Penh Values Research Articles

PI3K/AKT/mTOR and TLR4/MyD88/NF-κB Signaling Inhibitors Attenuate Pathological Mechanisms of Allergic Asthma.

Asthma is an inflammatory airway disease wherein bronchoconstriction, airway inflammation, and airway obstruction during asthma attacks are the main problems. It is recognized that imbalance of Th1/Th2 and Th17/Treg is a critical factor in asthma pathogenesis. Manipulation of these with signaling molecules such as mTOR, PI3K, Akt, and MyD88 can control asthma. Mouse model of allergic asthma was produced and treated with ketamine, metformin, metformin and ketamine, triciribine, LY294002, and torin2. MCh challenge test, BALf's Eos Count, the IL-4, 5, INF-γ, eicosanoid, total IgE levels were determined. The MUC5a, Foxp3, RORγt, PI3K, mTOR, Akt, PU.1, and MyD88 gene expressions and histopathology study were done. Asthma groups that were treated with all six components had reduced Penh value, total IgE, IL-4 and IL-5 levels, MUC5a, RORγt, MyD88 and mTOR expression, goblet cell hyperplasia, and mucus hyper-secretion. The eosinophil percentage and Cys-LT level were decreased by metformin and ketamine, triciribine, LY294002, and torin2. The level of IFN-γ was increased in triciribine, LY294002, and torin2. Metformin, metformin and ketamine, triciribine, LY294002, and torin2 reduced Akt and PI3K expression, peribronchial and perivascular inflammation, and increased expression of Foxp3. Torin2 had an effect on PU.1 expression. Inhibition of PI3K/AKT/mTOR and TLR4/MyD88/NF-κB signaling with targeted molecules can attenuate asthma pathology and play an important role in airways protection.

SET knockdown attenuated phenotype modulation and calcium channel associated markers of airway smooth muscle cells in asthmatic mice.

BackgroundDysfunctional phenotype modulation and calcium channels in airway smooth muscle cells (ASMCs) are important characteristics of airway remodeling in chronic asthma. However, the mechanisms underlying these pathological processes remain unclear. SET (I2PP2A, inhibitor-2 of protein phosphatase 2A) has many significant functions and is involved in various physiological and pathological processes. This study aimed to determine the function of SET in chronic asthma.MethodsBALB/c mice were sensitized by ovalbumin injection and repeated inhalation of ovalbumin. The Penh value was measured using the Buxco whole body plethysmography system. A short hairpin RNA of the SET gene was designed and transfected into ASMCs derived from asthmatic mice. Flow cytometry of Annexin-V/propidium iodide staining was used for evaluating cell apoptosis. Western blot was adopted to measure the expression levels of ASMCs phenotype modulation markers and calcium channel-associated proteins.ResultsThe results showed that shRNA targeting SET significantly decreased the expression of SET, and enhanced the apoptosis of ASMCs. SET knockdown promoted the expression of contractile phenotype markers such as α-SMA (alpha smooth muscle Actin), SM-MHC (smooth muscle Myosin heavy chain), and calponin, and inhibited the expression of synthetic phenotype markers including vimentin and CD44. The expression of the calcium channel-related proteins STIM1 (Stromal interaction molecule 1) and Orai1 were also inhibited after SET knockdown.ConclusionsThese data demonstrated that SET participated in the development of airway dysfunction in asthma, suggesting that the silencing of SET may be a new therapeutic target for the treatment of asthma patients.

Study effect of Baicalein encapsulated/loaded Chitosan-nanoparticle on allergic Asthma pathology in mouse model.

Asthma as chronic airway disease has high prevalence in children and imbalance of Th1/Th2 is a critical mechanism in pathogenesis of the asthma. Baicalein as a cell protective and anti-inflammatory flavonoid may have anti-asthma effect. Therefore, for better using lung, baicalein was used in chitosan-nanoparticle as anti-asthma treatment. Baicalein was loaded and encapsulated in chitosan nanoparticle. The morphology, physical characters (particle size, zeta potential and FT-IR) were analyzed. Drug encapsulation and loading capacity, accumulative release-time were studied. After asthma model producing, the mice were treated with L-B-NP and E-B-NP. At least, MCh challenge test, Cytokines measurement and Lung Histopathology were done. Nanoparticles had average size 285±25nm with negative charge -2.5mV. The L-B-NP decreased penh value and E-B-NP decreased inflammation. Both nanoparticles increased IL-12 and decreased IL-5. Also, L-B-NP decreased mucus secretion in bronchi. L-B-NP and E-B-NP control immune-allergo-inflammatory response of asthma. L-B-NP controlled AHR and E-B-NP controlled inflammation that can be used as controlling anti-asthma drug.

Oral Administration of Acrylamide Worsens the Inflammatory Responses in the Airways of Asthmatic Mice Through Agitation of Oxidative Stress in the Lungs.

Acrylamide is a toxic chemical substance produced when starch-rich foods are fried at high temperatures. Asthma is a chronic and complicated respiratory disease, of which genetic and environmental factors are the main triggers. Orally-received components may have an effect on asthma pathophysiology. The aim of this study was to investigate the role of AA as a stimulus in asthma. BALB/c mice were allocated into four groups as follows: two OVA-sensitized asthmatic groups, including one treated with AA by gavage feeding and one non-treated (asthma group), and two healthy (non-asthmatic) groups, one treated with AA by gavage feeding and one non-treated (negative control group). Airway hyperresponsiveness, cell count, cytokine levels in BAL fluid, lung histopathology, IgE levels, and oxidative stress indices including plasma level of MDA, pulmonary antioxidant enzymes (SOD and CAT) levels, HP content, and collagen fiber accumulation in lung tissue were measured. We found that the group of mice treated with both OVA and AA (asthmatic and AA-treated mice) experienced higher levels of asthma-associated biomarkers, including higher enhanced pause (Penh value), eosinophilic inflammation, mucus hyper secretion, goblet cell hyperplasia, total and OVA-specific IgE levels, IL-4, IL-5, and IL-13 levels than the group sensitized only with OVA (asthmatic mice). The OVA-AA-treated mice also experienced worsened levels of oxidative stress indicators. Healthy (non-asthmatic) mice that only received AA were in similar conditions to healthy untreated mice (negative control group). The OVA-AA-treated group showed more severe allergic asthma symptoms in comparison to the group only sensitized with OVA. Therefore, food/water contaminated with AA can act as a stimulant of allergic asthma and exacerbate the bronchial inflammatory responses.

3,4,5-Trihydroxycinnamic acid exerts anti-asthmatic effects in vitro and in vivo

3,4,5-Trihydroxycinnamic acid (THCA) has been reported to possess anti-inflammatory activity. However, the effect of THCA for treating allergic asthma was unknown. Therefore, in the present study, the anti-asthmatic effects of THCA were studied in both in vitro and in vivo studies. In phorbol 12-myristate 13-acetate (PMA)-stimulated A549 airway epithelial cells, THCA pretreatment decreased the mRNA expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecules 1 (ICAM-1), and reduced the mRNA expression of matrix metalloproteinase 9 (MMP-9). THCA also inhibited PMA-induced protein kinase B (AKT), mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activation in A549 cells. In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages, THCA pretreatment suppressed the mRNA expression of ICAM-1 and MMP-9. In addition, THCA suppressed the adhesion of EOL and A549 cells. In ovalbumin (OVA)-administered asthmatic mice, THCA exerted inhibitory activity on IL-5, IL-13, and MCP-1 in bronchoalveolar lavage fluid (BALF) and on OVA-specific immunoglobulin E (IgE) in serum. THCA attenuated the numbers of inflammatory cells in BALF and the influx of inflammatory cell in lung tissues. Furthermore, THCA downregulated the levels of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), and leukotriene B4 (LTB4) expression, mucus production and CREB phosphorylation as well as Penh value. These effects were accompanied by suppression of AKT, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and NF-κB activation. Therefore, the results of the current study suggest that THCA may be a valuable adjuvant or therapeutic in the prevention or treatment of allergic asthma.

In utero exposure to genistein decreased intranasal house dust mite-induced respiratory allergy in middle-aged male B6C3F1 offspring

Despite many hypothesized benefits of dietary isoflavone genistein (GEN) deriving from soy-based products, questions surrounding GEN’s developmental effects are increasing. To understand if in utero GEN exposure modulated postnatal respiratory allergies in the middle age, we conducted a time course study in the B6C3F1 offspring (PND 240-330) using a common household allergen (house dust mites: HDM; 10 μg/mouse for PND 240 and 290, and 50 μg/mouse for PND 330, a middle age in mice) following intranasal instillation, a physiological route of allergen exposure. GEN was administered to dams by gavage from gestational day 14 to parturition at a physiologically relevant dose (20 mg/kg body weight). Female and male offspring were sensitized with HDM allergens beginning about one month prior to sacrifice followed by challenges with three weekly dosings of HDM extracts, and they were euthanized at day 3 following the final HDM exposure. In utero exposure to GEN decreased HDM allergen-induced respiratory allergy in male B6C3F1 offspring at PND 330 as reflected by decreases in airway hyperresponsiveness (e.g., Penh value), HDM-specific IgG1 (a Th2 type Ab) and the activity of eosinophil peroxidase in the lung (an indication of eosinophil recruitment to the lungs). However, in utero exposure to GEN had minimal effects on HDM allergen-induced respiratory allergy in the middle-aged female offspring. Changes in serum total IgE, HDM-specific IgE, and lung histopathology scores in both male and female offspring were not biologically significant. Overall, in utero GEN exposure exerted a protective effect on respiratory allergy in the middle-aged male, but not female, B6C3F1 offspring following later-life HDM exposures.

Effect of neurokinin 1 receptor antagonist on airway inflammation and hyperresponsiveness in asthmatic mice

Objective To investigate the effects of neurokinin-1 receptor antagonist WIN 62, 577 on airway inflammation and airway hyperresponsiveness in asthmatic mice. Methods Thirty-two BALB/c mice(Specific-pathogen-free grade) were randomly divided into 4 groups: control group, asthmatic group, WIN 62, 577 intervention group and dexamethasone group.The asthmatic group, the WIN 62, 577 intervention group, and the dexamethasone group were given intraperitoneal injection of 0.2 ml of OVA sensitization solution at 0 d, 7 d, and 14 d, respectively.Then the asthmatic group, WIN 62, 577 group and dexamethasone group were given OVA challenge solution(4% OVA solution) by inhalation once a day for 30 min from 21 d to 28 d for 7 consecutive days.The WIN 62, 577 intervention group was given WIN 62, 577 300 μg intraperitoneal injection 1 h before each challenge; the dexamethasone group was given intraperitoneal injection of dexamethasone 2 mg/kg 1 h before each challenge.The airway responsiveness of each group of mice was detected by non-invasive pulmonary function test.The bronchoalveolar lavage fluid(BALF) was obtained for inflammatory count.The HE staining of lung tissue was used to observe airway inflammation in mice. Results Compared with the asthmatic group, the mice in the WIN 62, 577 intervention group showed less restlessness, standing upright, crouching back, scratching the ears and scratching the cheeks, shortness of breath and cyanosis of the lips.After inhaling different concentrations of acetylcholine, the Penh value of mice in the WIN 62, 577 intervention group and the dexamethasone group was significantly lower than that in the asthmatic group(P<0.05). Compared with the asthmatic group, the number of WBC and EOS in BALF decreased significantly in the WIN 62, 577 intervention group and the dexamethasone group(P<0.01). HE staining showed that the inflammatory changes in the lung tissue of mice in the WIN 62, 577 intervention group were significantly reduced, the bronchial epithelium did not fall off significantly, the mucosal edema was not obvious, the smooth muscle proliferation was reduced, and the inflammatory cell infiltration was reduced, similar to the airway changes in the dexamethasone group. Conclusion Neurokinin-1 receptor antagonist WIN 62, 577 can reduce airway inflammation and airway hyperresponsiveness in asthmatic mice. Key words: Asthma; Inflammation; Hyperresponsiveness; Substance P; Neurokinin-1 receptor

Silica dioxide nanoparticles aggravate airway inflammation in an asthmatic mouse model via NLRP3 inflammasome activation

Silica dioxide nanoparticles (SiONPs) are mainly used in the rubber industry; however, they are a major air pollutant in Asia. Thus, extensive research on this issue is required. In this study, we investigated the effects of SiONPs on asthma aggravation and elucidated the underlying mechanism using ovalbumin (OVA)-induced asthmatic mice model and in NCI–H292 cells. Mice exposed to SiONPs showed markedly increased Penh values, inflammatory cell counts, and inflammatory cytokine levels compared to OVA-induced asthmatic mice. Exposure to SiONPs also induced additional airway inflammation and mucus secretion with increases in protein expression levels of thioredoxin-interacting protein (TXNIP), NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, and interleukin (IL)-1β compared to those in OVA-induced asthmatic mice. Treatment of SiONPs in NCI–H292 cells also significantly increased mRNA expression levels of inflammatory cytokines accompanied with elevation in the levels of TXNIP, NLRP3 inflammasome, and IL-1β proteins in a concentration-dependent manner. Taken together, exposure to SiONPs aggravated asthma development, which is closely related to inflammasome activation. Our results provide useful information about the toxicological effects of SiONPs on asthma exacerbation and suggest the need to avoid SiONP exposure especially in individuals with respiratory diseases.

Physiological responses to cisplatin using a mouse hypersensitivity model

Background: The physiological mechanisms underlying the development of respiratory hypersensitivity to cisplatin (CDDP) are not well-understood. It has been suggested that these reactions are likely the result of type I hypersensitivity, but other explanations are plausible and the potential for CDDP to induce type I hypersensitivity responses has not been directly evaluated in an animal model. Objectives and Methods: To investigate CDDP hypersensitivity, mice were topically sensitized through application of CDDP before being challenged by oropharyngeal aspiration (OPA) with CDDP. Before and immediately after OPA challenge, pulmonary responses were assessed using whole body plethysmography (WBP). Results: CDDP did not induce an immediate response or alter the respiratory rate in sensitized mice. Two days later, baseline enhanced pause (Penh) values were significantly elevated (p < 0.05) in mice challenged with CDDP. When challenged with methacholine (Mch) aerosol, Penh values were significantly elevated (p < 0.05) in sensitized mice and respiratory rate was reduced (p < 0.05). Lymph node cell counts and immunoglobulin E levels also indicated successful sensitization to CDDP. Irrespective of the sensitization state of the mice, the number of neutrophils increased significantly in bronchoalveolar lavage fluid (BALF) following CDDP challenge. BALF from sensitized mice also contained 2.46 (±0.8) × 104 eosinophils compared to less than 0.48 (±0.2) × 104 cells in non-sensitized mice (p < 0.05). Conclusions: The results from this study indicate that dermal exposure to CDDP induces immunological changes consistent with type I hypersensitivity and that a single respiratory challenge is enough to trigger pulmonary responses in dermally sensitized mice. These data provide previously unknown insights into the mechanisms of CDDP hypersensitivity.

Effects of 3-methyladenine on airway inflammation, airway hyperresponsiveness and mucus secretion in asthmatic mice

Objective: To investigate the effects of 3-methyladenine on airway inflammation, airway hyperresponsiveness and mucus secretion in asthmatic mice, and to explore its mechanism. Methods: C57BL/6J female mice were randomly divided into normal control group (PBS), OVA group(OVA), OVA with 3-methyladenine group (OVA+3-MA), and OVA with 4-phenylbutyrate group (OVA+4-PBA). OVA group, OVA+3-MA group and OVA+4-PBA groups were all sensitized and challenged with OVA to establish asthmatic models, while PBS group was given PBS as a control. At 2 h before challenge, OVA+3-MA group was intraperitoneally injected with 3-methyladenine, and OVA+4-PBA group was intraperitoneally injected with 4-phenylbutyrate. Airway hyperresponsiveness, eosinophils, and pathological changes of pulmonary tissue (hematoxylin-eosin, HE staining) were measured to confirm the establishment of asthmatic models. Sections of pulmonary tissue were also stained with Masson and PAS. The expression level of LC3B was measured by immunofluorescence and Western blot. The Beclin1, Muc5ac, Atf6, Chop and Bip proteins in lung tissues were detected by Western blot. Results: The Penh value, and eosinophils in BALF in OVA group was significantly increased compared with PBS group (P<0.05). The Penh value in OVA+3-MA group and OVA+4-PBA group were significantly decreased compared with the OVA group at the concentration of 6.25 g/L, 12.50 g/L, 25.00 g/L, and 50.00 g/L of methacholine (all the P<0.05), and the eosinophils were also significantly decreased compared with the OVA group (P<0.05). Pulmonary histology revealed that OVA group showed high levels of inflammatory cell infiltration of bronchi and lung vessels, alveolar septal thickening, structural destruction, smooth muscle thickening, collagen deposition, and goblet cell hyperplasia. The levels of inflammatory cell infiltration of bronchi and lung vessels, alveolar septal thickening, structural destruction, smooth muscle thickening, collagen deposition, and goblet cell hyperplasia in OVA+3-MA group and OVA+4-PBA group were significantly lower than the OVA group, while the PBS group was normal. Compared with PBS group, the expression of LC3 Ⅱ/Ⅰ, Beclin1, Muc5ac, Atf6, Chop and Bip proteins in lung tissues in the OVA group were significantly increased (1.09±0.04 vs 0.34±0.09, P<0.05; 0.18±0.01 vs 0.06±0.01, P<0.05; 1.90±0.38 vs 0.46±0.11, P<0.05; 1.67±0.18 vs 0.41±0.08, P<0.05; 2.96±0.45 vs 1.11±0.10, P<0.05; 2.07±0.34 vs 0.49±0.17, P<0.05, respectively). Compared with the OVA group the expression of LC3 Ⅱ/Ⅰ, Beclin1, Muc5ac, Atf6, Chop and Bip proteins in lung tissues in the OVA+3-MA group and OVA+4-PBA group were significantly decreased (0.46±0.07 vs 1.09±0.04, 0.63±0.03 vs 1.09±0.04, both P<0.05; 0.11±0.02 vs 0.18±0.01, 0.12±0.02 vs 0.18±0.01, both P<0.05; 0.72±0.22 vs 1.90±0.38, 0.57±0.13 vs 1.90±0.38, both P<0.05; 1.06±0.12 vs 1.67±0.18, 1.02±0.12 vs 1.67±0.18, both P<0.05; 1.67±0.21 vs 2.96±0.45, 1.10±0.15 vs 2.96±0.45, both P<0.05; 1.03±0.11 vs 2.07±0.34, 0.97±0.10 vs 2.07±0.34, both P<0.05). Conclusion: 3-MA was shown to inhibit airway inflammation, airway hyperresponsiveness and mucus secretion in mice with bronchial asthma, and the mechanism may be related to inhibiting autophagy, and then inhibiting endoplasmic reticulum stress.

Repetitive antidotal treatment is crucial in eliminating eye pathology, respiratory toxicity and death following whole-body VX vapor exposure in freely moving rats.

Exposure to the chemical warfare nerve agent VX is extremely toxic, causing severe cholinergic symptoms. If not appropriately treated, death ultimately ensues. Based on our previously described whole-body vapor exposure system, we characterized in detail the clinical outcome, including respiratory dynamics, typical of whole-body exposure to lethal doses of VX vapor in freely moving rats. We further evaluated the efficacy of two different antidotal regimens, one comprising a single and the other repeated administration of antidotes, in countering the toxic effects of the exposure. We show that a 15min exposure to air VX concentrations of 2.34-2.42mg/m3 induced a late (15-30min) onset of obvious cholinergic signs, which exacerbated over time, albeit without convulsions. Marked eye pathology was observed, characterized by pupil constriction to pinpoint, excessive lacrimation with red tears (chromodacryorrhea) and corneal damage. Respiratory distress was also evident, characterized by a three-fourfold increase in Penh values, an estimate of lung resistance, and by lung and diaphragm histological damage. A single administration of TAB (the oxime TMB-4, atropine and the anticholinergic and antiglutamatergic benactyzine) at the onset of clinical signs afforded only limited protection (66% survival), with clinical deterioration including weight loss, chromodacryorrhea, corneal damage, increased airway resistance and late death. In contrast, a combined therapy of TAB at the onset of clinical signs and repeated administration of atropine and toxogonin (ATOX) every 3-5h, a maximum of five i.m. injections, led to 100% survival and a prompt recovery, accompanied by neither the above-described signs of eye pathology, nor by bronchoconstriction and respiratory distress. The necessity of recurrent treatments for successful elimination of VX vapor toxicity strongly supports continuous penetration of VX following termination of VX vapor exposure, most likely from a VX reservoir formed in the skin due to the exposure. This, combined with the above-described eye and respiratory pathology and absence of convulsions, are unique features of whole-body VX vapor exposure as compared to whole-body vapor exposure to other nerve agents, and should accordingly be considered when devising optimal countermeasures and medical protocols for treatment of VX vapor exposure.

Modulation of the airway smooth muscle phenotype in a murine asthma model and effects of nuclear factor-κB inhibition

Objective: Phenotype modulation of airway smooth muscle (ASM) is a unique characteristic of asthma and is considered to regulate airway remodeling, airway hyperresponsiveness (AHR) and inflammation. The nuclear factor-κB (NF-κB) signaling pathway plays a crucial role in phenotypic modulation. Thus, models of acute and chronic asthma were established and pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor was delivered by intraperitoneal injection. Methods: The Penh value was measured using the BUXCO WBP system. Lung tissues were subjected to histologic analysis. Phenotypic markers of ASM and COL1A1 mRNA levels were measured by RT-PCR. Expression levels of phosphorylated p65 (pP65) and α-SMA were detected by Western blot. Serum cytokine levels were quantified by RayBiotech ELISA array. Results: PDTC intervention decreased the Penh values in both the acute and chronic models. The ASM area and the airway collagen area were decreased in the PDTC intervention group. A decrease in phenotypic markers were detected in both the acute and chronic models in time-dependent manner, and PDTC intervention partially reversed the phenotypic modulation. The effect of PDTC intervention on systemic inflammation was also verified. Conclusion: These results revealed the existence of a dynamic ASM phenotype modulation procedure in asthma development and that targeting NF-κB by PDTC was effective to mitigate ASM phenotype modulation and major asthmatic pathological features.

Neonatal Streptococcus pneumoniae Pneumonia Induces an Aberrant Airway Smooth Muscle Phenotype and AHR in Mice Model.

Our previous study showed that neonatal S. pneumoniae infection aggravated airway inflammation and airway hyperresponsiveness (AHR) in an OVA-induced allergic asthma model. As airway smooth muscle (ASM) plays a pivotal role in AHR development, we aim to investigate the effects of neonatal S. pneumoniae pneumonia on ASM structure and AHR development. Non-lethal neonatal pneumonia was established by intranasally infecting 1-week-old BALB/C mice with the S. pneumoniae strain D39. Five weeks after infection, the lungs were collected to assess the levels of α-SMA and the contractile proteins of ASM. Our results indicate that neonatal S. pneumoniae pneumonia significantly increased adulthood lung α-SMA and SMMHC proteins production and aggravated airway inflammatory cells infiltration and cytokines release. In addition, the neonatal S. pneumoniae pneumonia group had significantly higher Penh values compared to the uninfected controls. These data suggest that neonatal S. pneumoniae pneumonia promoted an aberrant ASM phenotype and AHR development in mice model.

T CELL-INDUCED BRONCHOCONSTRICTION IN MICE

Introduction To investigate a role of helper T (Th) cells in asthma, T cell-transfer model was analyzed for late phase asthmatic responses. Methods Ovalbumin (OVA) specific Th clones were derived from splenocytes of DO11.10 transgenic mice expressing T cell receptor specific for OVA/H-2d. Th clones were adoptively transferred into unprimed mice. After intranasal or inhalation challenge with OVA, airway resistance was continuously monitored by either unrestrained whole body plethysmography (BUXCO) or resistance/compliance analyzer under anesthetized condition. Supernatants of stimulated Th clones were analyzed for contractile activity using collagen gels embedded with murine primary bronchial smooth muscle cells. Effects of H1R and LTR1 antagonist were analyzed both in vitro and in vivo. Results When unprimed mice were transferred with Th clones, T5-1, T6-2, T6-4, and T6-7, Penh values were significantly increased 6 hr after OVA challenge. In contrast, mice transferred with other Th clones, BF7, T6-1, or T6-10 did not show any change. Airflow limitation was confirmed by a direct measurement of airway resistance under anesthetized, restrained, and intubated conditions. The airflow limitation was also efficiently induced by the challenge with T cell epitope peptide, OVA323-339. Contractile activity was detected in the supernatants of T6-2 stimulated with immobilized anti-CD3. T cell-induced contraction was not affected by H1R or LTR1 antagonist. Conclusions Activation of Th cells resulted in an airflow limitation besides eosinophilic inflammation, AHR, and mucous hyperplasia. T cell-derived bronchoconstrictor might be a target for treatment-resistant asthma.

MiR-146a Mimics Attenuate Allergic Airway Inflammation by Impacted Group 2 Innate Lymphoid Cells in an Ovalbumin-Induced Asthma Mouse Model

Background: The prevalence of allergic asthma has increased dramatically. Previous studies have found that the microRNA 146a (miR-146a) expression in asthma inhibits cell proliferation and promotes apoptosis of bronchial smooth muscle cells. We aimed to investigate the effect of miR-146a mimics on ovalbumin (OVA)-induced asthma in a mouse model. Methods: Inflammatory cell infiltration in bronchoalveolar lavage fluid (BALF) was measured by flow cytometry. Levels of OVA-specific immunoglobulin E (IgE) in serum and cytokines in BALF were examined by enzyme-linked immunosorbent assay. For monitoring the airway, the Penh value (% baseline) was measured using a whole-body plethysmograph. Results: In OVA-induced asthmatic mice, miR-146a significantly suppressed the infiltration of inflammatory cells in BALF and decreased the levels of OVA-specific IgE and T helper 2 cell type cytokines. In addition, miR-146a inhibited the OVA-induced airway hyperresponsiveness and the group 2 innate lymphoid cell responses. Moreover, the effects of miR-146a mimics were dependent on interleukin 33 stimulation. Conclusions: Our results suggest that miR-146a mimics might serve as an attractive candidate for further preclinical studies as an anti-inflammatory treatment of asthma.

Dietary vitamin D3 deficiency exacerbates sinonasal inflammation and alters local 25(OH)D3 metabolism

RationalePatients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS).MethodsBalb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined.ResultsMice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A.ConclusionsVD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels.

Screening of Sceptridium ternatum for antitussive and antiasthmatic activity and associated mechanisms.

ObjectivesSceptridium ternatum is an expectorant in traditional Chinese medicine and is prescribed for the treatment of asthma. The study aim was to screen Sceptridium ternatum for ingredients with antitussive and antiasthmatic effects and to study their associated mechanisms.MethodsCough in mice was induced using ammonia. Cough latency and the number of coughs within 3 minutes were determined. Airway responsiveness was assessed using ovalbumin as a sensitizer and characteristic asthma indicators were measured.ResultsChloroform and ethyl acetate extracts significantly reduced the number of coughs within 3 minutes, tidal volume, and the percentage of eosinophilic granulocytes, lymphocytes and neutrophils. All extracts decreased airway responsiveness in asthmatic mice compared with the untreated group. Petroleum ether, chloroform and n-butanol extracts lowered the Penh values of asthmatic mice. Petroleum ether and ethyl acetate extracts greatly reduced interleukin-4 expression and the interleukin-4/interferon gamma ratio. Compared with the model group, all extracts reduced mRNA expression of the cysteinyl leukotriene receptor-1 (CysLT1).ConclusionsChloroform extract and ethyl acetate extract displayed obvious antitussive effects and reduced airway inflammation. Thus, these two extracts contain the effective ingredients of Sceptridium ternatum. The active mechanism was ascribed to inhibition of mRNA expression of the CysLT1 receptor in mice with bronchial asthma.

Guizhi-jia-houpu-xingzi decoction attenuates ovalbumin-induced allergic asthma via regulation of Toll-like receptor signal pathway

Purpose: To study the effect of Guizhi-jia-houpu-xingzi (GHX) on ovalbumin-induced allergic asthma in rats.Methods: An animal model of allergic asthma (AA) in rats was established by intraperitoneal injection (ip) of ovalbumin (OVA). Thereafter, GHX (375 mg/kg) was administered orally for 7 days. Pulmonary function, inflammatory cells, immunoglobulin E (Ig) E, interleukin-4 (IL)-4 and interferon-γ (IFN)-γ in serum and bronchoalveolar lavage fluids (BALF) were determined. Furthermore, mRNA expressions of Toll-like receptors (TLRs) signal pathway was determined using real time polymerase chain reaction PCR (q-RT-PCR).Results: GHX (375 mg/kg) significantly decreased respiratory rate (p &lt; 0.01) and Penh value (p &lt; 0.05) when compared with AA rats. The inflammatory cells (p &lt; 0.01) and levels of IL-4 (p &lt; 0.01) and IgE (p &lt; 0.01) were significantly decreased by GHX treatment when compared with AA rats; whereas IFN-γ (p &lt; 0.05) was significantly increased. Furthermore, GHX significantly decreased the mRNA expressions of GATA binding protein (GATA)-3 (p &lt; 0.01), TRL-2 (p &lt; 0.01), TRL-4 (p &lt; 0.01), myeloid differentiation factor 88 (MyD88) (p &lt; 0.01), TNF receptor associated factor 6 (TRAF6) (p &lt; 0.01) and β-arrestin (p &lt; 0.01) in lung tissues, relative to AA rats. However, GHX treatment led to significant up-regulation of mRNA expression of T-bet (p &lt; 0.01).Conclusion: These results demonstrate that GHX possesses a potential for treating allergic asthma via regulation of Toll-like receptor (TLR) signal pathway. They also provide a scientific basis for the probable use of GHX in clinical treatment of allergic diseases in future.Keywords: Guizhi-jia-houpu-xingzi decoction, Ovalbumin, Allergic asthma, Toll-like receptor

The regulative effects of Aspergillus fumigatus on expression of glucocorticoid receptor in asthmatic rats

Objective: To study the regulative effects of Aspergillus fumigatus (A.fumigatus) on expression of glucocorticoid receptor (GCR) in asthmatic rats. Methods: Wistar rats were randomly divided into 4 groups: a normal control group (UC), a normal control with A. fumigatus group (UC+ AF), an OVA group (OVA), and an OVA with A. fumigatus group (OVA+ AF). OVA and OVA+ AF groups were sensitized and challenged with OVA to establish asthmatic models. UC and UC+ AF groups were given normal saline as controls. After the last challenge, OVA+ AF and UC+ AF groups were given A. fumigatus spores intranasally. Airway hyper-responsiveness, eosinophil percentage (Eos%) and serum IgE level were measured to confirm the establishment of asthmatic models. Sections of pulmonary tissue were stained with hematoxylin-eosin (HE) and the expression of GCR mRNA and protein in lung tissues were measured by qRT-PCR and Western blot. Lung tissues and blood were plated on the potato dextrose agar(PDA)medium and cultured for 24 h to measure the number of colony. Results: The Penh value, Eos% in BALF and serum IgE level in UC+ AF group were slightly higher than those in the UC group (all the P>0.05). The Penh value, Eos% in BALF and serum IgE level in OVA group were significantly higher than those in the UC group (all the P<0.05). The Penh value in OVA+ AF group was significantly increased compared with the OVA group at the concentration of 25 g/L and 50 g/L of methacholine (all the P>0.05). Pulmonary histology revealed that both OVA group and OVA+ AF group showed high levels of inflammatory cell infiltration of bronchus and lung vessels, interstitial edema and smooth muscle thickening, while the UC and UC + AF groups were normal. Compared with the UC group, the expressions of GCR mRNA and protein in UC+ AF group and OVA group were decreased significantly (GCR mRNA in UC, UC+ AF and OVA group were 0.93±0.15, 0.65±0.10, 0.72±0.22, respectively, F=10.744, P<0.01; GCR protein in UC, UC+ AF and OVA group were 100±0, 89±8, 82±15, respectively, F=18.939, P<0.01). The expressions of GCR mRNA and protein in OVA+ AF group were further decreased than those in OVA group (GCR mRNA: OVA group: 0.72±0.22 vs OVA+ AF group: 0.52±0.08, t=2.462, P<0.05; GCR protein: OVA group: 81.88±15.41 vs OVA+ AF group: 59.09±7.60, t=2.997, P<0.05). The ratio of A. fumigatus colonization in lung tissues in OVA+ AF group (4/8) was higher than the UC+ AF group (0/8). Conclusion:A. fumigatus exposure can down-regulate the expression of GCR in the lung, which maybe an important mechanism of steroid-resistant asthma.

Co-inhalation of roflumilast, rather than formoterol, with fluticasone more effectively improves asthma in asthmatic mice.

Roflumilast is approved as an add-on therapy for chronic obstructive pulmonary disease. The inflammation in chronic obstructive pulmonary disease is mainly neutrophilic, while in asthma it is mainly eosinophilic, studies addressing role of roflumilast in eosinophilic inflammation are recommended. Also in severe asthma, the dominant inflammatory cells are neutrophils. Thus, roflumilast has a potential off-label use in the treatment of asthma. This study was designed to evaluate the effects of co-inhalation of roflumilast and fluticasone compared to that of formoterol and fluticasone in ovalbumin-sensitized and-challenged BALB/c mice. Besides normal control group, the ovalbumin-asthmatic mice were randomly divided into seven groups (n = 8): positive control, vehicle-treated, and five drug-treated groups. Treatments (µg/kg) were given as 15 min-inhalation once/day for five days as follows: roflumilast (500), formoterol (50), fluticasone (1000), roflumilast + fluticasone (500 + 1000), and formoterol + fluticasone (50 + 1000). Penh values were measured in conscious unrestrained mice using the single-chamber whole-body plethysmography. Airway hyperreactivity to inhaled methacholine was evaluated. Bronchoalveolar lavage fluid was used for the measurements of levels of IL-4, IL-5, TNF-α, OVA-specific IgE, and total and differential white cells. Lung sections were stained with hematoxylin and eosin and periodic acid-Schiff. The asthmatic mice showed significant increases in airway hyperreactivity which were significantly reversed by the combination treatments. The asthmatic mice showed significant increases in levels of IL-4, IL-5, TNF-α, ovalbumin-specific IgE, and total and differential white cells in bronchoalveolar lavage fluid. All treatments (except formoterol) significantly reversed these changes mainly with roflumilast + fluticasone. The asthmatic mice showed severe inflammatory infiltration and goblet cell hyperplasia which were maximally reversed by roflumilast + fluticasone, while minimally reversed by formoterol. In conclusion, co-inhalation of roflumilast + fluticasone more significantly improved inflammation and histopathological changes than co-inhalation of formoterol + fluticasone in ovalumin-asthmatic mice. Further studies are needed to help confirm the potential off-label add-on use of roflumilast in typical and atypical asthma and asthma-chronic obstructive pulmonary disease overlap syndrome. Impact statement Roflumilast, a selective phosphodiesterase-4 inhibitor, was approved for the treatment of chronic obstructive pulmonary disease (COPD). This study showed that co-inhalation of roflumilast and fluticasone significantly decreased airway hyperresponsiveness in ovalumin-asthmatic mice. Also, it more significantly improved inflammation and histopathological changes than co-inhalation of formoterol and fluticasone. The current results showed that inhaled roflumilast reduced counts of eosinophils, neutrophils, and macrophages in bronchoalveolar lavage fluid. Consequently, inhaled roflumilast might be of potential off-label benefit in treatment of eosinophilic and neutrophilic asthma and asthma-COPD overlap syndrome (ACOS). These results could also support other experimental and clinical studies addressing the same issue.