

DMPP attenuates lipopolysaccharide-induced lung injury by inhibiting glycocalyx degradation through activation of the cholinergic anti-inflammatory pathway.

The study aimed to investigate the therapeutic potential of 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptor (nAChR), in treating acute lung injury (ALI) induced by lipopolysaccharide (LPS). A murine ALI model was developed utilizing intraperitoneal injection of LPS. We evaluated the therapeutic efficacy of DMPP treatment in LPS-induced lung injury using various approaches, including pathohistological evaluation, appraisal of pulmonary edema, and measurement of inflammatory cytokine levels and their associated pathways within lung tissues. The gene chip data of LPS-induced acute lung injury mice were retrieved from the Gene Expression Omnibus (GEO) database for gene differential expression analysis and Gene Set Enrichment Analysis (GSEA) analysis. The impact of DMPP on glycocalyx shedding was assessed by measuring the expression levels of syndecan-1 (SDC-1) and matrix metalloproteinase-9 (MMP-9). DMPP treatment significantly improved pathomorphological changes and pathological lung injury scores in the LPS-induced ALI mouse model. The genes expressed differentially in the LPS-induced ALI group in GSE2411 were found to be involved in multiple processes, including the NF-κB signaling pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, as well as the JAK-STAT signaling pathway. DMPP treatment effectively downregulated pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and effectively restrained the LPS-induced upregulation of MMP-9 and shedding of syndecan-1, thereby contributing to the preservation of endothelial glycocalyx and attenuation of endothelial barrier dysfunction. The administration of DMPP has been shown to confer protection against LPS-induced acute lung injury via a cholinergic anti-inflammatory pathway, which effectively inhibits endothelial glycocalyx degradation.

Evaluation of the anti-inflammatory effects of PI3Kδ/γ inhibitors for treating acute lung injury

Phosphatidylinositol 3-kinase delta (PI3Kδ) and gamma (PI3Kγ) are predominantly located in immune and hematopoietic cells. It is well-established that PI3Kδ/γ plays important roles in the immune system and participates in inflammation; hence, it could be a potential target for anti-inflammatory therapy. Currently, several PI3K inhibitors are used clinically to treat cancers with aberrant PI3K signaling; however, their role in treating acute respiratory inflammatory diseases has rarely been explored. Herein, we investigated the potential anti-inflammatory activities of several pharmacological PI3K inhibitors, including marketed drugs idelalisib (PI3Kδ), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K with preferential α/δ) and the clinical drug eganelisib (PI3Kγ), for treating acute lung injury (ALI). In the lipopolysaccharide-induced RAW264.7 macrophage inflammatory model, the four inhibitors significantly suppressed proinflammatory cytokine expression by inhibiting the PI3K signaling pathway. Oral administration of PI3K inhibitors markedly improved lung injury in a murine model of ALI. PI3K pathway inhibition decreased inflammatory cell infiltration and totalprotein levels, as well as reduced the expression of associated lung inflammatory factors. Collectively, all four representative PI3K inhibitors exerted prominent anti-inflammatory properties, indicating that PI3K δ and/or γ inhibition could be ideal targets to treat respiratory inflammatory diseases by reducing the inflammatory response. The findings of the current study provide a new basis for utilizing PI3K inhibitors to treat acute respiratory inflammatory diseases.

Chromofungin, a chromogranin A-derived peptide, protects against sepsis-induced acute lung injury by inhibiting LBP/TLR4-dependent inflammatory signaling

Chromofungin (CHR) is a biologically active peptide derived from chromogranin A that exhibits anti-inflammatory effects. However, it remains unclear whether and how CHR protects against sepsis-induced acute lung injury (ALI). A murine model of sepsis-induced ALI was established through cecal ligation and puncture, with intraperitoneal injection of CHR. Lung inflammation and macrophage polarization were examined by measuring the levels of cytokines and markers of M1 (CD86, inducible nitric oxide synthase [iNOS]) or M2 macrophages (arginase-1 [Arg1], resistin-like molecule α1 [Fizz1] and CD206). In vitro, mouse MH-S cells pretreated with CHR was employed to explore the interplay between the lipopolysaccharide-binding protein (LBP)/toll-like receptor 4 (TLR4) signaling pathway and M1/M2 polarity. The results revealed CHR's ability to enhance the 7-day survival rate and protect lung pathological injury in sepsis-induced ALI. CHR increased the expression of interleukin-4 and interleukin-10 but decreased the expression of tumour necrosis factor-α and interleukin-1β. In addition, CHR notably facilitated M2 macrophage polarization, while significantly suppressingM1 polarization of alveolar macrophages. Mechanistic investigations delineated CHR's role in macrophage polarization by downregulating nuclear factor-κB expression through modulation of the LBP/TLR4 signaling pathway. Therefore, CHR may represent a novel strategy for the prevention of sepsis-induced ALI.

Chlorogenic acid reduces inflammation by inhibiting the elevated expression of KAT2A to ameliorate lipopolysaccharide-induced acute lung injury.

Respiratory diseases have become a global health problem and may lead to acute lung injury (ALI) in severe cases. ALI progression is associated with complex pathological changes; however, there are currently no effective therapeutic drugs. Excessive activation and recruitment of immunocytes in the lungs and the release of large amounts of cytokines are considered the primary causes of ALI, but the cellular mechanisms involved remain unknown. Therefore, new therapeutic strategies need to be developed to control the inflammatory response and prevent the further aggravation of ALI. Lipopolysaccharide was administered to mice via tail vein injection to establish an ALI model. Key genes regulating lung injury in mice were screened by RNA sequencing (RNA-seq), and their regulatory effects on inflammation and lung injury were assessed in in vivo and in vitro experiments. The key regulatory gene KAT2A up-regulated the expression of inflammatory cytokines and induced lung epithelial injury. Chlorogenic acid, a small natural molecule and KAT2A inhibitor, inhibited the inflammatory response and significantly improved the decreased respiratory function caused by lipopolysaccharide administration in mice by inhibiting the expression of KAT2A. Targeted inhibition of KAT2A suppressed the release of inflammatory cytokines and improved respiratory function in this murine model of ALI. Chlorogenic acid, a specific KAT2A-targeting inhibitor, was effective in treating ALI. In conclusion, our results provide a reference for the clinical treatment of ALI and contribute to the development of novel therapeutic drugs for lung injury.

Downregulation of ROR2 attenuates LPS-induced A549 cell injury through JNK and ERK signaling pathways.

We aimed to determine whether receptor tyrosine kinase-like orphan receptor 2 (ROR2) is involved in the occurrence of acute lung injury(ALI) by an animal study and explore the effect of ROR2 downregulation on lipopolysaccharide (LPS)-treated human lung carcinoma A549 cells by a cytological study. Murine models of ALI were successfully constructed by intratracheal instillation of LPS. Meanwhile, A549 cell line stimulated with LPS was used for a cytological study. The expression of ROR2 and its effect on proliferation, cell cycle, apoptosis, and inflammation were detected. It was found that LPS administration markedly inhibited the cell proliferation, resulted in cell cycle arrest at G1 phage, elevated levels of pro-inflammatory cytokines and apoptosis rate of A549 cells. However, LPS-mediated adverse effects mentioned above were significantly ameliorated by downregulation of ROR2 in comparison with LPS treatment. In addition, administration of ROR2 siRNA notably decreased the phosphorylation level of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in LPS-challenged A549 cells. Thus, the present data indicate that downregulation of ROR2 may decrease LPS-induced inflammatory responses and cell apoptosis through inhibiting JNK and ERK signaling pathway, which attenuates ALI.

Peptide-guided delivery improves the therapeutic efficacy and safety of glucocorticoid drugs for treating acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening conditions with excessive inflammation in the lung. Glucocorticoids had been widely used for ALI/ARDS, but their clinical benefit remains unclear. Here, we tackled the problem by conjugating prednisolone (PSL) with a targeting peptide termed CRV. Systemically administered CRV selectively homes to the inflamed lung of a murine ALI model, but not healthy organs or the lung of healthy mice. The expression of the CRV receptor, retinoid X receptor β, was elevated in the lung of ALI mice and patients with interstitial lung diseases, which may be the basis of CRV targeting. We then covalently conjugated PSL and CRV with a reactive oxygen species (ROS)-responsive linker in the middle. While being intact in blood, the ROS linker was cleaved intracellularly to release PSL for action. Invitro, CRV-PSL showed an anti-inflammatory effect similar to that of PSL. Invivo, CRV conjugation increased the amount of PSL in the inflamed lung but reduced its accumulation in healthy organs. Accordingly, CRV-PSL significantly reduced lung injury and immune-related side effects elsewhere. Taken together, our peptide-based strategy for targeted delivery of glucocorticoids for ALI may have great potential for clinical translation.

Regulation of CD18 stability by SIGIRR-modulated ubiquitination: new insights into the relationship between innate immune response and acute lung injury.

Inappropriate accumulation of alveolar macrophages (AMs) and subsequent excessive production of immune responses play critical roles in the pathogenesis of acute lung injury (ALI), but the core negative regulators governing innate signalling in AMs are ill defined. We have previously shown that single immunoglobin IL-1 receptor-related protein (SIGIRR), a negative regulator of IL-1 receptor and Toll-like receptor signalling, inhibits lipopolysaccharide (LPS)-induced inflammatory responses in AMs. To address the biological relevance of SIGIRR in vivo, we generated a murine ALI model via intratracheal instillation of LPS. Intriguingly, SIGIRR expression was observed to be decreased in resident and recruited macrophages during ALI. This decrease was associated with parallel induction in CD18 protein levels in LPS-challenged lung tissues. Through intranasal injection of SIGIRR lentiviral particles studies, we showed that the overexpression of SIGIRR attenuated recruitment of macrophages and neutrophils, decreased production of inflammatory cytokines and ameliorated pathological changes in lungs. Whilst exploring the basis for this phenotype, SIGIRR was found to be coexpressed with CD18 in AMs, and SIGIRR potentiated the instability of CD18 protein via enhancement of its ubiquitination and proteasome degradation. Conversely, by using CD18-/- mice, we further observed that CD18 deletion completely abolished the therapeutic effects of overexpression of SIGIRR on LPS-induced ALI. Mover, overexpression of CD18 in AMs promoted adhesion to ECM components, enhanced TLR4-mediated inflammasome activation and thereby potentiated IL-1β production. These data collectively identify SIGIRR/CD18 as a key negative regulatory circuit maintaining innate immune homeostasis in AMs along the pathogenesis of ALI.

Efficacy of Clinically Used PARP Inhibitors in a Murine Model of Acute Lung Injury

Poly(ADP-ribose) polymerase 1 (PARP1), as a potential target for the experimental therapy of acute lung injury (ALI), was identified over 20 years ago. However, clinical translation of this concept was not possible due to the lack of clinically useful PARP inhibitors. With the clinical introduction of several novel, ultrapotent PARP inhibitors, the concept of PARP inhibitor repurposing has re-emerged. Here, we evaluated the effect of 5 clinical-stage PARP inhibitors in oxidatively stressed cultured human epithelial cells and monocytes in vitro and demonstrated that all inhibitors (1-30 µM) provide a comparable degree of cytoprotection. Subsequent in vivo studies using a murine model of ALI compared the efficacy of olaparib and rucaparib. Both inhibitors (1-10 mg/kg) provided beneficial effects against lung extravasation and pro-inflammatory mediator production-both in pre- and post-treatment paradigms. The underlying mechanisms include protection against cell dysfunction/necrosis, inhibition of NF-kB and caspase 3 activation, suppression of the NLRP3 inflammasome, and the modulation of pro-inflammatory mediators. Importantly, the efficacy of PARP inhibitors was demonstrated without any potentiation of DNA damage, at least as assessed by the TUNEL method. These results support the concept that clinically approved PARP inhibitors may be repurposable for the experimental therapy of ALI.

Ferulic acid alleviates alveolar epithelial barrier dysfunction in sepsis-induced acute lung injury by activating the Nrf2/HO-1 pathway and inhibiting ferroptosis

Context Ferulic acid (FA) has antioxidative and anti-inflammatory effects, and is a promising drug to treat sepsis. Objective To study the therapeutic effect of FA in sepsis-induced acute lung injury (ALI) and its underlying mechanisms. Materials and methods The caecal ligation and puncture (CLP) manoeuvre was applied to establish a murine model of sepsis-induced ALI, and female BALB/c mice (6 mice per group) were subjected to 100 mg/kg FA or 0.8 mg/kg ferrostatin-1 (Fer-1, ferroptosis inhibitor) treatment to clarify the role of FA in preserving alveolar epithelial barrier function and inhibiting ferroptosis. Lipopolysaccharide (LPS; 500 ng/mL)-induced cell models were prepared and subjected to FA (0.1 μM), sh-Nrf2, and Fe (Fe-citrate, ferroptosis inducer; 5 M) treatment to study the in vitro effect of FA on LPS-induced alveolar epithelial cell injury and the role of the Nrf2/HO-1 pathway. Results We found that FA decreased the lung injury score (48% reduction), lung wet/dry weight ratio (33% reduction), and myeloperoxidase activity (58% reduction) in sepsis-induced ALI. Moreover, FA inhibited ferroptosis of alveolar epithelial cells and improved alveolar epithelial barrier dysfunction. The protective role of FA against alveolar epithelial barrier dysfunction could be reversed by the ferroptosis inducer Fe-citrate, suggesting that FA alleviates alveolar epithelial barrier dysfunction by inhibiting ferroptosis. Mechanistically, we found that FA inhibited ferroptosis of alveolar epithelial cells by activating the Nrf2/HO-1 pathway. Conclusion Collectively, our data highlighted the alleviatory role of ferulic acid in sepsis-induced ALI by activating the Nrf2/HO-1 pathway and inhibiting ferroptosis, offering a new basis for sepsis treatment.

The transcription factor KLF14 attenuates LPS-induced acute lung injury by ameliorating apoptosis of alveolar epithelial cells in mice

Lipopolysaccharide (LPS)-induced lung injury (ALI) is characterized by severe lung inflammation with high mortality. Pulmonary inflammation and apoptosis of alveolar epithelial cells cause the disruption of alveolar epithelial cell integrity, resulting in the failure of gas exchange, which is correlated with poor prognosis. However, the underlying regulatory mechanism remains unclear. In this study, we found that the Krüppel-like transcription factor KLF14 can suppress the apoptosis of alveolar epithelial cells in a LPS-mediated murine acute lung injury model by western blot and TUNEL assays. First, we found that the expression of KLF14 in alveolar epithelial cells of a murine ALI model was upregulated, and KLF14 deletion exacerbated lung injury, increased mortality, and promoted the apoptosis of alveolar epithelial cells. Furthermore, we identified that KLF14 protects lung tissue from LPS-mediated damage and suppresses the apoptosis of alveolar epithelial cells by inhibiting the NF-κB pathway. Our study provides further research insight and helps to explore the protective mechanism of KLF14 in inflammation and cell death. Our results suggested that the transcription factor KLF14 may act as a new target for the prevention and treatment of acute lung injury.

Melatonin Suppresses LPS-Induced Oxidative Stress in Dendritic Cells for Inflammatory Regulation via the Nrf2/HO-1 Axis.

Melatonin, an indoleamine synthesized in the pineal gland of mammals, is a natural bioactive compound with powerful antioxidant and anti-inflammatory properties. Here, we evaluated whether melatonin has the capacity to moderate the oxidative stress of dendritic cells (DCs) for inflammatory control in an acute lung injury (ALI) model. Our findings showed that melatonin remarkably inhibited total nitric oxide synthase (T-NOS) activity, nitric oxide (NO) production, intracellular reactive oxygen species (ROS) levels, and lipid peroxidation (MDA detection) levels in both an LPS-induced murine ALI model and LPS-induced DCs. Meanwhile, the reduced glutathione (GSH) level and the GSH/GSSG ratio were recovered. In addition, antioxidant enzymes, such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were increased in these processes. Moreover, melatonin also inhibited the LPS-induced secretions of IL-1β, IL-6, and TGF-β in vivo and in vitro. Finally, we found that the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) axis was required in the inhibition of LPS-induced oxidative stress in DCs by melatonin. Altogether, these data indicate that melatonin strongly suppresses the LPS-induced oxidative stress in DCs, which is a promising DC-targeted strategy via inflammatory control for ALI treatment.

Intermittent fasting attenuates lipopolysaccharide-induced acute lung injury in mice by modulating macrophage polarization

Acute lung injury (ALI) is a spectrum of acute and life-threatening pulmonary inflammatory conditions. Treatment of ALI remains a clinical challenge. Recently, intermittent fasting (IF) has been shown to improve health and alleviate many diseases. In this study, we tested whether IF attenuated ALI and investigated the mechanism underlying this process. In vivo, the effects of IF on ALI were evaluated in a lipopolysaccharide (LPS)-induced murine ALI model. We found that two times of 24-h fasting in a week before ALI efficiently ameliorated LPS-induced lung injury in mice, characterized by alleviated lung lesions, wet-to-dry weight ratio, myeloperoxidase activity, malondialdehyde content, and lower levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β. In vitro, functional assays were conducted to assess IF on the inflammatory response and macrophage polarization of bone marrow-derived macrophages (BMDMs) treated with LPS or IL-4. And PPARγ antagonist GW9662 and AMPK siRNA were used to test the role of PPARγ and AMPK in the IF-mediated improvement of ALI. The results showed that IF (serum deprivation) suppressed macrophage M1 activation and promoted M2 activation in LPS-treated BMDMs. While, IF also augmented macrophage M2 polarization in IL-4-treated BMDMs. Further mechanistic studies showed that the promotive effect of IF on M2 polarization was related to the activation of the PPARγ and AMPK pathways. In conclusion, this study suggests that IF enhances M2 polarization by activating the AMPK and PPARγ pathways, thus facilitating anti-inflammatory response and ameliorating ALI.

Lipopolysaccharide-induced murine lung injury results in long-term pulmonary changes and downregulation of angiogenic pathways

Acute respiratory distress syndrome is the most severe form of acute lung injury (ALI) and is associated with significant mortality. Lipopolysaccharide (LPS)-induced injury is a valuable murine model of ALI but there is a paucity of data on lung regeneration and the role of angiogenic signaling involving vascular endothelial growth factor (VEGF). Eight-week-old male C57BL/6J mice were randomized to receive intratracheal instillation of either LPS or isovolumetric phosphate buffered saline as a vehicle control. Mice were observed at a single follow-up time-point that was either short-term (24 h or 4 days) or long-term (7 days or 4 weeks). On pulmonary function testing, LPS-treated mice had increased compliance at 4 weeks post-instillation, which correlated with decreased vascularization and with time-dependent, progressive decrease in alveolarization. Treadmill exercise tolerance testing demonstrated impaired performance at 24 h, 4 days and 4 weeks following LPS exposure. On lung protein analysis, LPS instillation decreased VEGF expression at up to 4 weeks, and decreased activation of its key receptor, VEGFR2 at 7 days and 4 weeks post-instillation. Together, these data provide insight on long-term pulmonary functional outcomes 4 weeks after ALI and identify angiogenic proteins as possible therapeutic targets following lung injury.

The Alleviation of LPS-Induced Murine Acute Lung Injury by GSH-Mediated PEGylated Artesunate Prodrugs.

Acute lung injury (ALI) or its aggravated stage acute respiratory distress syndrome (ARDS) is a common severe clinical syndrome in intensive care unit, may lead to a life-threatening form of respiratory failure, resulting in high mortality up to 30–40% in most studies. Nanotechnology-mediated anti-inflammatory therapy is an emerging novel strategy for the treatment of ALI, has been demonstrated with unique advantages in solving the dilemma of ALI drug therapy. Artesunate (ART), a derivative of artemisinin, has been reported to have anti-inflammatory effects. Therefore, in the present study, we designed and synthesized PEGylated ART prodrugs and assessed whether ART prodrugs could attenuate lipopolysaccharide (LPS) induced ALI in vitro and in vivo. All treatment groups were conditioned with ART prodrugs 1 h before challenge with LPS. Significant increased inflammatory cytokines production and decreased GSH levels were observed in the LPS stimulated mouse macrophage cell line RAW264.7. Lung histopathological changes, lung W/D ratio, MPO activity and total neutrophil counts were increased in the LPS-induced murine model of ALI via nasal administration. However, these results can be reversed to some extent by treatment of ART prodrugs. The effectiveness of mPEG2k-SS-ART in inhibition of ALI induced by LPS was confirmed. In conclusion, our results demonstrated that the ART prodrugs could attenuate LPS-induced ALI effectively, and mPEG2k-SS-ART may serve as a novel strategy for treatment of inflammation induced lung injury.

Preclinical Pharmacokinetics and Safety of Intravenous RTD-1.

ABSTRACTSevere illness caused by coronavirus disease 2019 (COVID-19) is characterized by an overexuberant inflammatory response resulting in acute respiratory distress syndrome (ARDS) and progressive respiratory failure (A. Gupta, M. V. Madhavan, K. Sehgal, N. Nair, et al., Nat Med 26:1017–1032, 2020, https://doi.org/10.1038/s41591-020-0968-3). Rhesus theta (θ) defensin-1 (RTD-1) is a macrocyclic host defense peptide exhibiting antimicrobial and immunomodulatory activities. RTD-1 treatment significantly improved survival in murine models of a severe acute respiratory syndrome (SARS-CoV-1) and endotoxin-induced acute lung injury (ALI) (C. L. Wohlford-Lenane, D. K. Meyerholz, S. Perlman, H. Zhou, et al., J Virol 83:11385–11390, 2009, https://doi.org/10.1128/JVI.01363-09; J. G. Jayne, T. J. Bensman, J. B. Schaal, A. Y. J. Park, et al., Am J Respir Cell Mol Biol 58:310–319, 2018, https://doi.org/10.1165/rcmb.2016-0428OC). This investigation aimed to characterize the preclinical pharmacokinetics (PK) and safety of intravenous (i.v.) RTD-1. Based on the lack of adverse findings, the no observed adverse effect level (NOAEL) was established at 10 mg/kg/day in rats and 15 mg/kg/day in monkeys. Analysis of single ascending dose studies in both species revealed greater-than-dose-proportional increases in the area under the curve extrapolated to infinity (AUC0-∞) (e.g., 8-fold increase from 5 mg/kg to 20 mg/kg in rats) suggestive of nonlinear PK. The volume of distribution at steady state (Vss) ranged between 550 and 1,461 mL/kg, indicating extensive tissue distribution, which was validated in a biodistribution study of [14C]RTD-1 in rats. Based on interspecies allometric scaling, the predicted human clearance and Vss are 6.48 L/h and 28.0 L, respectively, for an adult (70 kg). To achieve plasma exposures associated with therapeutic efficacy established in a murine model of ALI, the estimated human equivalent dose (HED) is between 0.36 and 0.83 mg/kg/day. The excellent safety profile demonstrated in these studies and the efficacy observed in the murine models support the clinical investigation of RTD-1 for treatment of COVID-19 or other pulmonary inflammatory diseases.

A two-hit model of sepsis plus hyperoxia causes lung permeability and inflammation.

Mouse models of acute lung injury (ALI) have been instrumental for studies of the biological underpinnings of lung inflammation and permeability, but murine models of sepsis generate minimal lung injury. Our goal was to create a murine sepsis model of ALI that reflects the inflammation, lung edema, histological abnormalities, and physiological dysfunction that characterize ALI. Using a cecal slurry (CS) model of polymicrobial abdominal sepsis and exposure to hyperoxia (95%), we systematically varied the timing and dose of the CS injection, fluids and antibiotics, and dose of hyperoxia. We found that CS alone had a high mortality rate that was improved with the addition of antibiotics and fluids. Despite this, we did not see evidence of ALI as measured by bronchoalveolar lavage (BAL) cell count, total protein, C-X-C motif chemokine ligand 1 (CXCL-1) or by lung wet:dry weight ratio. Addition of hyperoxia [95% fraction of inspired oxygen ([Formula: see text])] to CS immediately after CS injection increased BAL cell counts, CXCL-1, and lung wet:dry weight ratio but was associated with 40% mortality. Splitting the hyperoxia treatment into two 12-h exposures (0-12 h and 24-36 h) after CS injection increased survival to 75% and caused significant lung injury compared with CS alone as measured by increased BAL total cell count (92,500 vs. 240,000, P = 0.0004), BAL protein (71 vs. 103 µg/mL, P = 0.0030), and lung wet:dry weight ratio (4.5 vs. 5.5, P = 0.0005), and compared with sham as measured by increased BAL CXCL-1 (20 vs. 2,372 pg/mL, P < 0.0001) and histological lung injury score (1.9 vs. 4.2, P = 0.0077). In addition, our final model showed evidence of lung epithelial [increased BAL and plasma receptor for advanced glycation end products (RAGE)] and endothelial (increased Syndecan-1 and sulfated glycosaminoglycans) injury. In conclusion, we have developed a clinically relevant mouse model of sepsis-induced ALI using intraperitoneal injection of CS, antibiotics and fluids, and hyperoxia. This clinically relevant model can be used for future studies of sepsis-induced ALI.