Lung Tissues Of Mice Research Articles

Comparative Automated Quantification of Tertiary Lymphoid Structures in Two Distinct Mouse Models of Inflammation.

Tertiary lymphoid structures (TLS) have been reported to form within nonlymphoid tissues upon various inflammatory conditions, such as tumors or bacterial infections. In particular, the lungs of patients with NSCLC or bacterial infections (such as tuberculosis or aspergillosis) are the sites of TLS neogenesis. An increasing number of preclinical models have been used over the years to recapitulate as accurately as possible the mechanisms and kinetics of TLS formation that are continuously reported in the clinic.We used herein two distinct murine models of pulmonary inflammation, relying on orthotopic lung tumor implantation, or intranasal administration of bacterial products, to generate TLS formation within mouse lung tissue. This review compares different workflows aiming at the detection and quantification of TLS using automated image analysis and artificial intelligence-assisted tissue classification. We also describe different methods for partitioning serial sections, depending on the tissue morphology and the experimental goal.

A simultaneous analysis strategy of glycerophospholipids and lipid mediators based on secondary extraction scheme.

Lipid peroxidation plays an important role in physiological and pathophysiological conditions; it occurs mainly in the vicinity of the membrane. Glycerophospholipids are indispensable for normal structure of membranes. Lipid mediators are final products of lipid peroxidation. Glycerophospholipids as a source of lipid mediators are closely related with lipid mediators in lipid peroxidation process. This study developed a strategy for simultaneous analysis of glycerophospholipids and lipid mediators in a sample based on a secondary extraction scheme. Due to differences in physicochemical properties of glycerophospholipids and lipid mediators, we used different solvents and methods to extract glycerophospholipids and lipid mediators based on a sample. In addition, we compared three methods of extracting glycerophospholipids to select the best extraction method. In this study, a UPLC-MRM absolute quantification strategy for three deuterated internal standards of lipid mediator was established. The calibration range of all standards is linear, and the correlation coefficients are greater than 0.999. Quantitative range, precision, and accuracy of all analytes-based analysis method meet requirements of method validation. And the accuracy and precision of this method for glycerophospholipids meet the requirements of method verification. It is satisfactory to apply this method in the analysis of the changes of enzymes in lipid peroxidation during the treatment of asthma with dexamethasone. The quantitative results of phospholipids and lipid mediators in mouse lung tissue were obtained by this method, indicating that dexamethasone may inhibit the activity of phospholipase As enzyme and thus slow down the lipid peroxidation process. This work may be of great significance for exploring the changes of enzymes in lipid peroxidation metabolism.

Molecular characterization of PANoptosis-related genes associated with immune infiltration and prognosis in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease with unknown pathogenesis and poor prognosis. PANoptosis, a newly identified form of inflammatory programmed cell death, has been implicated in various inflammatory lung diseases. This study aimed to identify differentially expressed PANoptosis-related genes (PRDEGs) associated with immune infiltration and prognosis in IPF, while also establishing a novel prognostic prediction model. A total of 63 PRDEGs were identified from GSE110147 dataset, with 31 exhibiting consistent expression trends in GSE213001. Enrichment analysis indicated that the majority of these PRDEGs were enriched in inflammatory and immune-related pathways. Three key PRDEGs—NLRP3, ATM, and VEGFA—were selected through univariate and multivariable Cox regression analyses. The prognostic prediction model developed from these key PRDEGs demonstrated robust predictive performance. Furthermore, the expression of most PRDEGs was positively correlated with pro-inflammatory immune cells, including macrophages, neutrophils, and CD4+ T cells. Validation of the expression levels of these key PRDEGs was conducted in fibrotic mouse lung tissue. This study suggests that PANoptosis plays a role in IPF, potentially linked to the infiltration of pro-inflammatory immune cells, and may influence disease progression through the regulation of inflammatory immune signaling pathway. A new prognostic prediction model for IPF based on PRDEGs was successfully developed.

Increasing coil temperature of a third-generation e-cigarette device modulates C57BL/6 mouse lung immune cell composition and cytokine milieu independently of aerosol dose

ABSTRACT Higher coil temperature in e-cigarette devices increases the formation of aerosols and toxicants, such as carbonyls. At present, the health implications of vaping at higher temperatures, including exacerbation of pulmonary inflammation, are largely unknown when aerosol dose is considered. To isolate the pulmonary effects of coil temperature, C57BL/6 mice were exposed to e-cigarette aerosols generated at lower (190°C) or higher (250°C) temperature for 3 days, while maintaining a similar chamber aerosol concentration. Increasing coil temperature did not markedly alter aerosol mass-normalized emissions of select carbonyls formed from thermal degradation pathways including formaldehyde, acetaldehyde, propionaldehyde, and acetone under the tested environment. Total bronchoalveolar cells, primarily macrophages, were significantly decreased in mice exposed to aerosols generated with higher coil temperatures compared to lower temperature exposures. The gene expression of IFNβ, IL-1β, TNFα, and IL-10 in mouse lung tissue was significantly reduced following e-cigarette exposure under both conditions, compared to filtered air exposure. Higher temperature exposures further exacerbated downregulation of IFNβ and IL-1β. Data suggest that higher temperature vaping might modulate acute pulmonary immune responses, potentially inducing immune suppression, even when normalized for aerosol dose exposure. Coil temperature thus appears to be an important parameter that needs to be regulated to ensure harm reduction for e-cigarette users.

Treatment of silicosis with quercetin depolarizing macrophages via inhibition of mitochondrial damage-associated pyroptosis

Macrophage polarization facilitates the inflammatory response and intensified fibrosis in the silicosis microenvironment by a mechanism related to macrophage pyroptosis, although the upstream target remains poorly defined. Currently, there are few reports on the development of drugs that alleviate macrophage polarization by dampening pyroptosis. The present study aims to explore the mechanics of silica mediating macrophage polarization and to investigate whether quercetin (Que) can depolarize macrophages with this mechanism. Silica processing led to prominent M1 polarization of macrophages. Additionally, significant macrophage polarization could be detected in the lung tissue of mice with airway-perfused silica. Further investigation turned out that pronounced mitochondria damage, mtDNA cytoplasmic ectomy, and pyroptosis occurred in response to silica. Nevertheless, Que treatment could effectively attenuate silica-induced mitochondria damage and pyroptosis as demonstrated in vitro and in vivo. Further exploration presented Que could bind to TOM70 and restore silica-induced mitochondrial damage. More importantly, the M1 polarization of macrophage was depressed with the co-treatment of Que and silica, wherein the inflammatory response and pulmonary fibrosis were also mitigated without obvious damage to vital organs. In conclusion, these findings proved that silica leads to mitochondrial damage, thereby evoking pyroptosis and promoting macrophage M1 polarization. Que could bind to TOM70 and restore its function, suppressing mitochondrial damage and pyroptosis, and depolarizing macrophages to reduce fibrosis, which provides a promising strategy for silicosis treatment in the future.

Synthesis and pharmacodynamic evaluation of 2-aminoindole derivatives against influenza A virus in vitro/vivo

Influenza virus is a kind of respiratory pathogen with high morbidity and mortality, which still threatens human health. Existing anti-influenza drugs have various limitations, such as the inability to alleviate body injury and side effects. There remains an urgent need to develop a novel antiviral drug to efficiently inhibit viral infection while avoiding body injury. A series of 2-aminoindole derivatives were synthesized via the TMSOTf-catalyzed reactions of N-arylynamides with sulfilimines and evaluated for their anti-influenza virus activity. The experimental results showed that 2-aminoindole 3h had significant antiviral activity (EC50 = 8.37 ± 0.65 μM) and the lowest cytotoxicity (CC50 = 669.26 ± 11.42 μM) in vitro. 2-Aminoindole 3h could inhibit viral replication by effectively binding to RNA-dependent RNA polymerase (RdRp), and could also directly target host cells to inhibit cytokine storms and apoptosis induced by viral infection, thereby improving host cell survival rate. In addition, viral load and organ injury in the lung tissue of infected mice were effectively reduced by 2-aminoindole 3h with satisfactory biosafety. These findings highlight the potential of a valuable therapeutic option against influenza infection while also laying the foundation for further research and development in this area.

Ginsenoside Rb1 targets to HO-1 to improve sepsis by inhibiting ferroptosis

Sepsis remains the leading cause of mortality among Intensive Care Unit (ICU) patients, with its pathogenesis and treatment not yet fully elucidated. Ferroptosis plays a critical role in sepsis, suggesting that ferroptosis-related genes may serve as potential therapeutic targets. This study aims to identify key ferroptosis-related genes in sepsis and explore targeted therapeutics. Through differential expression analysis of the GSE13940 and GSE26440 datasets, heme oxygenase-1 (HO-1) was identified as a hub gene associated with ferroptosis. Additionally, single-cell analysis of the GSE175453 dataset revealed a significant upregulation of HO-1 expression in monocyte lineages during sepsis. The cecal ligation and puncture (CLP) method was employed to induce sepsis in a mouse model, lung and intestinal tissues exhibited typical ferroptosis characteristics, with a significant increase in HO-1 expression. However, treatment with the HO-1 inhibitor zinc protoporphyrin (ZNPP) significantly ameliorated ferroptosis in CLP-induced lung and intestinal tissues, as well as in lipopolysaccharide (LPS)-induced THP-1 cells. Subsequently, molecular docking, surface plasmon resonance (SPR), and microscale thermophoresis (MST) experiments demonstrated that ginsenoside Rb1 specifically targets HO-1, identifying K18A as the key binding residue. Finally, experiments conducted both in vitro and in vivo verified that ginsenoside Rb1 significantly reduces HO-1 expression, inhibits ferroptosis in sepsis-induced lung, and intestinal tissues and THP-1 cells, and improves sepsis-induced pulmonary and intestinal damage. In conclusion, this study identifies HO-1 as a key ferroptosis target in sepsis and suggests ginsenoside Rb1 as a potential novel HO-1 inhibitor for the therapeutic approach of sepsis-induced organ dysfunction.

Preliminary proteomic analysis of mouse lung tissue treated with cyclophosphamide and Venetin-1

Cyclophosphamide (CPAm) is a widely used chemotherapeutic agent that exhibits potent anti-cancer properties but is often associated with debilitating side effects. Despite its efficacy, the management of CPAm-induced toxicities remains a significant clinical challenge. There has been growing interest in exploring complementary and alternative therapies to mitigate these adverse effects in recent years, and this may be a chance for the earthworm-derived preparation, Venetin-1. Its rich composition of bioactive compounds has demonstrated promising pharmacological properties, including anti-inflammatory, antioxidant, and immunomodulatory effects. These properties suggest its potential to counteract various systemic toxicities induced by CPAm. We conducted a comprehensive study to investigate the effect of Venetin-1 on cyclophosphamide-induced toxicity. Mice were administered CPAm for four days, followed by application of the earthworm preparation in two doses (50 mg/kg and 100 mg/kg b.w). Importantly, the preparation did not cause any side effects in all mice, ensuring the safety of the intervention. We then determined global changes in the proteome using proteomics and quantitative SWATH-MS analysis, which is a robust and reliable method. This allowed us to identify up- and downregulated proteins in each studied group, providing valuable insights into the mechanism of action of Venetin-1. As shown by the results, Venetin-1 had a significant effect on the proteome of mouse lung tissue. It was possible to determine quantitative changes in 400 proteins, and the analysis after administration of Venetin-1 showed a change in the global proteomic profile from upregulated to down-regulated. The stimulating properties of the preparation concerning the complement system were also confirmed in a separate validation experiment. Venetin-1 shows promise in reducing the harmful effects of cyclophosphamide on lung tissue. It encourages tissue regeneration, reduces inflammation, supports autophagy, and boosts the immune system. However, more research is needed to thoroughly elucidate and describe the benefits of Venetin-1.

Effect of leucine on mitochondria and oxidative stress to reduce virulence and pathogenicity of Acinetobacter baumannii

Elucidating the virulence mechanisms of A. baumannii is essential for developing strategies to mitigate pathogenicity. Although high-virulent strains are associated with increased mortality rate in severely infected patients, the underlying mechanisms remains not well understood. Our analysis revealed leucine as a pivotal biomarker, with the 11dP and paaK being significant contributors to virulence. The ATP-dependent activity and antioxidant activity were identified as the most important pathways in distinguishing the virulence of A. baumannii. Exogenous leucine was found to modulate mitochondria dysfunction and oxidative stress, thereby diminishing the pathogenicity of A. baumannii towards Beas 2B cells. Moreover, leucine reduced the virulence of A. baumannii to Galleria mellonella (G. mellonella) and alleviated pathological damage to lung tissues in mice. Our study offers a novel treatment strategy based on metabolomics, which may assist in the exploration and management of infections caused by highly virulent pathogens. It sets a new course for reducing the impact of highly virulent A. baumannii infections and has significant implications for the development of future therapeutic interventions.

MiR-125b-5p alleviates pulmonary fibrosis by inhibiting TGFβ1-mediated epithelial-mesenchymal transition via targeting BAK1

This study explores the role and potential mechanisms of microRNA-125b-5p (miR-125b-5p) in pulmonary fibrosis (PF). PF is a typical outcome of many chronic lung diseases, with poor prognosis and the lack of appropriate medical treatment because PF’s molecular mechanisms remain poorly understood. In this study, using in vitro and in vivo analyses, we find that miR-125b-5p is likely a potent regulator of lung fibrosis. The findings reveal that, on the one hand, miR-125b-5p not only specifically decreases in the epithelial-mesenchymal transition (EMT) of lung epithelial cells, but also shows a downregulation trend in the lung tissues of mice with PF. On the other hand, overexpression of miR-125b-5p on the cellular and animal levels downregulates EMT and fibrotic phenotypes, respectively. To clarify the molecular mechanism of the “therapeutic” effect of miR-125b-5p, we use the target prediction tool combined with a dual luciferase assay and complete a rescue experiment by constructing the overexpression vector of the target gene Bcl-2 homologous antagonist/ killer (BAK1), thus confirming that miR-125b-5p can effectively inhibit EMT and fibrosis process by targeting BAK1 gene. MiR-125b-5p inhibits the EMT in lung epithelial cells by negatively regulating BAK1, while overexpression of miR-125b-5p can alleviate lung fibrosis. The findings suggest that MiR-125b-5p/BAK1 can serve as a potential treatment target for PF.Graphical abstract

Novel TSPO Ligand 2-Cl-MGV-1 Can Counteract Lipopolysaccharide Induced Inflammatory Response in Murine RAW264.7 Macrophage Cell Line and Lung Models.

We assessed the anti-inflammatory activity of the TSPO ligand 2-Cl-MGV-1. Lipopolysaccharide (LPS) was used to induce inflammatory response in a murine RAW264.7 macrophage model (LPS: 100 ng/mL) and a mouse model (C57BL/6) of lung inflammation (LPS: 5 mg/kg). In the macrophage model, the presence of 2-Cl-MGV-1 (25 µM) caused the LPS-induced elevation in nitrite levels to decrease by 70% (p < 0.0001) and interleukin (IL)-6 by 50% (p < 0.05). In the mouse model, 2-Cl-MGV-1, administered 30 min before, or co-administered with, an LPS injection, significantly inhibited the elevation in serum IL-5 levels (both by 65%; p < 0.001 and p < 0.01, respectively). 2-Cl-MGV-1 administration to mice 30 min before LPS injection and 1 h thereafter significantly inhibited the elevation in IL-1β serum levels (both by 63%, p < 0.005). IL-6 elevation was inhibited by 73% (p < 0.005) when 2-Cl-MGV-1 was administered 30 min before LPS, by 60% (p < 0.05) when co-administered with LPS, and by 64% (p < 0.05) when administered 1 h after LPS. All cytokine assessments were conducted 6 h post LPS injection. Histological analyses showed decreased leukocyte adherence in the lung tissue of the ligand-treated mice. 2-Cl-MGV-1 administration 30 min prior to exposure to LPS inhibited inflammation-induced open field immobility. The beneficial effect of 2-Cl-MGV-1 suggests its potential as a therapeutic option for inflammatory diseases.

Upregulated DNMT3a coupling with inhibiting p62-dependent autophagy contributes to NNK tumorigenicity in human bronchial epithelial cells

NNK, formally known as 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanoe, is a potent chemical carcinogen prevalent in cigarette smoke and is a key contributor to the development of human lung adenocarcinomas. On the other hand, autophagy plays a complex role in cancer development, acting as a "double-edged sword" whose impact varies depending on the cancer type and stage. Despite this, the relationship between autophagy and NNK-induced lung carcinogenesis remains largely unexplored. Our current study uncovers a marked reduction in p62 protein expression in both lung adenocarcinomas and lung tissues of mice exposed to cigarette smoke. Interestingly, this reduction appears to be contingent upon the activity of extrahepatic cytochrome P450 (CYP450), revealing that NNK metabolic activation by CYP450 enzyme escalates its potential to induce p62 downregulation. Further mechanistic investigations reveal that NNK suppresses autophagy by accelerating the degradation of p62 mRNA, thereby promoting the malignant transformation of human bronchial epithelial cells. This degradation process is facilitated by the hypermethylation of the Human antigen R (HuR) promoter, resulting in the transcriptional repression of HuR - a key regulator responsible for stabilizing p62 mRNA through direct binding. This hypermethylation is triggered by the activation of ribosomal protein S6, which is influenced by NNK exposure and subsequently amplifies the translation of DNA methyltransferase 3 alpha (DNMT3a). These findings provide crucial insights into the nature of p62 in both the development and potential treatment of tobacco-related lung cancer.

Ruhao Dashi granules exert therapeutic effects on H1N1 influenza virus infection by altering intestinal microflora composition.

Antiviral medications for influenza could be ineffective due to the emergence of resistant influenza virus strains. Ruhao Dashi (RHDS) granules possess anti-inflammatory and antibacterial effects. The present study aimed to determine the efficacy of RHDS granules in treating influenza-infected mice and the mechanism underlying this treatment as well as its effect on the intestinal flora composition of the infected mice. The HPLC-UV method was used to identify the active components of RHDS granules. ICR mice were infected with influenza A virus (IAV) H1N1 subtype through a nasal drip. After the influenza mice model was successfully established, the pathological changes in the lungs were observed for 5 days after gavage treatment with 0.9% sterile saline and low, medium, and high doses (0.07, 0.14, and 0.28 g/mL, respectively) of RHDS granules. The serum levels of the cytokines IL-6 and TNF-α and sIgA were detected by ELISA. Real-time fluorescence quantitative PCR and western blotting assay were performed to determine the expression levels of the tight junction (TJ) proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) in colon tissues. Furthermore, 16S rRNA gene sequencing of feces samples was conducted to assess the effect of RHDS granules on the gut microbiota. RHDS granules exerted a protective effect on the lung tissues of IAV-infected mice; moreover, the granules reduced the synthesis of proinflammatory cytokines and increased the relative expression levels of claudin-1, occludin, and ZO-1 in colon tissues. Furthermore, RHDS granule treatment increased the relative abundance of Lactobacillus, Akkermansia, and Faecalibaculum and decreased the relative abundance of Muribaculaceae; thus, RHDS granules could stabilize the intestinal microbiota to some extent. RHDS granules exert a therapeutic effect on IAV-infected mice probably by modifying the structural composition of their intestinal microbiota.

Ozone-Induced Lung Injury are Mediated Via PPAR-Mediated Ferroptosis in Mice.

In recent years, the concentration of PM2.5 in China has decreased, while the concentration of ozone remains rising. Exposure to ozone contributes to respiratory illnesses; however, little is known about the underlying molecular mechanisms. The present study established an ozone-induced lung injury mice model to investigate potential molecular biomarkers and toxic mechanisms. Collected and analyzed the ozone pollution data in Xinxiang city from 2015 to 2022. At the same time, 24 male C57BL/6 mice were randomly assigned to control group and ozone exposure group. The ozone exposure concentration is 1ppm, with 4h of daily exposure for 33 consecutive days. HE staining was used to assess lung histological alterations and lung injury. High-throughput sequencing performed on the lung tissues of mice was used to analyze the differential expressed genes and signal transduction pathways. Xinxiang city is suffering from ozone pollution, especially in summer. HE staining showed that the ozone exposure could induce obvious inflammatory cell infiltration, alveolar wall thickening, or fracture. Transcriptome data revealed that there is a 145 differentially expressed genes between two groups and the genes enriched in PPAR signaling pathway, ferroptosis. The pivotal genes in the PPAR pathway including Adipoq, Lpl, Pck1, and Plin1 expression were significantly reduced. Additionally, the expression of Acsl6 and Scl7a11, which are close to PPAR pathway and ferroptosis has decreased. Ozone exposure could disrupt the lipid metabolism balance via downregulating lipid peroxidation-related genes through the PPAR signaling pathway, which further induced lung cell ferroptosis and aggravated lung injury in mice.

Protective effect and mechanism of rapamycin on pulmonary fibrosis induced by Chlormethine in mice

To evaluate the therapeutic effect and mechanism of rapamycin (RAPA) on pulmonary fibrosis induced by chlormethine in C57BL/6N mice. Based on body weight, the 18-20 g C57BL/6N mice were randomly divided into five groups: control group, chlormethine group, chlormethine+dexamethasone (1 mg/kg) group, chlormethine+RAPA (1 mg/kg) group and chlormethine+RAPA (2 mg/kg) group, with ten mice in each group. Mice were put to death on the 21st day after the first administration of chlormethine. HE staining and Masson staining were used to observe the pathological changes and degree of fibrosis in the lung tissue of mice, and RT-PCR was used to detect collagen Ⅰ, E-cadherin, vimentin, and α-SMA mRNA expression. After 21 days of administration of chlormethine to mice, significant pulmonary fibrosis characteristics were observed in the lung tissue of the mice. Compared with the chlormethine group, the weight of mice in the chlormethine+dexamethasone (1 mg/kg) group, chlormethine+RAPA (1 mg/kg) group and chlormethine+RAPA (2 mg/kg) group, significantly increased (P<0.05). Compared with the chlormethine group, the expression of pulmonary fibrosis-related indicators (collagen Ⅰ, E-cadherin, vimentin, and α-SMA) significantly improved (P<0.05) in the chlormethine+dexamethasone (1 mg/kg) group, chlormethine+RAPA (1 mg/kg) group and chlormethine+RAPA (2 mg/kg) group. Compared with the chlormethine group, the pathological changes and collagen deposition in the lung tissue of mice in the chlormethine+dexamethasone (1 mg/kg) group, chlormethine+RAPA (1 mg/kg) group and chlormethine+RAPA (2 mg/kg) group, were significantly improved. Transcriptome analysis of the lung tissue of mice revealed that RAPA treatment of chlormethine-induced pulmonary fibrosis might be related to NF-kappa B signaling pathway. Compared with the chlormethine group, the mRNA expression of p65 in the lung tissue of mice in the chlormethine+dexamethasone (1 mg/kg) group, chlormethine+RAPA (1 mg/kg) group and chlormethine+RAPA (2 mg/kg) group, significantly decreased (P<0.01). RAPA has a protective effect on pulmonary fibrosis induced by chlormethine in mice. Its efficacy is comparable to that of dexamethasone, which is currently being used in clinical practice. It is a new alternative therapy, and its mechanism may be related to inhibiting the activation of the NF-kappa B signaling pathway.

Cordyceps militaris Grown on Germinated Rhynchosia nulubilis (GRC) Encapsulated in Chitosan Nanoparticle (GCN) Suppresses Particulate Matter (PM)-Induced Lung Inflammation in Mice.

Cordyceps militaris grown on germinated Rhynchosia nulubilis (GRC) exerts various biological effects, including anti-allergic, anti-inflammatory, and immune-regulatory effects. In this study, we investigated the anti-inflammatory effects of GRC encapsulated in chitosan nanoparticles (CN) against particulate matter (PM)-induced lung inflammation. Optimal CN (CN6) (CHI: TPP w/w ratio of 4:1; TPP pH 2) exhibited a zeta potential of +22.77 mV, suitable for GRC encapsulation. At different GRC concentrations, higher levels (60 and 120 mg/mL) led to increased negative zeta potential, enhancing stability. The optimal GRC concentration for maximum entrapment (31.4 ± 1.35%) and loading efficiency (7.6 ± 0.33%) of GRC encapsulated in CN (GCN) was 8 mg/mL with a diameter of 146.1 ± 54 nm and zeta potential of +30.68. In vivo studies revealed that administering 300 mg/kg of GCN significantly decreased the infiltration of macrophages and T cells in the lung tissues of PM-treated mice, as shown by immunohistochemical analysis of CD4 and F4/80 markers. Additionally, GCN ameliorated PM-induced lung tissue damage, inflammatory cell infiltration, and alveolar septal hypertrophy. GCN also decreased total cells and neutrophils, showing notable anti-inflammatory effects in the bronchoalveolar lavage fluid (BALF) from PM-exposed mice, compared to GRC. Next the anti-inflammatory properties of GCN were further explored in PM- and LPS-exposed RAW264.7 cells; it significantly reduced PM- and LPS-induced cell death, NO production, and levels of inflammatory cytokine mRNAs (IL-1β, IL-6, and COX-2). GCN also suppressed NF-κB/MAPK signaling pathways by reducing levels of p-NF-κB, p-ERK, and p-c-Jun proteins, indicating its potential in managing PM-related inflammatory lung disease. Furthermore, GCN significantly reduced PM- and LPS-induced ROS production. The enhanced bioavailability of GRC components was demonstrated by an increase in fluorescence intensity in the intestinal absorption study using FITC-GCN. Our data indicated that GCN exhibited enhanced bioavailability and potent anti-inflammatory and antioxidant effects in cells and in vivo, making it a promising candidate for mitigating PM-induced lung inflammation and oxidative stress.

Sappanone A ameliorates acute lung injury through inhibiting the activation of the NF-κB signaling pathway

Acute lung injury (ALI) is a serious respiratory disease characterized by diffuse alveolar injury, and it has emerged as a major concern in clinical practice due to limited treatments. This study aimed to explore the pharmacological effects and regulatory mechanism of sappanone A (SA) on ALI. In vivo, mice were administered with SA followed by intratracheal injection of lipopolysaccharide (LPS) to establish an animal model of ALI. We observed that SA exerted comparable anti-inflammatory effects to dexamethasone, as evidenced by effectively mitigating histopathological abnormalities and suppressing the inflammatory response in the lung tissues of mice with ALI. RNA sequencing analysis revealed that SA significantly inhibited the activation of the nuclear factor kappa B (NF-κB) signaling pathway. In vitro, we found that SA protected BEAS-2B cells against LPS-induced cellular injury and reduced inflammatory cytokine generation. Furthermore, both in vivo and in vitro experiments demonstrated that SA effectively prevented LPS-induced oxidative stress and apoptosis. Consistent with the results of the RNA sequencing analysis, SA significantly inhibited the increased protein expressions of p105, p50, c-REL, as well as the ratios of p-p65/p65 and p-IκBα/IκBα in the lung tissues of mice with ALI and LPS-stimulated BEAS-2B cells. Additionally, SA inhibited the nuclear translocation of p65 in BEAS-2B cells stimulated with LPS. Importantly, specific blockade of the NF-κB signaling pathway using BAY11–7082 was identified to alleviate LPS-induced cellular injury in BEAS-2B cells. Collectively, these findings suggest that SA can ameliorate ALI, at least in part, through the inhibition of NF-κB signaling pathway activation.

Heparin-binding protein and sepsis-induced coagulopathy: Modulation of coagulation and fibrinolysis via the TGF-β signalling pathway

BackgroundHeparin-binding protein (HBP) levels have been linked to organ failure and may represent an inflammatory biomarker of sepsis. We found disseminated intravascular coagulation (DIC) is associated with higher HBP levels in patients and in in vivo and in vitro models. This prospective, single-center observational study investigated the effects and underlying mechanisms of HBP on the coagulation cascade in sepsis. Methods538 patients with sepsis from June 2016 to December 2019 were enrolled. Mechanisms underlying HBP and the coagulation system were investigated in human umbilical vein endothelial cells (HUVEC) and C57 mice. ResultsIncreased HBP was associated with sepsis-induced DIC. The optimal cutoff value was 37.5 ng/mL (sensitivity: 56 %, specificity: 65 %). Antithrombin-III (AT-III) activity, plasmin-a2 plasmin inhibitor complex (PIC), procalcitonin (PCT), hemoglobin, and HBP ≥37.5 ng/mL were associated with of DIC occurrence. In HUVECs &C57 mice models, Western blotting, qPCR, and immunohistochemistry analysis showed that the binding between HBP and TGF-β receptor 2 (TGFBR2) caused elevation of plasminogen activator inhibitor-1 (PAI-1) levels. Furthermore, we found that mice stimulated with HBP had higher levels of fibrinogen and D-dimer in the blood. HBP treatment caused the accumulation of fibrinogen in mice lung tissue. Treatment with TGFBR2-small interfering RNAs inhibited the effects. ConclusionPatients with sepsis having HBP ≥37.5 ng/mL at admission were more likely to develop DIC. HBP upregulates the expression of fibrinogen and PAI-1 via TGFBR2 and the TGF-β signalling pathway.

Human Umbilical Cord Mesenchymal Stem Cell Exosome-derived miR-335-5p Alleviated Lipopolysaccharide-induced Acute Lung Injury by Regulating the m6A Level of ITGβ4 Gene.

Acute lung injury (ALI) is a serious complication that may accompany severe pneumonia in children. Derived from human umbilical cord mesenchymal stem cell exosome (HucMSC-Exo) can contribute to the regeneration of damaged lung tissue. This study aims to investigate the impact of HucMSC-Exo on ALI and its potential mechanisms. Firstly, RT-qPCR was performed to assess the expression of miR-335-5p. Subsequently, Pearson correlation analysis was performed to examine the correlation between METTL14 and miR-335-5p, as well as the correlation between METTL14 and ITGβ4, while RNA immunoprecipitation (RIP) was used to determine the m6A modification level of ITGβ4. Additionally, molecular biology techniques were employed to evaluate the expression of glycolysis-related factors. Definitively, an LPS-induced ALI model was established to investigate the effect of miR-335-5p on mice lung tissue. miR-335-5p was found to be highly expressed in HucMSC-Exo. Transfection with miR-335-5p mimics resulted in increased glucose uptake. Pearson correlation analysis revealed a negative correlation between METTL14 and miR-335-5p, as well as between METTL14 and ITGβ4. The m6A level of ITGβ4 was elevated in ALI. Overexpression of METTL14 was found to reduce the expression of ITGβ4 and glucose levels, while overexpression of ITGβ4 reversed the effects of METTL14 overexpression. In vivo, results demonstrated that miR-335-5p could improve the extent of lung tissue lesions and reduce glycolytic levels. This study revealed the mechanism by which miR-335-5p derived from HucMSC-Exo could alleviate LPS-induced ALI by regulating the m6A modification of ITGβ4, providing a new direction for the treatment of ALI.

Targeting TLR4 and regulating the Keap1/Nrf2 pathway with andrographolide to suppress inflammation and ferroptosis in LPS-induced acute lung injury

Acute lung injury (ALI) is a severe inflammatory condition with a high mortality rate, often precipitated by sepsis. The pathophysiology of ALI involves complex mechanisms, including inflammation, oxidative stress, and ferroptosis, a novel form of regulated cell death. This study explores the therapeutic potential of andrographolide (AG), a bioactive compound derived from Andrographis, in mitigating Lipopolysaccharide (LPS)-induced inflammation and ferroptosis. Our research employed in vitro experiments with RAW264.7 macrophage cells and in vivo studies using a murine model of LPS-induced ALI. The results indicate that AG significantly suppresses the production of pro-inflammatory cytokines and inhibits ferroptosis in LPS-stimulated RAW264.7 cells. In vivo, AG treatment markedly reduces lung edema, decreases inflammatory cell infiltration, and mitigates ferroptosis in lung tissues of LPS-induced ALI mice. These protective effects are mediated via the modulation of the Toll-like receptor 4 (TLR4)/Kelch-like ECH-associated protein 1(Keap1)/Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Molecular docking simulations identified the binding sites of AG on the TLR4 protein (Kd value: -33.5 kcal·mol-1), and these interactions were further corroborated by Cellular Thermal Shift Assay (CETSA) and SPR assays. Collectively, our findings demonstrate that AG exerts potent anti-inflammatory and anti-ferroptosis effects in LPS-induced ALI by targeting TLR4 and modulating the Keap1/Nrf2 pathway. This study underscores AG's potential as a therapeutic agent for ALI and provides new insights into its underlying mechanisms of action.