Instillation Of Lipopolysaccharide Research Articles

17,18-Epoxyeicosatetraenoic Acid Inhibits TNF-α-Induced Inflammation in Cultured Human Airway Epithelium and LPS-Induced Murine Airway Inflammation.

17,18-Epoxyeicosatetraenoic acid (17,18-EpETE), an eicosapentaenoic acid metabolite, is generated from dietary oil in the gut, and antiinflammatory activity of 17,18-EpETE was recently reported. To evaluate the inhibitory effects of 17,18-EpETE in airway inflammation, we examined in vitro and in vivo effects on mucus production, neutrophil infiltration, and cytokine/chemokine production in airway epithelium. Nasal tissue localization of G protein-coupled receptor 40 (GPR40), a receptor of 17,18-EpETE, was determined by immunohistochemical staining. Expression of GPR40 mRNA in nasal mucosa of chronic rhinosinusitis (CRS) patients and control subjects was determined by reverse transcription-polymerase chain reaction (RT-PCR). The in vitro effects on airway epithelial cells were examined using normal human bronchial epithelial cells and NCI-H292 cells. To examine the in vivo effects of 17,18-EpETE on airway inflammation, we induced goblet cell metaplasia, mucus production, and neutrophil infiltration in mouse nasal epithelium by intranasal lipopolysaccharide (LPS) instillation. GPR40 is mainly expressed in human nasal epithelial cells and submucosal gland cells. RT-PCR analysis revealed that the expression of GPR40 mRNA was increased in nasal tissues from CRS patients compared with those from control subjects. 17,18-EpETE significantly inhibited tumor necrosis factor (TNF)-α-induced production of interleukin (IL)-6 , IL-8, and mucin from cultured human airway epithelial cells dose dependently, and these antiinflammatory effects on cytokine production were abolished by GW1100, a selective GPR40 antagonist. Intraperitoneal injection or intranasal instillation of 17,18-EpETE significantly attenuated LPS-induced mucus production and neutrophil infiltration in mouse nasal epithelium. Inflammatory cytokine/chemokine production in lung tissues and bronchoalveolar lavage fluids was also inhibited. These results indicate that 17,18-EpETE plays a regulatory role in mucus hypersecretion and neutrophil infiltration in nasal inflammation. Local or systemic administration may provide a new therapeutic approach for the treatment of intractable airway disease such as CRS.

Exploration of the Function of Ginsenoside RD Attenuates Lipopolysaccharide-Induced Lung Injury: A Study of Network Pharmacology and Experimental Validation.

Ginsenoside Rd (GSRd) displays a variety of pharmacological effects. However, the underlying role in acute lung injury (ALI) is not clear. In this study, the protective effect of GSRd on lipopolysaccharide (LPS)-induced ALI is investigated to explore the potential mechanisms. GSRd-target-ALI-related gene set was constructed. And bioinformatics tools were used to discover the potential mechanism. We observed the survival of subjects for 72 h. In addition, male BALB/c mice were intraperitoneal injected with GSRd (25 and 50 mg/kg) after received one intratracheal instillation of LPS. Inflammatory changes, oxidative stress, and phosphorylation were assessed to study the biological effects. A total of 245 interaction genes were collected. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were enriched in immune-inflammatory system. Among them, PI3K-Akt signaling pathway was the highest-ranked pathway of inflammatory response. In vivo study, it was found that GSRd improved survival in endotoxemic mice and inhibited the major characteristic of ALI. And the p-PI3K and p-Akt expression was significantly decreased by GSRd treatment. GSRd could protect mice against LPS-induced ALI effectively by inhibiting the PI3K-Akt signaling pathway.

Efficacy of intratracheal budesonide-surfactant combined therapy in surfactant-insufficient rat lungs with lipopolysaccharide insult.

Intratracheal steroid therapy for lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains challenging particularly in surfactant-insufficient lungs, a common problem of neonatal or pediatric ALI. Surfactant has been used as a vehicle for intratracheal steroid in the treatment of other types of ALI. This study investigated the efficacy of intratracheal budesonide (BUD) delivered by two concentrations of surfactant in the treatment of LPS-induced ALI in surfactant-insufficient rat lungs. Male adult rats were anesthetized and ventilated. Our ALI model was established by repeated saline lavage to produce surfactant insufficiency, followed by intratracheal LPS instillation. Five study groups (n = 5 for each) with different intratracheal treatments following ALI were used: control (no treatment), BUD (NS-BUD; BUD in saline), DS-BUD (BUD in diluted surfactant), FS-BUD (BUD in full-strength surfactant), FS (full-strength surfactant). Cardiopulmonary variables were monitored 4 hours post injury. Histological and immunohistochemical assessments of the lungs were performed. The FS-BUD and FS groups presented better gas exchange, less metabolic acidosis, less oxygen index, and more stable hemodynamic changes than the DS-BUD, NS-BUD, and control groups. The total lung injury scores assessed by histological examination were ordered as follows: FS-BUD < DS-BUD or FS < NS-BUD < control. The immunostaining intensities of lung myeloperoxidase showed the following order: NS-BUD, DS-BUD, or FS-BUD < control or FS. Only the FS-BUD group displayed a smaller immunostaining intensity of lung tumor necrosis factor (TNF)-α than the control group. Among our therapeutic strategies, intratracheal BUD delivered by full-strength surfactant confers an optimal protection against LPS-induced ALI in surfactant-insufficient rat lungs.

Effects of Ultrashort Wave Therapy on Inflammation and Macrophage Polarization after Acute Lung Injury in Rats.

Acute lung injury (ALI) features dysregulated pulmonary inflammation. Ultrashort waves (USWs) exert anti-inflammatory effectsbut no studies have evaluated their activity in ALI. Herein, we used an in vivo lipopolysaccharide (LPS)-induced ALI model to investigate whether the anti-inflammatory activity of USWs is mediated by altering the polarization of M1 to M2 macrophages. Twenty-four male Sprague-Dawley rats were randomly divided into control, untreated ALI, and ALI treated with USW groups (n = 8 in each group). ALI was induced by intratracheal LPS instillation. Rats in the USW group were treated for 15 min at 0, 4, and 8 h after a single LPS intratracheal instillation. Histopathologic examination, wet/dry lung weight ratio, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction, and western blot analyses were performed to evaluate the degree of lung injury and to determine macrophage phenotypes. Histopathologic examination disclosed attenuation of ALI, with reduced alveolar hemorrhage and neutrophilic infiltration in the USW group. Serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly decreased after USW therapy. Moreover, the messenger RNA (mRNA) expressions of TNF-α and IL-1β were significantly decreased in the USW group, whereas the mRNA expression of Arginase 1 (Arg1) and the protein expression of mannose receptor significantly increased in comparison with the untreated ALI group. We conclude that USW therapy may attenuate inflammation in LPS-induced ALI through the modulation of macrophage polarization. © 2021 Bioelectromagnetics Society.

Mesenchymal stromal (stem) cell therapy modulates miR-193b-5p expression to attenuate sepsis-induced acute lung injury.

Although mesenchymal stromal (stem) cell (MSC) administration attenuates sepsis-induced lung injury in pre-clinical models, the mechanism(s) of action and host immune system contributions to its therapeutic effects remain elusive. We show that treatment with MSCs decreased expression of host-derived microRNA (miR)-193b-5p and increased expression of its target gene, the tight junctional protein occludin (Ocln), in lungs from septic mice. Mutating the Ocln 3' untranslated region miR-193b-5p binding sequence impaired binding to Ocln mRNA. Inhibition of miR-193b-5p in human primary pulmonary microvascular endothelial cells prevents tumour necrosis factor (TNF)-induced decrease in Ocln gene and protein expression and loss of barrier function. MSC-conditioned media mitigated TNF-induced miR-193b-5p upregulation and Ocln downregulation in vitro When administered in vivo, MSC-conditioned media recapitulated the effects of MSC administration on pulmonary miR-193b-5p and Ocln expression. MiR-193b-deficient mice were resistant to pulmonary inflammation and injury induced by lipopolysaccharide (LPS) instillation. Silencing of Ocln in miR-193b-deficient mice partially recovered the susceptibility to LPS-induced lung injury. In vivo inhibition of miR-193b-5p protected mice from endotoxin-induced lung injury. Finally, the clinical significance of these results was supported by the finding of increased miR-193b-5p expression levels in lung autopsy samples from acute respiratory distress syndrome patients who died with diffuse alveolar damage.

Effect of houttuynia on improving lung injury in chronic obstructive pulmonary disease by regulating the TLR4 signaling pathway.

This study aimed to investigate the effects and mechanisms of houttuynia on lung pathological injury in rats with chronic obstructive pulmonary disease (COPD). Rats were randomly divided into the normal control, COPD model (model), low‐dose treatment (low, 5 mg/kg), middle‐dose treatment (middle, 10 mg/kg), and high‐dose treatment (high, 25 mg/kg) groups. The COPD rat model was induced by smoking combined with intratracheal instillation of lipopolysaccharide. The treatment groups were given Houttuynia by gavage at 30 min before smoking. The IL‐6, IL‐1β, and TNF‐α concentrations in serum and BALF were determined by ELISA. The pathological morphology was detected by HE staining. The apoptosis cell number was evaluated by TUNEL assay. Apoptotic proteins (caspase‐3 and caspase‐9) were measured by IHC assay in lung tissues. The relative proteins [TLR4, MyD88, and p‐NF‐κB(p65)] were evaluated by Western blot assay in lung tissues. Compared with the model group, the low, middle, and high groups could reduce pulmonary congestion, edema, inflammatory cell infiltration, and apoptosis cell number; downregulate the protein expression of caspase‐3, caspase‐9, TLR4, MyD88, and NF‐κB(p65) (p < .05); and inhibit the IL‐6, IL‐1β, and TNF‐α concentrations in serum and BALF. Houttuynia could improve the morphology and apoptosis cell number in lung tissues, thereby inhibiting the activation of the TLR4/MyD88/NF‐κB(p65) signaling pathway.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

The Cholinergic Drug Pyridostigmine Alleviates Inflammation During LPS-Induced Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a critical illness complication that is associated with high mortality. ARDS is documented in severe cases of COVID-19. No effective pharmacological treatments for ARDS are currently available. Dysfunctional immune responses and pulmonary and systemic inflammation are characteristic features of ARDS pathogenesis. Recent advances in our understanding of the regulation of inflammation point to an important role of the vagus-nerve-mediated inflammatory reflex and neural cholinergic signaling. We examined whether pharmacological cholinergic activation using a clinically approved (for myasthenia gravis) cholinergic drug, the acetylcholinesterase inhibitor pyridostigmine alters pulmonary and systemic inflammation in mice with lipopolysaccharide (LPS)-induced ARDS. Male C57Bl/6 mice received one intratracheal instillation of LPS or were sham manipulated (control). Both groups were treated with either vehicle or pyridostigmine (1.5 mg/kg twice daily, 3 mg/day) administered by oral gavage starting at 1 h post-LPS and euthanized 24 h after LPS administration. Other groups were either sham manipulated or received LPS for 3 days and were treated with vehicle or pyridostigmine and euthanized at 72 h. Pyridostigmine treatment reduced the increased total number of cells and neutrophils in the bronchoalveolar lavage fluid (BALF) in mice with ARDS at 24 and 72 h. Pyridostigmine also reduced the number of macrophages and lymphocytes at 72 h. In addition, pyridostigmine suppressed the levels of TNF, IL-1β, IL-6, and IFN-γ in BALF and plasma at 24 and 72 h. However, this cholinergic agent did not significantly altered BALF and plasma levels of the anti-inflammatory cytokine IL-10. Neither LPS nor pyridostigmine affected BALF IFN-γ and IL-10 levels at 24 h post-LPS. In conclusion, treatments with the cholinergic agent pyridostigmine ameliorate pulmonary and systemic inflammatory responses in mice with endotoxin-induced ARDS. Considering that pyridostigmine is a clinically approved drug, these findings are of substantial interest for implementing pyridostigmine in therapeutic strategies for ARDS.

Inhibiting Connexin43/Zonula Occludens‐1 Interactions Improves Alcoholic Lung Barrier Function in vivo

Chronic alcohol consumption is well known to contribute to an array of health complications and increases the incidence and severity of acute respiratory distress syndrome (ARDS), an acute lung injury marked by severe pulmonary edema and impairment of normal gas exchange. Despite advancements in understanding its pathogenesis, the mortality rate for ARDS remains over 40% and there are currently no effective pharmacological treatments for ARDS. Chronic alcohol consumption contributes to the pathogenesis of ARDS by altering the tight junction composition of the pulmonary epithelium which is essential to maintain normal barrier function. In this study, we investigate the use of a pharmacological peptide, aCT1, which mimics the cytoplasmic carboxy-terminal PDZ binding motif of the gap junction protein Connexin43 (Cx43) to disrupt endogenous Cx43 from binding scaffold proteins, including the tight junction protein Zonula Occludens-1 (ZO-1). By inhibiting ZO-1 binding to Cx43, we hypothesize that more ZO-1 is available to associate with tight junction proteins, including claudins, to enhance epithelial barrier function. We have previously shown that chronic alcohol use exacerbates acute endotoxin-mediated lung injury by compromising both the alveolar and pulmonary capillary epithelial barriers. Here, we tested whether treatment with aCT1 strengthens alveolar barrier function and mitigates the effects of alcohol-mediated impairment of tight junctions. Male C57BL/6J mice were pair-fed 20% ethanol or water for 14 weeks to establish a chronic alcohol model in vivo. Mice were sedated and given an intratracheal instillation of 5mg/kg of the bacterial endotoxin lipopolysaccharide (LPS) derived from E. coli O55:B5 to induce a second, direct insult in the alcohol-primed lung to impact the lung epithelium. Mice were administered via nebulizer 1h post-LPS instillation either 5mg/kg of the aCT1 peptide or a scrambled control. Evans Blue (EB) dye was injected into mouse tail veins 24h after LPS instillation. To measure alveolar flooding, bronchoalveolar lavage fluid (BAL) was collected 1h later and EB in BAL was measured and normalized to serum EB. We found that administration of aCT1 decreased the leakage of EB dye from the blood into the alveolar air space in both water-fed and alcohol-fed mice, thus improving lung barrier function. This improvement in pulmonary leak suggests that aCT1 has the capacity to mitigate ARDS. Further investigation will determine whether aCT1 has a positive impact on short-term mortality rates due to endotoxemia and whether it can improve pulmonary barrier dysfunction due to other causes of acute lung injury.

Impaired airway epithelial barrier integrity was mediated by PI3Kδ in a mouse model of lipopolysaccharide-induced acute lung injury

Cell-cell junctions are critical for the maintenance of cellular as well as tissue polarity and integrity. Dysfunction of airway epithelial barrier has been shown to be involved in the pathogenesis of acute lung injury (ALI). Yet the role of phosphatidylinositol 3-kinase delta (PI3Kδ) in dysregulation of airway epithelial barrier integrity in ALI has not been addressed. Mice were subjected to intratracheal instillation of lipopolysaccharide (LPS) to generate a ALI model. Two pharmacological inhibitors of PI3Kδ, IC87114 and AMG319, were respectively given to the mice. Expression of p110δ and its downstream substrate phospho-AKT (Ser473) was increased in LPS-exposed lungs. These increases were inhibited by IC87114 or AMG319. LPS led to pronounced lung injury that was accompanied by significant airway neutrophil recruitment and bronchial epithelial morphological alterations 72 h after exposure. We also found compromised expression of adherens junction protein E-cadherin and tight junction protein claudin-2 in the airway epithelial cells. Treatment with either IC87114 or AMG319 not only attenuated LPS-induced edema, lung injury and neutrophilc inflammation, reduced total protein concentration and IL-6, TNF-α secretion in BALF, but also restored epithelial E-cadherin and claudin-2 expression. In summary, our results showed that LPS can induce a delayed effect on airway epithelial barrier integrity that is mediated by PI3Kδ in a mouse model of ALI.

Distinct phenotypic expression levels of macrophages in neonatal lungs.

Alveolar macrophages are the front-line defense against environmental pathogens. However, to the best of our knowledge, differences in function and phenotypic expression levels of macrophages between neonatal and adult lungs have not previously been determined. The present study investigated lung tissues and analyzed blood samples to find cell markers of M1 and M2 macrophages in neonatal and adult rats. Pulmonary sepsis was induced by intrapleural instillation of lipopolysaccharide (LPS; 20 mg/kg) and survival time after administration of LPS was measured. In certain neonates, a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400w, was administered prior to induction of pulmonary sepsis. Compared with adults, fetal and neonatal lung tissues had significantly higher levels of iNOS and CD86 (M1 markers), whereas the expression levels of CD206 and arginase-1 (M2 markers) were lower in the neonatal lung. The circulating cells that co-expressed CD68 (monocytes and macrophages) and CD86 in the blood were also significantly higher in neonates than in adults (25.9±6.6 vs. 11.6±2.2%; P=0.007. At basal unstimulated conditions, lung tissue concentrations of nitrite and nitrate (NOx) were significantly lower in the neonates than in adults (112.1±55.9 vs. 340.9±124.9 µM/g; P<0.001). However, NOx was increased following administration of LPS. Administration of 1400w suppressed lung tissue levels of NOx and improved the survival time in neonatal rats treated with LPS. The present study demonstrated that M1 is the primary macrophage phenotype in the neonatal lung and that higher iNOS expression levels do not have a protective effect against pulmonary endotoxins in neonates. Overproduction of NO by iNOS in neonatal alveolar macrophages may result in detrimental effects during pulmonary inflammation.

Cell Wall Contents of Probiotics (Lactobacillus species) Protect Against Lipopolysaccharide (LPS)-Induced Murine Colitis by Limiting Immuno-inflammation and Oxidative Stress.

Currently, there are no effective therapeutic agents to limit intestinal mucosal damage associated with inflammatory bowel disease (IBD). Based on several clinical studies, probiotics have emerged as a possible novel therapeutic strategy for IBD; however, their possible mechanisms are still poorly understood. Although probiotics in murine and human improve disease severity, very little is known about the specific contribution of cell wall contents of probiotics in IBD. Herein, we investigated the protective effects of cell wall contents of three Lactobacillus species in lipopolysaccharide (LPS)-induced colitis rats. LPS-sensitized rats were rendered colitic by colonic instillation of LPS (500µg/rat) for 14 consecutive days. Concurrently, cell wall contents isolated from 106 CFU of L. casei (LC), L. acidophilus (LA), and L. rhamnosus (LA) was given subcutaneously for 21days, considering sulfasalazine (100mg/kg, p.o.) as standard. The severity of colitis was assessed by body weight loss, food intake, stool consistency, rectal bleeding, colon weight/length, spleen weight, and histological analysis. Colonic inflammatory markers (myeloperoxidase activity, C-reactive protein, and pro-inflammatory cytokines) and oxidative stress markers (malondialdehyde, reduced glutathione, and nitric oxide) were also assayed. Cell wall contents of LC, LA, and LR significantly ameliorated the severity of colitis by reducing body weight loss and diarrhea and bleeding incidence, improving food intake, colon weight/length, spleen weight, and microscopic damage to the colonic mucosa. The treatment also reduced levels of inflammatory and oxidative stress markers and boosted anti-oxidant molecule. In conclusion, cell wall contents of LC, LA, and LR attenuate LPS-induced colitis by modulating immuno-inflammation and oxidative stress.

Non-muscle myosin II knockdown improves survival and therapeutic effects of implanted bone marrow-derived mesenchymal stem cells in lipopolysaccharide-induced acute lung injury.

BackgroundBone marrow-derived mesenchymal stem cells (BMSCs) have been shown to have some beneficial effects in acute lung injury (ALI), but the therapeutic effects are limited due to apoptosis or necrosis after transplantation into injured lungs. Here, we aim to explore whether Non-muscle myosin II (NM-II) knockdown could enhance BMSCs survival and improve therapeutic effects in ALI.MethodsMSCs, isolated from rat bone marrow, were transfected with the small interfering RNA (siRNA) targeted to NM-II mRNA by a lentivirus vector. Rats were equally randomized to four groups: the control group was given normal saline via tail vein; the other three groups underwent intratracheal lipopolysaccharide (LPS) instillation followed by administration with either normal saline, BMSCs transduced with lentivirus-enhanced green fluorescent protein (eGFP) empty vector, or BMSCs transduced with lentivirus-eGFP NM-II siRNA. Hematoxylin and eosin staining was used to evaluate lung histopathologic changes and Masson trichrome staining was used to assess lung fibrosis. The myeloperoxidase activity was also tested in lung tissues. The mRNA expression of inflammatory cytokines in lung tissues was determined via quantitative reverse transcription PCR. Sex-determining region of the Y chromosome gene expression was measured by fluorescence in situ hybridization (FISH) assay. The expression of self-renewal activity and apoptosis-associated proteins were measured by Western blot.ResultsTransplantation of NM-II siRNA-modified BMSCs could improve histopathological morphology, decrease inflammatory infiltrates, down-regulate the expression levels of inflammatory cytokines, and reduce pulmonary interstitial edema. NM-II siRNA-modified BMSCs showed antifibrotic properties and alleviated the degrees of pulmonary fibrosis induced by endotoxin. In addition, NM-II knockdown BMSCs showed slightly better therapeutic effect on lung inflammation when compared with control BMSCs. The beneficial effects of NM-II siRNA-modified BMSCs may be attributed to enhanced self-renewal activity and decreased apoptosis.ConclusionsNM-II knockdown could inhibit the apoptosis of implanted BMSCs in lung tissues and improve its self-renewal activity. NM-II siRNA-modified BMSCs have a slightly enhanced ability to attenuate lung injury after LPS challenge.

Ameliorative Effect of Thymoquinone-Loaded PLGA Nanoparticles on Chronic Lung Injury Induced by Repetitive Intratracheal Instillation of Lipopolysaccharide in Rats.

Thymoquinone (TQ), the active constituent of Nigella sativa, possesses several benefits in traditional and modern medicines. This study examined the effect of a single dose of Nano-TQ on chronic lung injury induced by repetitive intratracheal installation of lipopolysaccharide (LPS). Rats received LPS twice weekly for 8 weeks via intratracheal installation and a single dose of TQ-PLGA NPs on the day after the last dose of LPS. Six rats from each group were sacrificed after 8 and 10 weeks, and samples were collected for analysis. Repetitive intratracheal installation of LPS caused histopathological alterations, including partial or complete obstruction of the alveoli, interstitial edema, mild fibroblastic proliferation, fibrous strands besides lymphocytes and plasma infiltrations, suffered fetalization, bronchiectasis, hypertrophied arterioles, and others. Investigation of the ultrastructure revealed prominent necrotic pneumocytes with destructed chromatin and remnant of necrotic debris in the narrowing alveolar lumen in LPS-induced rats. TQ-PLGA NPs effectively ameliorated LPS-induced histopathological and ultrastructural alterations in the lung of rats. In addition, TQ-PLGA NPs significantly alleviated serum levels of IL-10 and TGF-β1 in LPS-induced rats. In conclusion, TQ-PLGA NPs prevented inflammation and tissue injury in the lungs of rats challenged with repetitive intratracheal installation of LPS. Therefore, TQ-PLGA NPs represent a promising candidate for the prevention of lung injury induced by LPS, pending further studies to determine its safety and exact protective mechanism.

MCC950, a NLRP3 inhibitor, ameliorates lipopolysaccharide-induced lung inflammation in mice

Acute respiratory distress syndrome/chronic obstructive pulmonary disease (ARDS/COPD) is a diffuse inflammatory injury of the lung that is characterized by respiratory distress and vascular leakage and is caused by various factors. Although the treatment strategy for ARDS/COPD continues to be improved, it still lacks effective drugs. MCC950 is a potent and selective inhibitor ofthe nucleotide-binding oligomerization domain-like-receptor family pyrin domain-containing 3 (NLRP3) inflammasome. However, there have been no reports on the effects of MCC950 on lipopolysaccharide (LPS)-induced lung inflammation in mice. The objective of the present study was to evaluate the effects of MCC950 (given either intranasally or intraperitoneally) on inhibiting LPS-induced lung inflammation in mice. Acute lung inflammation was induced by intratracheal administration of LPS in ICR mice. The results showed that MCC950 at 50 mg/kg efficiently suppressed neutrophil lymphocytes (p < 0.001) and macrophage accumulation (p < 0.01) in bronchoalveolar lavage fluid (BALF) in LPS-instilled mice. In addition, hematoxylin and eosin (H&E) staining revealed that MCC950 at 50 mg/kg significantly inhibited pathological progress in the lung tissues (p < 0.01). Furthermore, treatment with MCC950 substantially reduced mRNA expression of IL-1β, IL-8, TGF-β1, and MMP-9 and also reduced protein levels of IL-1β, IL-18 and caspase-1 at 24 h after LPS instillation. The results of the present study indicate that MCC950 effectively inhibits LPS-induced lung inflammation in vivo, which can be considered for clinical translation.

Effect of SIS3 on Extracellular Matrix Remodeling and Repair in a Lipopolysaccharide-Induced ARDS Rat Model.

The remodeling of the extracellular matrix (ECM) in the parenchyma plays an important role in the development of acute respiratory distress syndrome (ARDS), a disease characterized by lung injury. Although it is clear that TGF-β1 can modulate the expression of the extracellular matrix (ECM) through intracellular signaling molecules such as Smad3, its role as a therapeutic target against ARDS remains unknown. In this study, a rat model was established to mimic ARDS via intratracheal instillation of lipopolysaccharide (LPS). A selective inhibitor of Smad3 (SIS3) was intraperitoneally injected into the disease model, while phosphate-buffered saline (PBS) was used in the control group. Animal tissues were then evaluated using histological analysis, immunohistochemistry, RT-qPCR, ELISA, and western blotting. LPS was found to stimulate the expression of RAGE, TGF-β1, MMP2, and MMP9 in the rat model. Moreover, treatment with SIS3 was observed to reverse the expression of these molecules. In addition, pretreatment with SIS3 was shown to partially inhibit the phosphorylation of Smad3 and alleviate symptoms including lung injury and pulmonary edema. These findings indicate that SIS3, or the blocking of TGF-β/Smad3 pathways, could influence remodeling of the ECM and this may serve as a therapeutic strategy against ARDS.

Echinacoside attenuates lipopolysaccharide-induced acute lung injury in newborn mice via inactivation of NF- κB/NLRP3 signaling pathway

Purpose: To investigate the effect of echinacoside (ECH) on acute lung injury (ALI) and the underlying mechanism of action.Methods: The ALI model was established through intranasal instillation of lipopolysaccharide (LPS). Lung tissue damage was determined using hematoxylin and eosin (H&amp;E) staining and lung wet-to-dry–weight ratio. Bronchoalveolar lavage fluid (BALF) protein concentration, cell count, and cytokine level were evaluated. Western blotting was used to determine protein expression level.Results: ECH attenuated lung tissue injury and lung wet-to-dry–weight ratio in the ALI model (p &lt; 0.01). The total protein content and number of total cells, neutrophils, and macrophages increased in BALF of mice treated with LPS, but these increases were reversed by ECH treatment (p &lt; 0.01). The levels of TNF-α and IL-1β increased in BALF and lung tissue of LPS-treated mice; however, ECH treatment decreased these changes (p &lt; 0.01). In addition, ECH inhibited the activation of the nuclear factor-κB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) pathway in LPS-treated mice (p &lt; 0.01).Conclusion: Echinacoside attenuates LPS-induced ALI via inactivation of the NF-κB/NLRP3 pathway, making echinacoside a potential drug for the treatment of ALI.&#x0D; Keywords: Echinacoside, Acute lung injury, Lipopolysaccharide, Nuclear factor-κB, NLR family pyrin domain containing 3

The BRD4 inhibitor JQ1 protects against chronic obstructive pulmonary disease in mice by suppressing NF-κB activation.

To examine the effect of the BRD4 inhibitor JQ1 on mice with chronic obstructive pulmonary disease (COPD) via NF-κB. COPD models constructed by exposure to cigarette smoke and intratracheal instillation of lipopolysaccharides (LPS) in mice were treated with JQ1 (15, 25 or 50 mg/kg). HE staining was performed to observe histopathological changes in the lung tissues. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of IL-10, IFN-γ, IL-17, IL-1β, IL-6, TNF-α, MMP-2, MMP-9, MDA, SOD, T-AOC and HO-1, and gelatin zymography assays were used to examine MMP-2 and MMP-9 activity. A TransAMTM NF-κB p65 detection kit was used to test NF-κB p65/DNA binding activity. Western blotting was conducted to analyze NF-κB p65 in the nucleus and its acetylation. JQ1 dose-dependently improved the histopathological changes in the lung tissues and decreased the mean linear intercept (MLI), destructive index and inflammatory score of the mice with COPD. The mice with COPD showed increased levels of MMP-2, MMP-9, IFN-γ, IL-17, IL-1β, IL-6 and TNF-α with decreased IL-10 level; these changes were reversed by JQ1 in a dose-dependent manner. In addition, JQ1 reduced the MDA level and increased the SOD, HO-1 and T-AOC levels in mice with COPD, with suppression of NF-κB p65 expression in the nucleus, NF-κB/p65 (Lys310) acetylation and NF-κB p65/DNA binding activity in the lung tissues. The BRD4 inhibitor JQ1 can downregulate MMP-2 and MMP-9 expression, reduce inflammatory responses, and alleviate oxidative stress in mice with COPD, and this mechanism might be related to the inhibition of NF-κB.

Apelin‑36 protects against lipopolysaccharide‑induced acute lung injury by inhibiting the ASK1/MAPK signaling pathway.

Apelin-36 is able to mediate a range of effects on various diseases, and is upregulated in lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, to the best of our knowledge, whether apelin-36 is able to regulate LPS-induced ALI has yet to be investigated. The present study aimed to investigate the role of apelin-36 in LPS-induced ALI, and the putative underlying mechanisms. Rats were assigned to one of four treatment groups: The Control group, apelin-36 group, LPS group and LPS + apelin-36 group. At 4 h after intratracheal instillation of LPS (5 mg/kg), rats were intraperitoneally treated with 10 nmol/kg apelin-36. Subsequently, pathological manifestations and the extent of inflammation and apoptosis of the lung tissues were assessed. Untransfected and apoptosis signal-regulating kinase 1 (ASK1)-overexpressing Beas-2B cells were treated with LPS in the absence or presence of apelin-36, and subsequently the levels of inflammation and apoptosis were assessed. The results obtained showed that the level of apelin-36 was increased in the bronchoalveolar lavage fluid (BALF) of LPS-treated rats. Co-treatment with apelin-36 alleviated LPS-induced lung injury and pulmonary edema, reduced the levels of pro-inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1 and tumor necrosis factor-α, in BALF, and inhibited apoptosis in the lung tissues. The presence of apelin-36 also blocked the activation of LPS-induced ASK1, p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase in lung tissues. In vitro studies performed with Beas-2B cells showed that the addition of apelin-36 led to an increase in the cell viability of LPS-induced Beas-2B cells in a concentration-dependent manner. Additionally, co-treatment with 1 µM apelin-36 prevented LPS-induced inflammation and apoptosis. However, overexpression of ASK1 significantly reversed the inhibitory effects of apelin-36 on LPS-induced inflammation and apoptosis. Taken together, the results of the present study demonstrated that apelin-36 was able to protect against LPS-induced lung injury both in vivo and in vitro, and these actions may be dependent on inhibition of the ASK1/mitogen-activated protein kinase signaling pathway.

&lt;p&gt;Adiponectin Protects Obese Rats from Aggravated Acute Lung Injury via Suppression of Endoplasmic Reticulum Stress&lt;/p&gt;

IntroductionEndoplasmic reticulum (ER) stress seems to mediate the obesity-induced susceptibility to acute lung injury (ALI). The present study was designed to evaluate the role of ER stress in adiponectin (APN)-induced lung protection in an obese rat model treated with lipopolysaccharide (LPS).MethodsFour-week-old male Sprague-Dawley rats fed either a normal chow diet or a high-fat diet for 12 weeks were randomly assigned to one of the following groups: lean rats, diet-induced obesity rats, lean rats with ALI, obese rats with ALI, obese rats pretreated with 4-phenylbutyric acid (4-PBA) before ALI or obese rats pretreated with APN before ALI. At 24 h after instillation of LPS into the lungs, cell counts in the bronchoalveolar lavage fluid (BALF) were determined. Lung tissues were separated to assess the degree of inflammation, pulmonary oedema, epithelial apoptosis and the expression of ER stress marker proteins.ResultsThe 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) expression in the lung tissues of obese rats was upregulated before ALI, as well as the elevated apoptosis in epithelial cells. During ALI, the expression of ER stress marker proteins was similarly increased in both lean and obese rats, while significant downregulation of Mitofusin 2 (MFN2) was detected in obese epithelial cells. The lung tissues of obese rats showed higher concentrations of tumor necrosis factor-alpha (TNF-α), Interleukin 6 (IL-6) and IL-10, enhanced neutrophil counts and elevated wet/dry weight ratios. APN and 4-PBA decreased the degree of ER stress and suppressed LPS-induced lung inflammation, pulmonary oedema and epithelial apoptosis.ConclusionAPN may exert protective effects against the exacerbated lung injuries in obese rats by attenuating ER stress, which operates as a key molecular pathway in the progression of ALI.