Establishment method and application of allergic asthma susceptible mouse model

Abstract

The invention belongs to the field of biomedicine, and relates to an establishment method and application of an allergic asthma susceptible mouse model. According to the method, a filial generation allergic asthma susceptible animal model is constructed through exposure of amoxicillin in a perinatal period, intragastric administration is performed on a female mouse from the 13th day of pregnancy to the 7th day of postpartum in an experiment, and a key window in the perinatal period is covered. And the filial generation is sensitized and excited by ovalbumin (OVA) to establish a standardized asthma model. It is found that exposure of amoxicillin in the perigenic period significantly reduces the richness and diversity of filial generation flora and inhibits taurine metabolism, resulting in significant increase of airway hyperreactivity and eosinophilic granulocytes in alveolar lavage fluid. The cycle of the model is shortened to 28 days, and by combining metabolome and 16S sequencing data, a new mechanism that antibiotics influence asthma susceptibility through a flora-metabolite axis is disclosed, and an experimental basis and a quantitative strategy are provided for clinical intervention.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for establishing a mouse model of allergic asthma susceptibility and its application. Background Art

[0002] Allergic asthma is a highly prevalent chronic respiratory disease worldwide, and its animal model construction and pathogenesis research are of great significance. Existing technologies mainly use ovalbumin (OVA) sensitization and stimulation methods, but there are problems such as long model construction cycle, unclear susceptibility mechanism and insufficient optimization of intervention measures. The traditional process requires complex steps such as perinatal administration of pregnant mice and multiple sensitization and stimulation of offspring after weaning. The entire cycle lasts for weeks to months, which seriously restricts research efficiency. Although epidemiological data show that perinatal antibiotic exposure is associated with an increased risk of asthma in offspring, the specific mechanism of action has not been fully elucidated. Existing studies mostly focus on the association between intestinal flora disorders and immune imbalance, and lack causal evidence at the metabolic level. Although intervention measures such as probiotics have shown improvement potential, key parameters such as strain selection, dosage window and duration of action lack standardized schemes, which limits clinical translation and application.

[0003] In order to solve the above problems, the applicant proposed a method for establishing and applying a mouse model of allergic asthma susceptibility. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for establishing a mouse model of allergic asthma susceptibility and its application, so as to solve the problems in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for establishing and applying a mouse model of allergic asthma susceptibility, comprising the following steps:

[0006] S1. 8-week-old C57BL / 6J mice were selected as experimental animals and raised in an SPF environment.

[0007] S2, from gestational day 13 to postpartum day 7, dams were given 200 mg / kg body weight amoxicillin / normal saline by gavage daily;

[0008] S3, pups were housed in separate cages according to sex after weaning and sensitized with three intraperitoneal injections of OVA on PND29, 36, and 43;

[0009] S4, challenged by nebulized inhalation of 1% OVA solution for 3 consecutive days starting from PND50;

[0010] S5. The Penh value after methacholine stimulation was measured by whole-body plethysmography system, and the asthma susceptibility was assessed by combining H&E and PAS staining pathological analysis of lung tissue.

[0011] Optionally, the OVA sensitization solution formula is: 7.2 mg OVA is mixed with 24 mL aluminum hydroxide adjuvant to prepare a sensitization solution with a volume of 10 mL / kg.

[0012] Optionally, the methacholine stimulation concentration gradient is 0, 3.125, 6.25, 12.5, 25 and 50 mg / mL, each nebulization is 1 minute, and data collection is performed at intervals of 9 minutes.

[0013] Optionally, the lung tissue pathological analysis includes: fixation with 4% paraformaldehyde for 24 hours and then embedding in paraffin, preparing 5 μm sections for H&E staining to evaluate inflammatory cell infiltration, and PAS staining to analyze goblet cell mucus secretion.

[0014] Optionally, during PND8-PND28, the young mice were orally administered with 100 μL of VSL#3 compound probiotic suspension containing 10^10 CFU / mL daily for intervention, wherein the probiotics included 4 types of lactobacilli, 3 types of bifidobacteria and thermophilic Streptococcus.

[0015] Optionally, the probiotic intervention groups include a control group, an amoxicillin exposure group, a probiotic intervention group, an OVA group, an amoxicillin+OVA group, and an amoxicillin+probiotic combined intervention group.

[0016] Beneficial effect: innovative elucidation of the mechanism

[0017] First study reveals that perinatal amoxicillin exposure increases asthma susceptibility through a dual pathway:

[0018] Directly destroy intestinal flora colonization, significantly reducing the abundance of probiotics such as Akkermansia, Faecalibacterium, and Lactobacillus (the flora richness decreased by 37.2%, and the diversity index decreased by 29.6%)

[0019] Inhibits the synthesis of key metabolites such as taurine (fecal taurine content dropped to 42.3% of the control group)

[0020] This discovery established a complete causal chain of antibiotics-microbiome-metabolites-asthma, filling the gap in mechanism research in the field.

[0021] Quantitative improvement of intervention effects

[0022] Supplementing VSL#3 compound probiotics during lactation can achieve multi-dimensional improvements:

[0023] Microbial flora recovery: The microbial flora imbalance index (MDI) of male offspring decreased from 4.8±1.2 to 2.1±0.7

[0024] Inflammation suppression: The total number of inflammatory cells in BALF decreased by 63.4%, and the proportion of eosinophils decreased by 71.8%

[0025] Improved airway responsiveness: Penh value decreased by 58.3% after methacholine challenge (P<0.01)

[0026] Model optimization and reproducibility

[0027] Through a standardized dosing regimen (continuous amoxicillin gavage from GD13 to PND7) and an evaluation system (Penh value + dual pathological indicators), the model construction cycle was shortened to 28 days, and the experimental repetition success rate was increased to 92.6% (about 75% for traditional models).

[0028] Clinical translational value

[0029] It provides a clear intervention window (the first 4 weeks of lactation) and dosage regimen (10^9 CFU / day), offering a feasible clinical solution for preventing the risk of antibiotic-related asthma, and has been proven to be safe and effective through animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of an embodiment of the present invention; DETAILED DESCRIPTION

[0031] The following describes preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0032] The present invention provides an asthma susceptibility animal model construction, and the specific implementation methods are as follows:

[0033] Eight-week-old SPF female C57BL / 6J mice were used as experimental subjects. Pregnant mice, after confirmation of pregnancy by vaginal smear, were randomly divided into four groups, with 6-8 pregnant mice per group. After weaning, the offspring were housed in cages separated by sex, with 4-5 mice per cage. The experiment lasted until the offspring were 8 weeks old. The control group received an equal volume of normal saline by gavage during pregnancy and lactation. The amoxicillin group received 200 mg / kg amoxicillin daily by gavage from day 13 of pregnancy to day 7 postpartum. The probiotic group received 10^9 CFU of the compound probiotic VSL#3 daily by gavage from day 8 to day 28 postpartum, in addition to the amoxicillin treatment. The combined intervention group received both amoxicillin exposure and VSL#3 intervention.

[0034] Perinatal antibiotic exposure employed a customized dosing strategy. Amoxicillin sodium powder was dissolved in saline to a 20 mg / mL solution, prepared immediately. A 1 mL syringe connected to a gavage needle was used for daily gavage at a fixed time. The dosing volume was calculated based on the mother's weight, ensuring accuracy to 0.01 mL. A standardized asthma model was established in offspring mice starting on postnatal day 29. During the sensitization phase, OVA sensitization solution containing 7.2 mg OVA and 24 mL aluminum hydroxide adjuvant was injected intraperitoneally on postnatal days 29, 36, and 43. The injection volume was 10 mL / kg. During the challenge phase, 1% OVA solution was inhaled via nebulization for 3 consecutive days from postnatal days 50 to 52, with a daily challenge time of 30 minutes. The control group underwent the same procedure, using an equal volume of saline instead of OVA.

[0035] For probiotic intervention, commercially available VSL#3 freeze-dried bacterial powder containing four strains of Lactobacillus, three strains of Bifidobacterium, and Streptococcus thermophilus was used. The powder was diluted to 1010 CFU / mL in sterile PBS and prepared immediately before use. Oral gavage was performed using a 100-μL pipette at a fixed time daily. Samples of each batch of bacterial solution were collected for plate counts to ensure viable counts ≥109 CFU / mL.

[0036] Key endpoints included airway hyperresponsiveness, measured using a whole-body plethysmography system. Patients were administered 0, 3.125, 6.25, 12.5, 25, and 50 mg / mL MCh solutions, each nebulized for 1 minute. Penh values ​​were recorded for 9 minutes after each nebulization, and the mean plateau value was used as the final result. The Penh value after 50 mg / mL MCh challenge was used as the primary outcome measure. Lung histopathology analysis involved fixation of the right middle lobe with 4% paraformaldehyde for 24 hours, dehydration with graded ethanol, and paraffin embedding for 5-μm-thick sections. Inflammatory cell infiltration was assessed using H&E staining, and goblet cell proliferation was analyzed using PAS staining. Semi-quantitative scoring was performed using Image-Pro Plus 6.0 software.

[0037] For 16S rRNA sequencing of the intestinal microbiome, fecal DNA was extracted using the QIAamp Fast DNA Stool Mini Kit. The V3-V4 region was amplified and a sequencing library was constructed. PE300 paired-end sequencing was performed on the Illumina MiSeq platform. Bioinformatics analysis included sequence assembly, OTU clustering, and species annotation. For metabolomics analysis, 50 mg of feces was added to 500 μL of 70% methanol-water solution, vortexed, and then placed on ice. After centrifugation, the supernatant was filtered through a 0.22 μm filter. LC-MS / MS analysis was performed using an ACQUITY UPLC HSS T3 column with an electrospray ionization source, and SCIEX Multiquant 3.0.3 software was used for peak area integration and relative quantification.

[0038] Statistical analysis was performed using Excel 2019, with double-checked data entry. Statistical analysis was performed using SPSS 26.0. One-way analysis of variance was used for multiple group comparisons, independent sample t-tests were used for two-group comparisons, and repeated measures analysis of variance was used for repeated measurement data. GraphPad Prism 9.0 was used to generate various charts. The implementation of this technical solution shortened the model construction cycle to 28 days and increased the experimental replication success rate to 92.6%. The sensitivity of the intestinal flora α-diversity index detection reached 0.01 units, and metabolomics testing could quantitatively analyze more than 500 metabolites. After probiotic intervention, the Penh value of male offspring decreased by 58.3%, and the total number of inflammatory cells in the BALF decreased by 63.4%. The relevant technical parameters were methodologically validated and met the standards of biomedical research, providing a reliable technical platform for studying the impact of perinatal environmental factors on offspring asthma susceptibility.

[0039] The present invention specifically illustrates the implementation and effects of the technical solution through the following two embodiments:

[0040] Example 1: Effect of perinatal amoxicillin exposure on offspring asthma susceptibility

[0041] Eight-week-old SPF female C57BL / 6J mice were randomly divided into a control group and an amoxicillin group, with six pregnant mice in each group. The amoxicillin group received daily oral administration of 200 mg / kg amoxicillin from gestational day 13 to postpartum day 7, while the control group received an equal volume of normal saline. After weaning, the offspring were housed separately by sex and sensitized with intraperitoneal injection of OVA solution on postpartum days 29, 36, and 43. Asthma was induced by nebulized 1% OVA solution for three consecutive days from postpartum days 50 to 52. 16S rRNA sequencing analysis of the gut microbiome revealed a 37.2% decrease in microbial richness and a 29.6% decrease in diversity in the offspring treated with amoxicillin. The dysbiosis index increased 2.1-fold, with Akkermansia, Faecalibacterium, and Lactobacillus abundances decreasing by 68.3%, 72.5%, and 61.8%, respectively. Metabolomics analysis revealed a decrease in fecal taurine content to 42.3%, and a 1.8-fold increase in sarcosine levels. Inflammatory markers included a 2.3-fold increase in lung tissue IL-5 levels and a 3.1-fold increase in the proportion of eosinophils in the BALF. Airway hyperresponsiveness assays showed that after 50 mg / mL methacholine challenge, the Penh value reached 1.8 times that of the control group. Experiments have shown that perinatal amoxicillin exposure significantly increases offspring susceptibility to asthma by disrupting intestinal flora and taurine metabolism.

[0042] Example 2: Composite probiotic intervention reverses the negative effects of amoxicillin exposure

[0043] Based on Example 1, a combined intervention group was set up, and 10^9 CFU VSL#3 compound probiotics were gavaged daily on days 8-28 after birth. Intestinal flora analysis showed that the bacterial richness of the male offspring in the combined intervention group recovered to 83.6% of that in the control group, the diversity returned to 76.4%, and the microbial imbalance index decreased by 41.2%. Metabolomics testing found that fecal taurine levels returned to 1.7 times that of the amoxicillin group, and sarcosine decreased to 1.2 times that of the control group. In terms of inflammatory indicators, the IL-5 level in lung tissue decreased by 42.7%, and the proportion of eosinophils in the BALF decreased by 58.3%. Airway hyperresponsiveness measurement showed that the Penh value of male offspring after 50 mg / mL methacholine stimulation decreased to 58.3% of that in the amoxicillin group. Experiments have confirmed that supplementation of VSL#3 during lactation can effectively reverse the bacterial flora and metabolic abnormalities caused by perinatal antibiotic exposure, significantly reducing the susceptibility of offspring to asthma.

[0044] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from all points of view, the embodiments should be regarded as illustrative and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and range of equivalents of the claims are included in the present invention. Any reference signs in the claims should not be construed as limiting the claim to which they relate.

[0045] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for establishing a mouse model of allergic asthma susceptibility and its application, characterized in that: The following steps are involved: S1. 8-week-old C57BL / 6J mice were selected as experimental animals and raised in an SPF environment. S2. From gestational day 13 to postpartum day 7, dams were given 200 mg / kg body weight of amoxicillin by oral gavage daily; S3, pups were housed in separate cages according to sex after weaning and sensitized with three intraperitoneal injections of OVA on PND29, 36, and 43; S4, challenged by nebulized inhalation of 1% OVA solution for 3 consecutive days starting from PND50; S5. The Penh value after methacholine stimulation was measured by whole-body plethysmography system, and the asthma susceptibility was assessed by combining H&E and PAS staining pathological analysis of lung tissue.

2. The method according to claim 1, wherein: The OVA sensitization solution formula is: 7.2 mg OVA is mixed with 24 mL aluminum hydroxide adjuvant to prepare a sensitization solution with a volume of 10 mL / kg.

3. The method according to claim 1, wherein: The methacholine stimulation concentration gradient was 0, 3.125, 6.25, 12.5, 25 and 50 mg / mL, each nebulization was 1 minute, and data were collected at intervals of 9 minutes.

4. The method according to claim 1, wherein: The lung tissue pathological analysis includes: fixation with 4% paraformaldehyde for 24 hours and then embedding in paraffin, preparing 5 μm sections for H&E staining to evaluate inflammatory cell infiltration, and PAS staining to analyze goblet cell mucus secretion.

5. The method according to any one of claims 1 to 4, characterized in that: During PND8-PND28, the young mice were gavaged daily with 100 μL of VSL#3 compound probiotic suspension containing 10^10 CFU / mL for intervention. The probiotics included 4 types of Lactobacillus, 3 types of Bifidobacterium and Streptococcus thermophilus.

6. The method according to claim 5, characterized in that: The probiotic intervention groups included a control group, an amoxicillin exposure group, a probiotic intervention group, an OVA group, an amoxicillin+OVA group, and an amoxicillin+OVA+probiotic intervention group.

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