Interleukin-6 In Bronchoalveolar Lavage Fluid Research Articles

Key inflammatory markers in bronchoalveolar lavage predict bronchiectasis progression in young children with CF

IntroductionInflammation appears early in cystic fibrosis (CF) pathogenesis, with specific elevated inflammatory markers in bronchoalveolar lavage fluid (BALF) correlating with structural lung disease.Our aim was to identify markers of airway inflammation able to predict bronchiectasis progression over two years with high sensitivity and specificity. MethodsChildren with CF with two chest computed tomography (CT) scans and bronchoscopies at a two-year interval were included (n= 10 at 1 and 3 years and n= 27 at 3 and 5 years). Chest CTs were scored for increase in bronchiectasis (Δ%Bx), using the PRAGMA-CF score. BALF collected with the first CT scan were analyzed for neutrophil% (n= 36), myeloperoxidase (MPO) (n= 25), neutrophil elastase (NE) (n= 26), and with a protein array for inflammatory and fibrotic markers (n= 26). ResultsMPO, neutrophil%, and inducible T-cell costimulator ligand (ICOSLG), but not clinical characteristics, correlated significantly with Δ%Bx. Evaluation of neutrophil%, NE, MPO, interleukin-8 (IL-8), ICOSLG, and hepatocyte growth factor (HGF), for predicting an increase of > 0.5% of Δ%Bx in two years, showed that IL-8 had the best sensitivity (82%) and specificity (73%). Neutrophil%, ICOSLG and HGF had sensitivities of 85, 82, and 82% and specificities of 59, 67 and 60%, respectively. The odds ratio for risk of >0.5% Δ%Bx was higher for IL-8 (12.4) than for neutrophil%, ICOSLG, and HGF (5.9, 5.3, and 6.7, respectively). Sensitivity and specificity were lower for NE and MPO). ConclusionsHigh levels of IL-8, neutrophil%, ICOSGL and HGF in BALF may be good predictors for progression of bronchiectasis in young children with CF.

Lipocalin-2 promotes acute lung inflammation and oxidative stress by enhancing macrophage iron accumulation.

Lipocalin-2 (LCN2) is an acute-phase protein that regulates inflammatory responses to bacteria or lipopolysaccharide (LPS). Although the bacteriostatic role of LCN2 is well studied, the function of LCN2 in acute lung damage remains unclear. Here, LCN2 knockout (KO) mice were used to investigate the role of LCN2 in LPS-treated mice with or without recombinant LCN2 (rLCN2). In addition, we employed patients with pneumonia. RAW264.7 cells were given LCN2 inhibition or rLCN2 with or without iron chelator deferiprone. LCN2 KO mice had a higher survival rate than wild-type (WT) mice after LPS treatment. In addition to elevated LCN2 levels in serum and bronchoalveolar lavage fluid (BALF), LPS treatment also increased LCN2 protein in alveolar macrophage lysates of BALF. LCN2 deletion attenuated neutrophil and macrophage infiltration in the lungs of LPS-treated mice as well as serum and BALF interleukin-6 (IL-6). Circulating proinflammatory cytokines and LCN2-positive macrophages were prominently increased in the BALF of pneumonia patients. In addition to increase of iron-stained macrophages in pneumonia patients, increased iron-stained macrophages and oxidative stress in LPS-treated mice were inhibited by LCN2 deletion. In contrast, rLCN2 pretreatment aggravated lung inflammation and oxidative stress in LPS-treated WT mice and then resulted in higher mortality. In RAW264.7 cells, exogenous LCN2 treatment also increased inflammation and oxidative stress, whereas LCN2 knockdown markedly diminished these effects. Furthermore, deferiprone inhibited inflammation, oxidative stress, and phagocytosis in RAW264.7 cells with high LCN2 levels, as well as LPS-induced acute lung injury in WT and LCN2 KO mice. Thus, these findings suggest that LCN2 plays a key role in inflammation and oxidative stress following acute lung injury and that LCN2 is a potential therapeutic target for pneumonia or acute lung injury.

Protective effect of Lindera aggregata on lipopolysaccharide-induced mice acute respiratory distress syndrome by regulating p38MAPK/ERK pathway

To explore the protective effect of Lindera aggregata on acute respiratory distress syndrome (ARDS) induced by lipopolysaccharide (LPS) in mice and its possible mechanism. Forty C57BL/6 mice were randomly divided into sham operation group, ARDS model group, low-dose Lindera aggregata (L-LA) group and high-dose Lindera aggregata (H-LA) group, with 10 mice in each group. ARDS model was established by injecting 5 mg/kg LPS through the trachea. The L-LA group and H-LA group were orally administrated 1 g/kg and 5 g/kg of the Lindera aggregate extract once a day, respectively, while the ARDS model group was given the same volume of normal saline, the sham group received no treatment. The Lindera aggregata was preadministered for 3 days before modeling, and continued for 2 days after modeling, then the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The pathological changes of lung tissue in each group of mice were observed under the microscope and the wet/dry weight ratio (W/D) of the lung were measured. Enzyme linked immunosorbent assay (ELISA) was used to examine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mice serum and BALF, and flow cytometry was used to detect the expression rate of CD40 on the surface of BALF macrophages. The phosphorylation levels of p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) proteins in lung tissue were measured by Western blotting. Lung histopathology under light microscope showed that the damage of alveolar structure, thickening of alveolar septum and infiltration of inflammatory cells in the H-LA group were less severe than those in the ARDS model group, while the pathological characteristics of ARDS in the L-LA group were not significantly different from those in the ARDS model group. Compared with the sham operation group, the lung W/D ratio, TNF-α and IL-6 protein contents in serum and BALF, BALF macrophage CD40 expression rate and lung tissue p38 and ERK1/2 protein phosphorylation levels were significantly increased in ARDS model group. The W/D ratio, the concentrations of TNF-α and IL-6 in serum and BALF, the expression rate of CD40 in BALF macrophages, and the phosphorylation levels of p38 and ERK1/2 protein in lung tissue in the L-LA group were not significantly different from those in the ARDS model group. The above indexes in the H-LA group were significantly lower than those in the ARDS model group and the L-LA group [W/D ratio: 5.70±0.19 vs. 6.20±0.31, 6.01±0.17; serum TNF-α (ng/L): 83.63±15.04 vs. 111.75±18.45, 108.12±13.98; serum IL-6 (ng/L): 111.38±8.75 vs. 244.13±26.85, 227.50±9.37; BALF TNF-α (ng/L): 36.25±2.82 vs. 51.13±5.44, 47.50±5.78; BALF IL-6 (ng/L): 35.63±2.20 vs. 49.63±4.90, 46.38±3.50; CD40 expression rate (%): 23.28±2.45 vs. 30.32±2.40, 28.17±1.98; p-p38/p38: 0.50±0.04 vs. 0.74±0.07, 0.69±0.04; p-ERK1/2/ERK1/2: 0.47±0.07 vs. 0.72±0.07, 0.68±0.05; all P < 0.01]. Lindera aggregata can inhibit LPS-induced lung inflammation and alleviate lung injury in ARDS mice. The mechanism may be related to the inhibition of the activation of p38 mitogen activated protein kinase/ERK (p38MAPK/ERK) signaling pathway.

Icariin Improves Glucocorticoid Resistance in a Murine Model of Asthma with Depression Associated with Enhancement of GR Expression and Function.

Icariin, a flavonoid glycoside isolated from Epimedium brevicornum, exerts a variety of biological activities. However, its effects on depression-induced glucocorticoid resistance in asthma and the underlying mechanisms have not been elucidated. In this study, a murine model of asthma with depression was established by exposure to ovalbumin combined with chronic unpredictable mild stress, and icariin was given orally during ovalbumin challenge and chronic unpredictable mild stress exposure. Depression-like behaviors were assessed by the open field test, forced swim test, and tail suspension test. The characteristic features of allergic asthma, including airway hyperreactivity, histopathology, inflammatory cytokine levels in bronchoalveolar lavage fluid, and immunoglobulin E and corticosterone levels in serum, were examined. Following splenocyte isolation in vitro, the inhibitory effects of corticosterone on the proliferation and cytokine secretion of splenocytes, glucocorticoid receptor DNA-binding activity, and expression of p-glucocorticoid receptor s226, glucocorticoid receptor α, and p-p38 mitogen-activated protein kinase in splenocytes were determined. We found that icariin had limited effects on depression-like behaviors, however, it markedly suppressed airway hyperresponsiveness, inflammatory infiltration in lung tissues, levels of interleukin-4, interleukin-5, and interleukin-6 in bronchoalveolar lavage fluid, and immunoglobulin E in serum. Furthermore, icariin improved the inhibitory effects of corticosterone on lipopolysaccharide-stimulated splenocytes, increased the glucocorticoid receptor expression and glucocorticoid receptor DNA-binding activity, and inhibited the phosphorylation of glucocorticoid receptors S226 and p38 mitogen-activated protein kinase. Taken together, icariin improved glucocorticoid resistance in a murine model of asthma with depression associated with enhancement of glucocorticoid receptor function and glucocorticoid receptor expression, and its effects on the glucocorticoid receptor function were related to decreased phosphorylation of glucocorticoid receptors S226 and p38 mitogen-activated protein kinase.

Antibiotic-induced microbiome depletion improves LPS-induced acute lung injury via gut-lung axis

AimsAcute lung injury (ALI) is an acute inflammatory disorder. However, the precise mechanisms underlying the pathology of ALI remain elusive. An increasing evidence suggests the role of the gut-microbiota axis in the pathology of lung injury. This study aimed to investigate whether antibiotic-induced microbiome depletion could affect ALI in mice after lipopolysaccharide (LPS) administration. Main methodsThe effects of antibiotic cocktail (ABX) on ALI in the mice after intratracheally administration of LPS (5 mg/kg) were examined. Furthermore, 16s rRNA analysis and measurement of short-chain fatty acids in feces samples and metabolomics analysis of blood samples were performed. Key findingsLPS significantly increased the interleukin-6 (IL-6) levels in the bronchoalveolar lavage fluid (BALF) of water-treated mice. Interestingly, an ABX significantly attenuated the LPS-induced increase in IL-6 in BALF and lung injury scores. Furthermore, ABX and/or LPS treatment markedly altered the α- and β-diversity of the gut microbiota. There were significant differences in the α- and β-diversity of the water + LPS group and ABX + LPS group. LEfSe analysis identified Enterococusfaecalis, Clostriumtertium, and Bacteroidescaecimyris as potential microbial markers for ABX + LPS group. Untargeted metabolomics analysis identified several plasma metabolites responsible for discriminating water + LPS group from ABX + LPS group. There were correlations between the relative abundance of the microbiome and plasma metabolites. Integrative network analysis showed correlations between IL-6 levels in BALF and several gut microbes (or plasma metabolites). SignificanceThese data suggest that ABX-induced microbiome depletion could protect against LPS-induced ALI via the gut-microbiota-lung axis.

Value of autotaxin in predicting refractory Mycoplasma pneumoniae pneumonia in children and its correlation with inflammatory cytokines

To study the value of autotaxin (an autocrine motility factor) level in serum and bronchoalveolar lavage fluid (BALF) in predicting refractory Mycoplasma pneumoniae pneumonia (RMPP) in children and its correlation with interleukin-6 (IL-6), interleukin-8 (IL-8), and C-reactive protein (CRP). A retrospective analysis was performed on 238 children with Mycoplasma pneumoniae pneumonia who were admitted from January 2019 to December 2021. According to disease severity, they were divided into two groups: RMPP (n=82) and general Mycoplasma pneumoniae pneumonia (GMPP; n=156). The two groups were compared in terms of the levels of autotaxin, IL-6, IL-8, and CRP in serum and BALF to study the value of autotaxin level in serum and BALF in predicting RMPP in children, as well as the correlation of autotaxin level with IL-6, IL-8, and CRP in children with RMPP. Compared with the GMPP group, the RMPP group had significantly higher levels of autotaxin, IL-6, IL-8, and CRP in serum and BALF (P<0.05). For the children with RMPP, the levels of autotaxin, IL-6, IL-8, and CRP in serum and BALF in the acute stage were significantly higher than those in the convalescent stage (P<0.05). The receiver operating characteristic (ROC) curve showed that the level of autotaxin in serum and BALF had a good value in predicting RMPP in children, with an area under the curve of 0.874 (95%CI: 0.816-0.935) and 0.862 (95%CI: 0.802-0.924), respectively. The correlation analysis showed that the level of autotaxin in serum and BALF was positively correlated with IL-6, IL-8, and CRP levels (P<0.001). The level of autotaxin in serum and BALF increases and is correlated with the degree of disease recovery and inflammatory cytokines in children with RMPP. Autotaxin can be used as a predictive indicator for RMPP in children.

BMSCs mediates endothelial cell autophagy by upregulating miR-155-5p to alleviate ventilator-induced lung injury.

In this study, we explored to detect the effects and mechanism of bone-marrow-derived mesenchymal stem cells (BMSCs) on ventilator-induced lung injury (VILI). We transplanted BMSCsin mice and then induced VILI using mechanical ventilation (MV) treatment. The pathological changes, the content of PaO2 and PaCO2 , wet/dry weight ratio (W/D) of the lung, levels of tumor necrosis factor-α and interleukin-6 in bronchoalveolar lavage fluid, and apoptosis were detected. The autophagy-associated factor p62, LC3, and Beclin-1 expression were analyzed by western blot. The quantitativepolymerase chain reaction was applied to detect abnormally expressed microRNAs, including miR-155-5p. Subsequently, we overexpressed miR-155-5p in VILI mice to detect the effects of miR-155-5p on MV-induced lung injury. Then, we carried out bioinformatics analysis to verify the BMSCs-regulated miR-155-5p that target messenger RNA. It was observed that BMSCs transplantation mitigated the severity of VILI in mice. BMSCs transplantation reduced lung inflammation, strengthened the arterial oxygen partial pressure, and reduced apoptosis and the W/D of the lung. BMSCs promoted autophagy of pulmonary endothelial cells accompanied by decreased p62 and increased LC3 II/I and Beclin-1. BMSCs increased the levels of miR-155-5p in VILI mice. Overexpression of miR-155-5p alleviated lung injury in VILI mice following reduced apoptosis and increased autophagy. Finally, TAB2 was identified as a downstream target of miR-155-5p and regulated by miR-155-5p. BMSCs may protect lung tissues from MV-induced injury, inhibit lung inflammation, promote autophagy through upregulating of miR-155-5p.

Protective effect of the effective part of Andrographis paniculata (Burm.f.) Nees on PM2.5-induced lung injury in rats by modulating the NF-κB pathway

Ethnopharmacological relevanceAndrographis paniculata (Burm.f.) Nees, a traditional Chinese herb, has been widely used in various Asian countries as a treatment for upper respiratory tract infections for centuries. Aim of the studyContinuous inhalation of fine particulate matter (PM2.5) may induce various respiratory diseases. This study elucidated the protective effect of the effective part of Andrographis paniculata (Burm.f.) Nees (AEP) against PM2.5-induced lung injury and detailed the underlying mechanism. Materials and methodsMale Wistar rats were orally administered 0.5% sodium carboxymethylcellulose (CMC-Na), andrographolide (AG) (200 mg/kg) and AEP (100 mg/kg, 200 mg/kg and 400 mg/kg) once a day for 28 days. The rats were intratracheally instilled with PM2.5 suspension (8 mg/kg) every other day beginning on the 24th day for a total of 3 times. On the 29th day, bronchoalveolar lavage fluid (BALF) was collected to analyze the levels of lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (AKP), total proteins (TP), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6). Hematoxylin & eosin staining was conducted to evaluate the pathological changes in the lung tissues. The protein expression of NF-κB p65 in the lung tissues was analyzed by immunohistochemistry staining. Moreover, the nuclear translocation of NF-κB p65 and the phosphorylation of IκBα were analyzed by western blotting. ResultsPM2.5 exposure caused lung toxicity, which was characterized by pathological injury and increased levels of LDH, ACP, AKP and TP in BALF. Meanwhile, PM2.5 exposure induced lung inflammatory response, including infiltration of inflammatory cells and increased levels of inflammatory factors, such as TNF-α and IL-6 in BALF. AEP treatment significantly ameliorated the PM2.5-induced lung toxicity and the inflammatory response in rats. Moreover, AEP significantly inhibited the PM2.5-induced upregulation of NF-κB p65 protein expression, phosphorylation of IκBα and nuclear translocation of NF-κB p65 in lung tissue. Compared to AG, AEP exhibited a better ability to alleviate PM2.5-induced pathological damage and decrease the TP level in the BALF. ConclusionAEP could be used to improve PM2.5-induced lung injury by modulating the NF-κB pathway, and multicomponent therapy with traditional Chinese medicine may be more effective than single-drug therapy.

Protective effects of different anti‑inflammatory drugs ontracheal stenosis following injury and potential mechanisms.

Tracheal stenosis following injury cannot be effectively treated. The current study compared the protective effects of different anti-inflammatory drugs on tracheal stenosis and investigated their possible mechanisms. Rabbit tracheal stenosis models following injury were constructed and confirmed using hematoxylin and eosin (H&E) staining. A total of 30 rabbits were divided into the control (CON), penicillin (PEN), erythromycin (ERY), budesonide (BUD) and PEN + ERY + BUD groups (n=6). Stenotic tracheal tissue, serum and bronchoalveolar lavage fluid (BALF) were collected 10 days after continuous treatment. Pathological changes in the tracheas were observed by H&E staining. Histone deacetylase 2 (HDAC2) expression in tracheal tissues was detected by immunofluorescence. Immunohistochemistry was performed to detect collagen I (Col-I) and collagen III (Col-III) levels in tracheal tissues. Transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF) and interleukin 8 (IL-8) levels in serum and BALF samples were determined using ELISA kits. Western blotting detected HDAC2, IL-8, TGF-β1 and VEGF levels in tracheal tissues. H&E staining demonstrated that tracheal epithelial hyperplasia and fibroblast proliferation in the ERY and PEN + ERY + BUD groups markedly improved compared with the CON group. Furthermore, in tracheal tissues, HDAC2 expression was significantly increased and IL-8, TGF-β1, VEGF, Col-I and Col-III levels were significantly decreased in the ERY and PEN + ERY + BUD groups compared with the CON group. Additionally, the results for the PEN + ERY + BUD were more significant compared with the ERY group. In serum and BALF samples, IL-8, TGF-β1 and VEGF levels in the ERY and PEN + ERY + BUD groups were significantly lower compared with the CON group, with the results of the PEN + ERY + BUD group being more significant compared with the ERY group. There were no significant differences between the PEN, BUD and CON groups. ERY inhibited tracheal granulation tissue proliferation and improved tracheal stenosis following injury and synergistic effects with PEN and BUD further enhanced these protective effects. The mechanism may involve HDAC2 upregulation and inhibition of local airway and systemic inflammatory responses.

Lycorine attenuates lipopolysaccharide-induced acute lung injury through the HMGB1/TLRs/NF-κB pathway.

Lung injury associated with systemic inflammatory response is a common problem affecting human health. Previous studies have shown that lycorine exerts a anti-inflammatory effect. However, whether lycorine alleviates lung injury remains unclear. To explore this issue, BALB/c mice and MLE-12 cells were treated with lipopolysaccharide (LPS) to establish lung injury mouse model and cell model, respectively. Glycyrrhizic acid, known as an inhibitor of ALI, was also used to study the effects of lycorine in vitro. Our results showed that after LPS treatment, the lung injury score, lung wet-to-dry weight ratio, and malondialdehyde (MDA) production in the lung tissues and the expression levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in bronchoalveolar lavage fluid were significantly increased, whereas their levels were decreased by lycorine. Additionally, LPS injection activated the high-mobility group box 1 (HMGB1)/Toll-like receptors (TLRs)/NF-κB pathway. However, lycorine treatment attenuated the activity of the HMGB1/TLRs/NF-κB pathway in the lung tissues. In vitro studies showed that lycorine administration significantly decreased the levels of inflammatory cytokines and MDA and attenuated the activity of the HMGB1/TLRs/NF-κB pathway in LPS-treated cells. Moreover, the inhibitory effects of lycorine on the inflammatory response and oxidative stress in LPS-treated lung cells were similar with that of glycyrrhizic acid, and this inhibition was intensified by both lycorine and glycyrrhizic acid treatment. We suggest that lycorine could alleviate LPS-induced lung injury of inflammation and oxidative stress by blocking the HMGB1/TLRs/NF-κB pathway, which gives a new perspective for ALI therapy to treat lycorine as a potential treatment clinically.

Effects of indoor coal fine particulate matter on the expression levels of inflammatory factors in ovalbumin-induced mice.

Objective: Cooking and heating with coal is the main source of household air pollution in acid rain-plagued areas of China and is a leading contributor to disease burden. In this study, we investigated the adverse effects of exposure to indoor fine particulate matter emission from coal combustion on the expression levels of inflammatory factors in ovalbumin (OVA)-induced mice. Methods: Forty BALB/c male mice were randomly divided into four groups (control group, PM2.5 group, OVA group, and OVA + PM2.5 group; n = 10) and treated with ovalbumin (OVA) or PM2.5, alone or together. Interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-17 (IL-17), transforming growth factor β1 (TGF-β1), and interferon γ (IFN-γ) protein expression levels were measured in bronchoalveolar lavage fluid (BALF). Additionally, serum immunoglobulin (Ig) IgE and IgG1 levels were measured. The mRNA expression levels of IL-7 and IFN-γ in pulmonary tissue were also analyzed. Bronchoalveolar lavage (BAL), inflammatory cell counts, and histopathological examinations were also performed. Results: Exposure to PM2.5 + OVA induced abnormal pathological changes and inflammatory responses in lungs compared to the control. The levels of IL-4, IL-7, IL-8 and IL-17 in BALF from the OVA + PM2.5 group were higher than those in BALF from the control group, OVA group, and PM2.5 group (P < 0.05). PM2.5 plus OVA significantly raised the serum IgE and IgG1 levels compared with the control group. An increasing IL-7 mRNA trend was found among the treatment groups (P < 0.05). The expression level of IFN-γ mRNA was significantly higher in the control group than in the other 3 groups (P < 0.05). Conclusion: Indoor coal PM2.5 was sufficient by itself to cause inflammatory cellular infiltration of pulmonary tissue, leading to organelle injury and physiological structure change. Additionally, it promoted the occurrence and development of asthma by influencing the expression levels of IL-7 and various relevant inflammatory factors (such as IL-4 and IL-8) and changing the equilibrium between Treg and Th17 cells.

Autophagy-dependent release of zinc ions is critical for acute lung injury triggered by zinc oxide nanoparticles

Pulmonary exposure to zinc oxide nanoparticles (ZnONPs) could cause acute lung injury (ALI), but the underlying molecular mechanism remains unclear. Herein, we established a ZnONPs-induced ALI mouse model, characterized by the histopathological changes (edema and infiltration of inflammatory cells in lung tissues), and the elevation of total protein and cytokine interleukin-6 in bronchoalveolar lavage fluid in time- and dose-dependent manners. This model also exhibited features like the disturbance of redox-state (reduced of glutathione to glutathione disulfide ratio, elevation of heme oxygenase-1 and superoxide dismutase 2), the decrease of adenosine triphosphate synthesis and the release of zinc ions in the lung tissues. Interestingly, we found that ZnONPs exposure caused the accumulation of autophagic vacuoles and the elevation of microtubule-associated proteins 1A/1B light chain (LC)3B-II and p62, indicating the impairment of autophagic flux. Our data indicated that the above process might be regulated by the activation of AMP-activated protein kinase but not the mammalian target of rapamycin pathway. The association between ZnONPs-induced ALI and autophagy was further verified by a classical autophagy inhibitor, 3-methyladenine (3-MA). 3-MA administration reduced the accumulation of autophagic vacuoles, the expression of LC3B-II and p62, followed by a significant attenuation of histopathological changes, inflammation, and oxidative stress. More importantly, 3-MA could directly decrease the release of zinc ions in lung tissues. Taken together, our study provides the evidence that ZnONPs-induced pulmonary toxicity is autophagy-dependent, suggests that limiting the release of zinc ions by inhibiting autophagy could be a feasible strategy for the prevention of ZnONPs-associated pulmonary toxicity.

The role of TLR3/NF-kappa B signaling pathway in paraquat-induced acute lung injury

Objective To investigate the pathogenesis of paraquat (PQ) induced acute lung injury through Toll like receptor 3 (Toll-like receptor-3, TLR3), TLR induced nuclear transcription factors (Nuclear Factor- kappa B, NF-κB) and its downstream pro-inflammatory factors TNF-α, IL-1β, IL-6. Methods The acute lung injury model of mice and the acute injury model of type II alveolar epithelial cells (A549) induced by PQ were established. The PQ mediated pathological changes of lung tissue, the cell count and cytospin of bronchoalveolar lavage fluid (BALF) were evaluated, and the pro-inflammatory factors in the lung tissue of mice were determined by ELISA the viability of A549 cells mediated by PQ was detected by CCK8 assay, and the mRNA expression and protein level of TLR3, Phospho-NF-kBp65, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in the lung tissues and A549 cells were observed by Real-time PCR and Western-blotting. In control group, the mice received normal saline (NS) instead of PQ. Results Compared with the control group, the mice in PQ group showed difficulty breathing, decreased activity, reduced food intake, and weight lost. The total number of BALF cells in the PQ group was significantly increased [NS: (0.018 8±0.102 1)×105vs. PQ: (0.237 4 ± 0.121 7)×105, t=9.804, P<0.01] with macrophage [NS: (0.162 8±0.086 5)×105vs. PQ: (1.063 3 ± 0.343 3)×105, t=8.043, P<0.01], lymphocyte [NS: (0.006 6±0.005 2)×105vs. PQ: (0.171 2 ± 0.099 1)×105, t=5.243, P<0.01] and neutrophils [NS: (0.000 04±0.000 1)×105vs. PQ: (0.901 9 ± 0.652 5)×105, t=4.370, P<0.01]. In PQ group, the appearance and volume of lung tissue increased with hyperemia and edema. HE slices showed inflammatory cell infiltration and pulmonary interstitial hemorrhage. Moreover, the expressions of TNF-α, IL-1β, IL-6 in BALF of PQ group was significantly higher than those of the control group by the ELISA assay [TNF-α: NS: (2.782 1 ± 3.521 5) vs. PQ: ( 7.512 6 ± 3.459 8) pg/mL, t=3.030, P<0.05; IL-1β: NS: (22.687 5 ± 14.229 3) vs.PQ: (163.100 4 ± 81.118 3) pg/mL, t=5.391, P<0.01; IL-6: NS: (1.653 3±0.442 7) vs. PQ: (648.565 6 ± 422.606 1) pg/mL, t=4.841, P<0.01]. CCK8 results indicated that the viability of A549 cells decreased by 25.3% and 36.4% at 24h after 200~400 μmol/L PQ treatment (all P<0.05). The mRNA expressions of TNF-α, IL-1β, IL-6 in the lung tissue and A549 cells in PQ group were higher than those in the control group as well (all P< 0.05). Furthermore, Western-blotting results revealed that the protein levels of TLR3 and Phospho-NF-κBp65 in the lung and A549 cells mediated by PQ were significantly higher than those in the control group (all P < 0.05). Conclusions PQ may induce acute lung injury by up-regulation of the expressions of inflammatory factors TNF-α, IL-1β, IL-6 through the TLR3/NF-κB signaling pathway. Key words: Paraquat; Acute lung injury; Toll-like receptor-3; Nuclear Factor-κappa B; Signal pathway; Inflammatory factors; Animal model; A549 cell

Nebivolol attenuates oxidative stress and inflammation in a guinea pig model of ovalbumin-induced asthma: a possible mechanism for its favorable respiratory effects.

An experimental model of ovalbumin (OVA) induced asthma was used to assess the effects of nebivolol, the third-generation selective β1-adrenergic receptor blocker, on airway reactivity, lung inflammation, and oxidative stress markers. The asthma induction protocol was done by OVA sensitization and challenge. Guinea pigs were classified into control, asthmatic, or asthmatic receiving nebivolol either 7.5 or 15 mg·kg-1·day-1 orally. At the end of the study respiratory, the anti-inflammatory and antioxidative effects of nebivolol were assessed. The asthmatic group exhibited a significant increase in early and late airway resistance, airway hyperreactivity to histamine, total and absolute leucocytic count, tumor necrosis factor-α, and interleukin-6 in bronchoalveolar lavage fluid and lung lipid peroxidation and a significant decrease in superoxide dismutase and glutathione compared to the control group. Additionally, there was a significant decrease in lung endothelial nitric oxide synthase (eNOS) and a significant increase in inducible nitric oxide synthase (iNOS) mRNA expression compared to the control group. The high dose of nebivolol counteracted the increased airway resistance induced by OVA, whereas it had no effect on airway hyperresponsiveness. Moreover, nebivolol exhibited significant anti-inflammatory and antioxidant effects and restored the altered levels of eNOS and iNOS compared to the asthmatic group. Collectively, these results suggest a beneficial effect of nebivolol in asthma.

Circulating and broncho-alveolar interleukin-6 in relation to body temperature in an experimental model of bovine Chlamydia psittaci infection.

In rodent models of experimentally induced fever, the important role of interleukin-6 (IL-6) as a circulating endogenous pyrogen is well established. Studies employing larger animal species and real infections are scarce. Therefore, we assessed bioactive IL-6 in peripheral blood and in broncho-alveolar lavage fluid (BALF) of calves after intra-bronchial inoculation with vital Chlamydia psittaci (Cp), with inactivated Cp, or with BGM cells. Only calves inoculated with vital Cp developed fever (peak at 2–3 days after challenge) and significantly increased IL-6 activity. Controls inoculated with either inactivated Cp or BGM cells also expressed increased bioactive IL-6, but no fever developed. Activity of IL-6 in BALF was significantly higher compared to blood serum. This experimental model of Cp infection revealed no apparent relation between IL-6 in blood and body temperature, but did reveal a relation between IL-6 and other markers of inflammation in BALF. We conclude that a local inflammatory response in the lungs of infected calves caused fever, which developed by mechanisms including other mediators besides IL-6.

Evaluation of LPS-Induced Acute Lung Injury Attenuation in Rats by Aminothiazole-Paeonol Derivatives.

Paeonol is a key phenolic compound in the root bark of Moutan Cortex Radicis that has been used in traditional Chinese Medicine to ameliorate inflammation. A series of aminothiazole-paeonol derivatives (APDs) were synthesized in this work and subjected to preliminary evaluation in cells followed by verification in animals. Quantification of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) in culture media of LPS-activated A549 cells, a lung epithelial adenocarcinoma cell line, were used to investigate the anti-inflammatory capability of APDs. ALI-bearing rats were employed to verify therapeutic efficacy of APDs according to observations of total cells, protein amounts, MCP-1 and IL-6 in bronchoalveolar lavage fluid (BALF). Histopathological examinations of lung tissues were consequently applied for validation of APDs. Among these compounds, 2-(2-aminothiazol-4-yl)-5-methoxyphenol (4) had the most potent activity, showing comparable inhibition of MCP-1/IL-6 and superior elimination of neutrophil infiltration and protein exudation in lungs compared to others as well as dexamethasone. This study demonstrated a comprehensive strategy to evaluate APDs through integration of cell-based screening and animal-based verification. In order to fulfill unmet needs of treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), APDs introduced in this work could be promising lead compounds to develop high potent anti-inflammation agents.

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role of CCN1 in inflammatory microenvironment during ALI/ARDS and focus on a potential communication between CCN1 and interleukin-6 (IL-6) in the airway epithelial cells. Our data illustrated that the expression levels of CCN1 and IL-6 in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide (LPS)-induced ALI mouse model were significantly elevated and the pulmonary expression of CCN1 was restricted to bronchial epithelial cells. Interestingly, both endogenous and exogenous CCN1 stimulated IL-6 production in vitro. Furthermore, LPS-induced IL-6 production in a bronchial epithelial cell line was blocked by CCN siRNA whereas CCN1 induced by LPS was sensitive to PI3K inhibition. Together, our data indicate a linear signal pathway, LPS-CCN1-IL-6, existing in bronchial epithelial cells after LPS exposure. This finding may represent an additional mechanism and a novel target for development of therapy and biomarker on ALI/ARDS.

Suppression of P2X7/NF-κB pathways by Schisandrin B contributes to attenuation of lipopolysaccharide-induced inflammatory responses in acute lung injury.

The aim of the present study was to assess the effects and mechanisms of Schisandrin B (SchB) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS (1mg/kg), and SchB (25, 50, and 75mg/kg) was injected 1h before LPS challenge by gavage. After 12h, bronchoalveolar lavage fluid (BALF) samples and lung tissues were collected. Histological studies demonstrated that SchB attenuated LPS-induced interstitial edema, hemorrhage, and infiltration of neutrophils in the lung tissue. SchB pretreatment at doses of 25, 50, and 75mg/kg was shown to reduce LPS-induced lung wet-to-dry weight ratio and lung myeloperoxidase activity. In addition, pretreatment with SchB lowered the number of inflammatory cells and pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interleukin-6 in BALF. The mRNA and protein expression levels of nuclear factor kappa B (NF-κB) signaling-related molecules activated by P2X7 were investigated to determine the molecular mechanism of SchB. The findings presented here suggest that the protective mechanism of SchB may be attributed partly to the decreased production of pro-inflammatory cytokines through the inhibition of P2X7/NF-κB activation.

Apios americana Medik Extract Alleviates Lung Inflammation in Influenza Virus H1N1- and Endotoxin-Induced Acute Lung Injury.

Apios americana Medik (hereinafter Apios) has been reported to treat diseases, including cancer, hypertension, obesity, and diabetes. The therapeutic effect of Apios is likely to be associated with its anti-inflammatory activity. This study was conducted to evaluate the protective effects of Apios in animal models of acute lung injury induced by lipopolysaccharide (LPS) or pandemic H1N1 2009 influenza A virus (H1N1). Mice were exposed to LPS or H1N1 for 2-4 days to induce acute lung injury. The treatment groups were administered Apios extracts via oral injection for 8 weeks before LPS treatment or H1N1 infection. To investigate the effects of Apios, we assessed the mice for in vivo effects of Apios on immune cell infiltration and the level of pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. After induction of acute lung injury, the numbers of neutrophils and total cells were lower in the Apios-treated groups than in the non-Apios-treated LPS and H1N1 groups. The Apios groups tended to have lower levels of tumor necrosis factor-a and interleukin-6 in BAL fluid. In addition, the histopathological changes in the lungs were markedly reduced in the Apios-treated groups. These data suggest that Apios treatment reduces LPS- and H1N1-induced lung inflammation. These protective effects of Apios suggest that it may have therapeutic potential in acute lung injury.

Esculin Inhibits the Inflammation of LPS-Induced Acute Lung Injury in Mice Via Regulation of TLR/NF-κB Pathways.

In this study, we investigated anti-inflammatory effects of esculin (ESC) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS, and ESC (20 and 40mg/kg) was given orally 1h prior to LPS administration. After 6h, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. ESC pretreatment decreased LPS-induced evident lung histopathological changes, lung wet-to-dry weight ratio, and lung myeloperoxidase activity. In addition, pretreatment with ESC inhibited inflammatory cells and proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β, and interleukin-6 in BALF. Furthermore, we demonstrated that ESC inhibited the Toll-like receptor-2 (TLR2), Toll-like receptor-4 (TLR4), myeloid differentiation primary response gene-88 (MyD88), and nuclear factor-κB (NF-κB) p65 in LPS-induced ALI. The results indicated that the ESC had a protective effect on LPS-induced ALI in mice.