Sensitizing agent for constructing neutrophilic asthma animal model, construction method and application thereof

Abstract

The present application relates to the field of biological medicine, in particular to a sensitizing agent for constructing a neutrophilic asthma animal model, a construction method and application. The present application uses Haemophilus influenzae outer membrane vesicles and ovalbumin as a sensitizing agent to construct a neutrophilic asthma animal model, and the test results show that the level of neutrophils in the blood and lung lavage fluid of mice is significantly increased compared with the treatment of ovalbumin as a sensitizing agent alone. The constructed mouse neutrophilic asthma model can be used for the research on the mechanism of clinical neutrophilic asthma and the development of neutrophilic asthma drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a sensitizing agent for constructing a neutrophilic asthma animal model, a construction method and application thereof. BACKGROUND

[0002] Asthma is a common chronic inflammatory disease of the airways, characterized by variable and recurring symptoms, reversible airflow obstruction, and bronchial hyperresponsiveness. Glucocorticoids are the most effective treatment for asthma. Asthma is classified into eosinophilic, neutrophilic, mixed granulocytic, and paucigranulocytic phenotypes based on the predominant inflammatory cell type in induced sputum. Neutrophilic asthma is associated with severe asthma, late-onset asthma, and glucocorticoid-insensitivity. Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacillus that colonizes the upper respiratory tract and is a conditional pathogen of the upper respiratory tract in children and adults. More and more evidence shows that NTHi infection is closely related to the occurrence of neutrophilic asthma. Bisgaard et al. found that children with early colonization of NTHi in the nasopharynx within 4 weeks after birth had a much higher incidence of wheezing than those without early colonization of NTHi. McCann et al. found that NTHi colonization led to a significant increase in airway mucus in a mouse model of allergen challenge. Wang et al. found that NTHi infection could promote the expression of MUC5AC mucin in airway epithelial cells through the MAPK signaling pathway and promote the secretion of MUC2 through the NF-κB signaling pathway, leading to airway mucus accumulation and airway stenosis. NTHi induces oxidative stress to produce reactive oxygen species, which kills intracellular NTHi, penetrates tissues, and causes airway epithelial shedding, mucosal damage, airway smooth muscle contraction, vascular leakage, and induction of airway hyperresponsiveness and airway remodeling. NTHi also uses its surface structures such as outer membrane proteins and adhesins to form biofilms in the lower airways, which helps NTHi evade host mucosal immune surveillance and clearance, creating conditions for chronic NTHi infection. Studies have shown that NTHi infection promotes Th17 immune responses and suppresses Treg immune responses, releasing large amounts of IL-17 to recruit blood neutrophils to infiltrate lung tissue, and IL-17 also causes an imbalance in the expression of GRα / GRβ in various cells, leading to glucocorticoid resistance. Outer membrane vesicles (OMVs) are 20-250 nm spherical lipid bilayers actively secreted by the outer membrane of gram-negative bacteria, which are the main carriers of virulence factors to the host. OMVs have the functions of intercellular and intracellular communication, biofilm formation, and immune regulation, and can achieve communication and symbiosis between pathogenic microorganisms and the host. OMVs contain the outer membrane, periplasm, and some cytoplasmic components of gram-negative bacteria.Therefore, the hemophilus influenza outer membrane vesicle-induced neutrophilic asthma mouse model is constructed, the neutrophilic asthma mode caused by bacterial infection is better simulated, and a valuable biological model is provided for exploring the mechanism of disease occurrence and development.

[0003] So far, ovalbumin OVA is the most commonly used sensitizing agent for preparing a neutrophilic asthma animal model, and there are two kinds of simple OVA and complex OVA (containing an adjuvant), and the complex OVA is more commonly used. In addition to the sensitizing agent, many non-allergic factors such as bacterial endotoxin (LPS), environmental pollution, cigarette smoke, mycoplasma infection, etc. can also cause the airway NEU (neutrophil) of asthma patients or mice to increase and the activity to enhance, and are related to AHR (airway hyperresponsiveness), and therefore, the OVA combined with non-allergic factors is usually used to establish a neutrophilic asthma experimental animal model. The closest method is to use the OVA and lipopolysaccharide (LPS)-induced NA model: LPS is a commonly used adjuvant, which is a common component of the cell wall of gram-negative bacilli, and it can efficiently induce cytokine synthesis and release by activating multiple signaling pathways, stimulate inflammatory response, aggravate AHR, and also can induce the recruitment of NEU and EOS (eosinophil count), thereby aggravating asthma, and therefore, LPS is commonly used to replicate the NA animal model.

[0004] However, LPS can only represent the cell wall component of bacteria and activate the immune signaling pathway by interacting with the cell surface receptor, and cannot represent the overall composition of the bacteria. At the same time, LPS does not have species specificity, and the mechanism of disease occurrence caused by different bacteria is not the same, and only using LPS instead of bacteria to induce neutrophilic asthma related to infection is relatively inaccurate. Hemophilus influenza is a common bacterium in NA sputum culture, and plays an important role in the occurrence and development of NA, and therefore, the construction of a neutrophilic asthma model induced by hemophilus influenza extracellular vesicles will provide a more powerful biological model for exploring the NA induced by hemophilus influenza. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a sensitizing agent for constructing a neutrophilic asthma animal model, a construction method and application.

[0006] The present application provides the application of hemophilus influenza outer membrane vesicles in the preparation of a neutrophilic asthma animal model.

[0007] The present application provides a sensitizing agent for constructing a neutrophilic asthma animal model, which is hemophilus influenza outer membrane vesicles.

[0008] The preparation method of the hemophilus influenza outer membrane vesicles comprises the following steps: obtaining the supernatant after culturing and first centrifuging hemophilus influenza, and obtaining the hemophilus influenza outer membrane vesicles by concentrating, filtering and second centrifuging the supernatant.

[0009] Further,

[0010] The culture medium is sBHI broth medium;

[0011] The culture condition is 37℃, 180rpm for 12h-16h;

[0012] The first centrifugation condition is 10,000xg, 4℃ for 10min-30min, preferably 10min;

[0013] The concentration of the concentrated filtrate is 100kDa;

[0014] The filter diameter of the filtration is 0.22μm;

[0015] The second centrifugation condition is 100,000xg, 4℃ for 0.5h-2h, preferably 1h.

[0016] The present application provides a combined sensitizer, which comprises ovalbumin and the sensitizer of the present application.

[0017] The present application uses Haemophilus influenzae outer membrane vesicles as a sensitizer combined with ovalbumin for the construction of a neutrophilic asthma animal model. The results show that, compared with other sensitizers, Haemophilus influenzae outer membrane vesicles combined with ovalbumin can aggravate mouse asthma and airway inflammation, and the level of neutrophils in serum and bronchoalveolar lavage fluid is also significantly increased, which is more conducive to the accurate simulation of neutrophilic asthma model and the research of neutrophilic asthma mechanism.

[0018] The present application provides a reagent combination, which comprises mixture 1, mixture 2, mixture 3, the sensitizer of the present application and / or any two or more combinations of the combined sensitizer of the present application;

[0019] The mixture 1 comprises Haemophilus influenzae outer membrane vesicles and physiological saline;

[0020] The mixture 2 comprises ovalbumin and physiological saline;

[0021] The mixture 3 comprises ovalbumin and physiological saline.

[0022] Further, in the reagent combination,

[0023] In the mixture 1, the concentration of Haemophilus influenzae outer membrane vesicles is 250μg / mL;

[0024] In the mixture 2, the concentration of ovalbumin is 50μg / mL;

[0025] In the mixture 3, the concentration of ovalbumin is 20mg / mL.

[0026] Further, the kit of the present application further comprises adjuvants, including but not limited to adjuvants, buffers, disinfectants, staining agents or culture solution, etc.

[0027] The present application provides a method for preparing a neutrophilic asthma animal model, which comprises treating a model animal with at least one of a), b) and c) as shown below to obtain a neutrophilic asthma animal model:

[0028] a) a sensitizer of the present application;

[0029] b) a combined sensitizer of the present application;

[0030] c) a reagent combination of the present application.

[0031] Further, the method comprises the following steps:

[0032] The model animal is treated with mixture 1 by atomization, then injected with mixture 2, and then treated with mixture 3 by atomization to obtain the neutrophilic asthma animal model.

[0033] Further, in the method,

[0034] The model animal comprises a mouse, a rat, a big white rabbit, a pig, a dog, a monkey or a sheep; in some embodiments of the present application, the model animal is a mouse, further a BALB / c mouse, and further a BALB / c female mouse.

[0035] The volume of mixture 1 treated by atomization is 10 mL;

[0036] The volume of mixture 2 injected is 200 μL;

[0037] The volume of mixture 3 treated by atomization is 15 mL;

[0038] The time of mixture 1 treated by atomization is day -10; the mouse is 5-6 weeks old when mixture 1 is treated by atomization;

[0039] The time of injecting mixture 2 is day 0;

[0040] The time of mixture 3 treated by atomization is day 12, day 13, day 14 or day 15.

[0041] The present application provides a neutrophilic asthma animal model prepared by the method.

[0042] The present application provides at least one of A) to D) as shown below for use in preparing a neutrophilic asthma animal model and / or a neutrophilic asthma drug:

[0043] A) The sensitizers described in this invention;

[0044] B) The combined sensitizers described in this invention;

[0045] C) The reagent combination described in this invention;

[0046] D) The preparation method described in this invention.

[0047] This invention utilizes Haemophilus influenzae outer membrane vesicles and ovalbumin as sensitizers to construct an animal model of neutrophilic asthma. Experimental results show that compared with treatment using ovalbumin alone as a sensitizer, the levels of neutrophils in the blood and bronchoalveolar lavage fluid of mice are significantly increased. The constructed mouse neutrophilic asthma model can be used for research on the pathogenesis of clinical neutrophilic asthma and for the development of drugs for neutrophilic asthma. Attached Figure Description

[0048] Figure 1 Analysis of outer membrane vesicle size of Haemophilus influenzae;

[0049] Figure 2 Electron microscopy analysis of outer membrane vesicles of Haemophilus influenzae;

[0050] Figure 3 Flowchart illustrating Haemophilus influenzae-induced norepinephrine (NA) activity;

[0051] Figure 4 Nebulized Haemophilus influenzae outer membrane vesicles showed increased airway resistance in mice;

[0052] Figure 5 The study showed decreased dynamic lung compliance in mice treated with Haemophilus influenzae outer membrane vesicles.

[0053] Figure 6 Nebulized Haemophilus influenzae outer membrane vesicles significantly increased lung tissue inflammation and airway narrowing in mice.

[0054] Figure 7 The study showed increased airway mucus secretion in the lung tissue of mice treated with nebulized Haemophilus influenzae outer membrane vesicles.

[0055] Figure 8 The study showed elevated levels of neutrophils in the blood of mice treated with aerosolized Haemophilus influenzae outer membrane vesicles.

[0056] Figure 9 The levels of neutrophils in the bronchoalveolar lavage fluid of mice were significantly increased after aerosolization of Haemophilus influenzae outer membrane vesicles. Detailed Implementation

[0057] The present application provides a sensitizing agent for constructing a neutrophilic asthma animal model, a construction method and application. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to achieve and apply the present application technology.

[0058] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0059] The present application is further described below in conjunction with examples:

[0060] Example 1 Construction of the model

[0061] I. Extraction, separation and identification of NTHi OMVs

[0062] NTHi strain (ATCC 49766 standard strain) was cultured in sBHI broth medium at 37°C, 180 rpm overnight. The bacterial body precipitate was removed by centrifugation at 10,000 x g, 4°C for 10 min. The supernatant was enriched to one sixth of the original volume using a 100 kDa concentration column (Millipore). The concentrated supernatant was filtered with a 0.22 μm filter to further remove bacteria. The supernatant was placed in a thick-walled ultracentrifuge tube and centrifuged at 100,000 g, 4°C for 1 h. After centrifugation, the supernatant was removed and the precipitate was OMVs (outer membrane vesicles).

[0063] The particle size distribution of OMVs was detected using Nanosight NS300 (Malvern Instruments), and the results are shown in Figure 1 .

[0064] At the same time, the OMVs were negatively stained, and the size and shape of the OMVs were observed using a transmission electron microscope, and the results are shown in Figure 2 .

[0065] Figure 1 The results show that the particle size distribution of OMVs is about 100 nm to 500 nm, and the particle size is relatively uniform; Figure 2 The results show that the OMVs extracted by us have a double-layer membrane structure of vesicles, and the scale represents 100 nm.

[0066] II. NTHi OMVs aggravate mouse asthma and airway inflammation

[0067] The NA procedure induced by Haemophilus influenzae is as follows Figure 3 . 5-week-old SPF BALB / c female mice were selected:

[0068] On day 10, the mice in the OVA+OMVs group were given 10 mL of 250 pg / mL OMVs (OMVs concentration was adjusted with normal saline) aerosol treatment in a closed space, and the mice in the other two groups (OVA group and control group) were given the same amount of 0.9% normal saline aerosol treatment;

[0069] On day 0, the mice in the OVA group and the OVA+OMVs group were injected intraperitoneally with 200 pL of 0.9% normal saline containing 50 pg / mL OVA, and the mice in the control group were injected with the same amount of 0.9% normal saline;

[0070] On days 12-15 of the experiment, the mice in the OVA group and the OVA+OMVs group were given 15 mL of 20 mg / mL OVA (OMVs concentration was adjusted with normal saline) aerosol inhalation treatment, and the mice in the control group were given the same amount of 0.9% normal saline;

[0071] On day 16, the RC small animal lung function instrument (DSI Buxco RC) was used to detect the effects of Haemophilus influenzae aerosol on the airway resistance ( Figure 4 ) and dynamic lung compliance ( Figure 5 ) of the mice.

[0072] As shown in Figure 4 , the aerosol of Haemophilus influenzae outer membrane vesicles increased the airway resistance of the mice; as shown in Figure 5 , the aerosol of Haemophilus influenzae outer membrane vesicles decreased the dynamic compliance of the mice.

[0073] The lung tissues of the mice in each group were taken for H&E (hematoxylin and eosin staining) and PAS staining (periodic acid-Schiff staining or glycogen staining) to detect the level of airway inflammation ( Figure 6 ) and mucus secretion ( Figure 7 ).

[0074] As shown in Figure 6 , the aerosol of Haemophilus influenzae outer membrane vesicles significantly aggravated the inflammation level and airway stenosis of the lung tissues of the mice; as shown in Figure 7 , the aerosol of Haemophilus influenzae outer membrane vesicles increased the mucus secretion of the airway of the lung tissues of the mice.

[0075] III. NTHi OMVs induced neutrophil infiltration

[0076] The serum and alveolar lavage fluid of the mice in the Control group, the OVA group and the OVA+OMVs group were collected, and the neutrophils were counted on the Wright-Giemsa stained slides according to the morphological criteria to detect the number of neutrophils in the blood and alveolar lavage fluid of the mice, as shown in Figure 8 and Figure 9 .

[0077] As shown in Figure 8The level of neutrophil in mouse blood is increased by atomized H. influenzae outer membrane vesicles; as shown in Figure 9 As shown, the level of neutrophil in mouse alveolar lavage fluid is significantly increased by atomized H. influenzae outer membrane vesicles

[0078] In summary, we successfully constructed a neutrophilic asthma mouse. The present application uses H. influenzae outer membrane vesicles as a sensitizing agent to construct a mouse model, which is more conducive to the accurate simulation of a neutrophilic asthma model and the study of the mechanism of neutrophilic asthma. Compared with OVA sensitization alone, the animal model constructed by the present application is more accurate in simulating atomized H. influenzae infection, and the use of outer membrane vesicles and ovalbumin together for atomization has stronger immunogenicity and better effects.

[0079] The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A method for preparing a neutrophilic asthma animal model, characterized by, Comprise the following steps as shown below: The mice are treated with 2.5 mg of Haemophilus influenzae outer membrane vesicles by atomization, and then 10 μg of ovalbumin is injected, and then the mice are treated with 300 mg of ovalbumin by atomization to obtain the neutrophilic asthma animal model.

2. The preparation method of claim 1, wherein, The mice are treated with 2.5 mg of Haemophilus influenzae outer membrane vesicles by atomization at the age of 5-6 weeks; The time of injecting 10 μg of ovalbumin is the 10th day after the mice are treated with 2.5 mg of Haemophilus influenzae outer membrane vesicles by atomization; The time of treating with 300 mg of ovalbumin by atomization is the 12th day, the 13th day, the 14th day and the 15th day after injecting 10 μg of ovalbumin.

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