IL-6 In Bronchoalveolar Lavage Fluid Research Articles

Hesperidin Regulates Jagged1/Notch1 Pathway to Promote Macrophage Polarization and Alleviate Lung Injury in Mice with Bronchiolitis

Objective To explore the effect and mechanism of hesperidin in treating the lung injury in the mouse model of respiratory syncytial virus (RSV)-induced bronchiolitis. Methods A mouse model of RSV-induced bronchiolitis was established,and 60 BALB/c mice were assigned into a control group,a model group,a low-dose hesperidin (18 mg/kg) group,a high-dose hesperidin (36 mg/kg) group,and a high-dose hesperidin (36 mg/kg)+Jagged1(1 mg/kg) group by random number table method,with 12 mice in each group. Corresponding doses of drugs were administrated for intervention,and the control group and model group were administrated with the same amount of saline.The bronchoalveolar lavage fluid (BALF) samples were collected and alveolar macrophages were isolated.ELISA was employed to detect the levels of interleukin (IL)-4,IL-6,tumor necrosis factor-α (TNF-α),and IL-10 in BALF,and flow cytometry to detect the M1/M2 polarization of macrophages.qRT-PCR and Western blotting were respectively conducted to detect the mRNA and protein levels of inducible nitric oxide synthase (iNOS),arginase 1 (Arg-1),Jagged1,and Notch1 in the lung tissue. Results Compared with the control group,the modeling of RSV-induced bronchiolitis elevated the IL-4,IL-6,and TNF-α levels,increased the proportion of M1-type macrophages and the lung inflammation and mucus secretion scores,and up-regulated the mRNA and protein levels of iNOS,Jagged1,and Notch1 in BALF (all P<0.001).Meanwhile,the modeling lowered the IL-10 level,decreased the proportion of M2-type macrophages,and down-regulated the mRNA and protein levels of Arg-1 (all P<0.001).Compared with the model group,low- and high-dose hesperidin lowered the IL-4,IL-6,TNF-α levels,decreased the proportion of M1-type macrophages and the lung inflammation and mucus secretion scores,and down-regulated the mRNA and protein levels of iNOS,Jagged1,and Notch1 in BALF (all P<0.05).Moreover,hesperidin elevated the IL-10 level,increased the proportion of M2-type macrophages,and up-regulated the mRNA and protein levels of Arg-1 (all P<0.001).Using recombinant Jagged1 protein to activate Notch1 signaling pathway can significantly attenuate the promotion of high-dose hesperidin on M2 macrophage polarization and amelioration of lung inflammation damage (all P<0.01). Conclusion Hesperidin may alleviate the lung inflammation damage in mice with RSV-induced bronchiolitis by inhibiting the Jagged1/Notch1 signaling pathway and promoting the M2-type polarization of macrophages.

LncRNA H19 alleviates sepsis-induced acute lung injury by regulating the miR-107/TGFBR3 axis

ObjectiveAcute lung injury (ALI) increases sepsis morbidity and mortality. LncRNA H19 plays a critical role in sepsis. miR-107 is highly-expressed and TGFβ type III receptor (TGFBR3) is poorly-expressed in sepsis, yet their roles in sepsis development require further investigation. This study aimed to investigate the mechanism of H19 in alleviating sepsis-induced ALI through the miR-107/TGFBR3 axis.MethodsMice were intravenously injected with Ad-H19 adenovirus vector or control vector one week before establishing the mouse model of cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with oe-H19 or oe-NC plasmids and then stimulated by lipopolysaccharide (LPS). Lung injury was assessed via hematoxylin–eosin staining, measurement of wet-to-dry (W/D) ratio, and TUNEL staining. Levels of H19, miR-107, and TGFBR3 were determined by RT-qPCR. Apoptosis of PMVECs was evaluated by flow cytometry. Levels of Bax and Bcl-2 in lung tissues and PMVECs were measured using Western blot. Total protein concentration and the number of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) were quantified. Levels of TNF-α, IL-1β, IL-6, and IL-10 in BALF, lung tissues, and PMVECs were measured by ELISA. Cross-linking relationships among H19, miR-107 and TGFBR3 were verified by dual-luciferase and RIP assays.ResultsH19 was poorly-expressed in CLP-operated mice. H19 overexpression attenuated sepsis-induced ALI, which was manifested with complete alveolar structure, decreased lung injury score and lung W/D ratio, and inhibited apoptosis in CLP-operated mice, which was manifested with decreased number of TUNEL-positive cells and Bax level and increased Bcl-2 level. CLP-operated mice had increased concentration of total protein and number of total cells, neutrophils, and macrophages in BALF, which was nullified by H19 overexpression. H19 overexpression declined levels of TNF-α, IL-1β, and IL-6 and elevated IL-10 levels. H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production. H19 targeted TGFBR3 as the ceRNA of miR-107. miR-107 overexpression or silencing TGFBR3 partially averted the inhibition of H19 overexpression on LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production.ConclusionLncRNA H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production and attenuated sepsis-induced ALI by targeting TGFBR3 as the ceRNA of miR-107.

Interleukin-6 in blood and bronchoalveolar lavage fluid of hospitalized children with community-acquired pneumonia.

BackgroundHost biomarkers and cytokines help in the prediction of disease severity in adults with community-acquired pneumonia (CAP). Accurate assessment of pathogens and disease severity is essential to clinical decision-making. There are few validated prognostic tools in blood and bronchoalveolar lavage for children with CAP to assist with proper decision and management.MethodsWe performed a retrospective study of 118 children under 18 years of age, hospitalized for CAP with bronchoalveolar lavage management within the first 2 days. The primary outcome was disease severity: mild (with no complications), moderate (with mild to moderate complications), and severe (with severe complications). Comparison and performance analysis of biomarkers and cytokines in the blood or bronchoalveolar lavage fluid (BALF) across different severity categories/different pathogens were performed.ResultsAnalysis of 118 CAP cases revealed significant differences in the BALF levels of IL-6 (p = 0.000), CRP (p = 0.001), and ESR (p = 0.004) across different severity categories, while BALF IL-6 level was indicated as the best indicator to discriminate mild from moderate-to-severe cases with highest AUC (0.847, 95% CI: 0.748–0.946), fair sensitivity (0.839), and specificity (0.450), and severe from non-severe cases with highest AUC (0.847), sensitivity (0.917), and specificity (0.725). ALL biomarkers and cytokines exhibited no significant differences across different pathogen categories (p > 0.05), while BALF IL-6 (p = 0.000), blood ANC (p = 0.028), and ESR (p = 0.024) levels were obviously different in comparison to single Mycoplasma pneumoniae (MP)-, bacteria-, or virus-positive group vs. non-group. Blood CRP (r = 0.683, p = 0.000) and ESR (r = 0.512, p = 0.000) levels revealed significant correlation with the hospitalization course (HC). Among all the BALF cytokines, only BALF IL-6 showed a significant difference (p = 0.004, p < 0.01) across different severity categories, with good performance for predicting CAP severity in hospitalized children (AUC = 0.875, P = 0.004). Blood IL-6 and BALF IL-6 levels showed no significant correlation; in addition, BALF IL6 was better at predicting CAP severity in hospitalized children (AUC = 0.851, p = 0.011, p < 0.05) compared to blood IL-6.ConclusionBALF IL-6 and blood CRP levels, and ESR may have the ability for discriminating disease severity in hospitalized children with CAP, whereas WBC count and ANC have limited ability. No biomarkers or cytokines seemed to have the ability to predict the pathogen category, while BALF IL-6, blood ANC, and ESR may assist in the diagnosis of single MP, bacteria, and virus infections, respectively.

Taraxasterol Inhibits Hyperactivation of Macrophages to Alleviate the Sepsis-induced Inflammatory Response of ARDS Rats.

To explore the effect and mechanism of taraxasterol on sepsis-induced acute respiratory distress syndrome (ARDS). Twenty-four male SD rats were randomly divided into four groups: the control group, model (lipopolysaccharide, LPS) group, lipopolysaccharide+taraxasterol (LPS + TXL) group, and lipopolysaccharide+ulinastatin (LPS + UTI) group. The model of sepsis-induced ARDS was established by intraperitoneal injection of LPS. The lung water content of the rats in each group was determined by the dry/wet ratio. Pathology of rat lung tissue was observed through H&E staining. Wright staining was applied to count the number of neutrophils, macrophages, and total cells. ELISA was utilized to measure the levels of the inflammatory factors TNF-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid (BALF). Biochemical detection was adopted to check the levels of myeloperoxidase (MPO), superoxide dismutase (SOD) and catalase (CAT) in lung tissue. Western blotting was performed to check the protein expression of IL-12, iNOS, Arg-1, and Mrc1 in lung tissue. Compared with the LPS group, both taraxasterol and ulinastatin significantly decreased lung tissue water content, improved lung tissue injury, reduced the number of neutrophils, macrophages and total cells, and decreased the level of inflammatory factors. In addition, taraxasterol and ulinastatin also reduced the content of MPO and the expression of IL-12 and iNOS and increased the activity of SOD and CAT as well as the protein expression of Arg-1 and Mrc1. Taraxasterol can suppress macrophage M1 polarization to alleviate the inflammatory response and oxidative stress, thereby treating sepsis-induced ARDS.

Integrated plasma pharmacochemistry and network pharmacology to explore the mechanism of Gerberae Piloselloidis Herba in treatment of allergic asthma

Ethnopharmacological relevanceGerberae Piloselloidis Herba (GPH), a commonly used traditional medicine in China, is derived from Gerbera piloselloides (Linn.) Cass. It is featured by its special bioactivities as antitussive, expectorant, anti-asthma, anti-bacterial, anti-tumor, uterine analgesia, and immunity-enhancing. With a long history of medication in ethnic minority areas in China, it is often used as an effective treatment for cough and sore throat as well as allergic asthma. Although our previous investigation also has discovered GPH performed effective treatment on allergic asthma, its underlying mechanism remains unclear. Aim of the studyThis research aims to reveal the pharmacological mechanism of GPH in the treatment for allergic asthma through combination of plasma pharmacology and network pharmacology. Materials and methodsFirstly, the components of GPH in blood samples were identified using UHPLC- Q-Orbitrap HRMS. An interaction network of "compound-target-disease" was constructed based on the compounds confirmed in blood and on their corresponding targets of allergic asthma acquired from disease gene databases, predicting the possible biological targets and potential signal pathways of GPH with the network pharmacology analysis. Then, a molecular docking between the blood ingredients and the core targets was carried out using the Autodock Vina software. Subsequently, after establishing a mouse model with allergic asthma induced by ovalbumin (OVA), the effect of GPH on allergic asthma was evaluated by analyzing a series of indicators including behavior, lung pathological changes, inflammatory factors in serum and bronchoalveolar lavage fluid (BALF). Finally, the key pathway and targets predicted by network pharmacology and molecular docking were further verified using Western blot analysis. ResultsEleven chemical constituents (such as arbutin, neochlorogenic acid, chlorogenic acid, etc.) were identified through the analysis of plasma samples, on which basis a total of 142 genes intersecting GPH and allergic asthma were collected by network pharmacology. After performing enrichment analysis of these genes in gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG), it was found that arbutin-related targets mainly focused on phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signal pathway, while luteolin and marmesin -related targets tended to locate at Interleukin-17 (IL-17) signal pathway. Meanwhile, the findings of molecular docking suggested that such components as arbutin, luteolin and marmesin entering into blood had good binding with the core targets related to PI3K/Akt and IL-17 pathways. In addition, GPH improved the OVA-induced asthma symptoms, the alveolar septa thickening and the infiltration of inflammatory cell around bronchi and bronchioles as well as reduced the levels of IgE, IL-8 and TNF-α in serum or BALF. Furthermore, GPH could inhibit the phosphorylation level of Akt and the expression of PI3K, an efficacy supported by the findings by way of Western blot which suggests that GPH in the treatment of allergic asthma was linked to PI3K/Akt signal pathway. ConclusionIn this study, a comprehensive strategy to combine the UPLC-Q-Orbitrap HRMS with network pharmacology was employed to clarify the mechanism of GPH against allergic asthma, a finding where GPH may inhibit PI3K/Akt signal pathway to protect mice from OVA-induced allergic asthma. This study provides a deeper understanding of the pharmacological mechanism of GPH in treatment of asthma, offering a scientific reference for further research and clinical application of GPH in terms of allergic asthma.

Effect and Mechanism of Running on Mouse Emphysema Model Induced by Cigarette Extract

<bold>Objective</bold> To observe the effect of running on mouse emphysema model induced by cigarette extract, and to explore its mechanism based on signal transducer and activator of transcription 3 (STAT3). <bold>Methods</bold> A total of 40 male C57BL/6 mice were randomly divided into control group, exercise group, emphysema group and emphysema + exercise group, with 10 mice in each group. The control group and the exercise group were intraperitoneally injected with 0.3 mL phosphate buffer saline (PBS) at the 0, 11th and 22th day in the experiment; the emphysema group and the emphysema + exercise group were intraperitoneally injected with 0.3 mL cigarette extract at the 0, 11th and 22th day in the experiment. The control group and the emphysema group were free to move in the cage from the 23rd to 29th day; the exercise group and the emphysema + exercise group received aerobic running training from the 23rd day to the 29th day (15 m/min, 30 minutes a day). Hematoxylin eosin (HE) staining was used to observe the pathological changes of lung tissue and evaluate the severity of emphysema in the four groups; the expression levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid (BAL) and serum were analyzed to evaluate the pulmonary and systemic inflammation of the four groups; Western blot method was used to analyze the expression of STAT3 in lung tissues. <bold>Results</bold> 1) Severity of emphysema: compared with the control group, the alveolar chord length of mice in the emphysema group and the emphysema + exercise group significantly increased, the difference was statistically significant (<italic>P</italic>&lt;0.05). Compared with the emphysema group, the alveolar chord length in the emphysema + exercise group significantly decreased，the difference was statistically significant (<italic>P</italic>&lt;0.05). 2) Lung inflammation: compared with the control group, the contents of white blood cells, neutrophils and lymphocytes and the expression levels of IL-1β, IL-6, and IL-17 in BAL of the emphysema group and the emphysema + exercise group significantly increased, while the IL-10 level significantly decreased (<italic>P</italic>&lt;0.05). Compared with the emphysema group, the counts of white blood cells, neutrophils and lymphocytes and the expression levels of IL-1β, IL-6, and IL-17 in BAL of the emphysema + exercise group were statistically lower, and the IL-10 level was statistically higher, the difference was statistically significant (<italic>P</italic>&lt;0.05). 3) Systemic inflammation: compared with the control group, the counts of white blood cells, neutrophils and lymphocytes in the emphysema group statistically increased (<italic>P</italic>&lt;0.05), the expression levels of IL-1β, IL-17, and TNF-α in the emphysema group and the emphysema + exercise group statistically increased, and the IL-10 level statistically decreased (<italic>P</italic>&lt;0.05). Compared with emphysema group, the counts of white blood cells, neutrophils and lymphocytes and the expression levels of IL-1β, IL-17, and TNF-α in the emphysema + exercise group were lower, and the IL-10 level was statistically higher, the difference was statistically significant (<italic>P</italic>&lt;0.05). 4) STAT3 expression level in lung tissues: compared with the control group, p-STAT3 expression level in lung tissues of the emphysema group and the emphysema + exercise group statistically increased (<italic>P</italic>&lt;0.05). Compared with the emphysema group, the expression level of p-STAT3 in lung tissues of the emphysema + exercise group statistically decreased, the difference was statistically significant (<italic>P</italic>&lt;0.05). <bold>Conclusion</bold> Running could reduce the pulmonary and systemic inflammation, and alleviate the pathological manifestations of emphysema in the mice model of emphysema, and its mechanism may be related to the inhibition of STAT 3 activation.

Suppression of airway allergic eosinophilia by Hp-TGM, a helminth mimic of TGF-β.

Type 2-high asthma is a chronic inflammatory disease of the airways which is increasingly prevalent in countries where helminth parasite infections are rare, and characterized by T helper 2 (Th2)-dependent accumulation of eosinophils in the lungs. Regulatory cytokines such as TGF-β can restrain inflammatory reactions, dampen allergic Th2 responses, and control eosinophil activation. The murine helminth parasite Heligmosomoides polygyrus releases a TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β despite bearing no structural similarity to the mammalian protein. Here, we investigated if Hp-TGM could alleviate allergic airway inflammation in mice exposed to Alternaria alternata allergen, house dust mite (HDM) extract or alum-adjuvanted ovalbumin protein (OVA). Intranasal administration of Hp-TGM during Alternaria exposure sharply reduced airway and lung tissue eosinophilia along with bronchoalveolar lavage fluid IL-5 and lung IL-33 cytokine levels at 24 h. The protective effect of Hp-TGM on airway eosinophilia was also obtained in the longer T-cell mediated models of HDM or OVA sensitisation with significant inhibition of eotaxin-1, IL-4 and IL-13 responses depending on the model and time-point. Hp-TGM was also protective when administered parenterally either when given at the time of allergic sensitisation or during airway allergen challenge. This project has taken the first steps in identifying the role of Hp-TGM in allergic asthma and highlighted its ability to control lung inflammation and allergic pathology. Future research will investigate the mode of action of Hp-TGM against airway allergic eosinophilia, and further explore its potential to be developed as a biotherapeutic in allergic asthma.

Effect of Agmatine on a mouse model of allergic airway inflammation: A comparative study

Introduction Asthma is a chronic lung disease that injures and constricts the airways. This study evaluates the effects of agmatine on ovalbumin (OVA)-induced allergic inflammation of the airways. Methods OVA sensitization by intraperitoneal injection was used to induce airway inflammation in mice on days 0 and 7; then the mice were challenged using beclomethasone (150 µg/kg, inhalation), a standard anti-asthmatic drug, from day 14 to day 16. Furthermore, agmatine (200 mg/kg) was intraperitoneally injected on day 0 and then daily for 16 days, followed by OVA challenge. The lung weight ratio, total and differential cell counts, TNF-α, interleukin-5 (IL-5) and IL-13 in bronchoalveolar lavage fluid (BALF), lung nitrite/nitrate (NO), and oxidative parameters were determined. Moreover, histopathological and immunohistochemical staining was employed. Results Injection of agmatine (200 mg/kg) for 16 days significantly attenuated inflammation of the airways. The levels of BALF inflammatory cells, TNF-α, IL-5, IL-13, lung NO, and malondialdehyde (MDA), significantly decreased with concomitant elevation of superoxide dismutase (SOD) levels. Histological and immunohistochemical analyses of mast cells paralleled to biochemical improvements. Conclusion Finally, this study illustrated that agmatine attenuates the allergic inflammation of airways caused by OVA by mitigating cytokines release, NO expression, and oxidative stress.

Increased Oxidative Stress in Asthma-Relation to Inflammatory Blood and Lung Biomarkers and Airway Remodeling Indices.

Airway inflammation in asthma is related to increased reactive oxygen species generation, potentially leading to tissue injury and subsequent airway remodeling. We evaluated oxidative stress in peripheral blood from asthmatic subjects (n = 74) and matched controls (n = 65), using recently developed real-time monitoring of the protein hydroperoxide (HP) formation by the coumarin boronic acid (CBA) assay. We also investigated the relation of the systemic oxidative stress response in asthma to disease severity, lung function, airway remodeling indices (lung computed tomography and histology), and blood and bronchoalveolar lavage fluid (BAL) inflammatory biomarkers. We documented enhanced systemic oxidative stress in asthma, reflected by 35% faster and 58% higher cumulative fluorescent product generation in the CBA assay (p < 0.001 for both). The dynamics of HP generation correlated inversely with lung function but not with asthma severity or histological measures of airway remodeling. HP generation was associated positively with inflammatory indices in the blood (e.g., C-reactive protein) and BAL (e.g., interleukin [IL]-6, IL-12p70, and neutrophil count). Bronchial obstruction, thicker airway walls, increased BAL IL-6, and citrullinated histone 3 in systemic circulation independently determined increased HP formation. In conclusion, a real-time CBA assay showed increased systemic HP generation in asthma. In addition, it was associated with inflammatory biomarkers, suggesting that proper disease control can also lead to a decrease in oxidative stress.

Processed product (Pinelliae Rhizoma Praeparatum) of Pinellia ternata (Thunb.) Breit. Alleviates the allergic airway inflammation of cold phlegm via regulation of PKC/EGFR/MAPK/PI3K-AKT signaling pathway

Ethnopharmacological relevancePinelliae Rhizoma Praeparatum (PRP) is a traditional processed product of Pinellia ternata (Thunb.) Berit., which mainly used for treating cold asthma (CA). However, the mechanism of action of PRP for treating CA have not been fully elucidated. Aim of the studyTo investigate the core active constituents and the pharmacological mechanism of PRP against CA. Materials and methodsOvalbumin (OVA) and cold water-induced cold asthma model were established in male mice. The effects of water extract from PRP were evaluated by general morphological observation, expectorant activity, airway hyperresponsiveness, mucus hypersecretion, inflammatory cytokines, etc. Additionally, the mRNA and protein expression of mucin 5AC (MUC5AC) and aquaporin 5 (AQP5) in vivo and in vitro were detected by immunohistochemistry (IHC), qRT-PCR, and western blotting. The mechanisms of action were investigated through network pharmacology and transcriptomic, and validated through western blotting and molecular docking. ResultsPRP exhibited a favorable expectorant activity, and significantly reduced the airway inflammation, mucus secretion, and hyperresponsiveness in cold asthma model. It also reduced the levels of IL-4, IL-5, IL-8, and IL-13 in bronchoalveolar lavage fluid (BALF) and IL-4 and total IgE in serum, while obviously increased the levels of IL-10 and IFN-γ in serum for asthmatic mice. Meanwhile, PRP also attenuated the pathological changes and mucus production in cold asthmatic mice. Moreover, the downregulation of MUC5AC and upregulation of AQP 5 were detected by western blotting and qRT-PCR after administration with PRP both in vivo and in vitro. PRP expectedly inhibited the protein expression of PKC-α, SRC, p-EGFR, p-ERK1/2, p-JNK, p-p38, p-PI3K, and p-Akt levels in vivo. ConclusionsThese combined data showed that PRP suppressed the allergic airway inflammation of CA by regulating the balance of Th1 and Th2 cytokines and the possible involvement of the PKC/EGFR/MAPK/PI3K-Akt signaling pathway. Pentadecanoic acid, licochalcone A, β-sitosterol, etc. were considered as main active ingredients of PRP against CA. This study provides a novel perspective of the classical herbal processed product PRP in the treatment of CA.

Shp2 in Alveolar Macrophages Regulates Macrophage I Phenotype in Acute Lung Injury.

Macrophage play important role in acute lung injury (ALI). This study aims to explore the possible role of Shp2 in regulating macrophage 1 (M1) in ALI progression. ALI was induced in rats by intravenous injection of lipopolysacharide (LPS). Lentivirus was used to knock down Shp2 expression. Lungs from LPS-induced ALI rats were evaluated by H&amp;E staining and wet/dry lung weight ratio (W/D ratio) measurement. The expression of inflammatory cytokines IL-1β, TNF-α and IL-6 in bronchoalveolar lavage fluid were detected by ELISA. The expressions of M1 biomarker (iNOS) and macrophage 2 (M2) biomarker (Arg-1) in lung tissues and macrophages were measured by immunofluorescence and western blot. The ratio of M2/M1 was detected by flow cytometry. Inflammatory cytokines were highly expressed in ALI rat models, in which elevated expression of iNOS and decreased Arg-1 expression were detected. Shp2 was found to be highly expressed in lung tissues of ALI rat models. LPS treatment in NR8383 cells lead to increased M1 phenotype and elevated expression of Shp2. Suppression on Shp2 expression can counteract the LPS-induced effect and further attenuate ALI progression. Evidence collected from ALI rat and cell models showed that suppression Shp2 expression in macrophages can inhibit M1 phenotype to attenuate ALI progression.

Paeoniflorin Can Improve Acute Lung Injury Caused by Severe Acute Pancreatitis through Nrf2/ARE Pathway.

Objective To evaluate the potential therapeutic effect of paeoniflorin on acute lung injury induced by severe acute pancreatitis (SAP) and to initially explore the possible protective mechanisms of paeoniflorin. Method The SAP lung injury rat model was established by retrograde injection of 5% sodium taurocholate to the cholangiopancreatic duct. H&E staining was used to detect pathological changes in rat lung tissue. W/D ratio method, serum amylase (AMY), and lipase activity were used to assess the degree of lung injury in rats. Oxidation indicators such as LDH, MDA, and SOD in lung tissue were measured. Levels of inflammatory factors TNF-α, IL-6, and IL-10 were measured in bronchoalveolar lavage fluid (BALF). At the same time, Western blot was used to detect the expression of related proteins in the Nrf2/ARE signaling pathway. Results In SAP rats, paeoniflorin treatment could significantly alleviate lung injury conditions such as pulmonary edema and inflammatory cell infiltration in lung tissue and reduce serum amylase and lipase activities. Paeoniflorin can reduce the content of LDH and MDA in lung tissue and increase the content of SOD. In addition, ELISA results showed that paeoniflorin could inhibit the levels of TNF-α and IL-6 in BALF and upregulate the levels of IL-10. Paeoniflorin could upregulate the expression of Nrf2/ARE signaling pathway proteins Cyt-Nrf2, HO-1, and NQO1 in lung tissue of SAP rats. Conclusion Paeoniflorin may improve acute lung injury in rats with severe pancreatitis by inhibiting inflammation and oxidative stress response. These effects may be related to activating the Nrf2/ARE signaling pathway.

G9a promotes inflammation in Streptococcus pneumoniae induced pneumonia mice by stimulating M1 macrophage polarization and H3K9me2 methylation in FOXP1 promoter region

BackgroundStreptococcus pneumoniae has become a leading cause of pneumonia in recent years. Here, we investigated the mechanism of histone methylase G9a in Streptococcus pneumoniae-induced pneumonia (Spn).MethodsG9a expression in Spn mouse tissue was measured. G9a lentivirus interference vector was injected into Spn mice to evaluate the wet and dry weight of the right upper lobe and the total lung water content (TLW) and wet/dry ratio (W/D). The number of neutrophils, macrophages, and lymphocytes in bronchoalveolar lavage fluid (BALF) was detected, and the levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and IL-10 in BALF were assessed. The expressions of M1 and M2 macrophage markers were also detected. The enrichment of histone 3 lysine 9 dimethylation (H3K9me2) in the Forkhead Box P1 (FOXP1) promoter was detected by chromatin immunoprecipitation (ChIP) assay, and the transcription level of FOXP1 was detected. Mouse macrophage RAW264.7 was induced by lipopolysaccharide (LPS) following G9a interference.ResultsG9a in the lung tissue of Spn mice was increased. After G9a knockdown, the mouse weight increased, the infiltration of inflammatory cells was decreased, levels of pro-inflammatory cytokines in BALF were decreased, CD86 and inducible nitric oxide synthase (iNOS) were decreased, and CD206 and arginase-1 (Arg-1) were elevated. In LPS-induced RAW264.7, G9a inhibited macrophage polarization to M1 and promoted macrophage polarization to M2. G9a promoted H3K9me2 methylation in the FOXP1 promoter region and inhibited its transcription, while FOXP1 downregulation reversed the inhibition of G9a knockdown on macrophage polarization to M1 and the inflammatory effect on Spn mice.ConclusionsG9a promotes M1 polarization of macrophages by promoting H3K9me2 methylation in the FOXP1 promoter region, promoting an inflammatory response in Spn mice.

Respiratory MUC5B disproportion is involved in severe community-acquired pneumonia

BackgroundMucus production is a process involved in the pathogenesis of Community-acquired pneumonia (CAP). The study is to determine Mucin 5B (MUC5B) protein concentration and its proportion in the bronchoalveolar lavage fluid (BALF) of CAP patients and evaluate its value to help assess disease severity.MethodsA total of 118 patients were enrolled in this cross-sectional study, including 45 with severe CAP (SCAP) and 73 with non-severe CAP (NSCAP). MUC5B concentration in BALF were determined by immunoblotting analysis. Total protein concentration of BALF was detected by Pierce BCA kit. Cytokines IL6, IL10, IFNγ, IL13, and IL17 in BALF were measured using commercial enzyme-linked immunosorbent assay (ELISA). Spearman’s correlation analysis was applied to evaluate the relationships between MUC5B concentration or MUC5B/total protein ratio and the CURB-65 score, as well as cytokines. Logistic regression analysis was used to identify the independent factors associated with severe CAP. Receiver operating characteristic (ROC) curve was used to evaluate the assessment value of MUC5B/total protein ratio and other indexes for CAP severity.ResultsMUC5B concentration in the BALF of NSCAP group was higher than that in SCAP group [NSCAP 13.56 µg/ml (IQR 5.92–25.79) vs. SCAP 8.20 µg/ml (IQR 4.97–14.03), p = 0.011]. The total protein concentration in the BALF of NSCAP group was lower than that in SCAP group [NSCAP 0.38 mg/ml (IQR 0.15–1.10) vs. SCAP 0.68 mg/ml (IQR 0.46–1.69), p = 0.002]. The MUC5B/total protein ratio was remarkably higher in NSCAP group than that in SCAP groups [NSCAP 3.66% (IQR 1.50–5.56%) vs. SCAP 1.38% (IQR 0.73–1.76%), p < 0.001]. MUC5B/total protein ratio was negatively correlated with total protein concentration (rs = − 0.576, p < 0.001), IL6 (rs = − 0.312, p = 0.001), IL10 (rs = − 0.228, p = 0.013), IL13 (rs = − 0.183, p = 0.048), IL17 (rs = − 0.282, p = 0.002) and CURB-65 score (rs = − 0.239, p = 0.009). Logistic regression identified that MUC5B/total protein ratio, IL6 level and CURB-65 score as independent variables related to CAP severity. ROC curve demonstrated best assessment value of MUC5B/total protein ratio for SCAP (AUC 0.803, p < 0.001), with a sensitivity of 88.9% and a specificity of 64.4%.ConclusionsRespiratory MUC5B disproportion is related to CAP severity. MUC5B/total protein ratio may serve as an assessment marker and a potential therapeutic target for severe CAP.

Delicate regulation of IL-1β-mediated inflammation by cyclophilin A.

The inflammatory response is tightly regulated, but its regulatory principles are still incompletely understood. Cyclophilin A (CypA) has long been considered as a pro-inflammatory factor. Here, we discover how CypA precisely regulates interleukin-1β (IL-1β)-mediated inflammatory responses. In lipopolysaccharide-treated mice, CypA deficiency initially inhibits and then promotes lung inflammation, which is closely related to IL-1β production. Mechanistically, CypA not only facilitates pro-IL-1β processing by increasing Smurf1-mediated K63-linked ubiquitination in an ATP-dependent manner but also accelerates pro-IL-1β degradation, depending on Smurf1-mediated K48-linked ubiquitination. Moreover, in IL-1β-treated mice, CypA exacerbates lung injury by enhancing cytokine production. It also upregulates the ILK/AKT pathway by inhibiting Cyld-mediated K63-linked ILK deubiquitination, which promotes the epithelial-mesenchymal transition (EMT) to facilitate lung repair. Collectively, CypA promotes inflammation activation by increasing IL-1β production and then promotes inflammation resolution by enhancing redundant pro-IL-1β degradation and IL-1β-induced EMT, indicating the complex and delicate regulation of inflammatory response.

DNA Methyltransferase 3b in Myeloid Cells Does Not Affect the Acute Immune Response in the Airways during Pseudomonas Pneumonia.

DNA methyltransferase 3b (Dnmt3b) has been suggested to play a role in the host immune response during bacterial infection. Neutrophils and other myeloid cells are crucial for lung defense against Pseudomonas (P.) aeruginosa infection. This study aimed to investigate the role of Dnmt3b in neutrophils and myeloid cells during acute pneumonia caused by P. aeruginosa. Neutrophil-specific (Dnmt3bfl/flMrp8Cre) or myeloid cell-specific (Dnmt3bfl/flLysMCre) Dnmt3b-deficient mice and littermate control mice were infected with P. aeruginosa PAK via the airways. Bacteria burdens, neutrophil recruitment, and activation (CD11b expression, myeloperoxidase, and elastase levels), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) were measured in bronchoalveolar lavage fluid (BALF) at 6 and 24 h after infection. Our data showed that the bacterial loads and neutrophil recruitment and activation did not differ in BALF obtained from neutrophil-specific Dnmt3b-deficient and control mice, whilst BALF IL-6 and TNF levels were lower in the former group at 24 but not at 6 h after infection. None of the host response parameters measured differed between myeloid cell-specific Dnmt3b-deficient and control mice. In conclusion, dnmt3b deficiency in neutrophils or myeloid cells does not affect acute immune responses in the airways during Pseudomonas pneumonia.

Glibenclamide Alleviates LPS-Induced Acute Lung Injury through NLRP3 Inflammasome Signaling Pathway

Glibenclamide displays an anti-inflammatory response in various pulmonary diseases, but its exact role in lipopolysaccharide- (LPS-) induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) remains unknown. Herein, we aimed to explore the effect of glibenclamide in vivo and in vitro on the development of LPS-induced ALI in a mouse model. LPS stimulation resulted in increases in lung injury score, wet/dry ratio, and capillary permeability in lungs, as well as in total protein concentration, inflammatory cells, and inflammatory cytokines including IL-1β, IL-18 in bronchoalveolar lavage fluid (BALF), and lung tissues, whereas glibenclamide treatment reduced these changes. Meanwhile, the increased proteins of NLRP3 and Caspase-1/p20 after LPS instillation in lungs were downregulated by glibenclamide. Similarly, in vitro experiments also found that glibenclamide administration inhibited the LPS-induced upregulations in cytokine secretions of IL-1β and IL-18, as well as in the expression of components in NLRP3 inflammasome in mouse peritoneal macrophages. Of note, glibenclamide had no effect on the secretion of TNF-α in vivo nor in vitro, implicating that its anti-inflammatory effect is relatively specific to NLRP3 inflammasome. In conclusion, glibenclamide alleviates the development of LPS-induced ALI in a mouse model via inhibiting the NLRP3/Caspase-1/IL-1β signaling pathway, which might provide a new strategy for the treatment of LPS-induced ALI.

Lung injury caused by aspiration of organophosphorus insecticide and gastric contents in pigs

Introduction Patients who require mechanical ventilation after self-poisoning with ingested organophosphorus (OP) insecticides often die. Aspiration of stomach contents may contribute to lung injury and lethality. This study was designed to assess the severity of direct and indirect pulmonary injury created by pulmonary instillation of mixtures of OP insecticide, solvent (Solv) and porcine gastric juice (GJ) compared to controls. Methods Terminally anaesthetised minipigs (groups n = 5) were exposed to sham bronchoscopy or given mixtures (0.5 mL/kg) of: saline, GJ, OP insecticide and GJ (OP + GJ), or Solv and GJ (Solv + GJ), placed into the right lung, and monitored for 48 h. Lung injury was assessed through analysis of bronchoalveolar lavage fluid (BALF), computed tomography and histopathology. Results OP + GJ created a direct lung injury consisting of neutrophil infiltration, oedema and haemorrhage, as well as indirect injury to the other lung. OP + GJ directly-injured lung parenchyma had increased concentrations of BALF protein, albumin, IL-6, IL-8 and C-reactive protein (CRP) at 24 h (p < 0.05), and BALF protein, albumin and CRP at 48 h (p < 0.05), when compared with controls. Aspiration of GJ produced similar direct effects to OP + GJ but less indirect lung injury. Lung injury was less severe after Solv + GJ, for combined lung histopathology scores (vs. OP + GJ, p < 0.05) and for the proportion of directly-injured lung that was poorly/non-aerated at 48 h. Conclusion Pulmonary instillation of OP + GJ created more lung damage than controls or Solv + GJ. In patients with severe OP insecticide poisoning and reduced consciousness, early airway protection is likely to reduce pulmonary damage.

Mechanism of miR-181a-5p in Regulatory T/T-Helper 17 Immune Imbalance and Asthma Development in Mice with Allergic Rhinitis

Introduction: Allergic rhinitis (AR) is an immune disorder and also a risk factor of asthma. microRNAs (miRNAs) are implicated in autoimmune diseases, including RA. This study investigated effect of miR-181a-5p on regulatory T (Treg)/ T-helper (Th) 17 immune imbalance in AR. Methods: A murine model of AR was established and treated with lentivirus modified miR-181a-5p. The allergic symptoms of mice were examined. The contents of Th17-related cytokines (interferon [IFN]-γ and interleukin [IL]-6), Treg-related cytokine (IL-10), and Treg-specific nuclear transcription factor (Foxp3) in nasal mucosa and lung tissues were determined. The proportion of Treg and Th17 cells was analyzed by flow cytometry. The level of ovalbumin-specific immunoglobulin E in the serum, and the contents of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and IFN-γ, IL-6, and IL-10 in nasal lavage fluid were measured. The targeting relationship between miR-181a-5p and high mobility group box chromosomal protein 1 (HMGB1) was verified. HMGB1 and receptor for advanced glycation end products (RAGE) expression in RA were determined, and the interaction between HMGB1 and RAGE was detected. Results: miR-181a-5p expression was reduced in AR mice. miR-181a-5p overexpression attenuated allergic behaviors, alleviated Treg/Th17 imbalance, and delayed asthma development. HMGB1 and RAGE were elevated in AR mice. miR-181a-5p targeted HMGB1, and HMGB1 bound to RAGE, while miR-181a-5p overexpression reduced the binding between them. Activating HMGB1/RAGE reversed the protective effect of miR-181a-5p overexpression on AR and induced the development of asthma. Conclusion: miR-181a-5p overexpression reduced the binding of HMGB1 and RAGE by inhibiting HMGB1, thus alleviating Treg/Th17 immune imbalance and blocking AR from developing into asthma.

Prenatal and Postnatal Cigarette Smoke Exposure Is Associated With Increased Risk of Exacerbated Allergic Airway Immune Responses: A Preclinical Mouse Model

Increased exposure to household air pollution and ambient air pollution has become one of the world’s major environmental health threats. In developing and developed countries, environmental cigarette smoke (CS) exposure is one of the main sources of household air pollution (HAP). Moreover, results from different epidemiological and experimental studies indicate that there is a strong association between HAP, specifically CS exposure, and the development of allergic diseases that often persists into later life. Here, we investigated the impact of prenatal and postnatal CS exposure on offspring susceptibility to the development of allergic airway responses by using a preclinical mouse model. Pregnant BALB/c mice were exposed to either CS or air during pregnancy and lactation and in order to induce allergic asthma the offspring were sensitized and challenged with house dust mite (HDM). Decreased lung function parameters, like dynamic compliance and pleural pressure, were observed in PBS-treated offspring born to CS-exposed mothers compared to offspring from air-exposed mothers. Maternal CS exposure significantly increased the HDM-induced airway eosinophilia and neutrophilia in the offspring. Prenatal and postnatal CS exposure increased the frequency of Th2 cells in the lungs of HDM-treated offspring compared to offspring born to air-exposed mothers. Offspring born to CS-exposed mothers showed increased levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid compared to offspring from air-exposed mothers. Ex-vivo restimulation of lung cells isolated from HDM-treated offspring born to CS-exposed mothers also resulted in increased IL-4 production. Finally, serum immunoglobulins levels of HDM-specific IgE and HDM-specific IgG1 were significantly increased upon a HDM challenge in offspring born to CS-exposed mothers compared to offspring from air-exposed mothers. In summary, our results reveal a biological plausibility for the epidemiological studies indicating that prenatal and postnatal CS exposure increases the susceptibility of offspring to allergic immune responses.