A method for constructing an acute gout mouse model

Abstract

The present research aims to successfully establish an acute gout mouse model, and to observe the joint and organ pathological changes of the animal and detect the joint and blood related indexes to evaluate the stability of the model, so as to make an acute gout mouse model with good stability, good repeatability, practicability and closeness to clinic, facilitate further research on the pathogenesis and pathophysiological changes of acute gout, and provide a certain basis for how to implement the protective intervention measures of acute gout. The present application induces an acute gout mouse model with good stability and repeatability, practicability and closeness to clinic through injecting aromatic oil and benzopyrene into the mouse, electric shock stimulation, lithium hydroxide stimulation, hydroxyacetic acid gavage and blue light irradiation, and the acute gout model induced by multiple factors can better simulate the characteristics of human acute gout.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal disease model construction, and particularly relates to a method for constructing an acute gout mouse model. BACKGROUND

[0002] Gout is a disease caused by the accumulation of monosodium urate crystals in the joints, mainly manifested as hyperuricemia, gouty arthritis, urate deposition and joint deformity. High uric acid content in blood lymph can usually cause gouty arthritis, the formation of nodules, hyperuricemia, uric acid kidney stones and kidney diseases. In the past few decades, the incidence and prevalence of gout have increased significantly, resulting in a significant decrease in the quality of life of patients with the disease. As an independent risk factor, gout can induce and aggravate cardiovascular diseases such as coronary heart disease, seriously affecting the health of the population. Gout is a series of inflammatory reactions caused by the deposition of urate in the body due to the disorder of purine metabolism or the decrease of uric acid excretion in the kidney. When the serum uric acid is elevated to the supersaturation state, it is deposited in the joints, surrounding tissues and organs of the extracellular fluid, causing a group of gout syndromes (including urate stones, uric acid kidney stones, gouty arthritis and gout nephropathy), which not only can involve the bone joints, skin and soft tissue system, but also can involve the endocrine system, cardiovascular and cerebrovascular system, urinary system. Clinically, it can manifest as hyperuricemia, acute gouty arthritis, repeated urate deposition, chronic arthritis and joint dysfunction, and can cause gouty nephropathy, uric acid kidney stones and acute kidney injury, often with obvious heterogeneity. At present, the main treatments for acute gout in clinical practice are colchicine, non-steroidal anti-inflammatory drugs and glucocorticoids, but long-term use of these drugs often leads to many adverse reactions, including gastrointestinal discomfort, ulcers, liver and kidney damage, neurotoxicity and immunosuppression. Recent clinical new selective cyclooxygenase-2 inhibitors, such as etoricoxib, are very popular among gout patients, but studies have shown that long-term use of such drugs may have serious side effects on the heart, thereby limiting the application of such drugs. Therefore, the research on the treatment of acute gout has important clinical significance. At present, the treatment of gout is still a serious problem at home and abroad, and the research on the pathogenesis of various gout and the screening of treatment drugs largely depend on the establishment and application of gout animal models. Therefore, how to combine the clinical symptoms of gout with the characteristics of the animal model to simulate the clinical state and provide important basis for the pathogenesis and pathophysiological changes of gout is of great significance for promoting clinical diagnosis and the development of new drugs.

[0003] The related research of gout needs the help of animal models with similar performance of gout. The common methods for constructing gout animal models include: supplementing uric acid or uric acid precursors, intraperitoneal injection or gavage of uric acid or adenine, hypoxanthine and xanthine; adding yeast powder in feed to feed animals to form a model; intraperitoneal injection of oxypurinol or feeding animals with feed containing oxypurinol; gavage or intraperitoneal injection of ethambutol, nicotinic acid and the like; embryonic stem cell homologous recombination to destroy the uricase gene (EC1.7.3.3) of mice and then use gene recombination to obtain an animal model lacking uricase.

[0004] However, the currently available acute gout model still has many shortcomings, and does not truly simulate the characteristics of acute gout, which affects the research of the pathogenesis, pathophysiology and drug intervention measures screening of acute gout. In order to solve the above problems, the present application provides a method for stably and efficiently constructing an animal model with symptoms of acute gout, which can successfully establish a stable acute gout animal model, and the model can highly simulate the gout symptoms and gout-related inflammatory mechanisms of human gouty arthritis. SUMMARY

[0005] The present application aims to successfully establish an acute gout mouse model, observe the pathological changes of joints and organs of animals and detect joint and blood-related indicators to evaluate the stability of the model, and the purpose is to make an acute gout mouse model with good stability, good repeatability, practicality and close to clinical practice, which is convenient for further research on the pathogenesis and pathophysiological changes of acute gout, and provides a certain basis for how to implement protective interventions for acute gout.

[0006] The present application aims to develop an animal model that can focus on the classic symptoms of acute gout and can well evaluate the efficacy of drugs for acute gout. The acute gout mouse model constructed by the present application shows a high success rate and stability in inducing classic symptoms of acute gout (hyperuricemia, joint inflammation and deformity, gout stone deposition, kidney parenchymal lesions, etc.), as well as accompanying symptoms such as inflammatory cell infiltration of joint synovial tissue, gait change and hepatocyte edema. The present application can well simulate the clinical onset of acute gout and lay a good foundation for rapid screening of therapeutic drugs and interventions. The modeling method is stable and reliable, and close to clinical practice.

[0007] The present application is implemented by the following technical solutions:

[0008] A preparation method of an acute gout mouse model, characterized in that it comprises the following steps:

[0009] (1) Adaptive feeding: select the development of normal, good activity, body weight (20 ± 2) g of male KM mice, adaptive feeding 7 days; mice were raised in normal living environment, feeding, free diet, drinking water; the mouse breeding environment temperature (26 ± 1) ℃, humidity (50 ± 5) %, 12h / 12h light / dark cycle alternation.

[0010] (2) Drug injection: after adaptive feeding for 7 days, modeling began; on the first and third day of modeling in the morning, the mice were injected with aromatic oil 15 mg / kg (based on mouse weight), and on the second and fourth day of modeling in the morning, the mice were injected with benzo[a]pyrene 10 mg / kg (based on mouse weight).

[0011] (3) Electric shock stimulation: during the six days of modeling, the mice were electrically shocked for 30 minutes every day; the bottom of the mouse cage was provided with a foot electric stimulator and an electric shock floor, the electric shock floor was electrically connected to the foot electric stimulator, the foot electric stimulator was used to connect the electric shock floor to apply foot electric shock to the mice, and a controller was connected to the foot electric stimulator to control the electric shock floor to electrically shock the feet of the mice; when electric shock was performed every day, the controller released a current of 1.5 mA through the foot electric stimulator and the electric shock floor, each electric shock lasted for 10 seconds, and the intermittent stop lasted for 30 seconds, and the cycle was repeated for 30 minutes.

[0012] (4) Water stimulation: during the six days of modeling, lithium hydroxide was added to the drinking water of the mice, and the concentration of lithium hydroxide in the drinking water was 150 mg / L.

[0013] (5) Hydroxyacetic acid gavage: on the 5th-6th day of modeling, the mice were gavaged with a hydroxyacetic acid aqueous solution, once a day, with a dose of 0.2 ml per mouse, and the concentration of the hydroxyacetic acid aqueous solution was 70%.

[0014] (6) Blue light irradiation: on the 5th-6th day of modeling, the mice were irradiated with a blue light lamp, 4 hours a day; the wavelength generated by the blue light lamp was 460-480 nm, and the light intensity was 1200-1500 lx; after six days of modeling, an acute gout mouse model was obtained.

[0015] After the modeling, the model is evaluated by observing the physiological state of the mice, determining the swelling degree of the ankle joint, analyzing the gait, determining the blood uric acid, detecting the enrichment degree of neutrophils, detecting related indicators in the synovial tissue of the ankle joint by ELISA, detecting oxidation stress related molecules, observing the pathology of the synovial tissue section of the ankle joint, performing pathological examination on the kidney and liver, and determining the expression of TLR2, TLR4 and MyD88 molecules in the synovial tissue, etc. The results show that the mice have typical symptoms of acute gout, which meets the reliability and effectiveness of the determination of the animal model of acute gout, i.e. the pathogenesis, symptom manifestation and pathophysiology of the disease, and the acute gout mouse model can be successfully prepared according to the construction method of the application.

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

[0017] 1. The acute gout animal model commonly used at present has the problems of poor clinical simulation effect, poor stability and repeatability, etc., and the acute gout mouse model construction method of the application can induce a stable and repeatable, practical and clinically close acute gout mouse model by injecting aromatic oil and benzopyrene into the mice, electric shock stimulation, lithium hydroxide stimulation, hydroxyacetic acid gavage and blue light irradiation. This multi-factor induced acute gout model can better simulate the characteristics of human acute gout. The non-single factor induced acute gout animal model of the application is an ideal animal model from the perspective of pathophysiology theory, and from the perspective of simulating typical clinical symptoms and treatment prediction, which provides support for exploring the pathogenesis of acute gout and provides a theoretical basis for developing new solutions for treating acute gout.

[0018] 2. The acute gout mouse model constructed by the application has stable effect and small individual difference; the multiple factors synergize with each other, and the constructed disease model is more accurate. The model shows a high success rate and stability in inducing classical symptoms of acute gout (hyperuricemia, arthritis, deformity, gout stone deposition, kidney parenchymal lesions, etc.), as well as inflammatory cell infiltration in the synovial tissue of the joint, gait change, hepatocyte edema and other accompanying symptoms.

[0019] 3. The present invention injects 15 mg / kg of aromatic oil into the mouse peritoneal cavity on the morning of the 1st and 3rd day of modeling, and injects 10 mg / kg of benzopyrene into the mouse subcutaneously on the morning of the 2nd and 4th day of modeling. Aromatic oil can induce chemotaxis and necrosis of mouse leukocytes to a certain extent, thereby releasing a variety of inflammatory factors such as 5-HT and bradykinin, causing congestion and edema of the local tissue of inflammation, and increasing the concentration of K+ and H+, causing redness, swelling, heat and pain in the joints and periarticular tissues. Benzopyrene can activate the phosphatidylinositol 3-kinase / protein kinase B signaling pathway in mice, inhibit synovial tissue cell apoptosis, and enhance synovial tissue hyperplasia. Intraperitoneal injection of aromatic oil and subcutaneous injection of benzopyrene, the two of which cooperate with each other, can induce joint synovial tissue inflammation and tissue hyperplasia to a large extent, thereby effectively inducing acute gout in mice.

[0020] A certain degree of electric shock stimulation in mice can stimulate TLRs on the surface of macrophages to activate the NF-κB signaling pathway and produce inflammasomes, inducing the production of IL-1β and other pro-inflammatory cytokines, increasing the production of matrix metalloproteinases in the inflammatory region, aggravating cartilage dissolution and degradation, and thus exacerbating joint swelling. Electric shock stimulation after intraperitoneal injection of aromatic oil and subcutaneous injection of benzopyrene not only consolidates and strengthens the induced effects of drug injection, but also further aggravates cartilage degradation and joint swelling, greatly facilitating the induction of acute gout in mice.

[0021] Adding lithium hydroxide to the drinking water of mice can, to a certain extent, increase the levels of Notch1 receptor protein in the mouse serum and joint tissue, exacerbating the symptoms of acute gouty arthritis and the degree of articular cartilage damage. Based on drug injection and electric shock stimulation, adding lithium hydroxide to the drinking water can further aggravate the joint inflammation and articular cartilage damage of mice.

[0022] Gavage with glycolic acid in the late stage of modeling can increase the expression of adenosine deaminase and xanthine oxidase in tissues, enhance uric acid production, upregulate the intestinal epithelial urate transporter ABCG2, downregulate the expression of the glucose transporter GLUT9, increase uric acid reabsorption, inhibit uric acid excretion, and aggravate urate deposition in the kidneys. Building on the previous drug injections, electric shock stimulation, and lithium hydroxide stimulation, gavage with glycolic acid in the late stage of modeling can play a connecting role, further increasing blood uric acid levels and aggravating urate deposition.

[0023] Blue light irradiation on the 5th and 6th day of modeling can, to a certain extent, induce the production of free radicals and malondialdehyde in mouse tissues, leading to weakened mitochondrial oxidation of fatty acids, abnormal liver fat metabolism, imbalanced fatty acid regulation, increased purine metabolism, and uric acid accumulation. Blue light irradiation plays a finishing touch in the modeling steps of this application, synergizing with other steps to maximize the increase in mouse blood uric acid levels, ensuring a mouse acute gout model that can highly simulate the gout symptoms and gout-related inflammatory mechanisms of human gouty arthritis and has stable characteristics.

[0024] 4、The study can provide a good theoretical support and model reference for in-depth analysis of the immune mechanism of human acute gout and evaluation of new treatment methods. The research team has repeatedly verified the model, and the results are reliable. The model has been used to evaluate the efficacy of various drugs, and the application value of the model has been verified. The animal model shows the typical characteristics of acute gout: hyperuricemia, joint inflammation and deformity, gout stone deposition, and renal parenchymal lesions. The model can be used for basic research in the field of acute gout, and lays the foundation for experimental animals to explore the pathogenesis of acute gout. The study can provide a reliable animal model for the evaluation of acute gout drug efficacy, which has the advantages of high efficiency, stability and reliability, practicality, and low animal mortality. DETAILED DESCRIPTION

[0025] A preparation method of an acute gout mouse model, characterized in that it comprises the following steps:

[0026] (1) Adaptive feeding: select male KM mice with normal development, good activity, and body weight of (20±2) g, and adaptively feed them for 7 days; the mice are fed in a normal living environment, fed with feed, and given free diet and drinking water; the mouse feeding environment temperature is (26±1) ℃, humidity is (50±5) %, and the light / dark cycle alternates for 12h / 12h.

[0027] (2) Drug injection: start modeling after 7 days of adaptive feeding; on the first and third days of modeling, inject 15mg / kg (based on mouse body weight) of aromatic oil into the mouse abdominal cavity in the morning, and on the second and fourth days of modeling, inject 10mg / kg (based on mouse body weight) of benzopyrene subcutaneously into the mouse in the morning.

[0028] (3) Electric shock stimulation: during the six days of modeling, electric shock the mouse for 30min every day; the bottom of the mouse cage is provided with a foot electric stimulator and an electric shock bottom plate, the electric shock bottom plate is electrically connected to the foot electric stimulator, the foot electric stimulator is used to connect and cooperate with the electric shock bottom plate to apply foot electric shock to the mouse, and a controller is connected to the foot electric stimulator to control the electric shock bottom plate to electrically shock the mouse's feet; when electric shock is implemented every day, the controller releases a 1.5mA current through the foot electric stimulator and the electric shock bottom plate, each electric shock lasts for 10s, and the interval stops for 30s, and the cycle is repeated for 30min.

[0029] (4) Water stimulation: during the six days of modeling, add lithium hydroxide to the drinking water of the mouse, and the concentration of lithium hydroxide in the drinking water is 150mg / L.

[0030] (5), hydroxyacetic acid gavage: on the 5-6th day of modeling, hydroxyacetic acid aqueous solution was used for gavage to mice, once a day, the dose was 0.2 ml per mouse, and the concentration of the hydroxyacetic acid aqueous solution was 70%.

[0031] (6), blue light irradiation: on the 5-6th day of modeling, the mice were irradiated by turning on the blue light, 4h per day; the wavelength generated by the blue light was 460-480nm, and the light intensity was 1200-1500lx; the acute gout mouse model was obtained after six days of modeling.

[0032] After modeling, the model was evaluated by observing the physiological state of the mice, determining the swelling degree of the ankle joint, analyzing the gait, determining the blood uric acid, determining the enrichment degree of neutrophils, detecting related indicators in the synovial tissue of the ankle joint by ELISA, detecting oxidative stress related molecules, observing the pathology of the synovial tissue of the ankle joint, pathological examination of the kidney and liver, and determination of the expression of TLR2, TLR4 and MyD88 molecules in the synovial tissue of the ankle joint (the control group was injected with normal saline, gavaged with normal saline and normally fed), and the results were as follows:

[0033] 1. Physiological state observation

[0034] The control group mice were in good spirits, with normal diet and water intake, and normal weight gain. The model group mice were in poor spirits, with yellow and dry fur, and gout nodules could be felt at the peak of inflammation. The mice could be seen limping, and the amount of food and water intake were significantly reduced, and the weight gain was significantly slowed down.

[0035] 2. Swelling degree of ankle joint

[0036] The distance between the medial malleolus and the lateral malleolus of the mice was measured before and after modeling at different time points using a digital vernier caliper, and the mean value was taken after continuous measurement for 3 times. Swelling degree of ankle joint = current measurement joint diameter - initial measurement diameter

[0037] Compared with the control group, the right ankle joint of the model mice was significantly swollen.

[0038] 3. Gait analysis of mice

[0039] The gait behavior of mice was detected by DigiGait imaging system 24h after modeling, including various gait mechanics and posture indexes such as paw area, stride length, etc.

[0040] Compared with the control group, the gait behavior of the model group mice changed significantly, that is, the area of the affected side foot was significantly reduced compared with the healthy side paw. Further analysis found that the stride length of the model group mice was also significantly reduced. This result suggests that the model group mice showed significant changes in gait behavior.

[0041] 4. Blood uric acid determination

[0042] The collected upper serum was detected by biochemistry analyzer.

[0043] The results showed that the blood uric acid level of the mouse model group was significantly higher than that of the normal control group.

[0044] 5, the degree of neutrophil enrichment

[0045] Take the joint fluid smear of each group of mice, dry first with Wright's staining solution to fully cover the film surface, 2 min later, add 2-3 drops of Giemsa staining solution, blow evenly with ear ball, then add phosphate buffer to form surface tension on the film surface, continue staining for about 30 min, rinse with running water, dry and examine under a microscope. Randomly select 5 fields under a microscope to count the number of neutrophils, and take the average value. Take the number of neutrophils in the model group as the standard, calculate the enrichment degree of neutrophils in the joint fluid of each group of mice. The enrichment degree of neutrophils = the average number of neutrophils in this group / the average number of neutrophils in the model group x 100%.

[0046] Almost no neutrophils were found in the joint fluid of the control group mice, while a large number of neutrophils were found in the joint fluid of the model group.

[0047] 6, ELISA detection of related indicators in synovial tissue

[0048] The mice were sacrificed by cervical dislocation, cut off 0.5 cm above the right ankle joint on ice, remove the fur, put into a centrifuge tube, cut open the joint cavity, dilute with pre-cooled 0.9% NS 0.5 mL, ultrasonic for 15 min at 0℃, centrifuge at 3000 r / min for 10 min at low temperature, take the supernatant, and determine the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) according to the corresponding ELISA kit instructions.

[0049] Compared with the control group, the model group can significantly up-regulate the levels of IL-1β, IL-6 and TNF-α, and significantly down-regulate the level of IL-10.

[0050] 7, oxidative stress related molecule detection

[0051] 24 hours after modeling, the mice were anesthetized with isoflurane, and the affected ankle joint was quickly removed for biochemical analysis. The activities of superoxide dismutase (SOD), reduced glutathione (GSH) and malondialdehyde (MDA) were determined according to the kit.

[0052] Compared with the control group, the content of SOD and GSH in the ankle joint tissue of the model group mice decreased significantly, while the content of oxidative stress product MDA increased significantly. It is suggested that there is a significant oxidative stress reaction in the ankle joint tissue of the affected side of the model group mice.

[0053] 8. Pathological observation of ankle synovial tissue section

[0054] After blood sampling, the right hind ankle of the mouse was cut off, the skin was removed, and the ankle synovial tissue was obtained. The ankle synovial tissue was routinely processed, paraffin-embedded, and sectioned, and the pathological changes of the ankle synovial tissue of the mouse were observed by HE staining.

[0055] The ankle synovial tissue morphology and structure of the control group mice were normal; the synovial membrane of the model control group was swollen, and the cells showed obvious proliferation, the structure was disordered, and a large number of leukocyte infiltration, small blood vessel formation and fibroblasts were observed.

[0056] 9. Pathological examination of kidney and liver

[0057] The kidney and liver of the mouse were removed and fixed with 4% tissue cell fixative. The cut kidney and liver samples were ultrasonically treated, paraffin-embedded, and quickly cooled in a 4°C refrigerator and sectioned. After n-butanol dewaxing treatment, the sections were rehydrated with different concentrations of alcohol, stained and washed with water, and dehydrated with different concentrations of alcohol, and then sealed with neutral balsam. The pathological changes of the kidney and liver were observed under a microscope and photographed.

[0058] The kidney tissue morphology and structure of the control group mice were normal, the glomerular size was normal, the structure was clear, and no obvious pathological changes such as inflammatory cell infiltration were observed. Compared with the control group, the glomerulus of the model group was obviously atrophic, the renal interstitium was edematous, the renal tubules were obviously dilated with epithelial cell edema, and inflammatory cell infiltration was observed.

[0059] The liver tissue structure of the control group mice was clear, the liver cell size was normal, and the arrangement was neat, and no obvious pathological changes were observed. Compared with the control group, the liver cells around the central vein of the model group were obviously edematous

[0060] 10. Determination of TLR2, TLR4 and MyD88 molecule expression in mouse ankle synovial tissue

[0061] The expression of Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4) and Myeloid Differentiation Factor 88 (MyD88) molecules in the ankle synovial tissue of the mouse was determined by immunohistochemical method. The measurement method was to take 5 high-power fields (×400) for microscopic examination for each section, and the average integral optical density value (IOD) of each group was determined by Image-ProPlus6.0 image analysis software, and the average IOD value was calculated.

[0062] The expression of TLR2, TLR4 and MyD88 molecules in the ankle synovial tissue of the model group mice was significantly higher than that of the control group. It is proved that the modeling is successful.

Claims

1. A method for preparing an acute gout mouse model, characterized by, Comprising the following steps: (1) adaptive feeding: select the development of normal, good activity, body weight (20 ± 2) g of male KM mice, adaptive feeding for 7 days; the mice were fed in normal living environment, feed feeding, free diet, drinking water; the mouse breeding environment temperature (26 ± 1) ℃, humidity (50 ± 5) %, 12h / 12h light / dark cycle alternation; (2) drug injection: after adaptive feeding for 7 days, start modeling; on the first and third day of modeling in the morning, the mice were injected with aromatic oil 15mg / kg intraperitoneally, and on the second and fourth day of modeling in the morning, the mice were injected with benzo[a]pyrene 10mg / kg subcutaneously; (3) electric shock stimulation: during the six days of modeling, the mice were electrically shocked for 30min every day; the bottom of the mouse cage was provided with a foot electric stimulator and an electric shock floor, the electric shock floor was electrically connected to the foot electric stimulator, the foot electric stimulator was used to connect the electric shock floor to apply foot electric shock to the mice, and a controller was connected to the foot electric stimulator to control the electric shock floor to electrically shock the feet of the mice; when electric shock was implemented every day, the controller released a current of 1.5mA through the foot electric stimulator and the electric shock floor, each electric shock lasted for 10s, and the intermittent stop lasted for 30s, and the cycle was repeated for 30min; (4) water stimulation: during the six days of modeling, lithium hydroxide was added to the drinking water of the mice, and the concentration of lithium hydroxide in the drinking water was 150mg / L; (5) glycolic acid gavage: on the 5th-6th day of modeling, the mice were gavaged with glycolic acid solution, once a day, and the dose was 0.2ml per mouse, and the concentration of the glycolic acid solution was 70%; (6) blue light irradiation: on the 5th-6th day of modeling, the mice were irradiated with blue light, 4h a day; the wavelength generated by the blue light was 460-480nm, and the light intensity was 1200-1500lx; after six days of modeling, the acute gout mouse model was obtained.