Construction method and application of children inflammatory bowel disease rat model

Abstract

The invention discloses a construction method and application of a child inflammatory bowel disease rat model. The construction method comprises the following specific steps: smearing a 3% w / v oxazolone pre-sensitizing solution on the skin of a rat for pre-sensitization, and continuously performing repeated intermittent enema with a 1% w / v oxazolone enema. The model accords with the clinical manifestation and pathological characteristics of the children inflammatory bowel disease, and is a better model for researching the children inflammatory bowel disease.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a rat model of inflammatory bowel disease in children and an application thereof. Background Art

[0002] Pediatric Inflammatory Bowel Disease (PIBD) refers to a chronic, recurrent, nonspecific immune-mediated inflammatory bowel disease in children. It is a recognized fact that its global incidence rate is increasing year by year. The incidence of PIBD in Asia is about 0.5 to 21.6 / 100,000, and the prevalence is about 5 to 25.2 / 100,000, and it is still rising. The annual growth rate of PIBD incidence in children under 10 years old is 5% to 7%. A multicenter study in the Asia-Pacific region found that the average age of PIBD is 9.07 years old, and the proportion of extremely early PIBD (onset age less than 6 years old) and early PIBD (onset age less than 10 years old) is as high as 29.3% and 52.7%, respectively; the number of new PIBD cases has increased significantly, and nearly half (48.6%) of the population have been diagnosed in the past five years (2015-2019).

[0003] Unlike adult inflammatory bowel disease (IBD), PIBD has atypical symptoms, more severe intestinal inflammation, faster disease progression, and more relapses, more complex pathological immune features, and more common extraintestinal manifestations such as erythema nodosum. It also seriously affects the growth and development of children, leading to growth retardation or delayed puberty. Suspected PIBD can be diagnosed based on clinical history, physical examination, and laboratory test results. Endoscopic examination, especially histopathological evaluation, is considered the most reliable diagnostic standard. It is very important to identify suspected PIBD patients early and refer them for endoscopic evaluation and biopsy in a timely manner, because delayed diagnosis increases the risk of disease complications, such as stenosis or fistula complications and growth disorders.

[0004] In order to better study the pathogenesis and treatment measures of PIBD, it is necessary to construct a stable PIBD model, but there are currently very few studies involving PIBD animal models. Recently, researchers gave 3-week-old male C57 / BL6 mice 3% w / v dextran sulfate sodium (DSS) solution to drink for 5 consecutive days to induce acute colitis. It was found that young mice with colitis showed weight loss, significantly shortened colon length, and enlarged spleen. Histopathological analysis found colon epithelial degeneration and immune cell infiltration, but this model does not meet the chronic and recurrent characteristics of PIBD. A single use of chemical reagents can induce an acute inflammatory response, and multiple intermittent uses after skin pre-sensitization will lead to chronic inflammation. Therefore, a PIBD model can be constructed by repeated chemical induction of young SD rats.

[0005] There are three commonly used chemical agents to induce chronic IBD, including DSS, trinitrobenzenesulfonic acid (TNBS) and oxazolone, with modeling periods of 8 weeks, 6 weeks and 5 weeks respectively. DSS aqueous solution can be administered by free drinking or oral gavage, while TNBS and oxazolone are modeled by skin presensitization and repeated rectal administration.

[0006] Animal age and other factors affect the induction and severity of PIBD. The age of rats can be matched to human age through techniques such as the weight of the eye lens, the growth of molars, the count of the tibial endometrial layer, and the growth of muscles and bones accompanied by the closure and thickening of the epiphysis. The age of rats and humans is not linearly related. A 3-week-old rat is approximately equivalent to a 6-month-old infant, an 8-week-old rat corresponds to a 10-year-old child, and sexual maturity at 2-month-old male and 2.5-month-old female rats marks the entry into puberty, while 6 months of age in rats corresponds to 18 years old in humans. In the early preliminary experiments, it was found that the mortality rate of suckling rats given oxazolidinone enema was high, but the success rate of modeling of surviving suckling rats was low, which may be due to the effect of breast milk on intestinal inflammation in suckling rats.

[0007] Therefore, it is necessary to develop an animal model that conforms to the pathological characteristics of human PIBD, namely, chronic relapsing and progressive disease. Summary of the invention

[0008] The present invention provides a method for constructing a rat model of inflammatory bowel disease in children and application thereof. The model constructed by the present invention conforms to the clinical manifestations and pathological characteristics of inflammatory bowel disease in children and is a better model for studying inflammatory bowel disease in children.

[0009] In order to achieve the above purpose, this application adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for constructing a rat model of inflammatory bowel disease in children, the specific steps of the construction method are: the rats are pre-sensitized by applying 3% w / v oxazolone presensitization solution to their skin, and then continue to use 1% w / v oxazolone enema solution for repeated intermittent enema.

[0011] In the above technical scheme, the rats used were 3-week-old female SD rats.

[0012] In the above technical solution, the dosage used for repeated intermittent enema is 10 mg / kg.

[0013] In the above technical scheme, after pre-sensitization of rat skin by applying 3% w / v oxazolone presensitization solution on the first day, the rats were given enema on the 6th day, the 20th day and the 34th day respectively.

[0014] In the above technical scheme, the preparation method of the oxazolone presensitization solution is: acetone and olive oil are mixed in a volume ratio of 4:1 to form a solvent, and oxazolone powder is added to the solvent in a ratio of 60 mg: 2 mL to dissolve, that is, a 3% w / v oxazolone presensitization solution is prepared.

[0015] In the above technical scheme, the preparation method of the oxazolone presensitization solution is: the preparation method of the oxazolone enema solution is: add oxazolone powder to anhydrous ethanol in a ratio of 20 mg: 1 mL to dissolve it, then add pure water equal to the anhydrous ethanol and mix well to prepare a 1% w / v oxazolone enema solution.

[0016] In a second aspect, the present invention provides an application of a rat model of inflammatory bowel disease in children constructed by the above-mentioned construction method in the development of therapeutic drugs for inflammatory bowel disease in children.

[0017] The beneficial effect of the present invention is that the present invention first explores the influence of the sex of SD mice and the dose of oxazolone enema solution on the induction and severity of PIBD, and determines that the most efficient and economical method for constructing a stable PIBD mouse model is: 3-week-old female SD mice are pre-sensitized by applying 3% w / v oxazolone presensitization solution to the skin, and then continue to use 1% w / v oxazolone enema solution (at a dose of 10 mg / kg) for repeated intermittent enema. The model of the present invention conforms to the clinical manifestations and pathological characteristics of inflammatory bowel disease in children, and is a better model for studying inflammatory bowel disease in children. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Effects of sex and oxazolone enema dose on the survival rate and growth and development of SD rats. Figure A: Schematic diagram of the modeling process of oxazolone solution. Figure B: Survival curves of oxazolone enema of different sexes and different doses of SD rats. Figure C: Histogram of survival time of oxazolone enema of different sexes and different doses of SD rats. Figure D: Histogram of weight change of oxazolone enema of different sexes and different doses of SD rats. Figure E: Histogram of weight loss percentage of oxazolone enema of different sexes and different doses of SD rats. Figure F: Histogram of DAI score of oxazolone enema of different sexes and different doses of SD rats. *, P value less than 0.05; **, P value less than 0.01; ***, P value less than 0.001; the rest are statistically insignificant differences between the two groups.

[0019] Figure 2:Effects of different doses of oxazolone enema on colon and mesenteric lymph nodes in SD rats of different sexes. Figure A: Gross observation of the whole colon after different doses of oxazolone enema. Figure B: Comparison of colon length of SD rats of different sexes after different doses of oxazolone enema. Figure C: Beaded enlargement of mesenteric lymph nodes. Figure D: Gross observation of mesenteric lymph nodes after different doses of oxazolone enema. *, P value less than 0.05; **, P value less than 0.01; ***, P value less than 0.001; the rest are no statistically significant differences between the two groups.

[0020] Figure 3 :Effects of different doses of oxazolone enema on intestinal pathological changes in SD rats of different genders. Figure A: HE staining of colon after different doses of oxazolone enema in female rats. Figure B: HE staining of colon after different doses of oxazolone enema in male rats. Figure C: Comparison of CMDI of different doses of oxazolone enema in SD rats of different genders. Figure D: Comparison of CMDI of weight change after different doses of oxazolone enema in SD rats of different genders. *, P value less than 0.05; **, P value less than 0.01; ***, P value less than 0.001; the rest are no statistically significant differences between the two groups.

[0021] Figure 4 : Swiss roll HE staining of colon.

[0022] Figure 5 :The effect of VNS on intestinal immune cells in PIBD. Figure A: Whole intestine immunofluorescence images of colon T cells, B cells, NK cells, macrophages, dendritic cells, and neutrophils in each group. Figure BG: Local magnified immunofluorescence images of colon T cells, B cells, NK cells, macrophages, dendritic cells, and neutrophils in each group.

[0023] Figure 6 :Abnormal vagus nerve function in PIBD young mice. Figure A: Vagus nerve CAP waveform. Figure B: Vagus nerve ECG. Figure C: Heart rate variability curve. Figure D: Average heart rate evaluation chart. Figure E: Mean arterial blood pressure evaluation chart. Figure F: Colonic acetylcholine content evaluation chart. The scatter points on the left side of the evaluation chart are the original data, and the right side represents the mean difference and its 95% confidence interval. If the 95% confidence interval of the effect size does not include 0, the P value is less than 0.05.

[0024] Figure 7 :The symptoms of PIBD young mice were aggravated after vagotomy. Figure A: Weight change assessment chart. Figure B: DAI assessment chart. Figure C: Colon length assessment chart. Figure D: CDMI assessment chart. Figure E: HIS assessment chart. Figure F: The left side shows the gross observation of the mucosal surface of the distal colon, and the right side shows the HE staining of the middle colon. The scatter points on the left side of the assessment chart are the original data, and the right side shows the mean difference and its 95% confidence interval. If the 95% confidence interval of the effect size does not include 0, the P value is less than 0.05. DETAILED DESCRIPTION

[0025] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0026] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0027] In order to exclude the influence of breast milk on PIBD, the present invention selects 3-week-old weaned mice for modeling, and in order to make the age of onset closer to human PIBD, the oxazolone solution with the shortest modeling cycle is used for modeling. The present invention adopts 3-week SD mice of different genders to repeatedly and intermittently give different doses of oxazolone after skin presensitization of oxazolone solution, and repeats three times, and the control group replaces oxazolone with equal doses of normal saline. Draw a survival curve, monitor weight changes to evaluate its growth and development, evaluate its disease activity by weight loss, stool characteristics and occult blood, and evaluate and score intestinal pathological damage by gross observation and HE staining, so as to evaluate the influence of gender and oxazolone solution dosage on the degree of intestinal inflammatory damage in young mice. Evaluate the vagus nerve function of young mice with enteritis by heart rate variability analysis, nerve compound action potential, blood pressure, and intestinal acetylcholine content, and observe the effect of vagotomy on intestinal inflammation. The present invention explores the effects of gender and oxazolidinone dosage on the modeling of inflammatory bowel disease in children, and provides a rat model of inflammatory bowel disease in children. The model conforms to the clinical manifestations and pathological characteristics of inflammatory bowel disease in children, and is a better model for studying inflammatory bowel disease in children.

[0028] 1. Experimental process

[0029] 1. Experimental Animals

[0030] Three male and six female 9-week-old SPF SD rats were isolated and quarantined for one week and raised in the barrier system of the Animal Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The breeding environment complies with the national standard "Experimental Animal Environment and Facilities" (GB14925-2010). Two female and one male SD rats were bred together in a cage. When the pups were 3 weeks old, they were separated from the mother rats. Following the 3R principle, different numbers of 3-week-old SD rats of different genders were randomly sampled according to the experimental needs for subsequent experiments. They should be fed adaptively in a single cage for three days before surgery.

[0031] 2. Random Grouping

[0032] (1) Fifteen 3-week-old female SD mice and 15 male SD mice were divided into 6 different treatment groups:

[0033] Control group female: female young rats, 0.9% sodium chloride solution (normal saline) skin presensitization and multiple enema;

[0034] Control group: male young rats, saline skin presensitization and multiple enemas;

[0035] OXA1 female: female young mice, skin presensitization with oxazolone presensitization solution and multiple enemas of 30 mg / kg oxazolone enema solution;

[0036] OXA1 male: male young rats, skin presensitization with oxazolone presensitization solution and multiple enemas with 30 mg / kg oxazolone enema solution;

[0037] OXA2 female: female young mice, skin presensitization with oxazolone presensitization solution and multiple enemas of 10 mg / kg oxazolone enema solution;

[0038] OXA2 male: Male young rats were skin presensitized with oxazolone presensitization solution and multiple enemas of 10 mg / kg oxazolone enema solution were administered.

[0039] (2) Ten 3-week-old female SD mice were randomly divided into two different treatment groups:

[0040] PIBD: female young mice, the left vagus nerve was separated but not removed, PIBD model was established;

[0041] VNC: female young mice, left vagotomy, PIBD model.

[0042] (3) Ten 3-week-old female SD mice were randomly divided into two different treatment groups:

[0043] Control group: vagus nerve was isolated but no electrodes were implanted;

[0044] Electrode implantation group: The vagus nerve was isolated and electrodes were implanted.

[0045] 3. Oxazolone-induced chronic PIBD

[0046] (1) Solution preparation

[0047] ① Oxazolone presensitization solution: acetone and olive oil are vortexed and mixed in a volume ratio of 4:1 to make a solvent. 60 mg of oxazolone powder is added to 2 mL of solvent and dissolved to prepare a 3% w / v oxazolone presensitization solution. This solution is flammable and must be prepared in a fume hood and used immediately after preparation.

[0048] ② Oxazolone enema solution: add 20 mg of oxazolone powder into centrifuge tubes containing 1 mL of anhydrous ethanol to dissolve, then add an equal amount of pure water. Vortex mix and dilute to 1% w / v oxazolone enema solution, prepare and use immediately.

[0049] (2) Oxazolone skin sensitization and multiple enema (the control group was given an equal dose of normal saline)

[0050] ① Skin presensitization on the first day: mark the young mice, weigh and record the weight, and use isoflurane anesthetic according to the instructions for anesthesia. Remove the hair of the back 2.5cm×2.5cm exposed skin, disinfect with 75% ethanol and wipe with dry cotton, absorb 150μL of oxazolidinone presensitization solution and apply it to the hairless skin on the back.

[0051] ② Enema on the 6th day: Weigh and record, anesthetize with isoflurane anesthetic, and place the young mice in prone position. Draw oxazolidinone enema solution into a 1mL syringe and connect it to a 3.5F catheter. Moisten the catheter with paraffin oil and gently insert the catheter into the colon from the anus to a depth of about 8cm. Slowly inject at 10mg / kg and 30mg / kg, respectively, stand upside down for 2 minutes, and keep warm for resuscitation.

[0052] ③ Repeat the previous step twice every 14 days, that is, repeat the operation on day 6 on the 20th day and the 34th day.

[0053] ④ After enema, observe and record the weight, fecal characteristics, hair, mental state and other general conditions of the mice every day. Use the fecal occult blood qualitative detection kit to detect fecal occult blood. Use a sampling wood chip or toothpick to pick up a small amount of feces 10-50mg (mung bean size), apply it on sterilized cotton or white porcelain plate; add 2 drops of O-tolidine Solution (about 0.1mL) to different locations of the feces; add 2 drops of oxidant, immediately start timing and observe the color change. The result interpretation criteria are as follows:

[0054] After adding the reagent, it immediately turns blue-black-brown and is recorded as 4+;

[0055] After adding the reagent, it immediately turns blue-brown and gradually turns dark brown, which is recorded as 3+;

[0056] After adding the reagent, it initially appears light green, then gradually turns into obvious blue-brown, which is recorded as 2+;

[0057] After 10 seconds, it gradually changes from light green to green, which is recorded as +;

[0058] No color development within 2 minutes is negative.

[0059] ⑤ On the 38th day, anesthetize the young mice, open the abdominal cavity, and observe the gross condition of the mesenteric lymph nodes and colon, including congestion, edema, necrosis, stiffness, necrosis, intestinal adhesion, etc. Cut the mesentery from the cecum to the anus, carefully separate the entire colon and measure the length of the colon. Cut the colon along the mesentery to observe whether the intestinal mucosa is smooth and whether there is erosion, thickening, necrosis or ulcer.

[0060] (3)PIBD evaluation criteria

[0061] ① DAI was assessed based on body weight, stool characteristics, and fecal occult blood. The assessment criteria are shown in Table 1.

[0062] Table 1 DAI scoring criteria

[0063]

[0064]

[0065] ② The colon mucosal damage index (CMDI) was evaluated based on gross observation of the colon. The scoring criteria are shown in Table 2.

[0066] Table 2 CMDI scoring criteria

[0067]

[0068] ③ Inflammation-related histology score (IHS) was performed based on colon HE staining. The scoring criteria are as follows:

[0069] 0 points: no signs of inflammation;

[0070] 1 point: low-level inflammation with scattered infiltrating mononuclear cells (1 to 2 lesions);

[0071] 2 points: moderate inflammation, multifocal;

[0072] 3 points: severe inflammation, increased vascular density, and significant thickening of the vascular wall;

[0073] 4 points: extremely severe inflammation, accompanied by transmural immune cell infiltration and goblet cell loss.

[0074] 4. Vagotomy

[0075] After anesthesia, fix the neck, remove hair, disinfect with iodine and then deiodinate with 75% ethanol. Cut the skin along the midline of the neck from the sternum upwards, the incision is about 1 cm, and the subcutaneous fascia and skin are bluntly separated to expose the superficial muscles of the neck. The neck muscles are separated, and the carotid sheath is found on both sides of the sternohyoid muscle and below the sternomastoid muscle according to the carotid artery pulsation. The vagus nerve and carotid artery are carefully separated, the vagus nerve is removed, sutured, and warmed for resuscitation.

[0076] 5. Vagus nerve compound action potential (CAP) measurement

[0077] Using the BL-420N signal acquisition and processing system, after isolating the vagus nerve according to the previous step, the nerve stimulation electrode and the nerve discharge guidance electrode were connected and placed on the isolated vagus nerve to record the resting and post-stimulation CAP.

[0078] 6. Electrocardiogram (ECG) measurement

[0079] After anesthesia and fixation, the mice were connected to full-lead ECG wires using the BL-420N signal acquisition and processing system, and twelve-lead ECG connection electrodes were inserted subcutaneously into the limbs and left chest of the mice to record ECG.

[0080] 7. Arterial blood pressure measurement

[0081] After anesthesia and fixation, the carotid artery was isolated and cannulated according to step 3), and the pressure sensor was connected using the BL-420N signal acquisition and processing system to record the blood pressure and calculate the mean arterial blood pressure.

[0082] 8. Tissue homogenization

[0083] Rinse the tissue with pre-cooled phosphate buffered saline (PBS) (0.01M, pH=7.4) to remove residual blood and weigh. Cut the tissue into small pieces, add 9 mL PBS per 1 g of tissue and record, add protease inhibitors and grind thoroughly on ice. Ultrasonicate the homogenate, centrifuge at 5000×g for 5-10 minutes, and take the supernatant.

[0084] 9.ELISA

[0085] (1) Place the test kit and samples at room temperature for 20 minutes before the experiment to rewarm.

[0086] (2) Solution preparation

[0087] ①1× washing liquid: Calculate the required volume of 1× washing liquid and dilute 25× concentrated washing liquid with pure water to make 1× application liquid.

[0088] ② Gradient dilution of standard: centrifuge the freeze-dried standard for 1 minute, add 1mL of standard and sample diluent and let stand for 10 minutes, invert several times to fully dissolve it, and prepare a 1000pg / mL standard working solution. Perform multiple dilutions, add 500μL of sample diluent to each tube, draw 500μL from the 1000pg / mL standard working solution to the next centrifuge tube and mix and dilute to 500pg / mL, dilute in sequence, and the last tube is a blank tube. Finally, 8 tubes of isoproportionately diluted antibody standards are obtained, with a concentration gradient of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 0pg / mL. The last tube is a blank tube without adding standard working solution.

[0089] ③HRP substrate: The reagent should be prepared within 15 minutes before use. Protect from light. 100 μL of solution is required for each well.

[0090] ④HRP-streptavidin: Prepare according to the required amount for each test (calculated as 100 μL / well), dilute the 100-fold concentrated HRP-labeled streptavidin into 1-fold working solution with diluent (centrifuge before dilution), and use within 30 minutes.

[0091] (3) ELISA operation

[0092] ① Add 50 μL of standard working solution and test sample to each well, cover with sealing film, and incubate in a 37℃ incubator for 30 minutes.

[0093] ② Washing: Aspirate the sample from each well, then add 250 μL of washing buffer for washing. Wash three times in total. After the last wash, remove the residual liquid in the well and invert the 96-well plate on clean absorbent paper to pat dry.

[0094] ③Add 50 μL of 1:100 diluted detection antibody to each well, seal the plate and incubate at room temperature for 1 hour. Repeat step ②.

[0095] ④Add 100 μL HRP-streptavidin working solution to each well, seal the plate, and incubate at room temperature for 30 minutes. Repeat step ②.

[0096] ⑤ Add 90 μL of color developer to each well, seal the plate and incubate at 37°C in the dark for 15 minutes.

[0097] ⑥ Add 50 μL of stop solution to each well and tap the plate to mix.

[0098] ⑦ Preheat the full-wavelength microplate reader before use, set the measurement wavelength to 450nm, and the calibration wavelength to 540nm or 570nm. Measure and save the optical density (OD) value of each well within 30 minutes.

[0099] 10. Organ paraffin embedding and HE staining

[0100] (1) Wash the fresh tissue with physiological saline and remove excess water with absorbent paper. Fix the tissue in 4% paraformaldehyde fixative for more than 24 hours. Take out the tissue, trim it flat in a fume hood, and place it in a dehydration box.

[0101] (2) Dehydrate and make transparent in a gradient from low to high concentration of ethanol: the dehydration box is placed in 75% ethanol for 4 hours → 85% ethanol for 2 hours → 90% ethanol for 2 hours → 95% ethanol for 1 hour → anhydrous ethanol for 30 minutes → anhydrous ethanol for 30 minutes → 2 / 3 anhydrous ethanol + 1 / 3 xylene for 10 minutes → 1 / 2 anhydrous ethanol + 1 / 2 xylene for 10 minutes → 1 / 3 pure ethanol + 2 / 3 xylene for 10 minutes → xylene for 10 minutes → xylene for 10 minutes (until the tissue is transparent).

[0102] (3) Wax embedding from low temperature to high temperature: Place the transparent tissue in an oven and melt 1 / 2 paraffin + 1 / 2 xylene for 30 minutes → pure paraffin for 1 hour, repeat 3 times. After the paraffin is completely immersed in the tissue block, embed it in the embedding machine. Put the melted wax into the embedding frame and label it before the wax solidifies. Cool it in a -20℃ refrigerator, take out the wax block after solidification and trim it.

[0103] (4) Slice the sample on a paraffin slicer to a thickness of 4 μm. With the shiny side facing up, spread the sample on a slide in 40°C warm water in a slide spreader and place it in a 50°C oven for 30 minutes.

[0104] (5) Dewaxing and rehydration of paraffin sections: Place the sections in xylene for 20 minutes → 1 / 2 anhydrous ethanol + 1 / 2 xylene for 5 minutes → anhydrous ethanol for 5 minutes → anhydrous ethanol for 5 minutes → 95% ethanol for 5 minutes → 90% ethanol for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes, and wash with distilled water.

[0105] (6) Staining: Place the sections in hematoxylin solution for 5 minutes → wash with distilled water for 2 minutes → differentiate with differentiation solution for 3 seconds → wash with distilled water for 2 minutes, spin dry → blue with bluing solution for 3 seconds → wash with distilled water for 2 minutes, spin dry → place the sections in eosin solution for 5 minutes → wash with distilled water for 2 minutes, spin dry.

[0106] (7) Dehydration and sealing: Place the slices in 95% ethanol for 5 minutes → 95% ethanol for 5 minutes → anhydrous ethanol for 5 minutes → anhydrous ethanol for 5 minutes → xylene for 5 minutes → xylene for 5 minutes until transparent. Allow to dry and seal with neutral gum.

[0107] 11. Immunofluorescence

[0108] (1) Dewaxing of paraffin sections: Place the sections in xylene I for 15 minutes → xylene II for 15 minutes → anhydrous ethanol I for 5 minutes → anhydrous ethanol II for 5 minutes → 85% ethanol for 5 minutes → 75% ethanol for 5 minutes → wash with distilled water.

[0109] (2) Place the tissue sections in a repair pot filled with EDTA antigen repair buffer (pH = 9.0) or citric acid (pH = 6.0) antigen repair solution for antigen repair. Prevent excessive evaporation of the buffer and do not dry the slides. After natural cooling, place the slides in PBS (pH = 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time.

[0110] (3) Blocking endogenous peroxidase: Place the sections in 3% hydrogen peroxide (hydrogen peroxide: pure water = 1:9) and incubate at room temperature in the dark for 25 minutes. Place the slides in PBS and wash them three times on a decolorizing shaker for 5 minutes each time.

[0111] (4) BSA blocking: After the slices are slightly dried, a circle is drawn around the tissue with a histochemical pen (to prevent the antibody from flowing away), and 3% BSA is dripped into the circle to evenly cover the tissue, and the tissue is blocked at room temperature for 10 minutes.

[0112] (5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion to the slice, and incubate the slice flat in a light-proof humidified box at 4°C overnight. (Add a small amount of water to the humidified box to prevent the antibody from evaporating)

[0113] (6) Adding secondary antibody: Place the slide in PBS and wash on a decolorizing shaker three times, 5 minutes each time. After the slices are slightly dried, add a secondary antibody of the same species as the primary antibody in the circle, cover the tissue, and incubate at room temperature for 1 hour in the dark.

[0114] (7) Color development: After incubation, wash away the residual secondary antibody with PBS, and wash the sections in PBS on a decolorizing shaker three times, 5 minutes each time. After the sections are slightly dried, add freshly prepared fluorescent color development solution to the circle, and rinse the sections with PBS to stop color development.

[0115] (8) Repeat steps (2) to (7).

[0116] (9) DAPI counterstaining of cell nuclei: Place the slide in PBS and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add DAPI staining solution in the circle and incubate at room temperature for 10 minutes away from light.

[0117] (10) Scanning: The slices were fully scanned using a multi-channel fluorescence scanner.

[0118] 2. Experimental Results

[0119] 1. Gender and enema oxazolidinone dose affect the survival rate and growth of SD mice

[0120] SD young mice are in the growth and development period. Even if the intestines are damaged by oxazolidinone, the weight will decrease in the acute phase (1 to 3 days of oxazolidinone enema), but the weight will recover in the remission phase. Therefore, the percentage of weight loss is based on the average weight gain of the control group as the baseline, rather than the self-control before and after. Figure 1As shown in the data, within the same sex, the mortality rate after enema of 30 mg / kg oxazolone enema (OXA1) was as high as 50%, and its average survival time was significantly reduced compared with saline (control group) and 10 mg / kg oxazolone enema (OXA2) enema. The weight gain rate of OXA1 was also significantly lower than that of the other two groups, and the difference was statistically significant; the percentage of weight loss of OXA 1 was much higher than that of OXA2. The DAI of oxazolone was significantly higher than that of the control group, and the difference was statistically significant. The DAI of OXA1 was higher than that of OXA2, but the difference between the two groups was not statistically significant. When comparing the same dose of oxazolone enema, the mortality rate of males was significantly higher than that of females, and the average survival time was lower than that of females, but the difference was not statistically significant; the weight change and weight loss percentage of males were higher than those of females, and the difference was statistically significant; the DAI score of males was also higher than that of males, but the difference between the two was not statistically significant.

[0121] 2. Gender and enema oxazolidinone dose affect the degree of intestinal damage in SD mice

[0122] like Figure 2 As shown, compared with the control group, the OXA1 mesenteric lymph nodes increased and were beaded, the colon and small intestine formed adhesions and were difficult to separate, the colon was highly congested and edematous, the colon was significantly shortened, the intestinal wall was significantly thickened, the entire intestinal wall necrosis was visible in the distal part of the colon, and multiple ulcers were visible to the naked eye; the OXA2 mesenteric lymph nodes increased and enlarged but the number and size were lighter than OXA1, the intestine was adhered but could be bluntly separated, the colon was moderately congested and edematous, the colon was shortened, the intestinal wall was thickened and the mucosa was rough and granular, the intestinal mucosa was eroded and there was necrosis and ulceration on the surface.

[0123] 3. Effects of gender and enema oxazolidinone dose on intestinal pathological changes in SD mice

[0124] like Figure 3 As shown in the figure, the results of colon HE staining showed that the control group had clear structures of each layer of the colon, neatly arranged columnar cells and goblet cells, and arranged crypts, without obvious inflammatory cell infiltration; OXA1 had obvious inflammatory cell infiltration, normal proximal structure and crypt morphology, low-level inflammation and scattered infiltrating mononuclear cells, but the middle and especially distal colon had obvious destruction, unclear structure, crypt disappearance, transmural inflammation and goblet cell loss; OXA2 had clear proximal and middle colon structures and normal crypt morphology, but scattered immune cell infiltration in the mucosa, and severe inflammation in the distal colon, even transmural immune cell infiltration and goblet cell loss in some intestinal segments. Comparing the CMDI and HIS of each group, compared with the control group, the CMDI and HIS of OXA1 and OXA2 were significantly increased and the difference was statistically significant; the CMDI and HIS of OXA1 were higher than those of OXA2. Except for the statistically significant difference between OXA1 and OXA2 in males, there was no statistically significant difference between other groups.

[0125] 4. Intestinal damage and immune cell infiltration in PIBD mice

[0126] like Figure 4 As shown, the control group had clear structures of each layer of the colon, neatly arranged columnar cells and goblet cells, orderly arranged crypt morphology, and no inflammatory cell infiltration; PIBD had obvious inflammatory cell infiltration, normal proximal structure and crypt morphology, low-level inflammation and scattered infiltrating mononuclear cells, but the middle and especially distal colon morphology was obviously destroyed, the structure was unclear, the crypts disappeared, accompanied by transmural inflammation and goblet cell loss.

[0127] CD3, CD19, NKp46, CD68, CD103, and MPO were used to label T cells, B cells, NK cells, macrophages, dendritic cells, and neutrophils, respectively. Figure 5 As shown, the normal control group (Control) did not show obvious infiltration of adaptive immune cells such as T cells and B cells, while innate immune cells such as NK cells, macrophages and neutrophils were mainly scattered in the submucosal layer and muscular layer. The colon of PIBD mice was thickened, the colon crypts were destroyed, and the intestinal lymph nodes were significantly proliferated; local magnification showed a large number of T cell infiltrations in the mucosal layer and submucosal layer; isolated lymphoid follicles were scattered in the mucosal lamina propria, mainly composed of a large number of T cells and B cells; NK cells, macrophages, and neutrophil infiltrations were seen in the lamina propria, submucosal layer, and muscular layer; and a large number of neutrophils infiltrated the transmural inflammatory site; and dendritic cells were scattered throughout the entire layer. After VNS treatment, the degree of immune cell infiltration in each layer of the colon was significantly reduced.

[0128] This shows that low-dose oxazolone can induce stable PIBD in 3-week-old female SD mice. After enema of oxazolone solution, obvious growth and development disorders, diarrhea, bloody stools, loss of colonic epithelial crypts, and lymphocyte infiltration occurred. The disease characteristics and pathological changes were consistent with the clinical manifestations of children with PIBD. Intestinal inflammation is more severe in the distal colon and even forms transmural inflammation, which may be related to the distribution of the vagus nerve. Acetylcholine secreted by the terminal of the vagus nerve has an anti-inflammatory effect. The vagus nerve is more distributed in the ascending colon and transverse colon, and the more distal the colon, the less it is distributed. The mortality rate of SD mice after enema of 30mg / kg oxazolone was significantly increased compared with 10mg / kg. At the same enema dose, the survival rate of males was lower than that of females, and the difference in the degree of PIBD within the group was greater. Therefore, considering the stability of the model, it is reasonable to choose 3-week-old female SD mice at a dose of 10mg / kg for modeling for subsequent experiments. A dose of 10 mg / kg was used to model 3-week-old female SD mice, and it was found that the intestinal innate immunity and adaptive immunity of PIBD mice were aggravated. The overactivation of the immune system led to the destruction of the intestinal mucosal structure, infiltration of inflammatory cells such as cells, macrophages, and neutrophils, and crypt loss, and damage to the intestinal mucosal barrier. In addition, long-term and repeated chronic intestinal inflammation led to abdominal adhesions, intestinal fibrosis and stiffness, and a significant shortening of the colon.

[0129] 5. Abnormal vagus nerve function in PIBD mice

[0130] Three-week-old female SD rats were sensitized to the skin by applying oxazolone presensitization solution and repeatedly given 10 mg / kg oxazolone enema to establish a stable PIBD model and evaluate the vagus nerve function of PIBD rats. Figure 6 As shown in A, the vagus nerve CAP amplitude of PIBD mice was reduced and its response time was prolonged. Figure 6 As shown in B-D, the RR interval of PIBD mice was prolonged and the mean heart rate was increased, and the differences were statistically significant, indicating that the heart rate variability of PIBD mice was changed. In addition, the mean arterial blood pressure of PIBD mice increased ( Figure 6 E), colonic acetylcholine content was significantly reduced ( Figure 6 F), the differences were statistically significant. These manifestations all reflect the abnormal vagus nerve function in PIBD mice.

[0131] 6. Symptoms of PIBD mice worsen after vagotomy

[0132] 3-week-old female SD rats were subjected to vagotomy (VNC) and then PIBD model was established to observe the effect of vagus nerve integrity on the occurrence and development of PIBD. Figure 7As shown in the figure, compared with PIBD with intact vagus nerve, PIBD rats after vagotomy were listless, had significantly reduced weight gain, and suffered from severe diarrhea and bloody stools. Dissection revealed that the VNC group had severe abdominal adhesions that were difficult to separate, the colon was shortened, the intestinal wall was significantly thickened, and the range of transmural inflammatory necrosis extended to the middle part of the colon. HE staining showed that the destruction of the mucosal structure, inflammatory cell infiltration, and crypt loss were significantly aggravated, resulting in higher DAI and IHS scores and more obvious shortening of the colon, and the differences between the two groups were statistically significant. The CMDI score was also higher than that of PIBD, but the difference between the two groups was not statistically significant. This shows that the symptoms of growth disorder and intestinal damage in PIBD were aggravated after vagotomy.

[0133] This shows that the occurrence and development of PIBD is closely related to vagal nerve function. The vagus nerve negatively regulates cardiac chronotropic, inotropic and varicotropic functions. When the vagus nerve function is impaired, heart rate and blood pressure will increase. In PIBD rats, it was found that the vagus nerve function tension was reduced and the function was abnormal. After the vagotomy, the SD rats' susceptibility to PIBD increased and the condition worsened, suggesting that vagus nerve dysfunction may promote the occurrence and development of PIBD. Restoring vagus nerve function through bioelectric stimulation may alleviate the symptoms of PIBD. Similar findings have been found in adult IBD patients, namely, reduced heart rate variability and destruction of vagal nerve tension. In CD patients, it was observed that vagal nerve tension was negatively correlated with TNF-α levels, indicating that low vagal nerve tension is associated with a pro-inflammatory state. Many animal experiments and clinical studies have confirmed the therapeutic effect of VNS on IBD. It is speculated that VNS can restore vagal nerve tension and thus alleviate the occurrence and development of PIBD.

[0134] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a rat model of inflammatory bowel disease in children, characterized in that: The specific steps of the construction method are as follows: rats are pre-sensitized by applying 3% w / v oxazolone presensitization solution to their skin, and then repeatedly and intermittently enema with 1% w / v oxazolone enema solution.

2. The construction method according to claim 1, characterized in that: The rats used were 3-week-old female SD rats.

3. The construction method according to claim 1, characterized in that: The dose used for repeated intermittent enema is 10 mg / kg.

4. The construction method according to claim 1, characterized in that: After pre-sensitization of the rats' skin with 3% w / v oxazolone pre-sensitization solution on day 1, the rats were given enema on days 6, 20 and 34, respectively.

5. The construction method according to claim 1, characterized in that: The preparation method of the oxazolone presensitization solution is as follows: acetone and olive oil are mixed in a volume ratio of 4:1 to prepare a solvent, and oxazolone powder is added into the solvent in a ratio of 60 mg:2 mL to dissolve, so as to prepare a 3% w / v oxazolone presensitization solution.

6. The construction method according to claim 1, characterized in that: The preparation method of the oxazolone presensitization solution is as follows: the preparation method of the oxazolone enema solution is as follows: oxazolone powder is added into anhydrous ethanol at a ratio of 20 mg:1 mL to dissolve, and then pure water equal to the anhydrous ethanol is added and mixed to prepare a 1% w / v oxazolone enema solution.

7. Use of the rat model of inflammatory bowel disease in children constructed by the construction method according to any one of claims 1 to 6 in the development of therapeutic drugs for inflammatory bowel disease in children.

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