A method for preparing a rat acute pharyngitis model

Abstract

The present research aims to successfully establish a rat acute pharyngitis model, and to observe the pathological changes of the pharynx of the animal and detect the pharynx and blood related indexes to evaluate the stability of the model, so as to make a rat acute pharyngitis model which is simple to operate, long in symptom maintenance time, stable and unified, more close to the clinic, and to facilitate the further research on the pathogenesis and pathophysiological changes of the acute pharyngitis model, and to provide a certain basis for how to implement the pharynx protection intervention measures. The method for constructing the rat acute pharyngitis model of the present application is simple to operate, long in symptom maintenance time, stable and unified, more close to the clinic, and good in stability and repeatability. The rat acute pharyngitis model constructed by the present application is not only simple to operate, but also long in symptom maintenance time, which can be up to more than 1 month.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of disease animal model construction, and particularly relates to a preparation method of a rat acute pharyngitis model. BACKGROUND

[0002] Pharyngitis is a common upper respiratory tract disease, which is divided into acute and chronic pharyngitis according to the length of disease course and the degree of pathological changes. Modern medical research shows that acute pharyngitis is caused by infection of viruses and bacteria such as influenza virus, parainfluenza virus, coxsackie virus, adenovirus, rhinovirus, staphylococcus, streptococcus A, pneumococcus, or physical or chemical factors such as high temperature, dust and smoke, which stimulate the acute inflammation of pharyngeal mucosa. Chronic pharyngitis may be induced by acute pharyngitis multiple recurrence, upper respiratory tract chronic inflammation, reflux esophagitis, chronic bronchitis, etc., or caused by long-term physical and chemical factor stimulation or overuse of voice due to occupation. The common clinical manifestations of acute pharyngitis are local symptoms such as dry throat, throat itching, throat pain, burning sensation or foreign body sensation, and mild systemic symptoms. The clinical manifestations of chronic pharyngitis are local symptoms such as pharyngeal discomfort, throat itching, foreign body sensation, burning sensation, dry sensation, irritation, and some have mild pain. The secretions in the pharynx are not easy to cough out, and the systemic symptoms are not obvious. Acute pharyngitis is an acute inflammation of pharyngeal mucosa, submucosa and its lymphatic tissue, and its pathogenic factors are mainly bacterial infection, viral infection and physical and chemical stimulation. The clinical manifestations are redness, swelling, heat and pain in the throat, and dry and itchy throat. The disease has a rapid onset and rapid development, and if not treated in time, it can lead to complications such as otitis media, acute bronchitis and pneumonia. In recent years, the incidence of acute pharyngitis caused by irregular work and eating habits has also increased. It can evolve into chronic pharyngitis due to multiple relapses, which affects the quality of life of patients.

[0003] Acute pharyngitis brings great trouble to the normal life of patients, so it is necessary to study the drugs for treating acute pharyngitis. It is very important to study whether the drugs are effective for acute pharyngitis through animal experiment research. Replication or establishment of a successful acute pharyngitis animal model will provide scientific basis and valuable reference for studying the efficacy and mechanism of action of drugs for treating pharyngitis and upper respiratory tract inflammation.

[0004] The current common method for preparing acute pharyngitis model includes: ammonia direct spray method, ammonia fixed point quantitative smearing method, ammonia + turpentine compound factor method, bacterial injection infection method and castration method. Ammonia direct spray method is commonly used in the study of acute pharyngitis model because of its simple operation. Adult rats and rabbits are often used as experimental objects because adult pharyngitis is more common. This method uses the method of directly spraying ammonia on the pharyngeal mucosa to cause the pharynx to feel chemical stimulation, causing pharyngeal mucosal edema and inflammatory reaction. However, improper operation can cause oral mucosa burn and even lung injury, leading to respiratory depression. The bacterial injection infection method prepares an acute pharyngitis model by bacterial injection infection. The pharynx of the experimental animal appears red and swollen, the infected mucosa has a few punctate purulent secretions, or the mucosal epithelium is severely keratinized, the spicule is obvious, the gland cells are hypertrophic, and the interstitium of the mucosa, submucosa and muscle layer has obvious inflammatory cell infiltration, but subcutaneous hemorrhage and vascular dilation are not obvious. The levels of serum nitric oxide and myeloperoxidase are significantly higher than those in the blank group, and the levels of IL-1β, IL-6β and TNF-α are significantly higher than those in the blank group. Qibenming et al. removed the bilateral ovaries of rats after anesthesia, and the pharyngeal mucosa of the rats showed atrophic changes after 30 days, successfully preparing a rat atrophic pharyngitis model.

[0005] Although the acute pharyngitis models prepared by ammonia direct spray method, ammonia fixed point quantitative smearing method, ammonia + turpentine method and bacterial injection infection method have certain similarities with clinical indicators in terms of pathological damage and serum cytokine index changes, they have the shortcomings of difficult operation, short symptom maintenance time, and certain risks in pharyngeal spray smearing and pharyngeal injection. Improper operation of the above methods can cause oral mucosa burn and even lung injury, leading to respiratory depression. The ammonia direct spray method commonly used in China also has the problem of non-uniformity. The concentration of ammonia used is from 2.5%, 6%, 10%, 15% to 25%. Therefore, there is an urgent need for an acute pharyngitis model that is simple to operate, has a long symptom maintenance time, is stable and uniform, and is more close to the clinic. SUMMARY

[0006] The purpose of this study is to successfully establish a rat acute pharyngitis model, observe the pathological changes of the pharynx of the animal and detect the related indicators of the pharynx and blood to evaluate the stability of the model. The purpose is to make a rat acute pharyngitis model that is simple to operate, has a long symptom maintenance time, is stable and uniform, and is more close to the clinic, which is convenient for further studying the pathogenesis and pathophysiological changes of acute pharyngitis model and providing certain basis for how to implement pharyngeal protection intervention measures.

[0007] The present application aims to develop an animal model capable of focusing on the classic symptoms of acute pharyngitis and capable of well evaluating the drug efficacy of acute pharyngitis. The rat acute pharyngitis model constructed in the present application shows a high success rate and stability in inducing the classic symptoms of acute pharyngitis (pharyngeal redness, a large number of inflammatory cell infiltration, pharyngeal mucosa swelling and thickening, submucosal tissue edema, etc.). The present research can well simulate the clinical onset of acute pharyngitis, lay a good foundation for rapid screening of therapeutic drugs and intervention measures, and the modeling method is simple, stable and reliable, and close to the clinic.

[0008] The present application is realized by the following technical solutions.

[0009] A preparation method of a rat acute pharyngitis model, characterized in that it comprises the following steps:

[0010] (1) Adaptive feeding: select SPF level rats with normal development, good vitality, 6-8 weeks of age, and a body weight of (190±10) g, with half male and half female, and adaptively feed for one week; the temperature and humidity of the feeding room are kept basically constant, the temperature is controlled between 26-28 DEG C, the humidity is maintained at about 70%, and the feeding room is kept clean; the rat tray is changed every morning, and sufficient clean drinking water and feed are added every morning to ensure the daily drinking and eating of all rats.

[0011] (2) Intravenous injection: the rats are adaptively fed for one week before modeling; 10 μL of 5-hydroxytryptamine hydrochloride solution and 20 μL of sodium citrate solution are intravenously injected into the rats in the morning of the first day of modeling, the concentration of the 5-hydroxytryptamine hydrochloride solution is 50 mg / L, the concentration of the sodium citrate solution is 80 mg / L, and the interval time between the two injections is 30 minutes; then the rats are transferred to the modeling room.

[0012] (3) Dry heat stimulation: the temperature in the modeling room is maintained at about 37 DEG C, and the relative humidity of the air in the modeling room is less than 15%.

[0013] (4) Full light and sleep deprivation: during the three days of modeling, the modeling room is kept under 24-hour light, and the rats are placed in a rotating cylinder which is uniformly moved by a motor, so as to deprive the rats of sleep; the diameter and height of the rotating cylinder are both 40 cm.

[0014] (5) Diet stimulation: during the three days of modeling, the rats are fed with granular feed containing anisodamine, and lithium chloride is added to the drinking water of the rats, the content of anisodamine in the feed is 1 g / kg, and the concentration of lithium chloride in the drinking water is 15-20 mg / L; the rat acute pharyngitis model can be obtained after three days.

[0015] After the modeling, the model is evaluated by observing the behavior and the appearance of the pharynx of the rat, detecting the blood routine, HE staining, detecting the serum inflammatory factor level, detecting the protein expression of the pharynx by the immunohistochemical method, and detecting the gene expression by the fluorescence quantification, etc.

[0016] The technical scheme of the present application has the following advantages:

[0017] 1. The current commonly used acute pharyngitis model has the problems of large operation difficulty, short symptom maintenance time, and non-uniform method, while the acute pharyngitis model construction method of the present application can induce a rat acute pharyngitis model with simple operation, long symptom maintenance time, stable and uniform, more close to the clinic, good stability and repeatability, and the multi-factor induced acute pharyngitis model can better simulate the characteristics of human acute pharyngitis. The current commonly used ammonia direct spraying method and bacterial injection infection method have large artificial operation difficulty, and slight carelessness will cause animal injury, and the symptom maintenance time is short, only 1-2 weeks, while the acute pharyngitis model constructed by the present application has long symptom maintenance time, which can be more than 1 month.

[0018] 2. The non-single factor induced acute pharyngitis animal model of the present application is an ideal animal model from the perspective of pathophysiology theory, and is also ideal from the perspective of simulating typical clinical symptoms and treatment prediction, which can provide support for exploring the pathogenesis of acute pharyngitis, and provide a theoretical basis for developing new solutions for treating acute pharyngitis. The acute pharyngitis animal model constructed by the present application has high efficiency, stable effect, and small individual difference; the multiple factors are mutually synergistic, and the constructed disease model is more accurate. The model shows high success rate and stability in inducing the classic symptoms of acute pharyngitis (pharyngeal redness, a large number of inflammatory cell infiltration, pharyngeal mucosa swelling and thickening, submucosal tissue edema, etc.).

[0019] 3. In the morning of the first day of modeling, the present invention intravenously injects rats with 10 μL of 5-hydroxytryptamine hydrochloride solution and 20 μL of sodium citrate solution. 5-hydroxytryptamine hydrochloride can activate the NF-κB signaling pathway in rats and recruit more inflammatory cells by regulating the expression of the inflammatory factor COX-2 protein and the chemokine MCP-1, thereby inducing pharyngeal inflammation and damage. Sodium citrate can affect the leukocyte transendothelial migration signaling pathway in rats, thereby exacerbating the pharyngeal inflammation response in rats. 5-hydroxytryptamine hydrochloride and sodium citrate are injected intravenously into rats sequentially. The two work synergistically to induce pharyngeal tissue damage in rats to a large extent and increase the tissue infiltration of inflammatory cells.

[0020] Dry heat stimulation in rats can induce inflammatory cell aggregation, localized tissue edema, pathological epithelial hyperplasia, and mucosal dysfunction in the pharyngeal mucosa. After intravenous injection of 5-hydroxytryptamine hydrochloride and sodium citrate, continuous dry heat stimulation not only consolidates and strengthens the induction effect of intravenous injection but also further induces inflammatory cell aggregation and localized tissue edema in the pharyngeal mucosa, significantly facilitating the induction of osteoporotic acute pharyngitis in rats.

[0021] Full light exposure and sleep deprivation in rats can, to a certain extent, stimulate the release of specific inflammatory mediators and cytokines, leading to oxidative stress, ultimately leading to inflammatory proliferation of tissue and organ cells and fibrosis of epithelial and mesenchymal cells. Following intravenous injection of 5-hydroxytryptamine hydrochloride and sodium citrate, full light exposure and sleep deprivation in rats exposed to dry heat further exacerbate pharyngeal inflammation and oxidative stress.

[0022] The present invention feeds rats with pelleted feed containing scopolamine and adds lithium chloride to drinking water. Scopolamine can activate the mitogen-activated protein kinase p38 in the rat's MAPK signaling pathway, drive white blood cells to gather at the inflammation site, and release a variety of inflammatory factors. By adding scopolamine to the pelleted feed, scopolamine can better exert its effect in the pharynx when the rat swallows the pelleted feed. Lithium chloride can stimulate rat vascular endothelial cells to secrete adhesion molecules, stimulate monocytes-macrophages and other cells to secrete chemotactic cytokines, cause white blood cells to gather at the inflammation site, and promote inflammatory response. The scopolamine in the feed and the lithium chloride in the drinking water cooperate with each other to maximize the induction and aggravation of pharyngeal tissue inflammation and oxidative stress in the rat.

[0023] The multiple steps are coordinated with each other, the intravenous injection causes the inflammation damage of the pharynx, the dry heat stimulation and the whole light and sleep deprivation consolidate and strengthen the induction of the intravenous injection, at the same time, the dry heat stimulation further induces the inflammatory cell aggregation and local tissue edema of the pharyngeal mucosa, the whole light and sleep deprivation further induces the inflammatory hyperplasia and fibrosis, and the diet stimulation based on the above treatment further induces and aggravates the inflammation and oxidative stress of the pharyngeal tissue of the rats, aggravates the pathological reaction of the pharynx of the rats, and the multiple steps together ensure the stable effect of the acute pharyngitis model of the rats.

[0024] 4、The research can provide good theoretical support and model reference for in-depth analysis of the immune mechanism of human acute pharyngitis and evaluation of new treatment methods. The research team has repeatedly verified the model, the results are reliable, and has used the model to evaluate the efficacy of various drugs, verifying the application value of the model. The animal model shows the typical characteristics of acute pharyngitis: pharyngeal redness, a large number of inflammatory cell infiltration, pharyngeal mucosa swelling and thickening, submucosal tissue edema, etc. The model can be used for basic research in the field of acute pharyngitis, and lays the foundation for exploring the pathogenesis of acute pharyngitis. The research can provide a reliable animal model for the evaluation of the efficacy of acute pharyngitis drugs, which has the advantages of simple operation, long symptom maintenance time, stable and uniform, closer to clinical practice, good stability and repeatability, etc. DETAILED DESCRIPTION

[0025] I. A preparation method of a rat acute pharyngitis model, characterized in that it comprises the following steps:

[0026] (1) Adaptive feeding: select SPF grade rats with normal development, good vitality, 6-8 weeks of age, and body weight of (190±10) g, half male and half female, and adaptively feed for one week; the temperature and humidity of the feeding room are kept basically constant, the temperature is controlled between 26-28℃, and the humidity is maintained at about 70%; the feeding room is kept clean; the rat tray is changed every morning, and sufficient clean drinking water and feed are added every morning to ensure the daily drinking and eating of all rats.

[0027] (2) Intravenous injection: the rats are adaptively fed for one week before modeling; on the first day of modeling in the morning, the rats are intravenously injected with 10 μL of 5-hydroxytryptamine hydrochloride solution and 20 μL of sodium citrate solution, respectively, the concentration of the 5-hydroxytryptamine hydrochloride solution is 50 mg / L, the concentration of the sodium citrate solution is 80 mg / L, and the interval between the two injections is 30 minutes; then the rats are transferred to the modeling room.

[0028] (3) Dry heat stimulation: the temperature of the modeling room was maintained at about 37°C, and the relative humidity of the air in the modeling room was kept less than 15%.

[0029] (4) Full light and sleep deprivation: during the three-day modeling period, the modeling room was kept in 24-hour light, and the rats were placed in a rotating cylinder which was uniformly moved by a motor, so that the rats were subjected to sleep deprivation; the diameter and height of the rotating cylinder were both 40 cm.

[0030] (5) Diet stimulation: during the three-day modeling period, the rats were fed with granular feed containing scopolamine, and lithium chloride was added to the drinking water of the rats, the content of scopolamine in the feed was 1 g / kg, and the concentration of lithium chloride in the drinking water was 15-20 mg / L; the rat acute pharyngitis model was obtained after three days. The symptoms of the obtained rat acute pharyngitis model can be maintained for more than 1 month.

[0031] After modeling, the model was evaluated by observing the behavior and apparent state of the rats, blood routine test, HE staining, serum inflammatory factor level detection, immunohistochemical detection of pharyngeal tissue protein expression, and fluorescence quantitative detection of gene expression (the control group was injected with normal saline and conventionally fed), and the results were as follows:

[0032] 1. Behavior and apparent state observation

[0033] The body weight of each group of rats was recorded, and the behavior of the rats after modeling was observed and recorded every day according to the hair condition and pharyngeal redness of the rats. After modeling, the hair condition, mental state, activity, water consumption, etc. of each group of rats were observed, and the pharyngeal mucosa was observed under the extra-belt reflector.

[0034] From the first day of modeling, the model group rats showed scratching symptoms, and the symptoms were obvious on the second and third days of modeling. There were scratches around the lips, the hair was dull, there was rapid panting, the water consumption frequency and amount increased, the food intake decreased, and the pharyngeal secretion increased. On the third day of modeling, in addition to the above symptoms being more obvious, there were scratches around the lips, and there were ulcers in the oral cavity. The rats were in a depressed state, the body temperature was slightly low, and the activity was reduced.

[0035] 2. Blood routine test

[0036] 1 mL of blood was taken from the retinal venous plexus and placed in an EDTA-coated blood collection tube. The number of white blood cells (WBC) and lymphocytes (LYMP) in whole blood was determined by a blood system analyzer.

[0037] Compared with the normal control group, the number of white blood cells (WBC) in the whole blood of the model group rats increased significantly, and the number of lymphocytes (LYMP) did not change significantly.

[0038] 3. HE staining

[0039] The pharyngeal tissues of the rats in each group were taken, fixed with 4% paraformaldehyde, dehydrated with ethanol, transparentized with xylene, embedded with paraffin, sliced, and then prepared into paraffin sections, stained with HE, sealed with neutral resin, dried, and observed under a light microscope for morphological changes of the pharyngeal tissues.

[0040] The mucosa and muscle layers of the control group were clear in structure, with obvious boundaries, and the overall staining was uniform, and no inflammatory exudate was observed, and the epithelial cells were not degenerated, necrotic or inflamed, and the glands were not hyperplastic or atrophic. The structure of the model group was disorganized, the pharyngeal mucosa epithelium was obviously edematous, and the inflammatory hyperemia in the tissue was obvious, and a large number of inflammatory cells such as neutrophils and lymphocytes were observed.

[0041] 4. Detection of serum inflammatory factor levels

[0042] The abdominal aortic blood of the rats in each group was taken, and the upper serum was taken after standing. The levels of IL-1β, IL-6, TNF-α, PEG2 and IL-10 in the serum of each group were determined by ELISA.

[0043] Compared with the control group, the contents of TNF-α, IL-1β, PEG2 and IL-6 in the serum of the model group were significantly increased, and the level of IL-10 was significantly decreased.

[0044] 5. Detection of pharyngeal tissue NF-κB P65, COX-2 and F4 / 80 protein expression by immunohistochemical method

[0045] The paraffin sections of the pharyngeal tissues of the rats were taken, and after antigen repair, blocking, incubation of NF-κB P65, COX-2 and F4 / 80 primary antibodies at 4°C overnight, incubation of secondary antibodies at 37°C for 30 min, DAB coloration, hematoxylin staining, ethanol dehydration and sealing with neutral resin, the expression of NF-κB P65, COX-2 and F4 / 80 proteins in the pharyngeal tissues was observed under a microscope.

[0046] The results of immunohistochemical detection showed that compared with the normal control group, the expression of NF-κB P65, COX-2 and F4 / 80 proteins in the pharyngeal tissues of the model group was significantly increased.

[0047] 6. Fluorescent quantitative detection of gene expression

[0048] The pharyngeal tissues of the rats were taken, and total RNA extraction, cDNA synthesis and qPCR reaction were performed according to the operation method of the kit to analyze the expression level of NF-κB p65 mRNA.

[0049] Compared with the control group, the expression level of NF-κB p65 mRNA in the model group was significantly increased. It was proved that the model was successfully established.

[0050] II. Selection of modeling method

[0051] SPF level rats with normal development, good vitality, 6-8 weeks of age, and body weight of (190±10) g are selected, half male and half female, and after adaptive feeding for one week, they are divided into eight groups, which are a control group, an ammonia water spraying group, a bacteria injection group, a model group, a comparison group one, a comparison group two, a comparison group three, and a comparison group four. The rats in the control group are bred under conventional conditions. The ammonia water spraying group uses a laryngeal atomizer to spray 15% ammonia water on the throat of the rats, and the spraying is performed twice a day for three days. The bacteria injection group submucosally injects Staphylococcus aureus (containing 1×10 8 ~1×10 9 CFU·mL -1 ) into the oral cavity of the rats, and the injection amount is 20 μL per rat, and the injection is performed twice with an interval of 24 hours. The model group is bred according to the model preparation method of the application, that is, intravenous injection of 5-hydroxytryptamine hydrochloride and sodium citrate + dry heat stimulation + full light and sleep deprivation + diet stimulation. The comparison group one is not intravenously injected, and other treatment methods are the same as those of the model group, that is, dry heat stimulation + full light and sleep deprivation + diet stimulation. The comparison group two is not subjected to dry heat stimulation, and other treatment methods are the same as those of the model group, that is, intravenous injection of 5-hydroxytryptamine hydrochloride and sodium citrate + full light and sleep deprivation + diet stimulation. The comparison group three is not subjected to full light and sleep deprivation, and other treatment methods are the same as those of the model group, that is, intravenous injection of 5-hydroxytryptamine hydrochloride and sodium citrate + dry heat stimulation + diet stimulation. The comparison group four is not subjected to diet stimulation, and other treatment methods are the same as those of the model group, that is, intravenous injection of 5-hydroxytryptamine hydrochloride and sodium citate + dry heat stimulation + full light and sleep deprivation. After three days, the behavior, the appearance of the throat, and the throat tissue of the rats in each group are observed, and the throat tissue of the rats in each group is taken for HE staining.

[0052] The results show that the behavior and the appearance of the throat of the rats in the control group are normal, the structure of the mucosa layer and the muscle layer is clear, and the epithelial cells are normal. The rats in the model group have scratches around the lips, oral ulcer, and a depressed spirit, the mucosa epithelium of the throat is obviously edematous, the inflammatory hyperemia in the tissue is obvious, and a large number of inflammatory cells infiltrate. The rats in the ammonia water spraying group, the bacteria injection group, and the four comparison groups all have symptoms similar to those of the model group, but the degree of pharyngitis symptoms is lower than that of the model group. The duration of acute pharyngitis symptoms of the rats in the ammonia water spraying group and the bacteria injection group is 9 days and 14 days, respectively, and the duration of acute pharyngitis symptoms of the rats in the comparison group one, the comparison group two, the comparison group three, and the comparison group four is 16 days, 20 days, 19 days, and 18 days, respectively. The duration of the typical symptoms of the rats in the model group is 32 days. It can be seen that the duration of acute pharyngitis of the rats in the model group is obviously longer than that of the rats in the other groups.

Claims

1. A method for preparing a rat model of acute pharyngitis, characterized in that, The method comprises the following steps: (1) adaptive feeding; selecting SPF rats with normal development, good vitality, 6-8 weeks of age, and a body weight of (190±10) g, half male and half female, adaptive feeding for one week; the temperature and humidity of the feeding room are kept basically constant, the temperature is controlled between 26-28℃, the humidity is maintained at about 70%, and the feeding room is kept clean; the rat tray is replaced every morning, and sufficient clean drinking water and feed are added every morning to ensure that all rats have daily drinking water and food; (2) intravenous injection: the rats are adapted to feed for one week before modeling; on the first day of modeling in the morning, the rats are intravenously injected with 10 μL of 5-hydroxytryptamine hydrochloride solution and 20 μL of sodium citrate solution, respectively, the concentration of the 5-hydroxytryptamine hydrochloride solution is 50 mg / L, the concentration of the sodium citrate solution is 80 mg / L, and the interval between the two injections is 30 minutes; then the rats are transferred to the modeling room; (3) dry heat stimulation: the temperature in the modeling room is maintained at about 37℃, and the relative humidity of the air in the modeling room is less than 15%; (4) full light and sleep deprivation: during the three days of modeling, the modeling room is kept under 24-hour light, and the rats are placed in a rotating cylinder which is driven by a motor to rotate at a constant speed, thereby depriving the rats of sleep; the diameter and height of the rotating cylinder are both 40 cm; (5) diet stimulation: during the three days of modeling, the rats are fed with granular feed containing anisodamine, and lithium chloride is added to the drinking water of the rats, the content of anisodamine in the feed is 1 g / kg, and the concentration of lithium chloride in the drinking water is 15-20 mg / L; after three days, the rat acute pharyngitis model is obtained.

2. The method for preparing a rat acute pharyngitis model according to claim 1, wherein: The symptoms of the obtained rat acute pharyngitis model can be maintained for more than one month.