Intratracheal Administration Of LPS Research Articles

Oridonin alleviates inflammation and endoplasmic reticulum stress in pediatric pneumonia via regulating the SIRT1-mediated Wnt/β-catenin signaling pathway.

Pediatric pneumonia is a prevalent and significant health concern worldwide, with elevated morbidity and mortality rates among affected children. This study was designed to elucidate the therapeutic impact of Oridonin (Ori) on pediatric pneumonia and unravel the underlying mechanisms involved. A pediatric infantile pneumonia model was established in mice through intratracheal administration of LPS. Additionally, a cell damage model was created in WI-38 cells by administering LPS. Protein levels were assessed via western blotting, and cell viability was measured with CCK-8. Inflammatory cytokines were quantified through ELISA, and specific assays were employed to evaluate oxidative stress markers. Flow cytometry was utilized to assess cell apoptosis. Ori alleviated lung inflammation, oxidative stress, apoptosis, and endoplasmic reticulum stress (ERS) in LPS-induced pneumonia mice. In addition, Ori increased the viability of LPS-induced pneumonia cells but decreased cell apoptosis. Furthermore, Ori reduced oxidative stress, inflammation, and ERS in LPS-induced pneumonia cells by enhancing SIRT1 to activate the Wnt/β-catenin pathway. This study suggested that Ori inhibited pediatric pneumonia by dampening the inflammatory response, oxidative stress, cell apoptosis, and ERS via the SIRT1/Wnt/β-catenin pathway.

Effectiveness of remote ischaemic conditioning is not affected by hyper-inflammation in a rat model of stroke

The inflammation and coagulopathy during coronavirus disease (COVID-19) impairs the efficiency of the current stroke treatments. Remote ischaemic conditioning (RIC) has shown potential in recent years to protect the brain and other organs against pathological conditions. This study aimed to evaluate the efficiency of RIC in brain infarct size using TTC staining and lung injury reduction by H&E staining during the hyper-inflammatory response in rats. The inflammation and coagulopathy were assessed by sedimentation rate, haematocrit, systemic oxidative stress and clotting time. Moreover, we observed changes in the cytokine profile. The results of the first part of the experiment showed that the inflammation and lung injury are fully developed after 24 h of intratracheal LPS administration. At this time, we induced focal brain ischaemia and examined the effect of RIC pre- and post-treatment. Our results showed that RIPre-C reduced the infarct size by about 23%, while RIPost-C by about 30%. The lung injury was also reduced following both treatments. Moreover, RIC modulated systemic inflammation. The level of chemokines CINC-1, LIX and RANTES decreased after 24 h of post-ischaemic reperfusion in treated animals compared to non-treated. The RIC-mediated decrease of inflammation was reflected in improved sedimentation rate and hematocrit, as well as reduced systemic oxidative stress. The results of this work showed neuroprotective and lung protective effects of RIC with a decrease in inflammation response. On the basis of our results, we assume that immunomodulation through the chemokines CINC-1, LIX, and RANTES play a role in RIC-mediated protection.

Deciphering the Therapeutic Potential of Myeloid-Specific JAK2 Inhibition in Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by severe inflammation and pulmonary dysfunction. Despite advancements in critical care, effective pharmacological interventions for ARDS remain elusive. While Janus kinase 2 (JAK2) inhibitors have emerged as an innovative treatment for numerous autoinflammatory diseases, their therapeutic potential in ARDS remains unexplored. In this study, we investigated the contribution of JAK2 and its underlying mechanisms in ARDS utilizing myeloid-specific JAK2 knockout murine models alongside a pharmacological JAK2 inhibitor. Notably, myeloid-specific JAK2 knockout led to a notable attenuation of ARDS induced by intratracheal administration of LPS, accompanied by reduced levels of neutrophils and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and lung tissue. Intriguingly, the ameliorative effects were abolished upon the depletion of monocyte-derived alveolar macrophages (Mo-AMs) rather than tissue-resident alveolar macrophages (TR-AMs). JAK2 deficiency markedly reversed LPS-induced activation of STAT5 in macrophages. Remarkably, pharmacological JAK2 inhibition using baricitinib failed to substantially alleviate neutrophils infiltration, implying that specific inhibition of JAK2 in Mo-AMs is imperative for ARDS amelioration. Collectively, our data suggest that JAK2 may mitigate ARDS progression through the JAK2 pathway in Mo-AMs, underscoring JAK2 in alveolar macrophages, particularly Mo-AMs, as a promising therapeutic target for ARDS treatment.

Ophiopogonin D alleviates acute lung injury by regulating inflammation via the STAT3/A20/ASK1 axis

BackgroundAcute lung injury (ALI) is characterized by acute pulmonary inflammatory infiltration. Alveolar epithelial cells (AECs) release numerous pro-inflammatory cytokines, which result in the pathological changes seen in ALI. Ophiopogonin D (OD), extracted from the roots of Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), reduces inflammation; however, the efficacy of OD in ALI has not been reported and the underlying molecular mechanisms remain unclear. PurposeThis study investigated the anti-inflammatory effects of OD, as well as the underlying mechanisms, in AECs and a mouse ALI model. MethodsLipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were used to stimulate macrophages and A549 cells, and a mouse ALI model was established by intratracheal LPS administration. The anti-inflammatory effects and mechanisms of OD in the TNF-α-induced in vitro inflammation model was evaluated using real-time quantitative polymerase chain reaction qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting, nuclear and cytoplasmic protein extraction, and immunofluorescence. The in vivo anti-inflammatory activity of OD was evaluated using hematoxylin and eosin staining, qPCR, ELISA, and western blotting. ResultsThe bronchoalveolar lavage fluid and lung tissue of LPS-induced ALI mice exhibited increased TNF-α expression. TNF-α induced a significantly greater pro-inflammatory effect in AECs than LPS. OD reduced inflammation and mitogen-activated protein kinase (MAPK) and transcription factor p65 phosphorylation in vivo and in vitro and promoted signal transducer and activator of transcription 3 (STAT3) phosphorylation and A20 expression, thereby inducing apoptosis signal-regulating kinase 1 (ASK1) proteasomal degradation. ConclusionOD exerts an anti-inflammatory effect by promoting STAT3-dependent A20 expression and ASK1 degradation. OD may therefore have therapeutic value in treating ALI and other TNF-α-related inflammatory diseases.

Schisandrin B protects against LPS-induced inflammatory lung injury by targeting MyD88

BackgroundAcute lung injury (ALI) is a challenging clinical syndrome that manifests as an acute inflammatory response. Schisandrin B (Sch B), a bioactive lignan from Schisandra genus plants, has been shown to suppress inflammatory responses and oxidative stress. However, the underlying molecular mechanisms have remained elusive. Hypothesis/purposeThis study performed an in-depth investigation of the anti-inflammatory mechanism of Sch B in macrophages and in an animal model of ALI. MethodsqPCR array was used to probe the differential effects and potential target of Sch B. ALI was induced by intratracheal administration of LPS in experimental mice with or without Sch B treatment. ResultsOur studies show that Sch B differentially modulates inflammatory factor induction by LPS in macrophages by directly binding myeloid differentiation response factor-88 (MyD88), an essential adaptor protein in the toll-like receptor-4 (TLR4) pathway. Sch B spares non-MyD88-pathways downstream of TLR4. Such inhibition suppressed key signaling mediators such as TAK1, MAPKs, and NF-κB, and pro-inflammatory factor induction. Pull down assay using biotinylated-Sch B validate the direct interaction between Sch B and MyD88 in macrophages. Treatment of mice with Sch B prior to LPS challenge reduced inflammatory cell infiltration in lungs, induction of MyD88-pathway signaling proteins, and prevented inflammatory cytokine induction. ConclusionIn summary, our studies have identified MyD88 as a direct target of Sch B for its anti-inflammatory activity, and suggest that Sch B may have therapeutic value for acute lung injury and other MyD88-dependent inflammatory diseases.

Modeling of Chronic Lung Inflammation in Rats by Repeated Intratracheal Administration of LPS.

A model of a chronic lung inflammation in SPF Sprague-Dawley rats was developed by repeated intratracheal administration of LPS in a dose of 0.4 mg/kg. On day 22 of the study, male rats treated with LPS have relative monocytopenia and reduced mean concentration of hemoglobin in the erythrocyte and the mean platelet volume in comparison with the control animals (saline). Intratracheal administration of LPS induced an inflammatory process in the lungs characterized by focal atelectasis, compensatory emphysematous expansion of subpleural pulmonary acini, focal mononuclear and neutrophilic perivascular and peribronchial infiltration, and minor focal mononuclear and neutrophilic infiltration of the alveolar walls. Against the background of LPS administration, germinal centers appeared in the lymphoid follicles of the white pulp of the spleen, and focal mononuclear infiltration of the tracheal mucosa and/or submucosa was observed in some animals.

Emodin Protects Against Lipopolysaccharide-Induced Acute Lung Injury via the JNK/Nur77/c-Jun Signaling Pathway.

Background: Acute lung injury (ALI) is a serious inflammatory disease with clinical manifestations of hypoxemia and respiratory failure. Presently, there is no effective treatment of ALI. Although emodin from Rheum palmatum L. exerts anti-ALI properties, the underlying mechanisms have not been fully explored. Purpose: This study aimed to investigate the therapeutic effect and mechanism of emodin on LPS-induced ALI in mice. Methods: RAW264.7 cells and zebrafish larvae were stimulated by LPS to establish inflammatory models. The anti-inflammatory effect of emodin was assessed by ELISA, flow cytometric analysis, and survival analysis. In vitro mechanisms were explored by using Western blotting, luciferase assay, electrophoretic mobility shift assay (EMSA), and small interfering RNA (siRNA) approach. The acute lung injury model in mice was established by the intratracheal administration of LPS, and the underlying mechanisms were assessed by detecting changes in histopathological and inflammatory markers and Western blotting in lung tissues. Results: Emodin inhibited the inflammatory factor production and oxidative stress in RAW264.7 cells, and prolonged the survival of zebrafish larvae after LPS stimulation. Emodin suppressed the expression levels of phosphorylated JNK at Thr183/tyr182 and phosphorylated Nur77 at Ser351 and c-Jun, and increased the expression level of Nur77 in LPS-stimulated RAW264.7 cells, while these regulatory effects of emodin on Nur77/c-Jun were counteracted by JNK activators. The overexpression of JNK dampened the emodin-mediated increase in Nur77 luciferase activity and Nur77 expression. Moreover, the inhibitory effect of emodin on c-Jun can be attenuated by Nur77 siRNA. Furthermore, emodin alleviated LPS-induced ALI in mice through the regulation of the JNK/Nur77/c-Jun pathway. Conclusions: Emodin protects against LPS-induced ALI through regulation on JNK/Nur77/c-Jun signaling. Our results indicate the potential of emodin in the treatment of ALI.

Examination of Taurine Chloramine and Taurine on LPS-Induced Acute Pulmonary Inflammatory in Mice.

The novel coronavirus disease (COVID-19), which is prevalent in the world, develops severe pneumonia, of which 30% have fatal acute respiratory distress and acute lung injury. At present, there is no established treatment method for ARDS, and it is desired to develop a therapeutic drug as soon as possible. While TauCl has been reported to have anti-inflammatory effects on culture cells, little information is available concerning in vivo experiments. In the present study, we evaluated the anti-inflammatory effect of taurine chloramine (TauCl), a taurine derivative, against LPS-induced pneumonia in mouse. The mice were pretreated with TauCl intraperitoneally before intratracheal administration of LPS. Additionally, we evaluated the effect of taurine treatment by maintaining the mice on drinking water containing 0.5% taurine. Two days after LPS injection, body weight was decreased by 9.5 %, while lung weight was increased due to the infiltration of inflammatory cells; TauCl attenuated the gain in lung weight. LPS-induced acute pneumonia caused an increase in cytokine/chemokine mRNA expression, including that of IL-1β, -6, -17, TNF-α, and MCP-1. However, TauCl treatment attenuated IL-6 expression, but not that of the others although the induction of plasma IL-6 tended to be reduced by TauCl treatment. Importantly, a similar effect against LPS-induced acute lung inflammation was confirmed by taurine pretreatment. These findings suggest that TauCl treatment partially prevents IL-6 production induced by acute pneumonia in vivo.

IL-17 Receptor on Donor Cells Regulates Acute and Chronic Lung Allograft Rejection Potentiated by Repeated Endotoxin Inhalations

<h3>Purpose</h3> Chronic lung allograft dysfunction is a major complication after lung transplantation (LTx). Excessive IL17 receptor A (IL17RA) signaling has been recognized as a key factor; however, its precise mechanism is unclear. In a mouse minor alloantigen mismatched LTx model (C57BL/10 [B10, H-2^b] → C57BL/6 [B6. H-2^b]), we found that repeated airway endotoxin (lipopolysaccharide, LPS) challenge augments airway epithelial damage, acute and chronic rejection with airway obliteration, together with increased IL17A transcription in allografts but not isografts. We hypothesized that both donor and recipient cell responses to IL17A are important in mediating these processes. We therefore investigated the role of IL17RA on donor and recipient cells in the context of chronic rejection. <h3>Methods</h3> B10→B6 LTx and B6→B10 LTx were performed with or without intratracheal LPS administration (5 ug LPS/50 ul PBS, Day 3, 7, 10, 14, 17, 21). B6 IL17RA^−/− mice were used as donors or recipients. Grafts were assessed with micro CT scans on days 3 and 27; histological scoring and quantitative PCR were performed 28 days after LTx. <h3>Results</h3> Without LPS, neither donor nor recipient IL17RA deficiency modified histological scores or lung graft volume or density by CT scan. LPS-treated IL17RA^−/− recipients showed a similar degree of lung radiographic change and histological acute rejection compared to IL17RA^+/+ recipients; however airway obliteration was attenuated (Figure A). In contrast, recipients of IL17RA^−/− donor lungs showed reduced gain of graft density on CT, and diminished acute rejection and peri-airway fibrosis (Figure B-C). IL17RA^−/− donor grafts had fewer CXCL1 (a neutrophil chemoattractant) transcripts (Figure D). <h3>Conclusion</h3> IL17RA signaling on donor cells regulates acute and chronic rejection in LTx recipients experiencing airway inflammation.

Regulatory Role of STAT3-PLSCR2 Axis in Antiviral and Inflammatory Response

Abstract Cytokines are key factors for initiating inflammatory response and among them IFN-I is one of the most versatile because of its dual roles in antiviral and inflammatory activities. While STAT1 and STAT2 are critical for triggering antiviral response, the signaling events downstream of IFN-I receptor contributing to inflammation have yet to be defined. Here we demonstrate that STAT3 and one of its interacting partners PLSCR2 negatively regulate IFN-I-mediated antiviral and inflammatory response. Enhanced expression of IFN-I-stimulated genes (ISGs) is found in cells either absence of STAT3 or PLSCR2, leading to increased resistance to viral infection in vitro and in vivo. Moreover, STAT3 can cooperate with PLSCR2 to suppress IFN-I response. Interestingly, GSEA analysis for IFN-I-stimulated cells lacking PLSCR2 versus WT control displays an inflammatory signature with increased expression of inflammatory cytokines and chemokines, their receptors and TLRs. Stimulation of TLR ligands, including LPS, R848 or CpG, also results in enhanced expression of IFN-I and inflammatory cytokines and chemokines in PLSCR2KO cells as opposed to WT control. PLSCR2KO mice are also more susceptible to LPS-induced endotoxic shock in vivo with increased serum TFN. Similarly, PLSCR2KO mice also display increased acute lung injury in response to intratracheal administration of LPS with increased infiltration of macrophages and neutrophils and production of inflammatory cytokines and chemokines. Enhanced TLR response is also found in STAT3KO BM-derived macrophages. Therefore, these results demonstrate that STAT3 coordinates with PLSCR2 to regulate antiviral and inflammatory response and define a novel axis in modulating IFN-I-mediated response.

IL-17 Receptor on Donor Cells Regulates Acute and Chronic Lung Allograft Rejection Potentiated by Repeated Endotoxin Inhalations

Chronic lung allograft dysfunction is a major complication after lung transplantation (LTx). Excessive IL17 receptor A (IL17RA) signaling has been recognized as a key factor; however, its precise mechanism is unclear. In a mouse minor alloantigen mismatched LTx model (C57BL/10 [B10, H-2b] → C57BL/6 [B6. H-2b]), we found that repeated airway endotoxin (lipopolysaccharide, LPS) challenge augments airway epithelial damage, acute and chronic rejection with airway obliteration, together with increased IL17A transcription in allografts but not isografts. We hypothesized that both donor and recipient cell responses to IL17A are important in mediating these processes. We therefore investigated the role of IL17RA on donor and recipient cells in the context of chronic rejection. B10→B6 LTX and B6→B10 LTx were performed with or without intratracheal LPS administration (5ug LPS/ 50ul PBS, Day 3, 7, 10, 14, 17, 21). B6 IL17RA-/- mice were used as donors or recipients. Grafts were assessed with micro CT scans on days 3 and 27; histological scoring and quantitative PCR were performed 28 days after LTx. Without LPS, neither donor nor recipient IL17RA deficiency modified histological scores or lung graft volume or density by CT scan. LPS-treated IL17RA-/- recipients showed a similar degree of lung radiographic change and histological acute rejection compared to IL17RA+/+ recipients; however airway obliteration was attenuated (Figure A). In contrast, recipients of IL17RA-/- donor lungs showed reduced gain of graft density on CT, and diminished acute rejection and periairway fibrosis (Figure B-C). IL17RA-/- donor grafts had fewer CXCL1 transcripts (Figure D). IL17RA signaling on donor cells regulates acute and chronic rejection in LTx recipients experiencing airway inflammation.

Lipoxin A4 ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial\u2013mesenchymal transition

BackgroundAcute respiratory distress syndrome (ARDS) is characterized by alveolar epithelial disruption. Lipoxins (LXs), as so-called “braking signals” of inflammation, are the first mediators identified to have dual anti-inflammatory and inflammatory pro-resolving properties.MethodsIn vivo, lipoxinA4 was administrated intraperitoneally with 1 μg/per mouse after intra-tracheal LPS administration (10 mg/kg). Apoptosis, proliferation and epithelial–mesenchymal transition of AT II cells were measured by immunofluorescence. In vitro, primary human alveolar type II cells were used to model the effects of lipoxin A4 upon proliferation, apoptosis and epithelial–mesenchymal transition.ResultsIn vivo, lipoxin A4 markedly promoted alveolar epithelial type II cells (AT II cells) proliferation, inhibited AT II cells apoptosis, reduced cleaved caspase-3 expression and epithelial–mesenchymal transition, with the outcome of attenuated LPS-induced lung injury. In vitro, lipoxin A4 increased primary human alveolar epithelial type II cells (AT II cells) proliferation and reduced LPS induced AT II cells apoptosis. LipoxinA4 also inhibited epithelial mesenchymal transition in response to TGF-β1, which was lipoxin receptor dependent. In addition, Smad3 inhibitor (Sis3) and PI3K inhibitor (LY294002) treatment abolished the inhibitory effects of lipoxinA4 on the epithelial mesenchymal transition of primary human AT II cells. Lipoxin A4 significantly downregulated the expressions of p-AKT and p-Smad stimulated by TGF-β1 in primary human AT II cells.ConclusionLipoxinA4 attenuates lung injury via stimulating epithelial cell proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition.

Genipin protects rats against lipopolysaccharide-induced acute lung injury by reinforcing autophagy

Although the protective effects of genipin against acute lung injury (ALI) have been described previously, the associated mechanism remains unclear. We have previously reported that genipin exerts its pharmacological effects by regulating autophagy. Here, we hypothesized that the up-regulation of autophagy may contribute to the protective effects exhibited by genipin against ALI. In the present study, ALI was induced by intratracheal LPS administration in rats. Genipin treatment significantly reduced LPS-induced lung injury as evidenced by improved histopathology, decreased lung edema, total cells, and protein concentration in the bronchoalveolar lavage fluid (BALF). This protection was inhibited by 3-methyladenine (3-MA), an inhibitor of autophagy. Genipin treatment reduced the expression of P62 and increased the expression of Beclin-1 and LC3II, indicating increased autophagy. Genipin treatment also alleviated LPS-induced cell apoptosis (down-regulation of Bax, up-regulation of Bcl-2, and decreased number of terminal deoxynucleotidyl transferase dUTP nick end label-positive cells) and oxidative stress (increased SOD and decreased MDA content) in the lung. Furthermore, genipin attenuated LPS-induced production of TNF-α, IL-1β, and IL-6 in the lung and BALF. These protective effects induced by genipin were reversed by 3-MA treatment, indicating that autophagy was involved in the protective effects exerted by genipin against inflammation and apoptosis in ALI. In A549 cells incubated with LPS for 6 h, genipin treatment increased the number of GFP-LC3 punctae. 3-MA prevented the protective effects of genipin against mitochondrial dysfunction and cell death. These findings suggest that genipin protects against apoptosis and inflammation in LPS-induced ALI by promoting autophagy.

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.

Compounds of IL-6 Receptor Complex during Acute Lung Injury.

Soluble receptor of IL-6 (sIL-6R) and antagonist of the receptor complex, soluble glycoprotein 130 (sgp130) mediate opposite effects during inflammation. We measured the levels of these cytokines and their ratio in rat blood on the model of acute lung injury. The injury was modeled by the intratracheal administration of LPS. The levels of sgp130 and sIL-6R increased during the inflammatory process in the injured lungs. The sgp130/sIL-6R ratio increased or decreased depending on the intensity of the inflammatory process. sgp130/sIL-6R ratio might reflect the intensity of inflammation during lung injury.

Sodium butyrate alleviates LPS-induced acute lung injury in mice via inhibiting HMGB1 release

Sodium butyrate (SB) is a short chain 4-carbon fatty acid salt naturally exists in animal fats. Previous studies have proven that sodium butyrate has many beneficial functions such as anti-tumor and anti-inflammatory actions. In the current study we investigated the effect and possible mechanism of sodium butyrate in LPS-induced acute lung injury (ALI). ALI was induced by intratracheal administration of LPS (10 mg/kg) in male BALB/c mice. Sodium butyrate (500 mg/kg) was administered intraperitoneally 30 min prior to LPS exposure. We found that sodium butyrate significantly protected animals from LPS-induced ALI as evidenced by decreased the lung wet to dry weight ratio, total cells, neutrophils, macrophages, myeloperoxidase (MPO) activity, and lung histological damage compared to vehicle control. Sodium butyrate pretreatment markedly inhibited the production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, sodium butyrate pretreatment dramatically suppressed HMGB1 release and NF-κ B activation. Together, these results suggest that sodium butyrate pretreatment protects mice from LPS-induced acute lung injury, possibly through the modulation of HMGB1 and inflammatory responses.

Shikonin inhibits myeloid differentiation protein 2 to prevent LPS-induced acute lung injury.

Acute lung injury (ALI) is a challenging clinical syndrome, which manifests as an acute inflammatory response. Myeloid differentiation protein 2 (MD2) has an important role in mediating LPS-induced inflammation. Currently, there are no effective molecular-based therapies for ALI or viable biomarkers for predicting the severity of disease. Recent preclinical studies have shown that shikonin, a natural naphthoquinone, prevents LPS-induced inflammation. However, little is known about the underlying mechanisms. The binding affinity of shikonin to MD2 was analysed using computer docking, surface plasmon resonance analysis and elisa. In vitro, the anti-inflammatory effect and mechanism of shikonin were investigated through elisa, real-time quantitative reverse transcription PCR, Western blotting and immunoprecipitation assay. In vivo, lung injury was induced by intratracheal administration of LPS and assessed by changes in the histopathological and inflammatory markers. The underlying mechanisms were investigated by immunoprecipitation in lung tissue. Shikonin directly bound to MD2 and interfered with the activation of toll-like receptor 4 (TLR4) induced by LPS. In cultured macrophages, shikonin inhibited TLR4 signalling and pro-inflammatory cytokine production. These effects were produced through suppression of key signalling proteins including the NF-κB and the MAPK pathway. We also showed that shikonin inhibits MD2-TLR4 complex formation and reduces LPS-induced inflammatory responses in a mouse model of ALI. Our studies have uncovered the mechanism underlying the biological activity of shikonin in ALI and suggest that the targeting of MD2 may prove to be beneficial as a treatment option for this condition.

IL-35 Suppresses Lipopolysaccharide-Induced Airway Eosinophilia in EBI3-Deficient Mice

EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27-deficient mice or WSX-1/IL-27Rα subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-α and IL-1β. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.

Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes.

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

The Isosteroid Alkaloid Imperialine from Bulbs of Fritillaria cirrhosa Mitigates Pulmonary Functional and Structural Impairment and Suppresses Inflammatory Response in a COPD-Like Rat Model.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world. Present therapies for COPD have limited effect on reducing the progression of COPD and suppressing the inflammatory response in the lung. Bulbs of Fritillaria cirrhosa D. Don (BFC) have been used in many Asian countries for a long time to treat pulmonary diseases, such as cough, expectoration, and asthma. Steroidal alkaloids are the major biological active constituents in BFC, whereby imperialine is one of the important steroidal alkaloids. So far, there are no studies reporting the effect of imperialine on COPD. In this study, we investigated the effect of imperialine on pulmonary function and structure and inflammation in a COPD-like rat model which was induced by the combination of exposure to CS and intratracheal administration of LPS. Our data show that imperialine mitigates pulmonary functional and structural impairment and suppressed inflammatory response in a COPD-like rat model by mediating expression of related cytokines in lung tissues of the COPD-like rats, such as IL-1β, IL-6, IL-8, TNF-α, NF-κB, TGF-β1, MMP-9, and TIMP-1.