Study the mechanism of Dingchuan decoction in neutrophilic asthma mice on serum metabolomics / 中国医师杂志

Objective:Based on the method of metabonomics, the intervention effect of Dingchuan decoction on neutrophilic asthma and its possible mechanism were analyzed from the changes of endogenous metabolites.Methods:Forty C57BL/6 mice were randomly divided into five groups: normal group (group A), neutrophilic asthma model group (group B), Dingchuan decoction low dose treatment group (group C), Dingchuan decoction medium dose treatment group (group D), and Dingchuan decoction high dose treatment group (group E), with 8 mice in each group.B/C/D/E group used ovalbumin (OVA) and complete Freund′s adjuvant (CFA) to induce sensitized mice to establish neutrophilic asthma model, and C/D/E group used Dingchuan decoction with different concentrations of crude drugs for intervention treatment.Buxco small animal lung function tester was used to evaluate the airway reactivity of mice; The total number of white blood cells in bronchoalveolar lavage fluid (BALF) was counted by counting plate, and the number was classified by cell smear staining; The pathological changes of lung tissue were observed by hematoxylin and eosin (HE) staining.The serum metabolites of mice in each group were detected by high performance liquid chromatography coupled with four pole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS). The commonly used principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA) models were used for statistical analysis of the metabolite profiles in serum. The intervention effect of Dingchuan decoction and its possible mechanism were reflected from the changes of endogenous metabolites.Results:(1) General behavior observation: except mice in group A, mice in other groups showed asthma symptoms of different degrees during the challenge period. The symptoms of mice in each treatment group (group C, D, E) of Dingchuan decoction were less than those in group B. (2) Airway reactivity: the airway reactivity of mice in group B to methacholine (MCh) increased with the inhalation concentration, and the airway resistance at each concentration of MCh was significantly higher than that in group A (all P<0.01); the airway reactivity in group C, D and E was lower than that in group B (all P<0.01); the airway reactivity in group D and E was lower than that in group C (all P<0.05). There was no significant difference in airway reactivity between group D and E ( P>0.05). (3) Airway inflammatory cell infiltration: the total number of white blood cells (WBC) and percentage of neutrophil in BALF of group B were significantly higher than those of group A (all P<0.01). The total WBC and percentage of neutrophil in group C, D and E were lower than those in group B (all P<0.01). The total number of WBC and percentage of neutrophil in group D and E were lower than those in group C (all P<0.01). There was no significant difference between group D and group E (all P>0.05). (4) Pathological changes of lung tissues: no pathological changes were observed in the lung tissues of group A mice. In group B, typical pathological changes such as bronchial lumen stenosis, intraluminal mucosal folds hyperplasia, epithelial cell exfoliation, swelling, mucous embolus, alveolar and lung tissue structure destruction, massive inflammatory cell infiltration around bronchus and blood vessels were observed, among which neutrophil and lymphocyte infiltration were the most obvious. The damage of lung tissue structure, bronchial mucosa edema and inflammatory cell infiltration in Dingchuan decoction treatment groups were significantly improved compared with group B, and the pathological changes of lung tissue in group D were relatively light. (5) Metabolomics analysis: PCA, PLS-DA and OPLS-DA analysis of serum metabolites in each group showed that serum metabolites in group A and group B were significantly different. The metabolic pathway analysis showed that Dingchuan decoction with different crude drug concentrations could improve the metabolic disorders caused by asthma in different degrees. Conclusions:Dingchuan decoction can effectively reduce airway inflammation and airway hyperresponsiveness in mice with neutrophil asthma, and effectively regulate metabolic abnormalities caused by neutrophil asthma.

The role of neutrophils in asthma / 浙江大学学报·医学版

Bronchial asthma is a chronic respiratory disease，characterized by airway inflammation，airway hyperresponsiveness，reversible airway obstruction and airway remodeling，in which a variety of cells including airway inflammatory cells and structural cells are involved. Previous studies have shown that asthma is mainly driven by Th2 cytokines IL-4，IL-5，and IL-13，leading to airway eosinophil inflammation. With further research，however，it has been found that neutrophils are also closely related to asthma. Numbers of neutrophils are elevated in airway through increased chemotaxis and decreased apoptosis，which is earlier than eosinophils，leading to airway neutrophilic inflammation. Neutrophils can produce elastase，myeloperoxidase，neutrophil extra- cellular traps，chemokines and cytokines，participating in the occurrence and development of asthma. The antagonists against these molecules，such as anti-IL-8 receptor antibody，anti-IL-17 antibody，and DNase，have shown positive effects on neutrophilic asthma，but further studies are needed to support their clinical application. This article mainly reviews the role of neutrophils in asthma and related mechanisms.

Caracterización clínica e inmunológica del Asma Neutrofílica / Clinical and immunological characterization of Neutrophilic asthma

RESUMEN Introducción: El asma bronquial es una enfermedad mucho más compleja de lo que inicialmente se consideraba, es multifactorial y se manifiesta en diferentes fenotipos clínicos, entre ellos, el asma neutrofílica. Objetivo: Caracterizar clínica e inmunológicamente el asma neutrofílica. Material y Métodos: Se realizó una revisión bibliográfica actualizada. Se empleó el motor de búsqueda Google Académico y se consultaron artículos de libre acceso en las bases de datos Pubmed y Scielo y se revisaron 50 artículos. Desarrollo: Los neutrófilos están presentes en las vías respiratorias del paciente con asma, se activan y pueden liberar mediadores que promueven y prolongan los síntomas del asma, la presencia de neutrófilos puede ser mediada por interleucina 17(IL-17), la hipótesis de cambios en la microbiota, la obesidad y las infecciones, constituyen elementos primordiales en este tipo de enfermedad. Para aliviar los síntomas de estos pacientes, se necesita una terapia dirigida a disminuir la inflamación dominada por neutrófilos. Conclusiones: El asma neutrofílica es una enfermedad crónica fenotípicamente heterogénea de las vías respiratorias, que implica la participación de numerosas células inflamatorias y más de cien mediadores con múltiples efectos inflamatorios, como el broncoespasmo, la exudación plasmática, la hipersecreción de moco y la activación sensorial. Actualmente existen pocas drogas dirigidas contra la inflamación neutrofílica y la mayoría se encuentran en estudio con el objetivo de aliviar y mejorar las condiciones de vida de cada uno de los pacientes que sufren esta enfermedad(AU)

ABSTRACT Introduction: Bronchial asthma is a much more complex disease than initially thought; it is multifactorial and manifests itself in different clinical phenotypes; among them, we can mention neutrophilic asthma. Objective: To characterize neutrophilic asthma from a clinical and immunological point of view. Material and methods: An updated bibliographic review was made. Google Scholar search engine was used; free access articles were consulted in PubMed and SciELO databases and 50 of them were reviewed. Development: Neutrophils are present in the respiratory tract of patients suffering from asthma; once those neutrophils are activated, they can release mediators that promote and prolong the symptoms of the disease. The presence of neutrophils can be mediated by IL-17. The hypothesis on changes in the microbiota, obesity and infections are key points in this kind of disease. To alleviate the symptoms of these patients, targeted therapy for neutrophil-dominated inflammation is needed. Conclusions: Neutrophilic asthma is a phenotypically heterogeneous chronic disease of the respiratory tract which involves numerous inflammatory cells and more than a hundred mediators with multiple inflammatory effects such as bronchospasm, plasma exudation, mucus hypersecretion and sensory activation. Currently, there are few drugs targeting neutrophilic inflammation and most of them are under study with the aim of alleviating and improving the living conditions in patients suffering from this disease(AU)

Metabolomic Analysis of Serum Glycerophospholipid Levels in Eosinophilic and Neutrophilic Asthma / 生物医学与环境科学（英文）

OBJECTIVE@#To compare the serum glycerophospholipid levels in the inflammatory subtypes of asthma by using targeted metabolomic analysis.@*METHODS@#Demographic and clinical data were collected from 51 patients with asthma between January 2015 and December 2015. Routine blood and sputum induction tests were performed. Eosinophilic asthma was defined as induced sputum containing ⪖ 3% eosinophils, and neutrophilic asthma, as induced sputum containing ⪖ 71% neutrophils. Serum metabolic glycerophospholipid profile was determined by liquid chromatography-mass spectrometry. Differences in glycerophospholipid levels between eosinophilic and non-eosinophilic asthma and between neutrophilic and non-neutrophilic asthma were analyzed using partial least squares discriminant analysis.@*RESULTS@#The serum lysophosphatidylglycerol level was significantly higher in the group with ⪖ 3% eosinophils in sputum than in the group with < 3% eosinophils in sputum. The area under the receiver-operating characteristic curve was ⪖ 70%. There was no significant difference in the serum metabolic glycerophospholipid profile between the group with sputum neutrophils ⪖ 71% and the group with sputum neutrophils < 71%.@*CONCLUSION@#Serum lysophosphatidylglycerol is produced abundantly in eosinophilic asthma and may be a biomarker of eosinophilic asthma. This information is helpful for identifying and tailoring treatment for the common asthma subtypes.

Effects of Ligustrazine on Airway Inflammation in A Mouse Model of Neutrophilic Asthma / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of ligustrazine (LTZ) on airway inflammation in a mouse model of neutrophilic asthma (NA).</p><p><b>METHODS</b>Forty healthy C57BL/6 female mice were randomly divided into 4 groups using a random number table, including the normal control, NA, LTZ and dexamethasone (DXM) groups, with 10 rats in each group. The NA mice model was established by the method of ovalbumin combined with lipopolysaccharide sensitization. At 0.5 h before each challenge, LTZ and DXM groups were intraperitoneally injected with LTZ (80 mg/kg) or DXM (0.5 mg/kg) for 14 d, respectively, while the other two groups were given the equal volume of normal saline. After last challenge for 24 h, the aerosol inhalation of methacholine was performed and the airway reactivity was measured. The bronchoalveolar lavage fluid (BALF) was collected. The Wright-Giemsa staining was used for total white blood cells and differential counts. The levels of cytokines interleukin (IL)-17 and IL-10 were detected by enzyme-linked immunosorbent assay. The pathological change of lung tissue was observed by hematoxylin eosin staining.</p><p><b>RESULTS</b>The airway responsiveness of the NA group was signifificantly higher than the normal control group (P<0.05), while those in the LTZ and DXM groups were signifificantly lower than the NA group (P<0.05). The neutrophil and eosinophil counts in the LTZ and DXM groups were signifificantly lower than the NA group (P<0.05), and those in the LTZ group were signifificantly lower than the DXM group (P<0.05). There were a large number of peribronchiolar and perivascular inflammatory cells in fifiltration in the NA group. The airway inflflammation in the LTZ and DXM groups were signifificantly alleviated than the NA group. The infifiltration in the LTZ group was signifificantly reduced than the DXM group. Compared with the normal control group, the IL-17 level in BALF was signifificantly increased and the IL-10 level in BALF was signifificantly decreased in the NA group (P<0.05). LTZ and DXM treatment signifificantly decreased IL-17 levels and increased IL-10 levels compared with the NA group (P<0.05), and the changes in the above indices were more signifificant in the LTZ group (P<0.05).</p><p><b>CONCLUSION</b>LTZ could alleviate the airway inflflammation in the NA mice model through increasing the IL-10 level and decreasing the IL-17 level.</p>

Inhibitory effect of resveratrol on pulmonary inflammation in mice with neutrophilic asth ma and its mechanism / 吉林大学学报(医学版)

Objective; To investigate the inhibitory effect of resveratrol on the pulmonary inflammation in the mice with neutrophilic asthma, and to elucidate its mechanism. Methods; A total of 18 female C57BL/6J mice were selected. The mouse models of neutrophilic asthma were established by sensitization with lipopolysaccharide (LPS) and ovalbumin (OVA). The model mice were randomly divided into model group (LPS + OVA group), resveratrol group (Res group, intragastric administration of 30 mg. kg-1 resveratrol at 2 h before challenge) and N-acetylcysteine group (NAC group, intragastric administration of 3 mmol · kg-1 N-acetylcysteine at 2 h before challenge). Other 6 age-matched female C57BL/6J mice were choosen as normal control group (PBS group). The behavioral changes of mice in various groups were observed during the atomization with methacholine; the airway hyperresponsiveness (AHR) of mice was analyzed with a lung function instrument, and the total number of cells and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were observed with light microscope; the pathological changes of lung tissue were observe by HE staining; the levels of interleukin-17 (IL-17) in the supernatant of BALF of the mice in various groups were detected by enzyme-linked immunosorbent assay (ELISA); the level of reactive oxygen species (ROS) in lung tissue was analyzed by specific fluorescent probe DCF-DA staining; the level of malondialdehyde (MDA) in lung homogenate was measured by MDA kit. Results: Compared with PBS group, the Penh value, total number of cells, inflammatory cells, airway inflammation and scores of the mice in LPS+OVA group were significantly increased (P<0. 05 or P<0. 01); the IL-17 level in BALF supernatant was significantly increased (P<0. 01); the ROS fluorescence intensity in lung tissue was significantly increased, and the MDA level in lung homogenates was significantly increased (P<0. 01). Compared with LPS + OVA group, the Penh value, total number of cells, inflammatory cells, airway inflammation and scores in Res group and NAC group were significantly decreased (P<0. 05 or P<0. 01); the IL-17 levels in BALF supernatant were significantly decreased (P<0. 05 or P<0. 01); the ROS fluorescence intensities in lung tissue were decreased, and the MDA levels in lung homogenates were significantly decreased (P < 0.01). Conclusion: Resveratrol can effectively inhibit the inflammatory response of lung tissue in the mice with neutrophilic asthma, and its mechanism may be related to inhibiting the oxidative stress reaction.

Therapeutic effects of anti-B7-H3 monoclonal antibody in a murine model of neutrophilic asthma / 中华微生物学和免疫学杂志

Objective To investigate the therapeutic effects of anti-B7-H3 blocking monoclonal antibody(McAb) in a murine model of neutrophilic asthma. Methods Twenty-four female BALB/c mice were randomly divided into four groups: PBS control group (group A), neutrophilic asthma group (group B),anti-B7-H3 McAb group (group C) and IgG isotype control group (group D). Those in group A were sensitized by injection of PBS and challenged with PBS through inhalation,while the other mice were sensi-tized by injection of ovalbumin (OVA) plus airway instillation of lipopolysaccharide(LPS),and then chal-lenged with OVA. Moreover,mice in group C and group D were respectively injected with anti-B7-H3 McAb and IgG isotype control in the induction period. Each mouse′s performance was observed. Samples of bron-choalveolar lavage fluid (BALF) and lung tissues were collected. Total and differential cell counts in BALF were determined by using microscope. Levels of cytokines including IFN-γ,IL-4,IL-6,IL-17,tumor nec-rosis factor-α (TNF-α) and granulocyte colony stimulating factor (G-CSF) in BALF were measured by ELISA. Lung sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to identify tissue inflammation and mucus production,respectively. Immunohistochemistry was used to measure the expression of B7-H3 in frozen mouse lung sections. Results Mice in group B and group D showed asth-matic symptoms such as breathlessness,dysphoria and incontinence after nebulization,while these symptoms in group C were alleviated due to anti-B7-H3 McAb treatment. No abnormalities were observed in group A. Compared with group A,the other three groups showed increased total cell count in BALF and higher per-centages of neutrophil and eosinophil(P<0.05). These three indicators in group C were lower than those in group B and group D (P<0.05). With regard to lung infiltration by Th1,Th2 and Th17 cells,the levels of IFN-γ,IL-4,IL-6,IL-17,TNF-α and G-CSF in BALF were increased in group B,group C and group D as compared with those in group A. Compared with group C,group B and group D showed higher levels of IL-6, TNF-α,IL-17 and G-CSF(P<0.05),but lower level of IL-4(P<0.05). No statistical difference in the level of IFN-γ was observed among group B, group C and group D. Histological staining of lung sections showed that no obvious inflammatory cells and mucus secretion was observed in group A. Massive infiltration of inflammatory cells and neutrophils and mucus hypersecretion were detected in group B and group D. Treatment with anti-B7-H3 McAb inhibited the accumulation of neutrophils and mucus hypersecretion in lung tissues of group C. Compared with group A, levels of B7-H3-positive cells were significantly increased in group B and group D. Anti-B7-H3-treated mice showed reduced levels of B7-H3-positive cells in lung tissues as compared with those in group B and group D(P<0.05). Conclusion Treatment with anti-B7-H3 bloc-king McAb in an early stage can relieve the asthmatic syndrome, reduce airway inflammatory cells, inhibit mucus production and down-regulate Th17 cell-related cytokine secretion,which helps to alleviate airway and systematic inflammation in mice with NA,and partially inhibit the development of NA.

Effects of triptolide on neutrophils asthmatic mice of WBC and EOS in bron-choalveolar lavage fluid / 中国免疫学杂志

Objective:To study the influence of triptolide on neutrophils asthmatic mice of WBC and EOS in bronchoalveolar lavage fluid.Methods:Using ovalbumin ( OVA) combined with lipopolysaccharide ( LPS) method to establish sensitized asthmatic mice,BALB/c mice were randomly divided into 32 neutrophilic asthma group ( NA group ) , neutrophil triptolide intervention group (TLN group),neutrophil dexamethasone group (DXN group) and normal control group (NC group),n=8,hemocytometer calculated for each group of mice bronchoalveolar lavage fluid ( BALF) of the total number of WBC and EOS;smears stained Switzerland View in-flammatory cell infiltration.Results: In bronchioalveolar lavage fluid, numbers and infiltrations of WBC and EOS were significantly decreased in the DXN,TLN group than those in the NA group(P<0.05);but were significantly higher than the NC group (P<0.05), the DXN group above parameters were significantly higher than the TLN group ( all P<0.05 ) .Conclusion: Triptolide can reduce the total number of BALF WBC and EOS,inhibit lung WBC,EOS infiltration,ease neutrophilic airway inflammation.