Instillation Of Lipopolysaccharide Research Articles

Mitochondria of lung venular capillaries mediate lung-liver cross talk in pneumonia.

Failure of the lung's endothelial barrier underlies lung injury, which causes the high mortality acute respiratory distress syndrome (ARDS). Multiple organ failure predisposes to the mortality, but mechanisms are poorly understood. Here, we show that mitochondrial uncoupling protein 2 (UCP2), a component of the mitochondrial inner membrane, plays a role in the barrier failure. Subsequent lung-liver cross talk mediated by neutrophil activation causes liver congestion. We intranasally instilled lipopolysaccharide (LPS). Then, we viewed the lung endothelium by real-time confocal imaging of the isolated, blood-perfused mouse lung. LPS caused alveolar-capillary transfer of reactive oxygen species and mitochondrial depolarization in lung venular capillaries. The mitochondrial depolarization was inhibited by transfection of alveolar Catalase and vascular knockdown of UCP2. LPS instillation caused lung injury as indicated by increases in bronchoalveolar lavage (BAL) protein content and extravascular lung water. LPS or Pseudomonas aeruginosa instillation also caused liver congestion, quantified by liver hemoglobin and plasma aspartate aminotransferase (AST) increases. Genetic inhibition of vascular UCP2 prevented both lung injury and liver congestion. Antibody-mediated neutrophil depletion blocked the liver responses, but not lung injury. Knockdown of lung vascular UCP2 mitigated P. aeruginosa-induced mortality. Together, these data suggest a mechanism in which bacterial pneumonia induces oxidative signaling to lung venular capillaries, known sites of inflammatory signaling in the lung microvasculature, depolarizing venular mitochondria. Successive activation of neutrophils induces liver congestion. We conclude that oxidant-induced UCP2 expression in lung venular capillaries causes a mechanistic sequence leading to liver congestion and mortality. Lung vascular UCP2 may present a therapeutic target in ARDS.NEW & NOTEWORTHY We report that mitochondrial injury in lung venular capillaries underlies barrier failure in pneumonia, and venular capillary uncoupling protein 2 (UCP2) causes neutrophil-mediated liver congestion. Using in situ imaging, we found that epithelial-endothelial transfer of H2O2 activates UCP2, depolarizing mitochondria in venular capillaries. The conceptual advance from our findings is that mitochondrial depolarization in lung capillaries mediates liver cross talk through circulating neutrophils. Pharmacologic blockade of UCP2 could be a therapeutic strategy for lung injury.

Network pharmacology and transcriptomics to determine Danggui Yifei Decoction mechanism of action for the treatment of chronic lung injury

Ethnopharmacological relevanceSeveral children with pneumonia (especially severe cases) have symptoms of cough and expectoration during the recovery stage after standard symptomatic treatment, which eventually results in chronic lung injury. Danggui yifei Decoction (DGYFD), a traditional Chinese formula, has shown clinical promise for the treatment of chronic lung injury during the recovery stage of pneumonia, however, its mechanism of action is yet to be deciphered. Aim of this studyTo investigate the therapeutic mechanism of DGYFD for the treatment of chronic lung injury by integrating network pharmacology and transcriptomics. Materials and methodsBALB/c mice were used to establish the chronic lung injury mouse model by intratracheal instillation of lipopolysaccharide (LPS). Pathological analysis of lung tissue, lung injury histological score, lung index, protein levels in bronchoalveolar lavage fluid (BALF), immunohistochemical staining, blood rheology, inflammatory cytokines, and oxidative stress levels were used to evaluate the pharmacological effects of DGYFD. Chemical components of DGYFD were identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Integrated network pharmacology together with transcriptomics was used to predict potential biological targets. Western blot analysis was used to verify the results. ResultsIn this study, we demonstrated that DGYFD could improve lung injury pathological changes, decreases lung index, down-regulate NO and IL-6 levels, and regulate blood rheology. In addition, DGYFD was able to reduce the protein levels in BALF, up-regulate the expression levels of occludin and ZO-1, improve the ultrastructure of lung tissues, and reverse the imbalance of AT I and AT II cells to repair the alveolar-capillary permeability barrier. Twenty-nine active ingredients of DGYFD and 389 potential targets were identified by UPLC-MS/MS and network pharmacology, and 64 differentially expressed genes (DEGs) were identified using transcriptomics. GO and KEGG analysis revealed that the MAPK pathway may be the molecular target. Further, we found that DGYFD inhibits phosphorylation levels of p38 MAPK and JNK in chronic lung injury mouse models. ConclusionsDGYFD could regulate the imbalance between the excessive release of inflammatory cytokines and oxidative stress, repair the alveolar-capillary permeability barrier and improve the pathological changes during chronic lung injury by regulating the MAPK signaling pathway.

Yinma Jiedu Granule attenuates LPS-induced acute lung injury in rats via suppressing inflammation level.

Yinma Jiedu Granule (YMJD) is a traditional Chinese patent medicine (CPM), which has been proved to have anti-inflammatory effects and therapeutical effects on obstructive pulmonary disease. The purpose of the current investigation is to find out if YMJD can alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS) in rats and its underlying mechanisms. Rats were treated with either vehicle or YMJD for 14 consecutive days, and 2h after the last administration, the rat model of ALI was induced by the intratracheal instillation of LPS. High performance liquid chromatography (HPLC) was applied for the fingerprint analysis of YMJD. The efficacy and molecular mechanisms were investigated. The results showed that treatment with YMJD improved the general state of rats, reduced weight loss and serum lactate (LA) levels, attenuated pulmonary edema and pathological damage of the lung tissue. Moreover, we found that YMJD effectively decreased the infiltration of white blood cells (WBC), lymphocytes (LYM), mononuclear cells (MON) and neutrophils (NEUT) in bronchoalveolar lavage fluid (BALF), reduced the concentration of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the lung tissue. Additionally, we found that YMJD could significantly increase the activity of superoxide dismutase (SOD) and reduce the malondialdehyde (MDA) level in the lung tissue. By employing RNA-sequencing, we have identified that JAK2/STAT1 is an important pathway that is involved in the lung protection of YMJD, and further Western blot assay verified that YMJD could effectively inhibit the activation of the JAK2/STAT1 pathway. YMJD could attenuate LPS-induced ALI through suppressing inflammation and oxidative stress in the lung tissue of rats, associating with the inhibition of JAK2/STAT1 activation. These findings provide evidence for the clinical use of YMJD for treatment of inflammatory pulmonary diseases like ALI.

OMIP-92: Characterization of rat macrophage subsets, lymphocytes, and granulocytes in bronchoalveolar lavage fluid.

Airway inflammation is a defense mechanism against inhaled agents characterized by infiltration of circulating immune cells. Given the inconsistent cellular identification across pre-clinical rat model, we have developed a flow cytometry panel of six colors to characterize macrophages subsets, lymphocytes and granulocytes in bronchoalveolar lavage fluid (BAL). Rats were challenged with intratracheal instillation of lipopolysaccharide (LPS). BAL were harvested 24 h after one LPS exposure in rats. This flow cytometry panel involve the description of macrophage subsets, T and B lymphocytes and neutrophils, which are central to airway immune responses, as based on scientific literature. By using a relatively small number of parameters to identify multiple cell types, additional parameters can be used for project/disease-specific activation markers.

Strain-dependent lung transcriptomic differences in cigarette smoke and LPS models of lung injury in mice.

Cigarette smoking increases the risk of acute respiratory distress syndrome (ARDS; Calfee CS, Matthay MA, Eisner MD, Benowitz N, Call M, Pittet J-F, Cohen MJ. Am J Respir Crit Care Med 183: 1660-1665, 2011; Calfee CS, Matthay MA, Kangelaris KN, Siew ED, Janz DR, Bernard GR, May AK, Jacob P, Havel C, Benowitz NL, Ware LB. Crit Care Med 43: 1790-1797, 2015; Toy P, Gajic O, Bacchetti P, Looney MR, Gropper MA, Hubmayr R, Lowell CA, Norris PJ, Murphy EL, Weiskopf RB, Wilson G, Koenigsberg M, Lee D, Schuller R, Wu P, Grimes B, Gandhi MJ, Winters JL, Mair D, Hirschler N, Sanchez Rosen R, Matthay MA, TRALI Study Group. Blood 119: 1757-1767, 2012) and causes emphysema. However, it is not known why some individuals develop disease, whereas others do not. We found that smoke-exposed AKR mice were more susceptible to lipopolysaccharides (LPS)-induced acute lung injury (ALI) than C57BL/6 mice (Sakhatskyy P, Wang Z, Borgas D, Lomas-Neira J, Chen Y, Ayala A, Rounds S, Lu Q. Am J Physiol Lung Cell Mol Physiol 312: L56-L67, 2017); thus, we investigated strain-dependent lung transcriptomic responses to cigarette smoke (CS). Eight-week-old male AKR and C57BL/6 mice were exposed to 3 wk of room air (RA) or cigarette smoke (CS) for 6 h/day, 4 days/wk, followed by intratracheal instillation of LPS or normal saline (NS) and microarray analysis of lung homogenate gene expression. Other groups of AKR and C57 mice were exposed to RA or CS for 6 wk, followed by evaluation of static lung compliance and tissue elastance, morphometric evaluation for emphysema, or microarray analysis of lung gene expression. Transcriptomic analyses of lung homogenates show distinct strain-dependent lung transcriptional responses to CS and LPS, with AKR mice having larger numbers of genes affected than similarly treated C57 mice, congruent with strain differences in physiologic and inflammatory parameters previously observed in LPS-induced ALI after CS priming. These results suggest that genetic differences may underlie differing susceptibility of smokers to ARDS and emphysema. Strain-based differences in gene transcription contribute to CS and LPS-induced lung injury. There may be a genetic basis for smoking-related lung injury. Clinicians should consider cigarette smoke exposure as a risk factor for ALI and ARDS.NEW & NOTEWORTHY We demonstrate that transcriptomes expressed in lung homogenates also differ between the mouse strains and after acute (3 wk) exposure of animals to cigarette smoke (CS) and/or to lipopolysaccharide. Mouse strains also differed in physiologic, pathologic, and transcriptomic, responses to more prolonged (6 wk) exposure to CS. These data support a genetic basis for enhanced susceptibility to acute and chronic lung injury among humans who smoke cigarettes.

Effect and mechanism of amphiregulin on acute respiratory distress syndrome in mice

To explore the protective effect of amphiregulin (Areg) on acute respiratory distress syndrome (ARDS) in mice and its underlying mechanism. (1) Male C57BL/6 mice aged 6-8 weeks were selected for animal experiments and divided into 3 groups (n = 10) according to the random number table method, which includes sham-operated group (Sham group), ARDS model group [ARDS model in mice was established by intratracheal instillation of lipopolysaccharide (LPS) 3 mg/kg] and ARDS+Areg intervention group [recombinant mice Areg (rmAreg) 5 μg was injected intraperitoneally 1 hour after LPS modeling]. The mice were sacrificed at 24 h after LPS injection lung histopathological changes were observed under hematoxylin-eosin (HE) staining and scored for lung injury; oxygenation index and wet/dry ratio of lung tissue were measured; the content of protein in bronchoalveolar lavage fluid (BALF) was detected by bicinchoninic acid (BCA) method, the level of inflammatory factors interleukins (IL-1β, IL-6) and tumor necrosis factor-α (TNF-α) in BALF were measured by enzyme-linked immunosorbent assay (ELISA). (2) Mice alveolar epithelial cell line MLE12 cells were obtained and cultured for experiment in vitro. Blank control group (Control group), LPS group (LPS 1 mg/L) and LPS+Areg group (rmAreg 50 μg/L was added 1 hour after LPS stimulation) were set. The cells and culture fluid were collected at 24 hours after LPS stimulation, and the apoptosis level of MLE12 cells was detected by flow cytometry; the activation level of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and the expressions of apoptosis-related proteins Bcl-2 and Bax in MLE12 cells were detected by Western blotting. (1) Animal experiments: compared with the Sham group, the lung tissue structure of ARDS model group was destroyed, the lung injury score was significantly increased, the oxygenation index was significantly decreased, the wet/dry weight ratio of lung was significantly increased, and the levels of protein and inflammatory factors in BALF were significantly increased. Compared with ARDS model group, lung tissue structure damage was reduced, pulmonary interstitial congestion, edema and inflammatory cell infiltration were significantly reduced, and lung injury score was significantly decreased (scores: 0.467±0.031 vs. 0.690±0.034) in ARDS+Areg intervention group. In addition, oxygenation index in ARDS+Areg intervention group was significantly increased [mmHg (1 mmHg ≈ 0.133 kPa): 380.00±22.36 vs. 154.00±20.74]. Lung wet/dry weight ratio (5.40±0.26 vs. 6.63±0.25), protein and inflammatory factors levels in BALF [protein (g/L): 0.42±0.04 vs. 0.86±0.05, IL-1β (ng/L): 30.00±2.00 vs. 40.00±3.65, IL-6 (ng/L): 190.00±20.30 vs. 581.30±45.76, TNF-α (ng/L): 30.00±3.65 vs. 77.00±4.16], and the differences were statistically significant (all P < 0.01). (2) Cell experiments: compared with the Control group, the number of apoptotic MLE12 cells was significantly increased in the LPS group, and the levels of PI3K phosphorylation, anti-apoptotic gene Bcl-2 level and pro-apoptotic gene Bax level were increased in MLE12 cells. Compared with the LPS group, the number of apoptosis in MLE12 cells was significantly reduced in the LPS+Areg group after administration of rmAreg treatment [(17.51±2.12)% vs. (36.35±2.84)%], and the levels of PI3K/AKT phosphorylation and Bcl-2 expression in MLE12 cells were significantly increased (p-PI3K/PI3K: 2.400±0.200 vs. 0.550±0.066, p-AKT/AKT: 1.647±0.103 vs. 0.573±0.101, Bcl-2/GAPDH: 0.773±0.061 vs. 0.343±0.071), and Bax expression was significantly suppressed (Bax/GAPDH: 0.810±0.095 vs. 2.400±0.200). The differences were statistically significant (all P < 0.01). Areg could alleviate ARDS in mice by inhibiting the apoptosis of alveolar epithelial cells through activating PI3K/AKT pathway.

Polymer Lung Surfactants Attenuate Direct Lung Injury in Mice.

If not properly managed, acute lung injuries, either through direct or indirect causes, have the potential to present serious risk for many patients worldwide. One of the mechanisms for the transition from acute lung injury (ALI) to the more serious acute respiratory distress syndrome (ARDS) is the deactivation of the native lung surfactant by injury-induced infiltrates to the alveolar space. Currently, there are no surfactant replacement therapies that are used to treat ALI and subsequent ARDS. In this paper, we present an indepth efficacy study of using a novel polymer lung surfactant (PLS, composed of poly(styrene-block-ethylene glycol) (PS-PEG) block copolymer micelles), which has unique properties compared to other tested surfactant replacements, in two different mouse models of lung injury. The results demonstrate that pharyngeal administration of PLS after the instillation of either acid (HCl) or lipopolysaccharide (LPS) can decrease the severity of lung injury as measured by multiple injury markers.

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

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

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

Effect of allyl isothiocyanate on oxidative stress in COPD via the AhR / CYP1A1 and Nrf2 / NQO1 pathways and the underlying mechanism

BackgroundChronic obstructive pulmonary disease (COPD) is currently the third leading cause of death globally. Oxidative stress affects various molecular mechanisms and is the main driving factor of COPD. Ally isothiocyanate (AITC) is an effective component of Semen Sinapis Albae, which has favorable effects for the treatment of COPD, but its mechanism has not been fully elucidated. PurposeThis study aimed to elucidate the antioxidant effect of AITC on COPD and its molecular mechanism, and preliminarily determine the role of AhR in the progression of COPD. Study DesignThe COPD rat model was established by smoking combined with intratracheal instillation of lipopolysaccharide. Different doses of AITC, positive control drug acetylcysteine, AhR inhibitor alpha-naphthoflavone, and agonist beta-naphthoflavone were administered by gavage. Human bronchial epithelial cells induced by cigarette smoke extract (CSE) were used in an in vitro model to explore the molecular mechanisms of AITC. MethodsThe effects of AITC on lung function and oxidative stress in rats were evaluated in vivo using the respiratory function test, white blood cell count, enzyme-linked immunosorbent assay, and histological staining. The changes in protein expression in the lung tissue were detected by immunohistochemistry and Western blotting. RT-PCR, western blotting, and immunofluorescence were used to explore the molecular mechanisms of AITC. Enzyme-linked immunosorbent assay, reactive oxygen species probing, and flow cytometry were used to determine the antioxidant effect of AITC. ResultsAITC can improve the lung function of rats with COPD, restore lung tissue structure, improve oxidative stress, reduce inflammation, and inhibit lung cell apoptosis. AITC reversed the upregulation of AhR and CYP1A1 and the down-regulation of Nrf2 and NQO1 in the lung tissues of rats with COPD. CSE stimulation can increase the expressions of AhR and CYP1A1 and decrease the expressions of Nrf2 and NQO1 in 16HBE cells, leading to severe oxidative stress and inflammatory response and, ultimately, apoptosis. AITC inhibited AhR and CYP1A1 expressions, induced Nrf2 and NQO1 expressions, promoted Nrf2 nuclear translocation, and improved CSE-induced toxicological effects. ConclusionAITC may improve lung oxidative stress by inhibiting the AhR / CYP1A1 and activating the Nrf2 / NQO1 pathways, thereby delaying the pathological progression of COPD.

Inhibition of fatty acid synthase protects obese mice from acute lung injury via ameliorating lung endothelial dysfunction

BackgroundObesity has been identified as a risk factor for acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the underlying mechanisms remain elusive. This study aimed to investigate the role of fatty acid synthase (FASN) in lipopolysaccharide (LPS)-induced ALI under obesity.MethodsA high-fat diet-induced obese (DIO) mouse model was established and lean mice fed with regular chow diet were served as controls. LPS was intratracheally instilled to reproduce ALI in mice. In vitro, primary mouse lung endothelial cells (MLECs), treated by palmitic acid (PA) or co-cultured with 3T3-L1 adipocytes, were exposed to LPS. Chemical inhibitor C75 or shRNA targeting FASN was used for in vivo and in vitro loss-of-function studies for FASN.ResultsAfter LPS instillation, the protein levels of FASN in freshly isolated lung endothelial cells from DIO mice were significantly higher than those from lean mice. MLECs undergoing metabolic stress exhibited increased levels of FASN, decreased levels of VE-cadherin with increased p38 MAPK phosphorylation and NLRP3 expression, mitochondrial dysfunction, and impaired endothelial barrier compared with the control MLECs when exposed to LPS. However, these effects were attenuated by FASN inhibition with C75 or corresponding shRNA. In vivo, LPS-induced ALI, C75 pretreatment remarkably alleviated LPS-induced overproduction of lung inflammatory cytokines TNF-α, IL-6, and IL-1β, and lung vascular hyperpermeability in DIO mice as evidenced by increased VE-cadherin expression in lung endothelial cells and decreased lung vascular leakage.ConclusionsTaken together, FASN inhibition alleviated the exacerbation of LPS-induced lung injury under obesity via rescuing lung endothelial dysfunction. Therefore, targeting FASN may be a potential therapeutic target for ameliorating LPS-induced ALI in obese individuals.

Jinzhen Oral Liquid alleviates lipopolysaccharide-induced acute lung injury through modulating TLR4/MyD88/NF-κB pathway

BackgroundAcute lung injury (ALI) has the attribution of excessive inflammation of the lung. Jinzhen oral liquid (JO), a famous Chinese recipe used to treat ALI, has a favorable therapeutic effect on ALI. However, its anti-inflammatory mechanism has not been extensively studied. PurposeThis study was to elucidate the effects of JO on lipopolysaccharide (LPS)-induced ALI and its molecular mechanism. MethodsAn ALI model was established by intratracheal instillation of LPS (2 mg/50 μl). The open field experiment was carried out to explore the spontaneous movement and exploratory behavior of ALI mice. Cytokines levels concentrations (IL-6, IL-10 and TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Network pharmacology was used to predict the mechanism of JO against ALI. Immunofluorescence, co-immunoprecipitation, fluorescence resonance energy transfer (FRET), Western blot and RT-PCR were used to verify the molecular mechanisms of JO. ResultsThe in vivo results suggested that JO (1, 2, 4 g/kg) dose-dependently improved the exercise performance of mice and reduced the lung W/D weight ratio as well as the production of IL-6 and TNF-α, but increased the release of IL-10 in the ALI group. The network pharmacological analysis demonstrated that the Toll-like receptor (TLR) pathway might be the fundamental action mechanisms of JO against ALI. Immunofluorescence staining and co-immunoprecipitation analysis showed that JO decreased the expression levels of TLR4 and MyD88 and reduced their interaction in the lung tissue of ALI mice. Meanwhile, JO decreased nuclear translocation and phosphorylation of NF-κB P65. The results from cellular experiments were in line with those in vivo. The FRET experiment also confirmed that JO disturbed the interaction of TLR4 and MyD88. Subsequently, we also found that the six indicative components of JO have the similar therapeutic effect as JO. ConclusionsIn summary, we suggested that JO suppressed the TLR4/MyD88/NF-κB signaling pathway, thus inhibiting LPS-induced ALI in vitro and in vivo. The clarified mechanism provided an important theoretical basis and a novel treatment strategy for the ALI treatment of JO.

Pulmonary rehabilitation restores limb muscle mitochondria and improves the intramuscular metabolic profile.

Exercise, as the cornerstone of pulmonary rehabilitation, is recommended to chronic obstructive pulmonary disease (COPD) patients. The underlying molecular basis and metabolic process were not fully elucidated. Sprague-Dawley rats were classified into five groups: non-COPD/rest (n = 8), non-COPD/exercise (n = 7), COPD/rest (n = 7), COPD/medium exercise (n = 10), and COPD/intensive exercise (n = 10). COPD animals were exposed to cigarette smoke and lipopolysaccharide instillation for 90 days, while the non-COPD control animals were exposed to room air. Non-COPD/exercise and COPD/medium exercise animals were trained on a treadmill at a decline of 5° and a speed of 15 m/min while animals in the COPD/intensive exercise group were trained at a decline of 5° and a speed of 18 m/min. After eight weeks of exercise/rest, we used ultrasonography, immunohistochemistry, transmission electron microscopy, oxidative capacity of mitochondria, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI), and transcriptomics analyses to assess rectal femoris (RF). At the end of 90 days, COPD rats' weight gain was smaller than control by 59.48 ± 15.33 g (P = 0.0005). The oxidative muscle fibers proportion was lower (P < 0.0001). At the end of additional eight weeks of exercise/rest, compared to COPD/rest, COPD/medium exercise group showed advantages in weight gain, femoral artery peak flow velocity (Δ58.22 mm/s, 95% CI: 13.85-102.60 mm/s, P = 0.0104), RF diameters (Δ0.16 mm, 95% CI: 0.04-0.28 mm, P = 0.0093), myofibrils diameter (Δ0.06 μm, 95% CI: 0.02-0.10 μm, P = 0.006), oxidative muscle fiber percentage (Δ4.84%, 95% CI: 0.15-9.53%, P = 0.0434), mitochondria oxidative phosphorylate capacity (P < 0.0001). Biomolecules spatial distribution in situ and bioinformatic analyses of transcriptomics suggested COPD-related alteration in metabolites and gene expression, which can be impacted by exercise. COPD rat model had multi-level structure and function impairment, which can be mitigated by exercise.

Combination of Merocel sponge with Lipopolysaccharide to establish rat rhinosinusitis model

ObjectiveThe study aimed to investigate the feasibility of establishing rhinosinusitis model in rats combinated with Lipopolysaccharide (LPS) and merocel sponge. MethodsSD (Sprague Dawley) rats that underwent nasal obstruction using Merocel sponge packing, rats with LPS instillation alone, and rats with both nasal obstruction and LPS instillation were used to establish rat models of rhinosinusitis. After the models were established, the nasal symptoms of rats were recorded, the histopathological examination and Transmission Electron Microscopy (TME) of the sinus tissue were performed and the levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6) in the blood were also analyzed. The expressions of Aquaporin-5 (AQP5), Occludin, Toll-Like Receptor-4 (TLR4), Medullary differentiation factor 88 (MyD88) and phosphorylated (p)-p65 protein were detected by Western blot to evaluate the effect and mechanism of the experimental models. ResultsWe found that compared with the control group and LPS group, the sinusitis symptom scores in the Merocel sponge combined with LPS group were significantly increased; the respiratory epithelia of the maxillary sinus were degenerated, cilia were detached, and even inflammatory cell infiltration occurred; the levels of TNF-α and IL-6 were increased; the expression of AQP5 and Occludin protein was decreased; and the expressions of TLR4, MyD88, and p-p65 protein were increased. ConclusionFor the first time, we successfully established a rat rhinosinusitis model using Merocel sponge with LPS and explored the possible mechanism of LPS action.

Retinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice

Retinoids are potent transcriptional regulators that act in regulating cell proliferation, differentiation, and other cellular processes. We carried out studies in male mice to establish the importance of local cellular retinoid stores within the lung alveolus for maintaining its health in the face of an acute inflammatory challenge induced by intranasal instillation of lipopolysaccharide. We also undertook single cell RNA sequencing and bioinformatic analyses to identify roles for different alveolar cell populations involved in mediating these retinoid-dependent responses. Here we show that local retinoid stores and uncompromised metabolism and signaling within the lung are required to lessen the severity of an acute inflammatory challenge. Unexpectedly, our data also establish that alveolar cells other than lipofibroblasts, specifically microvascular endothelial and alveolar epithelial cells, are able to take up lipoprotein-transported retinoid and to accumulate cellular retinoid stores that are directly used to respond to an acute inflammatory challenge.

Sichen Formula Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Blocking the TLR4 Signaling Pathways.

Sichen (SC) formula is a classic prescription of Tibetan medicine. Due to its potential anti-inflammatory effect, the SC formula has been clinically used to treat respiratory diseases for many years in the Chinese Tibet region. The present study aimed to investigate the anti-inflammatory effect of SC and explore the underlying mechanisms. SC formula was characterized by HPLC analysis. The acute lung injury (ALI) mouse model was induced by direct intratracheal lipopolysaccharide (LPS) instillation, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Meanwhile, RAW264.7 macrophages were stimulated by LPS. The contents of inflammatory mediators in the culture medium were determined by ELISA. Protein levels were determined by immunohistochemical staining or Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was performed using immunofluorescence and Western blotting. In the LPS-induced ALI mouse model, SC treatment suppressed the secretion of inflammatory mediators (TNF-α, IL-6, IL-1β, MCP-1, MIP-1α, and RANTES) in BALF. SC treatment hindered the recruitment of macrophages. SC treatment also inhibited the expression of CD68, p-p65, and TLR4 in the lung tissue. In the LPS-exposed RAW264.7 cells, the cell viability was not changed up to 400 μg/mL of SC. SC concentration-dependently suppressed the production of nitric oxide, prostaglandin E2, TNF-α, IL-6, MCP-1, MIP-1α, and RANTES in LPS-challenged RAW264.7 cells. The expression levels of iNOS, COX-2, p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/β, p-IκB, p-p65, p-c-Jun, and p-IRF3 were decreased after SC treatment. Moreover, the nuclear translocation of p65, c-Jun, and IRF3 was also blocked by SC treatment. SC treatment inhibited the inflammatory responses in LPS-induced ALI mouse model/RAW264.7 macrophages. The underlying mechanism of this action may be closely associated with the suppression of TLR4 signaling pathways. These research findings provide further pharmacological justifications for the medicinal use of SC in the management of respiratory diseases.

The main e-cigarette component vegetable glycerin enhances neutrophil migration and fibrosis in endotoxin-induced lung injury via p38 MAPK activation

We investigated the effects of vegetable glycerin (VG), a main e-cigarette constituent, on endotoxin-induced acute lung injury (ALI). Mice received intratracheal administration of 30% VG in phosphate buffered saline (PBS) vehicle or only PBS (control) for 4 days. On Day 5, mice received an intratracheal instillation of lipopolysaccharide (LPS) (LPS group and VG + LPS group) or PBS (VG group and control group). Lung histopathology, expression of chemokine receptors, and regulatory signaling were analyzed 24 h after the Day 5 treatment. VG significantly increased ALI-associated histopathological and fibrotic changes in both the VG group and LPS-induced ALI mice (VG + LPS group). Immunohistochemistry (IHC) and western blot analyses revealed that VG administration resulted in upregulation of neutrophil markers [lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO)] as well as upregulation of the expression of transforming growth factor-β (TGF-β), a central mediator of fibrogenesis, in the lungs of both VG and VG + LPS groups. VG enhanced the expression of adhesion molecules [very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1)] and increased activation of p38 mitogen-activated protein kinase (p38 MAPK) to prompt neutrophil recruitment in the lungs of mice with ALI. Intraperitoneal administration of a p38 inhibitor attenuated these histopathological changes significantly as well as VG-induced upregulation in expression of Ly6G, MPO, VLA-4, VCAM-1, TGF-β, and collagen-1 in mice with ALI. In conclusion, VG enhances neutrophil chemotaxis and fibrosis and it amplifies the inflammatory response associated with LPS-induced ALI in the lungs via enhancement of p38 MAPK activity.

α1-adrenoceptor stimulation ameliorates lipopolysaccharide-induced lung injury by inhibiting alveolar macrophage inflammatory responses through NF-κB and ERK1/2 pathway in ARDS.

Catecholamines such as norepinephrine or epinephrine have been reported to participate in the development of acute respiratory distress syndrome (ARDS) by activating adrenergic receptors (ARs). But the role of α1-AR in this process has yet to be elucidated. In this study, ARDS mouse model was induced by intratracheal instillation of lipopolysaccharide. After treatment with α1-AR agonist phenylephrine or antagonist prazosin, lung pathological injury, alveolar barrier disruption and inflammation, and haemodynamic changes were evaluated. Cytokine levels and cell viability of alveolar macrophages were measured in vitro. Nuclear factor κB (NF-κB), mitogen-activated protein kinase, and Akt signalling pathways were analysed by western blot. It showed that α1-AR activation alleviated lung injuries, including reduced histopathological damage, cytokine expression, and inflammatory cell infiltration, and improved alveolar capillary barrier integrity of ARDS mice without influencing cardiovascular haemodynamics. In vitro experiments suggested that α1-AR stimulation inhibited secretion of TNF-α, IL-6, CXCL2/MIP-2, and promoted IL-10 secretion, but did not affect cell viability. Moreover, α1-AR stimulation inhibited NF-κB and enhanced ERK1/2 activation without significantly influencing p38, JNK, or Akt activation. Our studies reveal that α1-AR stimulation could ameliorate lipopolysaccharide-induced lung injury by inhibiting NF-κB and promoting ERK1/2 to suppress excessiveinflammatoryresponses of alveolar macrophages.

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

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

Knockout of S1PR3 attenuates acute lung injury in mice by inhibiting the MAPK pathway

To investigate whether knockout of S1PR3 improves lipopolysaccharide (LPS)-induced acute lung injury in mice by inhibiting mitogen activated protein kinases (MAPKs). Male C57BL/6J and S1PR3 knockout (S1PR3-/-) mice were both randomized into two groups (n=8) for intratracheal instillation of normal saline or LPS to induce acute lung injury. The expressions of S1PR3, IL-1β and IL-18 in the lung tissues were detected using RT-qPCR, lung tissue injury was observed with HE staining, and cell apoptosis was detected using flow cytometry. Western blotting was performed to detect the expression levels of caspase-1, GSDMD, p-JNK, p-ERK and p-p38 proteins. In the cell experiment, type II alveolar epithelial cells (MLE-12 cells) were treated with PBS, LPS, CYM5541 (a S1PR3 agonist), or CYM5541 + LPS, and the cell apoptosis and expression levels of MAPK signal pathway molecules were detected. The expression of S1PR3 was up-regulated and serum IL-1β and IL-18 levels were elevated significantly in the nontransgenic mice with acute lung injury (P < 0.001). By comparison, the elevation of IL-1β and IL-18 levels was obviously reduced in S1PR3 knockout mice with acute lung injury, which also showed significant improvement of pulmonary hemorrhage, inflammation and exudation, lowered wet-to-dry ratio of the lungs, and decreased cell apoptosis and expressions of cleaved caspase-1 and GSDMD (P < 0.05). In MLE-12 cells, treatment with the S1PR3 agonist significantly increased the expression of pyroptosis-associated proteins (P < 0.05). S1PR3 knockout strongly inhibited the activation of MAPKs family (JNK and ERK p38; P < 0.05), but their expressions were significantly increased following treatment with the S1PR3 agonist (P < 0.05). Inhibition of S1PR3 can improve LPSinduced acute lung injury in mice by inhibiting the activation of MAPK signaling.