Proteomic Alterations in B Lymphocytes of Sensitized Mice in a Model of Chemical-Induced Asthma

Introduction and Aim

The role of B-lymphocytes in chemical-induced asthma is largely unknown. Recent work demonstrated that transferring B lymphocytes from toluene diisocyanate (TDI)-sensitized mice into naïve mice, B cell KO mice and SCID mice, triggered an asthma-like response in these mice after a subsequent TDI-challenge. We applied two-dimensional difference gel electrophoresis (2D-DIGE) to describe the “sensitized signature” of B lymphocytes comparing TDI-sensitized mice with control mice.

Results

Sixteen proteins were identified that were significantly up- or down-regulated in B lymphocytes of sensitized mice. Particularly differences in the expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 could be correlated to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses.

Conclusion

This study revealed important alterations in the proteome of sensitized B cells in a mouse model of chemical-induced asthma, which will have an important impact on the B cell function.     

B-lymphocytes as Key Players in Chemical-Induced Asthma

T-lymphocytes and B-lymphocytes are key players in allergic asthma, with B-lymphocytes producing antigen-specific immunoglobulins E (IgE). We used a mouse model of chemical-induced asthma and transferred B-lymphocytes from sensitized animals into naïve wild type mice, B-lymphocyte knock-out (B-KO) mice or severe combined immunodeficiency (SCID) mice. On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% toluene diisocyanate (TDI) (20µl/ear). On day 15, mice were euthanized and the auricular lymph nodes isolated. B-lymphocytes (CD19⁺) were separated from the whole cell suspension and 175,000 cells were injected in the tail vein of naïve wild type, B-KO or SCID mice. Three days later, the mice received a single oropharyngeal challenge with 0.01% TDI (20µl) or vehicle (acetone/olive oil (AOO)) (controls). Airway reactivity to methacholine and total and differential cell counts in the bronchoalveolar lavage (BAL) fluid were measured 24 hours after challenge. B-lymphocytes of AOO or TDI-sensitized mice were characterized for the expression of surface markers and production of cytokines. We found that transfer of B-cells obtained from mice dermally sensitized to toluene diisocyanate (TDI) into naïve wild type mice, B-KO mice or SCID mice led, within three days, to an acute asthma-like phenotype after an airway challenge with TDI. This response was specific and independent of IgE. These B-lymphocytes showed antigen presenting capacities (CD80/CD86 and CD40) and consisted of B effector (Be)2- (IL-4) and Be1-lymphocytes (IFN-γ). The transferred B-lymphocytes were visualized near large airways, 24 hours after TDI challenge. Thus, B-lymphocytes can provoke an asthmatic response without the action of T-lymphocytes and without major involvement of IgE.     

Antibody to mCLCA3 Suppresses Symptoms in a Mouse Model of Asthma

Background

Asthma is a complex and heterogeneous chronic inflammatory disorder that is associated with mucous cell metaplasia and mucus hypersecretion. Functional genomic analysis indicates that mucous cell metaplasia and mucus hypersecretion depend on members of the calcium-activated chloride channel (CLCA) gene family. It has been reported that the inhibition of CLCAs could relieve the symptoms of asthma. Thus, the mCLCA3 antibody may be a promising strategy to treat allergic diseases such as asthma.

Methods

We constructed asthmatic mouse models of OVA-induced chronic airway inflammatory disorder to study the function of the mCLCA3 antibody. Airway inflammation was measured by HE staining; goblet cell hyperplasia and mucus hypersecretion were detected by PAS staining; muc5ac, IL-13, IFN-γ levels in bronchoalveolar lavage fluid (BALF) were examined by ELISA; Goblet cell apoptosis was measured by TUNEL assay and alcian blue staining; mCLCA3, Bcl-2 and Bax expression were detected by RT-PCR, Western blotting and immunohistochemical analysis.

Results

In our study, mice treated with mCLCA3 antibody developed fewer pathological changes compared with control mice and asthmatic mice, including a remarkable reduction in airway inflammation, the number of goblet cells and mCLCA3 expression in lung tissue. The levels of muc5ac and IL-13 were significantly reduced in BALF. We also found that the rate of goblet cell apoptosis was increased after treatment with mCLCA3 antibody, which was accompanied by an increase in Bax levels and a decrease in Bcl-2 expression in goblet cells.

Conclusions

Taken together, our results indicate that mCLCA3 antibody may have the potential as an effective pharmacotherapy for asthma.     

Overexpression of Dimethylarginine Dimethylaminohydrolase 1 Attenuates Airway Inflammation in a Mouse Model of Asthma

Levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are increased in lung, sputum, exhaled breath condensate and plasma samples from asthma patients. ADMA is metabolized primarily by dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2. We determined the effect of DDAH1 overexpression on development of allergic inflammation in a mouse model of asthma. The expression of DDAH1 and DDAH2 in mouse lungs was determined by RT-quantitative PCR (qPCR). ADMA levels in bronchoalveolar lavage fluid (BALF) and serum samples were determined by mass spectrometry. Wild type and DDAH1-transgenic mice were intratracheally challenged with PBS or house dust mite (HDM). Airway inflammation was assessed by bronchoalveolar lavage (BAL) total and differential cell counts. The levels of IgE and IgG1 in BALF and serum samples were determined by ELISA. Gene expression in lungs was determined by RNA-Seq and RT-qPCR. Our data showed that the expression of DDAH1 and DDAH2 was decreased in the lungs of mice following HDM exposure, which correlated with increased ADMA levels in BALF and serum. Transgenic overexpression of DDAH1 resulted in decreased BAL total cell and eosinophil numbers following HDM exposure. Total IgE levels in BALF and serum were decreased in HDM-exposed DDAH1-transgenic mice compared to HDM-exposed wild type mice. RNA-Seq results showed downregulation of genes in the inducible nitric oxide synthase (iNOS) signaling pathway in PBS-treated DDAH1-transgenic mice versus PBS-treated wild type mice and downregulation of genes in IL-13/FOXA2 signaling pathway in HDM-treated DDAH1-transgenic mice versus HDM-treated wild type mice. Our findings suggest that decreased expression of DDAH1 and DDAH2 in the lungs may contribute to allergic asthma and overexpression of DDAH1 attenuates allergen-induced airway inflammation through modulation of Th2 responses.     

Effect of Mouse Strain in a Model of Chemical-induced Respiratory Allergy

The inhalation of many types of chemicals is a leading cause of allergic respiratory diseases, and effective protocols are needed for the detection of environmental chemical–related respiratory allergies. In our previous studies, we developed a method for detecting environmental chemical–related respiratory allergens by using a long-term sensitization–challenge protocol involving BALB/c mice. In the current study, we sought to improve our model by characterizing strain-associated differences in respiratory allergic reactions to the well-known chemical respiratory allergen glutaraldehyde (GA). According to our protocol, BALB/c, NC/Nga, C3H/HeN, C57BL/6N, and CBA/J mice were sensitized dermally with GA for 3 weeks and then challenged with intratracheal or inhaled GA at 2 weeks after the last sensitization. The day after the final challenge, all mice were euthanized, and total serum IgE levels were assayed. In addition, immunocyte counts, cytokine production, and chemokine levels in the hilar lymph nodes (LNs) and bronchoalveolar lavage fluids (BALF) were also assessed. In conclusion, BALB/c and NC/Nga mice demonstrated markedly increased IgE reactions. Inflammatory cell counts in BALF were increased in the treated groups of all strains, especially BALB/c, NC/Nga, and CBA/J strains. Cytokine levels in LNs were increased in all treated groups except for C3H/HeN and were particularly high in BALB/c and NC/Nga mice. According to our results, we suggest that BALB/c and NC/Nga are highly susceptible to respiratory allergic responses and therefore are good candidates for use in our model for detecting environmental chemical respiratory allergens.     

Cellular Intrinsic Mechanism Affecting the Outcome of AML Treated with Ara-C in a Syngeneic Mouse Model

The mechanisms underlying acute myeloid leukemia (AML) treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.     

Pulmonary Inflammation and Airway Hyperresponsiveness in a Mouse Model of Asthma Complicated by Acid Aspiration

Several studies have indicated a strong association between asthma and aspiration of stomach contents. However, the complex association between these inflammatory processes has not been studied extensively in animal models. In the present study, we developed an animal model to evaluate the inflammatory cell, chemokine, and airway responses to asthma complicated by aspiration. The model was produced by sensitizing mice to cockroach allergens from house-dust extracts. Mice with asthma-like airway responses then were inoculated intratracheally with either an acidic solution or saline. Acid aspiration increased airway hyperresponsiveness in mice with asthma for at least 8 h. After 6 h, the combined injury caused an additive, not synergistic, increase in airway hyperresponsiveness and neutrophil recruitment to the airways. Although cysteinyl leukotrienes in bronchoalveolar lavage fluid were higher after acid aspiration, treatment with a receptor antagonist before aspiration did not diminish airway hyperresponsiveness. Vagal mechanisms reportedly mediate airway responses in acid aspiration; however, pretreatment with an anticholinergic agent did not reduce airway responses to acid. These results are consistent with an effective model of the acute effects of aspiration on the allergic lung. Further studies could examine how various forms of aspiration influence the severity of asthma.Abbreviations: BAL, bronchoalveolar lavage; MIP, macrophage inflammatory protein; Penh, enhanced pauseAsthma is an escalating public health problem in children and adults.⁴⁹ In patients with asthma, exaggerated immune responses to allergens produce lung inflammation and dysfunction. These responses lead to the characteristic airway hyperresponsiveness, obstructed airflow, and clinical symptoms associated with asthma.⁴⁹ Although several conditions aggravate asthma, much recent attention has focused on the provocative association between asthma and aspiration of stomach acid. The prevalence of gastroesophageal reflux in some asthma patient populations is greater than 50% ²¹ and significantly exceeds the prevalence in nonasthmatic populations.^20,47 This finding suggests an association between the 2 diseases and the possibility that gastroesophageal reflux promotes or aggravates symptoms that lead to the diagnosis asthma. In fact, several studies have shown a decrease in asthma symptoms after medical or surgical treatment of gastroesophageal reflux.^4,11,18,19Stomach acid may exacerbate asthma symptoms through 2 mechanisms. The first is a vagal reflex initiated in response to acid in the esophagus. Clinical studies in humans^20,50 and experimental studies in animals^34,48 have shown that acid in the esophagus promotes neurologic responses leading to bronchoconstriction and impaired airway function. Esophageal acid also may cause substance P- mediated neurogenic inflammation.¹⁶ The second mechanism is due to aspiration into the airways, which also has been documented to occur in asthma patients.²⁵ The presence of acid in the trachea increases airway hyperresponsiveness in anesthetized animals through vagal mechanisms,⁴⁸ particularly in the presence of preexisting lung inflammation.³² In addition to neurologic responses, aspiration of acid induces a pattern of pulmonary inflammation characterized by the release of proinflammatory cytokines and neutrophil recruitment.^26,31 That inflammation may also increase airway responsiveness.⁶Well-established models for both asthma^6,10,14,27 and aspiration^31,39 studies are available currently. However, the patterns of inflammation that occur after sequential insults are complex and may not be predicted by studies of the responses to individual insults.⁸ In addition, the mechanisms for exacerbation of airway hyperresponsiveness by aspiration in asthma have been limited to use of anesthetized animals. A model that allows recovery from the anesthesia after delivery of the aspirate permits the development and evaluation of pulmonary changes under more physiologic conditions. Therefore, the goals of this study were to: (1) describe acute exacerbation of asthma by acid aspiration in mice after recovery from anesthesia; (2) determine the effects of combined insults on airway hyperresponsiveness; and (3) profile the cellular and cytokine responses to the combined insults to assess potential mechanisms for the pulmonary responses to asthma complicated by aspiration.     

Modulation of Protease Activated Receptor 1 Influences Human Metapneumovirus Disease Severity in a Mouse Model

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.     

G-Protein-Coupled Estrogen Receptor Agonist Suppresses Airway Inflammation in a Mouse Model of Asthma through IL-10

Estrogen influences the disease severity and sexual dimorphism in asthma, which is caused by complex mechanisms. Besides classical nuclear estrogen receptors (ERαβ), G-protein-coupled estrogen receptor (GPER) was recently established as an estrogen receptor on the cell membrane. Although GPER is associated with immunoregulatory functions of estrogen, the pathophysiological role of GPER in allergic inflammatory lung disease has not been examined. We investigated the effect of GPER-specific agonist G-1 in asthmatic mice. GPER expression in asthmatic lung was confirmed by immunofluorescent staining. OVA-sensitized BALB/c and C57BL/6 mice were treated with G-1 by daily subcutaneous injections during an airway challenge phase, followed by histological and biochemical examination. Strikingly, administration of G-1 attenuated airway hyperresponsiveness, accumulation of inflammatory cells, and levels of Th2 cytokines (IL-5 and IL-13) in BAL fluid. G-1 treatment also decreased serum levels of anti-OVA IgE antibodies. The frequency of splenic Foxp3⁺CD4⁺ regulatory T cells and IL-10-producing GPER⁺CD4⁺ T cells was significantly increased in G-1-treated mice. Additionally, splenocytes isolated from G-1-treated mice showed greater IL-10 production. G-1-induced amelioration of airway inflammation and IgE production were abolished in IL-10-deficient mice. Taken together, these results indicate that extended GPER activation negatively regulates the acute asthmatic condition by altering the IL-10-producing lymphocyte population. The current results have potential importance for understanding the mechanistic aspects of function of estrogen in allergic inflammatory response.     

Evidence of Infectious Asthma Phenotype: Chlamydia-Induced Allergy and Pathogen-Specific IgE in a Neonatal Mouse Model

Asthma is a chronic respiratory disease whose etiology is poorly understood. Recent studies suggest that early-life respiratory infections with atypical bacteria may play an important role in the induction or exacerbation of chronic respiratory disease. The current study utilized a neonatal mouse ovalbumin (OVA) sensitization model of asthma to determine the course of early-life respiratory tract infection by Chlamydia. Neonatal (day 1) and adult (6 wks) BALB/c mice were infected intranasally with Chlamydia (MoPn) and 7 weeks later were sensitized and challenged with ovalbumin. Allergic airway disease was characterized by examination of serum and bronchoalveolar lavage fluid (BAL) cellularity, cytokine production and antibody response. The presence of Chlamydia was determined by PCR and culture. Ova-specific IgE was quantified by ELISA and Chlamydia-specific IgE was determined via Western blot analysis. Chlamydial infection in neonatal mice induced increased production of Th₂ cytokines (IL-4, 5, 10, and 13) in both BAL and serum, while infected adult mice produced increased Th₁ cytokines (IL-2, IFN-γ). The BAL from infected neonates contained significantly elevated levels of eosinophils compared to infected adult mice. Although adult mice cleared the infection ∼30 days post infection (pi), neonates were still infected 66 days after initial infection. Chlamydia-specific IgE was detected in both the BAL and serum of neonatal mice beginning 28 days post infection, however, infected adult mice did not produce Chlamydia-specific IgE antibodies over the course of the study. When allergic airway was induced using Ova, infected neonatal mice increased their production of IL-4, IL-5 and IL-13 by >2 fold compared to uninfected controls and infected adult groups. Our findings demonstrate that early-life Chlamydia infection induces a Th₂-dominant cytokine response in the airways of neonatal mice, leading to chronic infection. More significantly, early life respiratory colonization with Chlamydia elicits pathogen-specific IgE production, which further supports an infectious asthma phenotype.     

The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

Compartmental and Temporal Dynamics of Chronic Inflammation and Airway Remodelling in a Chronic Asthma Mouse Model

Background

Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes.

Methods

Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening.

Results

Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued.

Conclusions

Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy.     

Assessment of the Safety of Lactobacillus casei IMV B-7280 Probiotic Strain on a Mouse Model

Probiotics, in particular Lactobacillus (lactic acid bacteria, LAB) strains, are widely used in clinical practice. Despite that these probiotics have GRAS (generally regarded as safe) and qualified presumption of safety (QPS) statuses, the safety of particular strains still needs to be thoroughly studied. The aim of the study was to evaluate the safety of Lact. casei IMV B-7280 strain by investigating toxicity and the effects on gut microbiota in experimental animal model. Male BALB/c mice (7–8 weeks, weight 20–24 g) were treated with amounts of Lact. casei IMV B-7280 strain: 5 × 10⁶, 5 × 10⁸, or 5 × 10⁹ CFU/animal once per day during 7 days, or in the amount of 1 × 10¹⁰ CFU/animal once per day during 3 days (most of the proposed probiotic doses for humans—from 10⁸ to 10⁹ CFU) and monitored during 14 days. Blood tests and serum biochemistry were conducted; the cecal content from mice of the experimental and control groups were freshly collected and analyzed. At the end of the experiments (15th day), the presence of LAB in the heart, liver, kidney, and mesenteric lymph nodes and peripheral blood was determined; histology of the brain, liver, heart, fragments of the small and large intestine, and mesenteric lymph nodes was conducted. Survival rate of BALB/c mice treated with Lact. casei IMV B-7280 strain in different concentrations in toxicity experiments during 14 days was 100%. We observed no signs of toxicity as changes in gait, lethargy, sleep, somatomotor activity as well as changes in fur, eyes, skin and mucous membranes, tremors, behavior pattern, convulsions, salivation, diarrhea, and local injuries in mice from all experimental groups. After administration of probiotic strain, the number of opportunistic bacteria in cecal contents, such as Staphylococcus spp., Candida spp., Pseudomonas spp., and total aerobic and optionally anaerobic bacteria decreased compared to controls; the population of beneficial bacteria such as lactobacilli increased in cecal contents of these mice. LAB were not detected in the peripheral blood, heart, liver, kidneys, and mesenteric lymph nodes after administration of this strain to intact mice. Lact. casei IMV B-7280 strain is safe at dose up to 10¹⁰ CFU/animal during 3- and 7-day oral administration to mice and has a positive effect on the gut microbiota composition; it could be potentially considered as safe probiotic for humans.

     

小鼠胰岛素抵抗哮喘模型的建立与评估

目的:探讨小鼠胰岛素抵抗哮喘模型的建立方法,并进行评估。方法:C57BL/6J小鼠随机分为4组:正常对照组(HC)、哮喘组(NIRA)均给予普通饲料喂养;胰岛素抵抗组IRNA)、胰岛素抵抗+哮喘组(IRA)均给予高脂饲料(D12492)喂养。每周称重,第6-14周,每周检测各组小鼠空腹血糖(FPG)、空腹血清胰岛素(FINS)水平,计算稳态模型胰岛素抵抗评价指数(HOMA-IR)评估胰岛素抵抗程度;小鼠胰岛素抵抗模型建立成功后在其基础上诱导哮喘模型,NIRA组和IRA组小鼠给予卵清蛋白(OVA)致敏、激发;HC组和IRNA组小鼠给予生理盐水作为对照,末次激发24后,制作肺病理切片,计数肺泡灌洗液(BALF)中白细胞总数及分类,检测血清和BALF中相关炎性因子的水平,比较各组小鼠胰岛素抵抗指数与哮喘评价指标,评估模型。结果:(1)第9周末,IRNA组、IRA组小鼠的HOMA-IR值均2.5,表明胰岛素抵抗小鼠模型建立成功;(2)肺组织病理切片中,HC组、IRNA组小鼠肺组织无炎症改变,NIRA组、IRA组炎细胞浸润明显,尤以IRA组更甚。(3)与HC组比较,NIRA组(P0.01)、IRA组(P0.01)BALF中白细胞总数、嗜酸性粒细胞比例明显增高;(4)血清中抗OVA特异性Ig E(P0.01)和Ig G1(P0.05)水平显著升高;(5)血清和BALF中IL-4(P0.01)、IL-17(P0.05)的水平明显升高,且IRA组(P0.05)明显高于NIRA组;IFN-γ(P0.05)的水平明显降低,且IRA组(P0.05)明显低于NIRA组。结论:用高脂饲料喂养C57BL/6J小鼠9周,可建立稳定的胰岛素抵抗模型,从第10周开始用OVA致敏、激发诱发哮喘,可成功建立稳定的胰岛素抵抗哮喘小鼠模型,为进一步研究胰岛素抵抗与哮喘相关机制奠定基础。     

平喘固本合剂对昆系小鼠急性哮喘模型气道炎症作用影响

目的:探讨平喘固本合剂对昆系小鼠急性哮喘模型气道炎症作用影响及相关机制。方法:昆明系小鼠40只,随机分为对照组(A),哮喘模型组(B),喘固本合剂治疗组(C),布地奈德雾化治疗组(D)及平喘固本合剂联合布地奈德雾化治疗组(E)。用鸡卵蛋白(OVA)致敏建立昆系小鼠哮喘急性气道炎症模型,并给与药物治疗10天。对各组支气管肺泡灌洗液(BALF)中各种细胞进行分类并计数,观察肺组织的病理变化,同时应用ELISA法测定灌洗液中炎症相关因子的水平变化。结果:与B组比较,C组、D组、E组BALF中细胞总数、嗜酸性粒细胞数、巨噬细胞数及IL-4、IL-4/IFN-γ明显降低(P<0.05),IFN-γ明显升高(P<0.05)。对于IL-4、IL-4/IFN-γ水平的比较C组与D组无明显统计学差异,E组与两者具有明显统计学意义(P<0.05)。HE染色显示C组、D组、E组较单纯模型组炎症细胞浸润,平滑肌肥厚及黏膜肺组织水肿等炎症表现明显减轻。结论:平喘固本合剂对哮喘昆系小鼠气道炎症有明显的抑制作用,其作用机制可能与抑制IL-4的表达、炎性细胞聚积及促进IFN-γ的表达有关,并且可能与糖皮质激素有一定协同作用。     

小鼠动物实验方法系列专题(六) 卵清蛋白诱导129Sv小鼠建立哮喘模型的方法

近年来,哮喘发病率有逐年增长的趋势,因此利用动物模型研究哮喘发生的分子生物学机制及治疗方案具有重要的意义。利用卵清蛋白(ovalbumin,OVA)致敏诱导动物发生哮喘是比较成熟的方法。常用的实验动物有小鼠、大鼠、豚鼠、家兔等。该文主要介绍一种可以有效致敏129Sv品系小鼠、建立哮喘疾病模型的技术路线,并对模型指标进行了具体的描述,供从事相关研究的人员参考。     

三七总皂苷对支气管哮喘小鼠气道炎症及气道重建的影响

目的:探讨三七总皂苷对支气管哮喘小鼠气道炎症及气道重建的影响。方法:选择昆明种小鼠24只,将其随机分为三七总皂苷(PNS)治疗组、阳性对照组(哮喘组)和阴性对照组,每组8只。小鼠腹腔内注射氢氧化铝-卵清蛋白悬浊液(OVA-Al(OH)3)进行致敏,致敏完成后用2%卵清蛋白溶液(OVA)雾化激发,在激发前30分钟给药治疗,雾化激发6周。建模完成后,做心肺组织灌流,取左肺固定做HE染色,镜下观察评估气道病理学改变(气道狭窄阻塞率、气道上皮坏死糜烂率、上皮细胞杯状化生率、炎性细胞浸润率及平滑肌增生率);行免疫组织化学染色,用图像分析软件半定量测量转化生长因子-beta1(TGF-β1)的含量;行特殊染色,用图像分析软件测量基底膜厚度;取右肺组织,利用酶联免疫吸附法(ELISA)测定肺组织匀浆白介素-17(IL-17)因子的水平。结果:1各组小鼠经心肺组织灌流后,与哮喘组比较,三七治疗组小鼠的肺部红肿减轻,肺表颜色较正常组略微泛红。2三七治疗组气道狭窄率、上皮细胞坏死率、炎细胞浸润率、平滑肌增生率均显著低于哮喘组(P0.05),但与正常组比较无显著性差异(P0.05)。3三七治疗组气道上皮细胞TGF-β1含量显著低于哮喘组(P0.001),肺组织白介素-17含量降低至哮喘组一半以下(P0.05),与对照组相比均无显著性差异(P=0.94,P=0.23)。4哮喘组、三七组和对照组小鼠的支气管基底膜厚度分别为(0.7893±0.014)、(0.7216±0.016)、(0.5655±0.012)μm,哮喘组显著高于三七组和对照组,均具有显著性差异(P0.05)。结论:三七总皂苷可以有效改善支气管哮喘小鼠的气道重建,并抑制其气道炎性。     

Influence of Age and Strain on Striatal Dopamine Loss in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract : Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Affected individuals exhibit a characteristic pattern of neurological and behavioral features attributable in part to dysfunction of basal ganglia dopamine systems. In the current studies, striatal dopamine loss was investigated in five different HPRT-deficient strains of mice carrying one of two different HPRT gene mutations. Caudoputamen dopamine concentrations were significantly reduced in all five of the strains, with deficits ranging from 50.7 to 61.1%. Mesolimbic dopamine was significantly reduced in only three of the five strains, with a range of 31.6-38.6%. The reduction of caudoputamen dopamine was age dependent, emerging between 4 and 12 weeks of age. Tyrosine hydroxylase and aromatic amino acid decarboxylase, two enzymes responsible for the synthesis of dopamine, were reduced by 22.4-37.3 and 22.2-43.1%, respectively. These results demonstrate that HPRT deficiency is strongly associated with a loss of basal ganglia dopamine. The magnitude of dopamine loss measurable is dependent on the genetic background of the mouse strain used, the basal ganglia sub-region examined, and the age of the animals at assessment.