Method for constructing animal model of non-allergic rhinitis induced by temperature change

Abstract

The invention relates to the technical field of animal model construction, in particular to an animal model for inducing non-allergic rhinitis by temperature change, a method for constructing the animal model and a new scheme for researching an inflammation mechanism of the animal model, and particularly relates to a method for constructing the animal model for inducing non-allergic rhinitis by temperature change. The invention intends to re-cognize upper airway hyperresponsiveness diseases from the perspective of peripheral neuroimmunology, explore a new modeling method of NAR mice, and aims to induce non-allergic rhinitis by using temperature change, deepen cognition of NAR specific pathogenesis, study a specific mechanism of neuropeptide rise, strive to achieve the purpose of diagnosing and treating rhinitis according to the principle of precision medicine, and provide a new model for the NAR mice. An animal model and an animal experiment basis are provided for new clinical treatment in the future, and the method comprises a grouping step, an experiment step and an animal model evaluation setting step.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to an animal model of non-allergic rhinitis induced by temperature changes, and a method for constructing such a model. In particular, it relates to a method for constructing an animal model of non-allergic rhinitis induced by temperature changes. Background Technology

[0002] Upper airway hyperresponsiveness (AHR) is a heterogeneous disease, mainly including allergic rhinitis (AR) and non-allergic rhinitis (NAR). It is usually defined as "the occurrence of one or more nasal symptoms, such as runny nose, sneezing, and nasal congestion, when exposed to environmental stimuli (such as smoke, temperature / humidity changes, strong odors and other irritants)".

[0003] Epidemiological surveys show that the incidence of allergic rhinitis (AHR) has been gradually increasing over the past few decades, affecting not only quality of life but also becoming a global health problem. The pathogenesis of AHR is currently mainly based on the immunological theory of type I hypersensitivity mediated by allergen immunoglobulin E (IgE) and the inflammatory response of submucosal helper T cells 2 (Th2); however, the pathogenesis of non-allergic rhinitis remains unclear.

[0004] This invention aims to re-understand upper airway hyperresponsiveness from the perspective of peripheral neuroimmunology (peripheral neuroimmunology refers to the interaction between immune cells in tissues and neurons in the peripheral nervous system, which fits the research mechanism of this model). Specifically, temperature changes stimulate nerve endings in the nasal mucosa, transmitting nerve signals to the peripheral nervous system (trigeminal ganglion), causing an inflammatory response and nerve sensitization in the trigeminal ganglion. This leads to an increase in the release of neuropeptides from nerve endings in the nasal mucosa. The increased neuropeptides act on tissue immune cells, causing a further immune response that leads to rhinitis symptoms.

[0005] This invention explores a novel modeling method for NAR in mice, filling the gap in animal models of NAR induced by temperature changes, deepening our understanding of the specific pathogenesis of NAR, and striving to achieve the goal of diagnosing and treating rhinitis according to the principles of precision medicine.

[0006] Current research progress at home and abroad: AHR is a key feature of various upper airway disease phenotypes, namely, the increased response of nasal mucosal epithelial cells to various chemical and physical stimuli. As the first line of defense to sense external environmental stimuli and temperature changes, nasal mucosal epithelial cells can detect air humidity, temperature and chemical substances. While regulating and filtering the air, they also have a certain defense against pathogens.

[0007] Studies on the epithelial and sensory mechanisms of nasal hyperresponsiveness suggest that AHR is mainly caused by three types of mechanisms: (1) impaired epithelial barrier function; (2) hypersensitive response to endogenous and exogenous stimuli; and (3) enhancement of the efferent nervous system.

[0008] The literature “Neuroimmune mechanisms of upper airway hyperresponsiveness and its research progress” Zhao Changqing et al., Chinese Journal of Otorhinolaryngology Head and Neck Surgery, 2022, 57, suggests that the neuroimmune mechanisms involved in AHR mainly include: (1) neurogenic inflammatory response; (2) Th2-like response induced by type 2 innate lymphocyte cells (ILC2); (3) local and systemic immune responses of the nasal mucosa involving transient receptor potential (TRP); and (4) the biological response phase of the nasal mucosa under the regulation of the biological clock and its reset, etc.

[0009] In addition, TRP-mediated neurogenic inflammatory responses and local mucosal immune responses have been extensively studied in the upper respiratory tract, with TRPV1 (warmth sensation 40-55℃, pain, and inflammation defense) and TRPA1 (cold sensation 0-15℃, pain defense) being the most frequently studied. They are expressed in afferent neurons, epithelial cells, T cells, and mast cells.

[0010] TRP channels are a group of voltage-gated cation channels. Based on their amino acid sequence homology, six subfamilies of mammalian TRP proteins, comprising 28 members, have been identified: TRPC (Canonical 1-7), TRPV (Vanilloid 1-6), TRPM (Melastatin 1-8), TRPA (Ankyrin 1), TRPP (Polycystin 1-3), and TRPML (Mucolipin 1-3). They are part of a G protein-coupled receptor (GPCR)-mediated cellular signaling cascade and can be directly activated by cold, heat, mechanical, and chemical stimuli, including various harmful exogenous compounds and molecules associated with tissue damage. Activation of these channels depolarizes nerves and releases neuropeptides such as calcitonin-related peptide (CGRP) and substance P (SP), leading to vasodilation and increased capillary permeability, thereby triggering a series of inflammatory responses.

[0011] TRPA1 is a channel of great importance in the pathophysiology of acute respiratory distress syndrome (AHR). The TRPA1 protein was first successfully isolated from human lung fibroblasts in 1999. TRPA1 is expressed in afferent nerves and epithelial cells of the lower airway and is sensitive to low temperature, mechanical and chemical stimuli (H2O2, NO, ozone, LPS, etc.), leading to calcium and sodium ion influx, cell membrane depolarization, and the generation of neuronal action potentials, ultimately causing pain and inflammation. Therefore, the TRPA1 ion channel is crucial for maintaining normal physiological functions.

[0012] When the respiratory tract is exposed to external stimuli such as cigarette smoke, intracellular calcium... 2+ Increased concentrations stimulate the release of SP, CGRP, and other substances, which can cause bronchoconstriction and trigger the release of inflammatory factors from respiratory lymphocytes, thereby inducing bronchial inflammatory responses such as cough and asthma. Pharmacologically, blocking the TRPA1 channel in trigeminal sensory neurons can reduce the inflammatory response to allergic stimuli in mice, while also inhibiting the activity of other nociceptors in the inflammatory area.

[0013] Similarly, TRPA1 mRNA levels were significantly elevated in the nasal mucosa of IR patients, and they exhibited a lower stimulation threshold for the TRPA1 agonist, butene isothiocyanate. Furthermore, TRPA1 is one of the molecules mediating the airway cold stimulation response; in IR patients, elevated local H2O2 concentrations were found to enhance the activity of cold-sensitive TRPA1, and NO is expected to produce a similar effect. Therefore, it may be the molecular basis for the nasal mucosal contraction response induced by dry, cold air.

[0014] In summary, this demonstrates the important pathogenic role of TRP channel proteins in AHR; however, existing research mainly focuses on allergic diseases, and research on temperature-induced non-allergic rhinitis is still lacking, urgently requiring an animal model to fill this gap. Summary of the Invention

[0015] This invention aims to re-understand upper airway hyperresponsiveness from the perspective of peripheral neuroimmunology, explore new modeling methods in NAR mice, and use temperature changes (cold) to induce non-allergic rhinitis, thereby deepening the understanding of the specific pathogenesis of NAR, studying the specific mechanism of neuropeptide elevation, and striving to achieve the goal of diagnosing and treating rhinitis according to the principles of precision medicine, and providing animal models and animal experimental evidence for new clinical treatments in the future.

[0016] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0017] Methods for constructing animal models of non-allergic rhinitis induced by temperature changes include:

[0018] Step S1: Select a number of mice for practical application, the mice being SPF grade C57BL / 6J mice, and divide the mice into a healthy group (nc) and a cold group (cold).

[0019] The healthy mice (nc) were fed in a standard environment in an SPF-grade animal facility, and were fed for the same number of days as the cold mice.

[0020] The mice in the cold group and the mice in the healthy group were housed in the same environment, and modeling and stimulation experiments were conducted under different conditions.

[0021] Step S2, Modeling and Stimulation Experiment: The mice in the cold group were subjected to modeling and stimulation experiments. The process is as follows:

[0022] The mice were placed in an environmental simulation chamber with simulated environmental conditions of 0-4℃ and 50±5% humidity.

[0023] The mice were placed in the environmental simulation box for more than 20 minutes and less than or equal to 40 minutes each time. After being removed, they were placed in their daily breeding environment. The removal operation was repeated more than or equal to 2 times / day and less than or equal to 8 times / day for 7 consecutive days, which was considered to complete the modeling and stimulation experiment.

[0024] Step S3: During and after the modeling and stimulation experiment, the mice were observed by video recording, and the characteristics of the rhinitis in the mouse model were recorded by playing back the video.

[0025] Step S4, evaluation of the non-allergic rhinitis mouse model, the process is as follows:

[0026] Step S101: Refer to the qualitative indicators of the evaluation criteria for the allergic rhinitis mouse model, the qualitative indicators including: number of sneezes, nose-scratching behavior, and degree of runny nose;

[0027] Step S102: The qualitative indicators are supplemented with quantitative indicators to form quantitative evaluation items, which include:

[0028] The nose-scratching time T1 / T0 represents the continuous nose-scratching time T1 within a fixed observation time T0;

[0029] The number of sneezes, n / T0, represents the number of sneezes, n, within a fixed observation time T0.

[0030] The length L of the phenol red line is the length of the indicator marker L representing the continuous length of nasal secretions;

[0031] Step S103: Divide the quantitative evaluation items into score segments based on the actual quantitative evaluation data of mice to form semi-quantitative and semi-qualitative evaluation items;

[0032] The score segments are divided into corresponding score segments of the qualitative indicators, and the scores of the qualitative indicators are used as the scoring criteria to obtain the scoring criteria for animal models of non-allergic rhinitis induced by temperature changes.

[0033] The success rate of the non-allergic rhinitis mouse model was calculated using the same standard as that of the allergic rhinitis mouse model.

[0034] Alternatively, the quantitative indicators are recorded during the provocation experiment, and the count is performed by playing back the video, with the phenol red line marked separately; the length of the phenol red line is used to indicate the degree of nasal discharge.

[0035] More preferably, SPF-grade C57BL / 6J mice are placed in an environmental simulation chamber for 40 minutes each time, and then placed in their daily feeding environment, 4 times a day for 7 consecutive days.

[0036] Preferably, SPF-grade C57BL / 6J mice are placed in an environmental simulation chamber for 20 minutes each time, and then placed in their daily feeding environment, 8 times a day for 7 consecutive days.

[0037] The present invention has the following beneficial effects:

[0038] Firstly, this invention pioneers a new modeling method for NAR (non-allergic rhinitis) mice, filling the gap in animal models for NAR induced by temperature changes. Through this model, the neuroimmune mechanism of peripheral nerves in chronic rhinitis can be studied, striving to achieve the goal of diagnosing and treating rhinitis according to the principles of precision medicine.

[0039] Secondly, the temperature change (cold) induced non-allergic rhinitis animal model of the present invention is non-infectious, non-allergic, and non-chemically stimulated, providing an animal model with a single stimulus and simple pathogenic factors for studying the peripheral nerve immune mechanism, and also providing an effective animal model for exploring new clinical treatment options in the future.

[0040] Thirdly, the animal model of the present invention has the advantages of low modeling cost (no drug cost) and short time (7 days for modeling is one of the shortest among existing rhinitis animal models), which facilitates the expansion of the number of animal models and the large-scale statistical analysis of symptom data. It lays the foundation for proposing a quantitative standard evaluation system for rhinitis symptoms in mouse models and strives to provide the possibility of combining quantitative standards for symptoms with histopathological results in the future.

[0041] Fourthly, this invention explored the gradient of the number of stimulations and stimulation time of the model, and obtained the current optimal solution for constructing a model of non-allergic rhinitis induced by temperature change (cold). Attached Figure Description

[0042] Appendix Figure 1 This is a schematic diagram of the process of the method of the present invention;

[0043] Appendix Figure 2 This is the control data for the number of sneezes per 5 minutes obtained using the method of this invention;

[0044] Appendix Figure 3 Comparative data on nose-grabbing time (continuous description / 5 min) obtained using the method of the present invention;

[0045] Appendix Figure 4 The length of the phenol red line in nasal secretions obtained by the method of the present invention;

[0046] Appendix Figure 5 Statistics on the success rate of excitation experiments obtained using the method of this invention;

[0047] Appendix Figure 6 Detection of transcriptional levels in the nasal mucosa of a mouse model obtained using the method of this invention;

[0048] Appendix Figure 7 Detection of protein expression levels in the nasal mucosa of a mouse model;

[0049] Appendix Figure 8 Histological examination of nasal mucosa in mouse model (PAS staining);

[0050] Appendix Figure 9 Transcriptional level detection of the trigeminal ganglion in a mouse model of this invention;

[0051] Appendix Figure 10 Detection of trigeminal ganglion protein expression levels in a mouse model of this invention;

[0052] Appendix Figure 11 The evaluation criteria for a mouse model of non-allergic rhinitis.

[0053] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. Detailed Implementation

[0054] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0055] 1.1 Regarding upper airway hyperresponsiveness, AHR

[0056] Upper airway hyperresponsiveness (AHR) is a heterogeneous group of diseases, mainly including:

[0057] Allergic rhinitis (AR) and non-allergic rhinitis (NAR) are generally defined as "one or more nasal symptoms, such as runny nose, sneezing, and nasal congestion, triggered by environmental stimuli (such as smoke, temperature / humidity changes, strong odors, and other irritants)"; the purpose of this invention is to create an animal research model for non-allergic rhinitis (NAR).

[0058] In the information already known in this field, harmless external stimuli may trigger maladaptive responses and produce unpleasant sensations, such as nasal pain, itching, and burning. Epidemiological surveys show that the incidence of acute rhinitis (AR) has been gradually increasing over the past few decades, affecting not only quality of life but also becoming a global health problem. The pathogenesis of AR is currently mainly based on the immunological theories of IgE-mediated type I hypersensitivity reaction and submucosal Th2 inflammatory response.

[0059] The actual pathogenesis of non-allergic rhinitis is still unclear. This invention aims to re-understand upper airway hyperresponsiveness from the perspective of peripheral neuroimmunology, explore new modeling methods for NAR mice, fill the gap in animal models of NAR induced by temperature changes, deepen the understanding of the specific pathogenesis of NAR, and strive to achieve the goal of diagnosing and treating rhinitis according to the principles of precision medicine.

[0060] Furthermore, regarding the pathogenesis of AR, current findings indicate that it is primarily based on the immunological theory of type I hypersensitivity mediated by allergen immunoglobulin E (IgE) and the inflammatory response of submucosal helper T cells (Th2). However, the pathogenesis of non-allergic rhinitis (NAR) that is negative for serum specific IgE (sIgE) or skin prick tests is unclear, and there is a lack of international consensus on its unified definition and diagnostic criteria. It is usually treated as an exclusionary diagnosis and its treatment is often confused with AR.

[0061] 1.2 Modeling and Activation Experiments for Establishing a Mouse Animal Model

[0062] This invention relates to the field of animal model construction technology, specifically an animal model of non-allergic rhinitis induced by cold temperature changes. The invention also includes a method for constructing this model and a novel treatment regimen for it, namely, a method for constructing an animal model of non-allergic rhinitis induced by temperature changes.

[0063] First, SPF-grade C57BL / 6J mice were divided into groups of 9-10, and multiple groups were planned, including a blank control group and an experimental group.

[0064] SPF-grade C57BL / 6J mice in the healthy group were fed normally under normal experimental conditions and served as a blank control group.

[0065] SPF-grade C57BL / 6J mice in the cold group were used as experimental materials for the construction and stimulation of an animal model of temperature-induced non-allergic rhinitis.

[0066] It should be noted that, regardless of whether the mice are in the healthy group or the cold group, individual mice with large differences in body size need to be excluded after the feeding, modeling and stimulation experiments.

[0067] This scheme designs five modeling and excitation experimental processes with different simulated environmental conditions. Each batch in the cold group only enters one simulated environmental condition.

[0068] Example 1, First Modeling and Activation Experiment

[0069] SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber with simulated environmental conditions of 0-4℃ and 50±5% humidity.

[0070] The SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 20 minutes each time, and then returned to their normal living environment twice a day.

[0071] For 7 consecutive days;

[0072] After the modeling was completed, a stimulation experiment was conducted. The stimulation experiment was conducted at an ambient temperature of 4°C and a humidity of 50±5%. The symptoms of the mice were recorded on video for 20 minutes to assess the success of the modeling and the success rate.

[0073] Example 2, Second Modeling and Activation Experiment

[0074] SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber with simulated environmental conditions of 0-4℃ and 50±5% humidity.

[0075] The SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 20 minutes each time, and then placed in their daily rearing environment, 4 times a day for 7 consecutive days.

[0076] After the modeling was completed, a challenge experiment was conducted (ambient temperature 4℃, humidity 50±5%), and the symptoms of the mice were recorded on video for 20 minutes to assess the success of the modeling and the success rate.

[0077] Example 3, Third Modeling and Activation Experiment

[0078] SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber with simulated environmental conditions of 0-4℃ and 50±5% humidity.

[0079] The SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 40 minutes each time, and then placed in their daily rearing environment twice a day for 7 consecutive days.

[0080] After the modeling was completed, a challenge experiment was conducted (ambient temperature 4℃, humidity 50±5%), and the symptoms of the mice were recorded on video for 20 minutes to assess the success of the modeling and the success rate.

[0081] Example 4, Fourth Modeling and Activation Experiment

[0082] SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber with simulated environmental conditions of 0-4℃ and 50±5% humidity.

[0083] The SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 40 minutes each time, and then placed in their daily rearing environment, 4 times a day for 7 consecutive days.

[0084] After the modeling was completed, a challenge experiment was conducted (ambient temperature 4℃, humidity 50±5%), and the symptoms of the mice were recorded on video for 20 minutes to assess the success of the modeling and the success rate.

[0085] Example 5, Fifth Modeling and Activation Experiment

[0086] SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber with simulated environmental conditions of 0–4℃ and 50±5% humidity.

[0087] The SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 20 minutes each time, and then placed in their daily rearing environment, 8 times a day for 7 consecutive days.

[0088] After the modeling was completed, a challenge experiment was conducted (ambient temperature 4℃, humidity 50±5%), and the symptoms of the mice were recorded on video for 20 minutes to assess the success of the modeling and the success rate.

[0089] Among the above embodiments 1 to 5, the schemes of embodiments 4 and 5 are the most preferred experimental schemes, with the highest actual modeling evaluation success rate and success rate;

[0090] In Examples 1-5 above, the simulated environmental conditions are: temperature 0-4℃, humidity 50±5%. The lower limit of the selected temperature is 0℃, which is based on the social acceptance of animal experimentation humanitarianism and the avoidance of activation of skin receptors below 0℃. The upper limit of the temperature is 4℃, which is based on the consideration of temperature conditions for establishing animal models of non-allergic rhinitis (NAR) in the background art, and in reality (in Examples 1-5 above), the environmental simulation chamber involves repeated switching times and intervals, which will produce a certain degree of temperature change.

[0091] 1.3 Establishment of evaluation criteria for non-allergic rhinitis (NAR);

[0092] The current method, namely the internationally recognized evaluation criteria for allergic rhinitis (AR) mouse models, focuses on behavioral evaluation of the mice, mainly including: indicators (1) number of sneezes, indicator (2) nose-scratching behavior, and indicator (3) degree of runny nose; for example:

[0093] Number of sneezes: Record within 30 minutes, 0 points (no sneezing) to 3 points (≥11 sneezes);

[0094] Nose scratching behavior: 0 points (no nose scratching) to 3 points (continuous rubbing);

[0095] Runny nose severity: 0 points (no runny nose) to 3 points (runny nose);

[0096] Total score determination: ≥5 points is considered a successful model.

[0097] Post-provocation observation: Symptoms were recorded within 30 minutes after nasal provocation for 7 consecutive days. After excluding individual differences, the success rate of modeling in batches of mice was statistically analyzed.

[0098] The above examples illustrate the applicable criteria for allergic rhinitis (AR). The criteria of indicators (1) to (3) reflect the "qualitative" nature.

[0099] The mouse model provided by this invention aims to study the correlation between non-allergic rhinitis (NAR) and the interaction between immune cells and peripheral nervous system neurons. Allergic rhinitis (AR) and non-allergic rhinitis (NAR) show positive and negative results respectively in IgE detection, indicating a difference in the allergens causing inflammation. Existing qualitative evaluation standards for allergic rhinitis (AR) cannot be quantified. Therefore, the evaluation process for the quantitative evaluation standards for non-allergic rhinitis (NAR) provided in this technical solution is as follows:

[0100] Step S101: Select qualitative indicators of the evaluation criteria for the allergic rhinitis mouse model, including: number of sneezes, nose-scratching behavior, and degree of runny nose;

[0101] Step S102: Express the qualitative indicators as quantitative indicators to form quantitative evaluation items, wherein the quantitative evaluation items include:

[0102] The nose-scratching time T1 / T0 represents the continuous nose-scratching time T1 within a fixed observation time T0;

[0103] The number of sneezes, n / T0, represents the number of sneezes, n, within a fixed observation time T0.

[0104] The length L of the phenol red line is the length of the indicator marker L representing the continuous length of nasal secretions;

[0105] Step S103: Divide the quantitative evaluation items into score segments based on the quantitative evaluation data generated in actual mice;

[0106] The division score corresponds to the score of the qualitative indicator, and the score of the qualitative indicator is used as the scoring standard to obtain the scoring standard for the animal model of non-allergic rhinitis induced by the temperature change being scored.

[0107] In the evaluation examples, as shown in the appendix Figure 11 As shown in Table 1, Table 1 presents the evaluation criteria for the proposed non-allergic rhinitis mouse model.

[0108] (1) The quantitative indicators of non-allergic rhinitis (NAR) correspond to the qualitative indicators of allergic rhinitis (AR), but non-allergic rhinitis (NAR) is quantified by the nose scratching time T1 / T0, the number of sneezes n / T0, and the length of the phenol red line L.

[0109] (2) A mouse model with a score of 5 or higher in the evaluation criteria for non-allergic rhinitis (NAR) is considered a successful NAR model;

[0110] The success rate is calculated as follows: the index of mice with symptoms of 5 or above in the same batch is divided by the total number of mice in the same batch (excluding mice with excessively large differences in individual body weight).

[0111] (3) Among them, the quantitative indicators were recorded by video during the challenge experiment, and the count was made by playing back the video. The phenol red line (the degree of nasal discharge) was marked separately.

[0112] The following are examples of the excitation experiment process and related data;

[0113] Please see the appendix Figure 2 , 3 As shown in the figure, nc on the horizontal axis represents the healthy group of mice, and cold represents the cold group of mice.

[0114] Appendix Figure 2 The following is a control data point showing the number of sneezes per 5 minutes. Figure 3 For the nose-grabbing time (continuous description / 5 min) control data;

[0115] Appendix Figure 4 The length of the phenol red line in nasal secretions;

[0116] Appendix Figure 2 ~Attached Figure 10 Both studies show a comparison between the healthy group (nc) and the cold group (cold);

[0117] Appendix Figure 5To demonstrate the experimental results of the mouse model nasal mucosa, the success rate of five stimulation experiments was statistically analyzed. Specifically, five schemes with different stimulation times and durations were set up, including a control group of healthy mice and a cold group. Each scheme had 9-10 mice. The percentage of mice that achieved a modeling score of 5 or above was considered as the modeling success rate.

[0118] Appendix Figure 6 In this invention, the transcriptional level of the nasal mucosa in the mouse model was detected, including elevated levels of interleukin-25 (IL-25), interleukin-33 (IL-33), thymic stromal lymphopoietin (TSLP), interleukin-4 (IL-4), and interleukin-5 (IL-5), confirming that the nasal mucosa of the model underwent a Th2-type inflammatory response.

[0119] Appendix Figure 7 In this study, the expression levels of proteins in the nasal mucosa of the mouse model were detected. The expression of interleukin-25 (IL-25), thymic stromal lymphopoietin (TSLP), interleukin-4 (IL-4), and substance P (SP) proteins were elevated, confirming that the nasal mucosa of the model underwent a Th2-type inflammatory response and an increase in the release of neuropeptide SP from nerve endings.

[0120] Appendix Figure 8 The study showed that PAS staining in a mouse model revealed an increase in goblet cells in the nasal mucosa and hyperplasia of submucosal glands.

[0121] Appendix Figure 9 The transcriptional level detection of the trigeminal ganglion in the mouse model of the present invention is shown in the figure. The levels of interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and C-Cmotif chemokine ligand 2 (CCL2) are elevated, which confirms that an inflammatory response occurs in the trigeminal ganglion of the model.

[0122] Appendix Figure 10 The invention demonstrates the detection of protein expression levels in the trigeminal ganglion of the mouse model. Increased expression of interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and substance P (SP) proteins confirms the presence of inflammatory response and increased neuropeptides in the trigeminal ganglion of this model.

[0123] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing an animal model of non-allergic rhinitis induced by temperature changes, characterized in that, include: Step S1, grouping steps; A number of mice for practical applications were selected, namely SPF grade C57BL / 6J mice, and the mice were divided into a healthy group (nc) and a cold group (cold). The healthy mice (nc) were fed in a standard environment in an SPF-grade animal facility, and were fed for the same number of days as the cold mice. The mice in the cold group and the mice in the healthy group were housed in the same environment, and modeling and stimulation experiments were conducted under different conditions. Step S2, experimental procedure; The modeling and stimulation experiments were conducted on the mice in the cold group. The procedure was as follows: The mice were placed in an environmental simulation chamber with simulated environmental conditions of 0-4℃ and 50±5% humidity. The mice were placed in the environmental simulation box for more than 20 minutes and less than or equal to 40 minutes each time. After being removed, they were placed in their daily breeding environment. The removal operation was repeated more than or equal to 2 times / day and less than or equal to 8 times / day for 7 consecutive days, which was considered to complete the modeling and stimulation experiment. During and after the modeling and stimulation experiments, the mice were observed by video recording, and the characteristics of rhinitis in the mouse model were recorded by playing back the video. Step S3, Animal model evaluation setting steps; The evaluation process for the non-allergic rhinitis mouse model was as follows: Step S101: Refer to the qualitative indicators of the evaluation criteria for the allergic rhinitis mouse model, the qualitative indicators including: number of sneezes, nose-scratching behavior, and degree of runny nose; Step S102: The qualitative indicators are supplemented with quantitative indicators to form quantitative evaluation items, which include: The nose-scratching time T1 / T0 represents the continuous nose-scratching time T1 within a fixed observation time T0; The number of sneezes, n / T0, represents the number of sneezes, n, within a fixed observation time T0. The length L of the phenol red line is the length of the indicator marker L representing the continuous length of nasal secretions; Step S103: Divide the quantitative evaluation items into score segments based on the actual quantitative evaluation data of mice to form semi-quantitative and semi-qualitative evaluation items; The score segments are divided into corresponding score segments of the qualitative indicators, and the scores of the qualitative indicators are used as the scoring criteria to obtain the scoring criteria for animal models of non-allergic rhinitis induced by temperature changes. The success rate of the non-allergic rhinitis mouse model was calculated using the same standard as that of the allergic rhinitis mouse model.

2. The method for constructing an animal model of temperature-induced non-allergic rhinitis as described in claim 1, characterized in that, include: The quantitative indicators were determined by video recording of mice during the challenge experiment, and the counts were performed by playing back the video. The phenol red line was marked separately. The length of the phenol red line was used to indicate the degree of nasal discharge.

3. The method for constructing an animal model of temperature-induced non-allergic rhinitis as described in claim 1, characterized in that, SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 40 minutes each time, and then placed in their usual feeding environment, 4 times a day for 7 consecutive days.

4. The method for constructing an animal model of temperature-induced non-allergic rhinitis as described in claim 1, characterized in that, SPF-grade C57BL / 6J mice were placed in an environmental simulation chamber for 20 minutes each time, and then placed in their usual feeding environment, 8 times a day for 7 consecutive days.

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