The aim of the present study was to investigate the role of ferroptosis in acute lung injury (ALI) mouse model induced by oleic acid (OA). ALI was induced in the mice via the lateral tail vein injection of pure OA. The histopathological score of lung, lung wet-dry weight ratio and the protein content of bronchoalveolar lavage fluid (BALF) were used as the evaluation indexes of ALI. Iron concentration, glutathione (GSH) and malondialdehyde (MDA) contents in the lung tissues were measured using corresponding assay kits. The ultrastructure of pulmonary cells was observed by transmission electron microscope (TEM), and the expression level of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA was detected by quantitative polymerase chain reaction (q-PCR). Protein expression levels of glutathione peroxidase 4 (GPX4), ferritin and transferrin receptor 1 (TfR1) in lung tissues were determined by Western blot. The results showed that histopathological scores of lung tissues, lung wet-dry weight ratio and protein in BALF in the OA group were higher than those of the control group. In the OA group, the mitochondria of pulmonary cells were shrunken, and the mitochondrial membrane was ruptured. The expression level of PTGS2 mRNA in the OA group was seven folds over that in the control group. Iron overload, GSH depletion and accumulation of MDA were observed in the OA group. Compared with the control group, the protein expression levels of GPX4 and ferritin in lung tissue were down-regulated in the OA group. These results suggest that ferroptosis plays a potential role in the pathogenesis of ALI in our mouse model, which may provide new insights for development of new drugs for ALI.
Acute Lung Injury ; chemically induced ; pathology ; Animals ; Apoptosis ; Bronchoalveolar Lavage Fluid ; chemistry ; Cyclooxygenase 2 ; metabolism ; Ferritins ; metabolism ; Glutathione ; analysis ; Glutathione Peroxidase ; metabolism ; Iron ; analysis ; Iron Overload ; physiopathology ; Lung ; cytology ; pathology ; Malondialdehyde ; analysis ; Mice ; Microscopy, Electron, Transmission ; Mitochondrial Membranes ; ultrastructure ; Oleic Acid

OBJECTIVETo investigate the effects of ligustrazine (LTZ) on airway inflammation in a mouse model of neutrophilic asthma (NA).

METHODSForty 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.

RESULTSThe 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).

CONCLUSIONLTZ could alleviate the airway inflflammation in the NA mice model through increasing the IL-10 level and decreasing the IL-17 level.

Animals ; Asthma ; blood ; complications ; drug therapy ; pathology ; Bronchoalveolar Lavage Fluid ; cytology ; Disease Models, Animal ; Female ; Interleukin-10 ; metabolism ; Interleukin-17 ; metabolism ; Leukocyte Count ; Lung ; drug effects ; pathology ; Mice, Inbred C57BL ; Neutrophils ; drug effects ; pathology ; Pneumonia ; blood ; complications ; drug therapy ; pathology ; Pyrazines ; pharmacology ; therapeutic use ; Respiratory Hypersensitivity ; blood ; complications ; drug therapy ; pathology

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.
Acute Lung Injury/chemically induced/*therapy ; Angiopoietin-1/*genetics ; Animals ; Bronchoalveolar Lavage Fluid ; Cytokines/metabolism ; Endotoxins ; Genetic Therapy ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung/metabolism ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/metabolism ; Neutrophils/metabolism ; Rats ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/*cytology

To study the protective effect and mechanism of synthetic salidroside on acute lung injury (ALI) induced by lipopolysaccharide (LPS), male Sprague-Dawley (SD) rats were randomly divided into saline control group, 3 mg/kg LPS model group, different doses of salidroside groups (5, 20 and 80 mg/kg), and 5 mg/kg dexamethasone group. Intratracheal LPS instillation was used to establish the ALI model 0.5 h after intraperitoneal injection of salidroside or dexamethasone, and the rats were sacrificed 6 h later. Lung wet/dry weight ratio (W/D) was calculated. Lung tissue pathology and lung injury score (LIS) were observed and evaluated through hematoxylin and eosin (HE) staining. The centrifugal sediment of bronchoalveolar lavage fluid (BALF) was used to count the polymorphonuclear leukocyte (PMN) number by Wright's staining, and the centrifugal supernatant of BALF was used to determine the contents of protein and inflammatory factors (TNF-α, IL-1β and IL-6). The contents of myeloperoxidase (MPO) and malondialdehyde (MDA) in lung tissue were determined. Western blot was used to detect the expression levels of phosphorylated and total nuclear factor kappa B (NF-κB)/p65 protein in lung tissue. The results showed that, compared with LPS group, the intervention of synthetic salidroside alleviated the pathological damage in lung tissue, decreased the LIS and lung W/D ratio (P < 0.05), reduced the PMN number, the contents of protein and inflammatory factors in BALF (P < 0.05), reduced the contents of MPO and MDA in lung tissue (P < 0.05), and inhibited the expression of p-NF-κB in lung tissue (P < 0.05). The results suggest that synthetic salidroside has a protective effect on ALI induced by LPS, and its mechanism is related to inhibiting the phosphorylation of NF-κB and reducing the aggregation of PMN in the lung.
Acute Lung Injury ; drug therapy ; Animals ; Bronchoalveolar Lavage Fluid ; Dexamethasone ; pharmacology ; Glucosides ; pharmacology ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; Lung ; drug effects ; pathology ; Male ; Malondialdehyde ; metabolism ; NF-kappa B ; metabolism ; Neutrophils ; cytology ; Peroxidase ; metabolism ; Phenols ; pharmacology ; Phosphorylation ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

To investigate the effects of cigarette smoking in different manners on acute lung injury in rats.The commercially available cigarettes with tar of 1,5, 11 mg were smoked in Canada depth smoking (health canada method, HCM) manner, and those with tar of 11 mg were also smoked in international standard (ISO) smoking manner. Rats were fixed and exposed to mainstream in a manner of nose-mouth exposure. After 28 days, the bronchoalveolar lavage fluids from left lung were collected for counting and classification of inflammatory cells and determination of pro-inflammatory cytokines IL-1β and TNF-α. The right lungs were subjected to histological examination and determination of myeloperoxidase (MPO) and superoxide dismutase (SOD) activities and glutathione, reactive oxygen species (ROS) and malondialdehyde (MDA) levels.In both HCM and ISO manners, the degree of lung injury was closely related to the tar content of cigarettes, and significant decrease in the body weight of rats was observed after smoking for one week. In a HCM manner, smoking with cigarette of 11 mg tar resulted in robust infiltration of macrophages, lymphocytes and neutrophils into lungs, significant increase in IL-1β and TNF-α levels and MPO activities, and significant decrease in GSH levels and SOD activities and increase in ROS and MDA levels (all<0.05). Smoking with cigarette of 5 mg tar led to moderate increase in IL-1β and TNF-α levels, and MPO activities (all<0.05), and moderate decrease in GSH levels and SOD activities and increase of ROS and MDA levels (all<0.05). However, smoking with cigarette of 1 mg tar affected neither inflammatory cell infiltration nor IL-1β and TNF-α levels.Cigarette smoking in nose-mouth exposure manner can induce acute lung injury in rats; and the degree of lung injury is closely related to the content of tar and other hazards in cigarettes.
Acute Lung Injury ; etiology ; pathology ; physiopathology ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Chemotaxis, Leukocyte ; drug effects ; Glutathione ; analysis ; drug effects ; Interleukin-1beta ; analysis ; drug effects ; Lung ; chemistry ; pathology ; Lymphocytes ; drug effects ; pathology ; Macrophages ; drug effects ; pathology ; Male ; Malondialdehyde ; analysis ; Neutrophil Infiltration ; drug effects ; Neutrophils ; drug effects ; pathology ; Peroxidase ; analysis ; drug effects ; Rats ; Reactive Oxygen Species ; analysis ; Smoking ; adverse effects ; Superoxide Dismutase ; analysis ; drug effects ; Tobacco Products ; adverse effects ; classification ; Tumor Necrosis Factor-alpha ; analysis ; drug effects ; Weight Loss ; drug effects

BACKGROUND: Diffuse interstitial lung diseases (DILDs) form a part of a heterogeneous group of respiratory diseases. Bronchoalveolar lavage (BAL) analysis has been used for differential diagnosis of DILDs, but their clinical usefulness is controversial. The aim of this study was to investigate the clinical usefulness of BAL cellular analysis with lymphocyte subsets for the differential diagnosis of DILDs. METHODS: A total of 69 patients diagnosed with DILDs were enrolled. Basic demographic data, BAL cellular analysis with lymphocyte subsets, histology, and high resolution computed tomogram (HRCT) findings were analyzed and compared as per disease subgroup. RESULTS: Significant differences were found between groups in the proportion of neutrophils (P=0.0178), eosinophils (P=0.0003), T cells (P=0.0305), CD4 cells (P=0.0002), CD8 cells (P<0.0001), and CD4/CD8 ratio (P<0.0001). These findings were characteristic features of eosinophilic pneumonia and sarcoidosis. Other parameters were not significantly different between groups. At the cut-off value of 2.16 for sarcoidosis, CD4/CD8 ratio showed sensitivity of 91.7% (95% CI, 61.5-98.6%) and specificity of 84.2% (95% CI, 72.1-92.5%). CONCLUSIONS: Routine analysis of BAL lymphocyte subset may not provide any additional benefit for differential diagnosis of DILDs, except for conditions where BAL is specifically indicated, such as eosinophilic pneumonia or sarcoidosis.
Aged ; Aged, 80 and over ; Area Under Curve ; Bronchoalveolar Lavage Fluid/*cytology ; CD4-CD8 Ratio ; Demography ; Eosinophils/cytology ; Female ; Humans ; Immunophenotyping ; Lung Diseases, Interstitial/*diagnosis/diagnostic imaging ; Lymphocyte Subsets/*cytology ; Male ; Middle Aged ; Neutrophils/cytology ; ROC Curve ; Sarcoidosis/diagnosis ; T-Lymphocytes/cytology ; Tomography, X-Ray Computed

OBJECTIVETo evaluate the changes of CD(4)(+) IL-17+T (Th17) and CD(4)(+)Foxp3+regulatory T (Treg) cells in peripheral blood and bronchoalveolar lavage fluid (BALF) , and therefore to explore the role of Th17 and Treg in acrolein exposure airway inflammation in rats.

METHODSForty male Wistar rats were randomly divided into 4 groups: a 2 wk acrolein exposure group, a 4 wk acrolein exposure group, a 2 wk control group and a 4 wk control group (n=10 each). Cells in BALF were collected and analyzed by absolute and differential cell counts.IL-17 and IL-6 levels in serum and BALF were tested by enzyme linked immunosorbent assay (ELISA). The proportion of CD(4)(+)IL-17+T and CD(4)(+) Foxp3+Treg in peripheral blood and BALF were determined by flow cytometry.The mRNA expressions of IL-17 and Foxp3 were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK and Games-Howell test were used for comparison between 2 groups.

RESULTSLevels of IL-17 were remarkable increased in the 2 wk acrolein exposure group and the 4 wk acrolein exposure group in serum [(52.64 ± 1.89) ng/L, (76.73 ± 5.57) ng/L], and BALF [(79.07 ± 5.67) ng/L, (96.61 ± 6.44) ng/L] compared with the 2 wk control group [(40.05 ± 3.12) ng/L, (56.75 ± 4.37) ng/L] and the 4 wk control group [(38.75 ± 3.23) ng/L, (53.27 ± 4.48) ng/L], all P<0.01. IL-6 was increased in the 2 wk and the 4 wk acrolein exposure group [ (33.28 ± 2.27) ng/L, (46.24 ± 3.16) ng/L] compared with the 2 wk and the 4 wk control group [ (16.37 ± 1.49) ng/L, (17.02 ± 1.43) ng/L] in BALF.Ratio of Th17 was higher in the 2 wk and the 4 wk acrolein exposure groups in peripheral blood (1.82 ± 0.18) %, (3.75 ± 0.48) % and BALF [(7.23 ± 0.27) %, (8.12 ± 0.38) %] compared with the 2 wk [(0.96 ± 0.07) %, (5.64 ± 0.63) %] and the 4 wk control group [(1.01 ± 0.08) %, (5.86 ± 0.57) %]. Ratio of Treg in BALF was higher in the acrolein exposure groups [ (8.83 ± 0.52) %, (12.05 ± 0.74) %] compared with the control groups [(4.37 ± 0.27) %, (5.01 ± 0.37) %]. The level of IL-17 mRNA was increased in the 2 wk and the 4 wk acrolein exposure group in peripheral blood [(25.78 ± 2.31), (34.69 ± 2.01) ] and in BALF [(23.04 ± 1.78), (34.56 ± 3.12)] compared with the 2 wk [(11.04 ± 2.53), (11.08 ± 2.05)] and the 4 wk [(12.03 ± 2.34), (12.69 ± 2.69)] control groups. Foxp3 mRNA was increased in the acrolein exposure groups [ (26.37 ± 3.24), (33.19 ± 2.98)] (24.4 ± 2.7), (30.3 ± 2.7) compared with the control groups [(12.37 ± 2.56), (13.12 ± 3.08)]. Th17 in acrolein exposure groups was positively correlated with counts of total cells and macrophages (r=0.5126, 0.5437, all P<0.01).

CONCLUSIONSA changed expression of Th17 and Treg cells and an vary of inflammatory cytokines were evident in airway inflammation of acrolein exposed rats, suggesting that Treg was involved in the immunological regulation and Th17 was associated with the persistent inflammation in acrolein induced airway inflammation in rats.

Acrolein ; toxicity ; Animals ; Bronchoalveolar Lavage Fluid ; cytology ; Cytokines ; metabolism ; Forkhead Transcription Factors ; metabolism ; Inflammation ; metabolism ; Male ; Rats ; Rats, Wistar ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology

The aim of this study is to investigate the anti-inflammatory effect of the adenosine derivative N6-(3-hydroxylaniline) adenosine (WS070117M1) on cigarette smoke plus LPS (lipopolysaccharide)-induced chronic obstructive pulmonary disease (COPD) in mice and its mechanism. COPD model was established by exposing male BALB/c mice to cigarette smoke and challenged with LPS inhalation. Supernatants of bronchoalveolar lavage fluid (BALF) were harvested and IL-1β, IL-6, IL-8 and TGF-β1 levels were measured by ELISA (enzyme-linked immunesorbent assay). The number of total white blood cells and neutrophils in bronchoalveolar lavage fluid was counted separately. Lung tissue was stained with Mayer 's hematoxylin and eosin for histopathologic examination. pAMPKa protein expression and distribution of lung tissue were analyzed by immunohistochemistry method. In vitro, levels of AMPKα phosphorylation in phorbol-12- myristate-13-acetate (PMA) differentiated THP-1 cells was detected by immunohistochemistry, IL-8 level in supernatants of cigarette smoke condensate stimulating PMA differentiated THP-1 cells was measured by ELISA. The results showed that WS070117M1 treatment significantly activated AMPKa in the lung tissue. It also resulted in down regulation of IL-1β, IL-6, IL-8 and TGF-β1 levels in bronchoalveolar lavage fluid and IL-8 level in cigarette smoke condensate stimulating PMA differentiated THP-1 cells. In addition, WS070117M1 could inhibit the recruitment of total white blood cells and neutrophils. These results suggest that WS070117M1 may alleviate the airway inflammation by activating AMPK in the lung tissue.
AMP-Activated Protein Kinases ; metabolism ; Adenosine ; analogs & derivatives ; Animals ; Bronchoalveolar Lavage Fluid ; Cell Line, Tumor ; Disease Models, Animal ; Humans ; Inflammation ; drug therapy ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Leukocyte Count ; Lipopolysaccharides ; Male ; Mice ; Mice, Inbred BALB C ; Neutrophils ; cytology ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; Smoke ; adverse effects ; Tobacco ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of chloroquine on airway hyperresponsiveness in asthmatic mice and explore the possible mechanism.

METHODSBalb/c mouse models of asthma established using OVA received intraperitoneal injections of chloroquine, dexamethasone, or both prior to OVA challenge. Within 24 h after the final challenge, airway hyper- responsiveness (AHR) of the mice was assessed, and the total cell count and the counts of different cell populations in the bronchoalveolar lavage fluid (BALF) were determined under light microscopy. The severity of lung inflammation was evaluated using HE staining, and the concentrations of IL-6 and PGF2α in the BALF were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSChloroquine pretreatment significantly decreased AHR (P<0.001) in the asthmatic mice and reduced the total cell count (P<0.01), eosinophils (P<0.001), neutrophils (P<0.01), and PGF2α levels in the BALF. Chloroquine combined with low-dose dexamethasone significantly lessened inflammations around the bronchioles (P<0.05) and blood vessels (P<0.01) in the lung tissue, and obviously lowered IL-6 (P<0.05) and PGF2α (P<0.001) in the BALF in the asthmatic mice.

CONCLUSIONChloroquine can inhibit AHR in asthmatic mice and produce better anti-inflammatory effect when combined with dexamethasone for treatment of neutrophilic asthma.

Animals ; Asthma ; chemically induced ; drug therapy ; Bronchoalveolar Lavage Fluid ; cytology ; Chloroquine ; pharmacology ; Dexamethasone ; pharmacology ; Dinoprost ; metabolism ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophils ; cytology ; Inflammation ; pathology ; Interleukin-6 ; metabolism ; Leukocyte Count ; Lung ; pathology ; Mice ; Mice, Inbred BALB C ; Neutrophils ; cytology

OBJECTIVETo investigate whether emodin exerts protective effects on mouse with allergic asthma.

METHODSA mouse model of allergic airway inflflammation was employed. The C57BL/6 mice sensitized and challenged with ovalbumin (OVA) were intraperitoneally administered 10 or 20 mg/kg emodin for 3 days during OVA challenge. Animals were sacrificed 48 h after the last challenge. Inflammatory cell count in the bronchoalveolar lavage fluid (BALF) was measured. The levels of interleukin (IL)-4, IL-5, IL-13 and eotaxin in BALF and level of immunoglobulin E (IgE) in serum were measured with enzyme-linked immuno sorbent assay kits. The mRNA expressions of IL-4, IL-5, heme oxygenase (HO)-1 and matrix metalloproteinase-9 (MMP-9) were determined by real-time quantitative polymerase chain reaction.

RESULTSEmodin induced significant suppression of the number of OVA-induced total inflammatory cells in BALF. Treatment with emodin led to significant decreases in the levels of IL-4, IL-5, IL-13 and eotaxin in BALF and total IgE level in serum. Histological examination of lung tissue revealed marked attenuation of allergen-induced lung eosinophilic inflammation. Additionally, emodin suppressed IL-4, IL-5 and MMP-9 mRNA expressions and induced HO-1 mRNA expression.

CONCLUSIONEmodin exhibits anti-inflammatory activity in the airway inflammation mouse model, supporting its therapeutic potential for the treatment of allergic bronchial asthma.

Animals ; Bronchoalveolar Lavage Fluid ; cytology ; Chemokines ; metabolism ; Disease Models, Animal ; Emodin ; chemistry ; pharmacology ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Heme Oxygenase-1 ; genetics ; metabolism ; Immunoglobulin E ; blood ; Interleukins ; genetics ; metabolism ; Leukocytes ; drug effects ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice, Inbred C57BL ; Ovalbumin ; Pneumonia ; blood ; drug therapy ; pathology ; Protective Agents ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism