Method for constructing Yunnan pine pollen allergy animal model

Abstract

The invention discloses a method for constructing a Yunnan pine pollen allergy animal model, which belongs to the technical field of Yunnan pine pollen allergy prevention and treatment and comprises the following steps: preparing Yunnan pine pollen allergens and sensitizing non-human mammals. The sensitization treatment comprises a basic sensitization treatment stage and an excitation sensitization treatment stage: in the basic sensitization treatment stage, peritoneal sensitization is carried out once a week and is continued for four weeks; in the nasal-drop sensitization treatment stage, different test substances with the concentration 2 times that of the basic sensitization stage are used for continuous 7-day quantitative nasal-drop excitation, and the nasal drop dosage is 2 microliters / g, and the symptoms of the mouse are observed within 10 minutes after nasal-drop excitation is completed each time. The allergic animal model constructed by adopting the crude extract of the pinus yunnanensis pollen allergen is the first animal model research related to pinus yunnanensis allergy at present, and a reference basis can be provided for subsequent experimental research on the aspect of deep research on pinus yunnanensis pollen allergy; a research model is provided for clinical research on the Yunnan pine pollen allergy or screening of medicines for resisting the Yunnan pine pollen allergy.

Description

Technical Field

[0001] This invention relates to the field of prevention and treatment of pine pollen allergy in Yunnan, specifically to a method for constructing an animal model of pine pollen allergy in Yunnan. Background Technology

[0002] Allergic (or hypersensitivity) diseases are a common and prevalent group of illnesses triggered by abnormal immune system reactions, severely impacting the quality of life for approximately 30% of the global population. Pollen allergy accounts for a significant proportion, with about 40% of allergic diseases related to pollen exposure. Specific proteins in pollen (mainly distributed in the 10-70 kDa range) can stimulate the body to produce immunoglobulin E (IgE), mediating the release of histamine, leukotrienes, and other active substances, triggering acute reactions such as allergic rhinitis, conjunctivitis, asthma, and urticaria. Pollen, characterized by strong antigenicity, high yield, small size, easy wind dispersal, and strong adaptability, is a major allergen.

[0003] Since the link between pollen and respiratory allergies was discovered in the 19th century, pollen research has received widespread attention. Against the backdrop of global climate change and urbanization, the incidence of pollen allergies has risen significantly. The prevalence in Europe is as high as 15%-40%, and in the United States, the pollen allergy rate among children was close to 18.9% in 2021. The situation in my country is equally serious, with the rate rising rapidly from a conservative estimate of about 1% in 2010 (5% in high-incidence areas) to recent levels of 8.7%-24.1% for adults and 3.9%-16.8% for children. Pollen allergy has become a major public health challenge.

[0004] The World Allergy Organization (WAO) points out that tree pollen is a major allergen, especially from the orders Fagaceae, Lamiaceae, Proteales, and Coniferales. As a widely distributed genus within Coniferales, *Pinus* has become a significant airborne allergen globally due to its wind-borne pollen and large production volume. In pollen monitoring in my country, *Pinus* pollen dispersal is greater than that of other species. Although pine pollen is widely used in the food and health product industries as a "natural nutrient reservoir" (rich in protein, amino acids, vitamins, minerals, etc.), related allergy cases are increasing, and its safety is a major concern. Studies show that the positive rate of pine pollen sensitization can reach 34.4%, indicating significant sensitization. However, there may be significant differences in protein composition, enzyme activity, and sensitizing protein profiles among pollen from different *Pinus* species. For example, *Pinus tabuliformis* showed a stronger IgE induction ability than other tested pine species in specific models (such as mouse nasal inhalation). This suggests that the sensitization potential of different pine species is not entirely consistent, thus requiring sensitization identification of pine pollen from different sources.

[0005] Yunnan pine is a key native tree species and major pollen source unique to southwestern my country (such as Yunnan, Sichuan, Guizhou, and Guangxi). Its distribution is concentrated, and its pollen production is enormous, making it a significant component of atmospheric pollen in the region during spring. However, research on the allergenicity of Yunnan pine pollen is currently lacking. Whether Yunnan pine possesses allergenic properties, or whether its allergenicity is similar to that of species like red pine, remains unclear. To further simulate and reflect real allergic reactions triggered by Yunnan pine pollen exposure, this study specifically investigates the differences in allergenicity among pine pollen species from different sources. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for constructing an animal model of Yunnan pine pollen allergy. The method includes priming and challenging model animals with a crude extract of Yunnan pine pollen allergen to construct the animal model of Yunnan pine pollen allergy.

[0007] To achieve the above objectives, the present invention provides a method for constructing an animal model of Yunnan pine pollen allergy, including the preparation of Yunnan pine pollen allergen and sensitization treatment of model animals;

[0008] The preparation method of Yunnan pine pollen allergen is as follows:

[0009] Weigh 1.0g of fresh Yunnan pine pollen and grind it thoroughly in liquid nitrogen until completely broken. Transfer the ground pollen powder to a centrifuge tube, add acetone, and stir and soak at 4℃ with a magnetic stirrer for 3h to remove fat. Centrifuge at 7000rpm / min for 20min to obtain a precipitate. Repeat the defatting process 3 times until all lipids are removed.

[0010] The defatted pollen precipitate was air-dried to allow the organic reagents to evaporate completely, resulting in dry, defatted pollen powder. Pre-cooled PBS buffer at 4°C was added at a mass-to-volume ratio of 1:10, and the mixture was stirred overnight at 4°C using a magnetic stirrer. The mixture was dispensed into 1.5 mL EP tubes and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected using a 10 mL syringe, filtered through a 0.45 μm filter membrane, and stored at -80°C for later use.

[0011] Sensitization treatment includes basic sensitization and challenge sensitization:

[0012] The basic sensitization includes sensitizing experimental animals intraperitoneally once a week with Yunnan pine pollen allergen.

[0013] The day after the basic sensitization treatment, challenge sensitization was performed. The challenge sensitization method included: administering Yunnan pine pollen allergen at twice the concentration of the basic sensitization stage into each nostril using a micropipette, and performing quantitative nasal instillation for 7 consecutive days; observing the experimental animals for symptoms within 10 minutes after each nasal instillation challenge.

[0014] Furthermore, the model animal is a female BALB / c mouse.

[0015] Furthermore, in the basic sensitization step, the injection dose is 10 uL / g based on the weight of the experimental animal.

[0016] Furthermore, the nasal drop dosage during the stimulation and sensitization phase is 2 uL / g based on the body weight of the experimental animal.

[0017] Through the above technical solution, the animal model of allergy constructed using Yunnan pine pollen allergen is the first animal model research on Yunnan pine allergy to date. It can provide a reference for subsequent in-depth experimental research on Yunnan pine pollen allergy, and provide a research model for clinical research on Yunnan pine pollen allergy or drug screening for anti-Yunnan pine pollen allergy. Attached Figure Description

[0018] Figure 1 Allergic symptom scores were assigned to each group of mice; * in the figure means: compared with the control group, *: P < 0.05; **: P < 0.001; ***: P < 0.0001;

[0019] Figure 2 Serum immune factor scores were assigned to each group of mice. *In the figure: *P < 0.05; **P < 0.001; ***P < 0.0001 compared to the control group.

[0020] Figure 3 The results of pathological sections of the nasal mucosa of mice in each group are shown in the figure. In the figure: yellow arrow: local epithelial shedding and exposure of the lamina propria are visible; blue arrow: local hemorrhage is visible; green arrow: a small number of granulocytes are scattered; brown cells: a small number of granulocytes are infiltrated in both the mucosa and the lamina propria; purple arrow: a large number of serous gland necrosis are visible in the lamina propria; red arrow: few granulocytes are seen in the submucosa. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0023] Regarding the selection of animals for constructing animal models: In this application, "model animals" include, but are not limited to, mice, rats, rabbits, dogs, and monkeys. Mice, as the most commonly used animal model, have advantages such as small size, rapid reproduction, and low cost. The selected BALB / c mice are a highly IgE-responsive strain resulting from inbreeding, preferentially producing Th2-type immune responses and IgE antibodies. They are often used as animal models for pollen allergies and food allergies. Using BALB / c mouse models more closely mimics the physiological and immune characteristics of human allergic reactions.

[0024] Regarding the approach to constructing allergy models: The mechanism of plant protein sensitization is complex. While we have some understanding of the main pathological steps and immune mechanisms involved, the specific steps of the pathological process are not yet fully elucidated. Generally, the pathogenesis is divided into two stages. The first stage is the sensitization stage: the allergen enters the body through the skin or digestive tract, inducing the body to produce specific IgE. The second stage is the challenge stage: its induction is the same as the sensitization stage in which an oral allergen produces allergic symptoms. The challenge stage causes a series of allergic symptoms, such as urticaria, itching, difficulty breathing, and abdominal pain. These reactions may be caused by chemical mediators such as histamine released by stimulated mast cells and leukotrienes synthesized by basophils.

[0025] The animal model of Yunnan pine pollen allergy constructed in this invention can provide a means and basis for clinical research on the pathogenesis of Yunnan pine pollen allergy and food allergy symptoms caused by consuming Yunnan pine pollen.

[0026] The preparation method of Yunnan pine pollen allergen is as follows:

[0027] Weigh 1.0g of fresh Yunnan pine pollen and grind it thoroughly in liquid nitrogen until completely broken. Transfer the ground pollen powder to a centrifuge tube, add acetone, and stir and soak at 4℃ with a magnetic stirrer for 3h to remove fat. Centrifuge at 7000rpm / min for 20min to obtain a precipitate. Repeat the defatting process 3 times until all lipids are removed.

[0028] The defatted pollen precipitate was air-dried to allow the organic reagents to evaporate completely, resulting in dry, defatted pollen powder. Pre-cooled PBS buffer at 4°C was added at a mass-to-volume ratio of 1:10, and the mixture was stirred overnight at 4°C using a magnetic stirrer. The mixture was dispensed into 1.5 mL EP tubes and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected using a 10 mL syringe, filtered through a 0.45 μm filter membrane, and stored at -80°C for later use.

[0029] Regarding the immune process, the construction experiment in this embodiment is divided into two stages: "basic sensitization treatment stage of allergen" and "nasal provocation treatment of allergen":

[0030] Mice were intraperitoneally sensitized weekly for four consecutive weeks, weighed before injection, with an intraperitoneal injection dose of 10 μL / g per mouse (based on mouse body weight). After intraperitoneal sensitization, mice underwent nasal challenge experiments for seven consecutive days. The nasal instillation method was as follows: Yunnan pine pollen allergen at twice the concentration of the basal sensitization stage was administered into each nostril using a micropipette at a dose of 2 μL / g (based on mouse body weight). The experimental animals were observed for symptoms within 10 minutes after each nasal challenge.

[0031] The control group is an animal model that is not administered the test substance and is otherwise identical to the experimental group. By comparison, if the relevant sensitization indicators of the experimental group, such as behavioral changes, changes in the content of immune factors in serum, and pathological section results are significantly different from those of the control group at the statistical level, it indicates that our animal model has been initially successfully constructed.

[0032] The present invention will be further illustrated by the following examples, but the present invention is not limited to these specific embodiments.

[0033] Example 1

[0034] This embodiment provides a method for constructing an animal model and sensitizing it. The specific steps are as follows: Construction of the sensitized animal model:

[0035] (1) Animal requirements

[0036] BALB / c mice, SPF grade, 4-6 weeks old, female, weighing 20±5g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (Animal Quality Certificate No.: 430727230101815341, Experimental Animal Production License No.: SCXK (Xiang) 2019-0004).

[0037] (2) Test substance

[0038] Yunnan pine pollen allergen + Al(OH)3 adjuvant, Al(OH)3 adjuvant, PBS blank group.

[0039] (3) Grouping of animals

[0040] The experimental animals were randomly divided into three groups (n=6 per group) according to their weight: Yunnan pine pollen allergen + Al(OH)3 adjuvant experimental group, Al(OH)3 adjuvant group, and PBS blank group.

[0041] (4) Preparation of immune adjuvants and preparation of test substances

[0042] 1) Preparation of immune adjuvants

[0043] All immune adjuvant solutions should be prepared fresh for use. Dosage: 50-100 μg / injection.

[0044] 2) Preparation of test substance

[0045] According to the body weight, add the corresponding Yunnan pine pollen allergen extract and Al(OH)3 adjuvant, mix evenly, and then stir to prepare a mixture of allergen, adjuvant and PBS.

[0046] (5) Sensitization treatment

[0047] 1) During the basic sensitization phase, the experimental animals in each group were intraperitoneally injected with the corresponding test substance on days 1, 8, 15 and 22, according to their grouping.

[0048] 2) During the challenge and sensitization phase, mice in each group were challenged by continuous quantitative intranasal drops of different test substances at twice the concentration of the baseline sensitization phase on days 23, 24, 25, 26, 27, 28, and 29. Clinical allergic symptoms of the animals were observed within 10 minutes after each challenge test, and corresponding blood and tissue samples were collected.

[0049] Example 2

[0050] In the embodiments of this application, the sensitization indicators detected include allergy clinical symptom scores, serum immune factors: total IgE, IL-4 and IFN-γ detection, and pathological section results.

[0051] (1) Allergy clinical symptom score

[0052] According to Table 1, the clinical allergic symptoms of mice were evaluated within 10 minutes after allergic provocation. The allergic reaction symptoms were recorded and observed, and continuous observation and real-time monitoring and scoring were performed. The results are shown in Table 1:

[0053] Table 1. Observation and scoring indicators of clinical allergic symptoms in mice

[0054]

[0055] (2) Measurement of immune factors in serum:

[0056] The obtained serum was analyzed according to the ELISA kit instructions, as follows:

[0057] 1) 30 minutes before the experiment, take out the reagent kit and let it return to room temperature. Before the experiment, soak the microplate 3 times and pat it dry.

[0058] 2) Add 100 μL of standard and test sample to the reaction wells, seal the plate and incubate at 37°C for 90 min, wash the plate 4 times and pat dry.

[0059] 3) Add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, and incubate at 37°C for 60 min. Wash the plate 4 times and pat dry.

[0060] 4) Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, and incubate at 37°C for 30 min. Wash the plate 5 times and pat dry.

[0061] 5) Add 100 μL of chromogenic substrate to the reaction wells, seal the plate, and develop the color at 37°C in the dark for 15 min.

[0062] 6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 min).

[0063] (3) Statistical analysis

[0064] Graphs and statistical analyses were performed using GraphPad 8.0 software. Data are expressed as mean ± standard deviation and compared using the Kruskal-Wallis test. P < 0.05 was considered statistically significant, while P > 0.05 was not.

[0065] Example 3

[0066] Based on the animal model construction method in Example 1 and the experimental method in Example 2, the specific experimental results and analysis are as follows:

[0067] 1. Clinical symptom score

[0068] When the body is stimulated by a certain allergen, a series of clinical allergic reactions will occur. The experiment scored the clinical symptoms exhibited by BALB / c mice after induced allergy, and the results are as follows: Figure 1 As shown in Table 2.

[0069] Table 2 Statistical results of allergy symptom scores in mice

[0070]

[0071] In the experimental group, the scores for "sneezing" and "nose scratching" in the clinical symptoms of allergy were significantly higher than those in the blank group and the adjuvant group, while the score for "rapid breathing" did not differ significantly among the model groups (P>0.05).

[0072] 2. Serum immune factor score

[0073] From the ELISA detection results of serum immune factors ( Figure 2 It can be seen that, compared with the blank group and the control group, the serum levels of specific IgE, IL-4 and IFN-γ in the experimental group mice were increased.

[0074] 3. Pathological section results

[0075] Figure 3(In the figure, A is the blank group, B is the adjuvant group, and C is the experimental group): Yellow arrow: Local mucosal epithelial shedding and exposure of the lamina propria; Blue arrow: Local hemorrhage; Green arrow: A small number of granulocytes are scattered; Brown cells: A small number of granulocytes are infiltrated in both the mucosal layer and the lamina propria; Purple arrow: A large number of serous gland necrosis are visible in the lamina propria; Red arrow: Few granulocytes are seen in the submucosa.

[0076] HE-stained sections ( Figure 3 It can be seen that the cells in the nasal mucosa of the blank group mice are neatly arranged, the glandular lumen structure is distinct, and there is little infiltration of inflammatory cells. The adjuvant control group shows local granulocyte distribution, while the experimental group shows mucosal epithelial shedding, and a small number of granulocytes are infiltrated in both the mucosa and the lamina propria. Compared with the blank group, the changes in cellular pathological state between the experimental groups are more significant.

[0077] Based on the results in 1-3 above, the mouse model constructed using crude protein sensitization and challenge treatment with Yunnan pine pollen in this application showed that the results of multiple sensitization response indicators were significantly higher than those of the two blank control groups.

[0078] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for 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 embodiments that can be understood by those skilled in the art.

[0079] The detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of pine pollen allergy in Yunnan, characterized in that: This includes the preparation of Yunnan pine pollen allergens and the sensitization treatment of model animals; The preparation method of Yunnan pine pollen allergen is as follows: Weigh 1.0g of fresh Yunnan pine pollen and grind it thoroughly in liquid nitrogen until completely broken. Transfer the ground pollen powder to a centrifuge tube, add acetone, and stir and soak at 4℃ with a magnetic stirrer for 3h to remove fat. Centrifuge at 7000rpm / min for 20min to obtain a precipitate. Repeat the defatting process 3 times until all lipids are removed. The defatted pollen precipitate was air-dried to allow the organic reagents to evaporate completely, resulting in dry, defatted pollen powder. Pre-cooled PBS buffer at 4°C was added at a mass-to-volume ratio of 1:10, and the mixture was stirred overnight at 4°C using a magnetic stirrer. The mixture was dispensed into 1.5 mL EP tubes and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected using a 10 mL syringe, filtered through a 0.45 μm filter membrane, and stored at -80°C for later use. Sensitization treatment includes basic sensitization and challenge sensitization: Basic sensitization includes sensitizing experimental animals intraperitoneally once a week with Yunnan pine pollen allergen; The day after the basic sensitization treatment, challenge sensitization was performed; The method for triggering sensitization includes: administering Yunnan pine pollen allergen at twice the concentration of the basal sensitization stage into each nostril using a micropipette, and performing quantitative nasal drops for 7 consecutive days to trigger sensitization. Observe the experimental animals for symptoms within 10 minutes after each nasal stimulation.

2. The method for constructing an animal model of Yunnan pine pollen allergy according to claim 1, characterized in that: The model animal was a female BALB / c mouse.

3. The method for constructing an animal model of Yunnan pine pollen allergy according to claim 1, characterized in that: In the basic sensitization step, the injection dose is 10 uL / g based on the body weight of the experimental animal.

4. The method for constructing an animal model of Yunnan pine pollen allergy according to claim 1, characterized in that: The nasal drop dosage for the sensitization phase was 2 μL / g based on the body weight of the experimental animal.

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