This study investigated the protective effect of arbutin(Arb) in yam on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in a mouse model and revealed its possible mechanism of action by metabolomics technology, providing a theoretical basis for clinical treatment of ALI. SPF BALB/c mice were randomly divided into normal control group, model group, resveratrol(Rv)-positive control group, Arb low-dose(15 mg·kg~(-1)) group, and Arb high-dose(30 mg·kg~(-1)) group. The LPS-induced ALI model was established in all groups except the normal control group. Hematoxylin-eosin(HE) staining, TUNEL staining, and WBP whole-body non-invasive pulmonary function testing were used to evaluate the degree of lung tissue damage and lung function changes. Enzyme-linked immunosorbent assay(ELISA) was used to detect the level of inflammatory factors in lung tissue. Flow cytometry was used to analyze the M1/M2 polarization status of macrophages in lung tissue. Western blot was used to detect the expression levels of the TLR4 signaling pathway and related apoptotic proteins. Liquid chromatograph-mass spectrometer(LC-MS) metabolomics was used to analyze the changes in serum metabolic profile after Arb intervention. The results showed that Arb pretreatment significantly alleviated LPS-induced lung tissue injury, improved lung function, reduced the levels of pro-inflammatory factors(IL-6, TNF-α, IL-18, and IL-1β), and regulated the polarization status of M1/M2 macrophages. In addition, Arb inhibited the activation of the TLR4 signaling pathway, reduced the expression of pro-apoptotic proteins such as Bax, caspase-3, and caspase-9, up-regulated the level of Bcl-2 protein, and inhibited apoptosis of lung cells. Metabolomic analysis showed that Arb significantly improved LPS-induced metabolic abnormalities, mainly involving key pathways such as galactose metabolism, phenylalanine metabolism, and lipid metabolism. In summary, Arb can significantly reduce LPS-induced ALI by regulating the release of inflammatory factors, inhibiting the activation of the TLR4 signaling pathway, improving metabolic disorders, and regulating macrophage polarization, indicating that Arb has potential clinical application value.
Animals ; Acute Lung Injury/chemically induced* ; Mice ; Mice, Inbred BALB C ; Arbutin/administration & dosage* ; Male ; Toll-Like Receptor 4/immunology* ; Apoptosis/drug effects* ; Lung/metabolism* ; Signal Transduction/drug effects* ; Protective Agents/administration & dosage* ; Humans ; Macrophages/immunology* ; Drugs, Chinese Herbal/administration & dosage*

This study aims to investigate the therapeutic effects of the Coptidis Rhizoma-Scutellariae Radix on cytokine storm secondary lung injury(CSSLI) induced by CpG1826 in mice, and to elucidate the potential molecular mechanisms by which its major active components, i.e., coptisine and wogonin, alleviate CSSLI by inhibiting the mitochondrial permeability transition pore(mPTP)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3) inflammasome pyroptosis pathway. In vivo, a mouse model of CSSLI was established by CpG1826 induction. Pulmonary edema was assessed by lung wet-to-dry weight ratio(W/D), lung injury was evaluated by hematoxylin-eosin(HE) staining, and ultrastructural changes in lung tissue were observed by transmission electron microscopy(TEM). The levels of interleukin(IL)-1β, high mobility group box 1 protein(HMGB1), IL-18, and IL-1α in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay(ELISA). The results showed that the decoction of the Coptidis Rhizoma-Scutellariae Radix significantly reduced pulmonary edema, alleviated lung injury, and decreased the concentrations of related cytokines in BALF more effectively than either single herb alone, thereby improving CSSLI. In vitro, a CpG1826-induced CSSLI model was established in mouse alveolar macrophage MH-S cells. Calcein-AM quenching was used to screen for the most effective monomer components from the herb pair in inhibiting mPTP opening. Coptisine(5, 10, 20 μmol·L~(-1)) and wogonin(10, 20, 40 μmol·L~(-1)) markedly inhibited mPTP opening, with optimal effects and a clear dose-dependent pattern. These components suppressed mPTP opening, thereby reducing the release of mitochondrial DNA(mtDNA) and the accumulation of reactive oxygen species(ROS), effectively reversing the CpG1826-induced decrease in mitochondrial membrane potential(MMP). Further studies revealed that both coptisine and wogonin inhibited pyroptosis and downregulated the expression of key proteins in the NLRP3/Caspase-1/gasdermin D(GSDMD) pathway. In conclusion, the Coptidis Rhizoma-Scutellariae Radix improves CpG1826-induced CSSLI in mice, and this effect is associated with the inhibition of the mPTP/NLRP3 pyroptosis pathway, providing scientific evidence for its clinical application and further development.
Animals ; Mice ; Drugs, Chinese Herbal/administration & dosage* ; Pyroptosis/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology* ; Male ; Lung Injury/immunology* ; Cytokines/immunology* ; Scutellaria baicalensis/chemistry* ; Oligodeoxyribonucleotides/adverse effects* ; Mice, Inbred C57BL ; Coptis chinensis

Acute lung injury(ALI) is a critical clinical condition primarily characterized by refractory hypoxemia and infiltration of inflammatory cells in lung tissue, which can progress into a more severe form known as acute respiratory distress syndrome(ARDS). Immune cells and inflammatory cytokines play important roles in the progression of the disease. Due to its unclear pathogenesis and the lack of effective clinical treatments, ALI is associated with a high mortality rate and severely affects patients' quality of life, making the search for effective therapeutic agents particularly urgent. Ginseng Radix et Rhizoma, the dried root of the perennial herb Panax ginseng from the Araliaceae family, contains active ingredients such as saponins and polysaccharides, which possess various pharmacological effects including anti-tumor activity, immune regulation, and metabolic modulation. In recent years, studies have shown that ginsenosides exhibit notable effects in reducing inflammation, ameliorating epithelial and endothelial cell injury, and providing anticoagulant action, indicating their comprehensive role in alleviating lung injury. This review summarizes the pathogenesis of ALI and the molecular mechanisms through which ginsenosides act at different stages of ALI development. The aim is to provide a scientific reference for the development of ginsenoside-based drugs targeting ALI, as well as a theoretical basis for the clinical application of Ginseng Radix et Rhizoma in the treatment of ALI.
Ginsenosides/pharmacology* ; Humans ; Acute Lung Injury/immunology* ; Animals ; Panax/chemistry* ; Drugs, Chinese Herbal

Acute lung injury (ALI) is a severe disease caused by viral infection that triggers an uncontrolled inflammatory response. This study investigated the capacity of jasurolignoside (JO), a natural compound, to bind to Toll-like receptor 4 (TLR4) and treat ALI. The anti-inflammatory properties of JO were evaluated in vitro through Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and co-immunoprecipitation. The investigation utilized a lipopolysaccharide (LPS)-induced ALI animal model to examine the therapeutic efficacy and mechanism of JO in vivo. JO attenuated inflammatory symptoms in infected cells and tissues by modulating the NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome and the nuclear factor κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. Molecular docking simulations revealed JO binding to TLR4 active sites, confirmed by cellular thermal shift assay. Surface plasmon resonance (SPR) demonstrated direct interaction between JO and TLR4 with a Kd value of 35.1 μmol·L^-1. Moreover, JO inhibited tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 secretion and reduced leukocyte, neutrophil, lymphocyte, and macrophage infiltration in ALI-affected mice. JO also enhanced lung function and reduced ALI-related mortality. Immunohistochemical staining demonstrated JO's ability to suppress TLR4 expression in ALI-affected mouse lung tissue. This study establishes that JO can bind to TLR4 and effectively treat ALI, indicating its potential as a therapeutic agent for clinical applications.
Toll-Like Receptor 4/chemistry* ; Animals ; Acute Lung Injury/chemically induced* ; Mice ; Humans ; Ilex/chemistry* ; Molecular Docking Simulation ; Male ; NF-kappa B/immunology* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/immunology* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics* ; RAW 264.7 Cells ; Disease Models, Animal

Acute lung injury (ALI) is a significant complication of sepsis, characterized by high morbidity, mortality, and poor prognosis. Neutrophils, as critical intrinsic immune cells in the lung, play a fundamental role in the development and progression of ALI. During ALI, neutrophils generate neutrophil extracellular traps (NETs), and excessive NETs can intensify inflammatory injury. Research indicates that Taohe Chengqi decoction (THCQD) can ameliorate sepsis-induced lung inflammation and modulate immune function. This study aimed to investigate the mechanisms by which THCQD improves ALI and its relationship with NETs in sepsis patients, seeking to provide novel perspectives and interventions for clinical treatment. The findings demonstrate that THCQD enhanced survival rates and reduced lung injury in the cecum ligation and puncture (CLP)-induced ALI mouse model. Furthermore, THCQD diminished neutrophil and macrophage infiltration, inflammatory responses, and the production of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α). Notably, subsequent experiments confirmed that THCQD inhibits NET formation both in vivo and in vitro. Moreover, THCQD significantly decreased the expression of peptidyl arginine deiminase 4 (PAD4) protein, and molecular docking predicted that certain active compounds in THCQD could bind tightly to PAD4. PAD4 overexpression partially reversed THCQD's inhibitory effects on PAD4. These findings strongly indicate that THCQD mitigates CLP-induced ALI by inhibiting PAD4-mediated NETs.
Extracellular Traps/immunology* ; Acute Lung Injury/immunology* ; Animals ; Sepsis/immunology* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Neutrophils/immunology* ; Male ; Protein-Arginine Deiminase Type 4/genetics* ; Mice, Inbred C57BL ; Humans ; Disease Models, Animal ; Cytokines/metabolism*

OBJECTIVE: To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary infection and explore the possible mechanism. METHODS: Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (=5). In the former two groups, was inoculated intranasal administration to establish mouse models of pulmonary infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 μg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. RESULTS: infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation ( < 0.05) and reduced chlamydia load in the lung tissue ( < 0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells ( < 0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages ( < 0.05) and Th1 cells ( < 0.05). CONCLUSIONS Inhibition of CD96 alleviates pneumonia in -infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity.
Animals ; Antigens, CD ; metabolism ; Chlamydia Infections ; complications ; immunology ; physiopathology ; Chlamydia muridarum ; Interferon-gamma ; genetics ; metabolism ; Killer Cells, Natural ; metabolism ; Lung Injury ; etiology ; genetics ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

BackgroundVentilator-induced lung injury (VILI) is commonly associated with barrier dysfunction and inflammation reaction. Glutamine could ameliorate VILI, but its role has not been fully elucidated. This study examined the relationship between inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-10) and adherens junctions (E-cadherin, p120-catenin), which were ameliorated by glutamine in VILI, both in vitro and in vivo.

MethodsFor the in vivo study, 30 healthy C57BL/6 mice weighing 25-30 g were randomly divided into five groups with random number table (n = 6 in each group): control (Group C); low tidal volume (Group L); low tidal volume + glutamine (Group L + G); high tidal volume (Group H); and high tidal volume + glutamine (Group H + G). Mice in all groups, except Group C, underwent mechanical ventilation for 4 h. For the in vitro study, mouse lung epithelial 12 (MLE-12) cells pretreated with glutamine underwent cyclic stretching at 20% for 4 h. Cell lysate and lung tissue were obtained to detect the junction proteins, inflammatory cytokines, and lung pathological changes by the Western blotting, cytokine assay, hematoxylin and eosin staining, and immunofluorescence.

ResultsIn vivo, compared with Group C, total cell counts (t = -28.182, P < 0.01), the percentage of neutrophils (t = -28.095, P < 0.01), IL-6 (t = -28.296, P < 0.01), and TNF-α (t = -19.812, P < 0.01) in bronchoalveolar lavage (BAL) fluid, lung injury scores (t = -6.708, P < 0.01), and the wet-to-dry ratio (t = -15.595, P < 0.01) were increased in Group H; IL-10 in BAL fluid (t = 9.093, P < 0.01) and the expression of E-cadherin (t = 10.044, P < 0.01) and p120-catenin (t = 13.218, P < 0.01) were decreased in Group H. Compared with Group H, total cell counts (t = 14.844, P < 0.01), the percentage of neutrophils (t = 18.077, P < 0.01), IL-6 (t = 18.007, P < 0.01), and TNF-α (t = 10.171, P < 0.01) in BAL fluid were decreased in Group H + G; IL-10 in BAL fluid (t = -7.531, P < 0.01) and the expression of E-cadherin (t = -14.814, P < 0.01) and p120-catenin (t = -9.114, P < 0.01) were increased in Group H + G. In vitro, compared with the nonstretching group, the levels of IL-6 (t = -21.111, P < 0.01) and TNF-α (t = -15.270, P < 0.01) were increased in the 20% cyclic stretching group; the levels of IL-10 (t = 5.450, P < 0.01) and the expression of E-cadherin (t = 17.736, P < 0.01) and p120-catenin (t = 16.136, P < 0.01) were decreased in the 20% cyclic stretching group. Compared with the stretching group, the levels of IL-6 (t = 11.818, P < 0.01) and TNF-α (t = 8.631, P < 0.01) decreased in the glutamine group; the levels of IL-10 (t = -3.203, P < 0.05) and the expression of E-cadherin (t = -13.567, P < 0.01) and p120-catenin (t = -10.013, P < 0.01) were increased in the glutamine group.

ConclusionsHigh tidal volume mechanical ventilation and 20% cyclic stretching could cause VILI. Glutamine regulates VILI by improving cytokines and increasing the adherens junctions, protein E-cadherin and p120-catenin, to enhance the epithelial barrier function.

Animals ; Cadherins ; metabolism ; Catenins ; metabolism ; Glutamine ; metabolism ; Inflammation ; metabolism ; Interleukin-6 ; metabolism ; Lung ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Ventilator-Induced Lung Injury ; immunology ; metabolism

BACKGROUNDSepsis is a major cause of mortality in Intensive Care Units. Anesthetic dose isoflurane and 100% oxygen were proved to be beneficial in sepsis; however, their application in septic patients is limited because long-term hyperoxia may induce oxygen toxicity and anesthetic dose isoflurane has potential adverse consequences. This study was scheduled to find the optimal combination of isoflurane and oxygen in protecting experimental sepsis and its mechanisms.

METHODSThe effects of combined therapy with isoflurane and oxygen on lung injury and sepsis were determined in animal models of sepsis induced by cecal ligation and puncture (CLP) or intraperitoneal injection of lipopolysaccharide (LPS) or zymosan. Mouse RAW264.7 cells or human peripheral blood mononuclear cells (PBMCs) were treated by LPS to probe mechanisms. The nuclear factor kappa B (NF-κB) signaling molecules were examined by Western blot and cellular immunohistochemistry.

RESULTSThe 0.5 minimum alveolar concentration (MAC) isoflurane in 60% oxygen was the best combination of oxygen and isoflurane for reducing mortality in experimental sepsis induced by CLP, intraperitoneal injection of LPS, or zymosan. The 0.5 MAC isoflurane in 60% oxygen inhibited proinflammatory cytokines in peritoneal lavage fluids (tumor necrosis factor-alpha [TNF-β]: 149.3 vs. 229.7 pg/ml, interleukin [IL]-1β: 12.5 vs. 20.6 pg/ml, IL-6: 86.1 vs. 116.1 pg/ml, and high-mobility group protein 1 [HMGB1]: 323.7 vs. 449.3 ng/ml; all P< 0.05) and serum (TNF-β: 302.7 vs. 450.7 pg/ml, IL-1β: 51.7 vs. 96.7 pg/ml, IL-6: 390.4 vs. 722.5 pg/ml, and HMGB1: 592.2 vs. 985.4 ng/ml; all P< 0.05) in septic animals. In vitro experiments showed that the 0.5 MAC isoflurane in 60% oxygen reduced inflammatory responses in mouse RAW264.7 cells, after LPS stimulation (all P< 0.05). Suppressed activation of NF-κB pathway was also observed in mouse RAW264.7 macrophages and human PBMCs after LPS stimulation or plasma from septic patients. The 0.5 MAC isoflurane in 60% oxygen also prevented the increases of phospho-IKKβ/β, phospho-IκBβ, and phospho-p65 expressions in RAW264.7 macrophages after LPS stimulation (all P< 0.05).

CONCLUSIONCombined administration of a sedative dose of isoflurane with 60% oxygen improves survival of septic animals through reducing inflammatory responses.

Adult ; Anesthesia ; methods ; Animals ; Blotting, Western ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Female ; Humans ; Inflammation ; drug therapy ; Isoflurane ; therapeutic use ; Leukocytes, Mononuclear ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Lipopolysaccharides ; pharmacology ; Lung Injury ; drug therapy ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; metabolism ; Oxygen ; therapeutic use ; Peroxidase ; metabolism ; RAW 264.7 Cells ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

The present study was designed to synthesize 2-Cyano-3, 12-dioxooleana-1, 9(11)-en-28-oate-13β, 28-olide (1), a lactone derivative of oleanolic acid (OA) and evaluate its anti-inflammatory activity. Compound 1 significantly diminished nitric oxide (NO) production and down-regulated the mRNA expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further in vivo studies in murine model of LPS-induced acute lung injury (ALI) showed that 1 possessed more potent protective effects than the well-known anti-inflammatory drug dexamethasone by inhibiting myeloperoxidase (MPO) activity, reducing total cells and neutrophils, and suppressing inflammatory cytokines expression, and thus ameliorating the histopathological conditions of the injured lung tissue. In conclusion, compound 1 could be developed as a promising anti-inflammatory agent for intervention of LPS-induced ALI.
Acute Lung Injury ; drug therapy ; genetics ; immunology ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemical synthesis ; Bronchoalveolar Lavage Fluid ; immunology ; Cyclooxygenase 2 ; genetics ; immunology ; Female ; Humans ; Interleukin-1beta ; genetics ; immunology ; Interleukin-6 ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Lung ; drug effects ; immunology ; Macrophages ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Neutrophils ; drug effects ; immunology ; Oleanolic Acid ; administration & dosage ; analogs & derivatives ; chemical synthesis ; Peroxidase ; genetics ; immunology ; RAW 264.7 Cells ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVETo investigate the role of autophagy in acute lung injury (ALI) caused by multiple trauma in rats via pretreat with 3-methyladenine (3-MA).

METHODSForty-five Sprague-Dawley male rats, with age of 4 months and body weight of 250-300 g,were randomly divided into three groups. In the sham group, the rats received sphenotresia only;in the control group, the rats were made model of femur shaft fracture combined with brain injury, and treated with physiological saline by abdominal cavity at 1 hour before making model; in the 3-MA group, the rats were made model of femur shaft fracture combined with brain injury,and treated with 3-MA of 10 mg/kg by abdominal cavity at 1 hour before making model. Histologic changes and the concentration of related inflammatory factors in the damaged lung tissue were examined at 48 h after opteration, at the same time, the effect of 3-MA on the expression of LC-3 II and Beclin-1 was examined through reverse transcriptase polymerase chain reaction technique (RT-PCR).

RESULTSCompared with sham group, LC-3 II and Beclin-1 level in control group at 48 h after operation were obviously increased (P < 0.01). Compared with control group, LC-3 II and Beclin-1 level in 3-MA group at 48 h after operation were obviously decreased (P < 0.01). Compared with sham group, the level of proinflammatory cytokines (TNF-α and IL-6) in control group obviously enhanced (P < 0.01). Compared with control group, above items in 3-MA group was obviously lower (P < 0.01). Compared with control group,the histopathological damage of lung in 3-MA group obviously reduced (P < 0.01).

CONCLUSIONAutophagy can aggravate the acute lung injury caused by fracture of shaft of femur combined with brain injuries,but 3-MA can reduce tissue damage by inhibiting the autophagy and inflammatory response.

Acute Lung Injury ; prevention & control ; Adenine ; analogs & derivatives ; therapeutic use ; Animals ; Apoptosis Regulatory Proteins ; analysis ; Beclin-1 ; Interleukin-6 ; analysis ; Lung ; chemistry ; immunology ; pathology ; Male ; Multiple Trauma ; complications ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; analysis