Psoriasis cardiovascular co-disease mouse model and construction method thereof

Abstract

The invention relates to the technical field of animal model construction, in particular to a psoriasis cardiovascular co-disease mouse model and a construction method thereof. The invention provides a construction method of a psoriasis cardiovascular disease mouse model, which comprises the following steps: feeding a mouse with high fat diet for 5-7 weeks, and injecting a streptozotocin solution for 2-4 days; after the mouse is fed with high-fat diet for 14-16 weeks, imiquimod is smeared on the mouse for 6-8 days, and the psoriasis cardiovascular disease mouse model is obtained. According to the invention, a novel metabolic disorder aggravated psoriasis cardiovascular co-disease mouse model is successfully constructed, and a solid foundation is laid for mechanism exploration of later psoriasis cardiovascular co-disease.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and particularly relates to a psoriasis-cardiovascular comorbidity mouse model and a construction method thereof. Background Art

[0002] Psoriasis is a common chronic inflammatory skin disease jointly mediated by genetics, environment and immunity, characterized by keratinocyte proliferation and dermal inflammatory infiltration. It has a high incidence and is prone to recurrence. A large number of epidemiological studies have found that patients with psoriasis have a higher risk of developing other systemic diseases, such as cardiovascular diseases, metabolic diseases, liver and kidney diseases, autoimmune diseases, mental diseases, etc. Currently, these diseases significantly associated with psoriasis are called psoriasis comorbidities. It is reported that 57.9% of psoriasis patients have at least one psoriasis comorbidity. Comorbidities not only affect the process and severity of psoriasis in patients, but also affect the treatment and prognosis of patients.

[0003] The Chinese Psoriasis Diagnosis and Treatment Guidelines (2023 Edition) classify psoriasis comorbidities into five categories. Cardiovascular and metabolic diseases are the general terms for cardiovascular diseases and metabolic diseases that increase the risk of cardiovascular diseases. They are the most common psoriasis comorbidities, including cardiovascular diseases, diabetes, obesity, hypertension, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease. A number of clinical studies have confirmed the association between psoriasis and cardiovascular diseases. The 2018 American Heart Association guidelines clearly point out that chronic inflammatory diseases such as psoriasis are risk factors for cardiovascular diseases. A prospective study by Gelfand et al. confirmed that psoriasis is an independent risk factor for myocardial infarction, especially for severe psoriasis patients with onset age < 50 years. The relative risk of myocardial infarction in psoriasis patients is negatively correlated with age. Cardiovascular comorbidity is the main cause of heart failure and death in psoriasis patients, but there is currently no effective animal model.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a new mouse model of psoriasis-cardiovascular comorbidity with aggravated metabolic disorders.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a construction method of a psoriasis-cardiovascular comorbidity mouse model, comprising the following steps:

[0008] After feeding the mice with a high-fat diet for 5 - 7 weeks, streptozotocin solution is injected for 2 - 4 days; after feeding the mice with a high-fat diet for 14 - 16 weeks, imiquimod is applied to the mice for 6 - 8 days to obtain a psoriasis-cardiovascular comorbidity mouse model.

[0009] Preferably, the mouse is a male mouse.

[0010] Preferably, the fat calories of the high-fat diet are 50-70 kcal%.

[0011] Preferably, the streptozotocin solution is prepared by mixing streptozotocin and citrate buffer solution.

[0012] Preferably, the initial concentration of the citrate buffer solution is 0.04-0.06 mol / L; the initial pH of the citrate buffer solution is 4.0-5.0.

[0013] Preferably, the mass-volume ratio of the mixture of streptozotocin and citrate buffer solution is 8-12 mg:1 mL.

[0014] Preferably, the injection volume of the streptozotocin solution is 3.2-4.8 mL / kg / d.

[0015] Preferably, the application position of imiquimod is the back of the mouse; the application amount of imiquimod is 60-65 mg / animal / d.

[0016] Preferably, the initial concentration of imiquimod is 4-6%.

[0017] The present invention also provides a psoriasis-cardiovascular comorbidity mouse model constructed by the construction method described above.

[0018] Advantages of the present invention:

[0019] Since psoriasis patients often suffer from complications such as hypertension, diabetes, obesity and metabolic dysfunction, the present invention feeds mice with a high-fat diet, injects streptozotocin solution and applies imiquimod during the feeding period, and successfully constructs a psoriasis-cardiovascular comorbidity mouse model. This model can simulate various clinical characteristics of psoriasis-cardiovascular comorbidity patients. The novel metabolic disorder-aggravated psoriasis-cardiovascular comorbidity mouse model constructed by the present invention provides a solid foundation for subsequent research on the mechanism of psoriasis-cardiovascular comorbidity. Description of the drawings

[0020] Figure 1 It is a schematic diagram of four groups of mouse models, where from top to bottom are the mouse models of Comparative Example 1, the mouse models of Comparative Example 2, the mouse models of Comparative Example 3, and the mouse models of Example 1;

[0021] Figure 2Skin detection results of four groups of mouse models. Among them, A is the dorsal skin map of four groups of mouse models, B is the change of PASI score of four groups of mouse models, C is the HE staining and Ki67 staining map of four groups of mouse models, D is the quantitative result of epidermal thickness of four groups of mouse models, E is the quantitative result of ear thickness of four groups of mouse models, F is the quantitative result of epidermal Ki67 staining of four groups of mouse models, and G is the quantitative result of ear Ki67 staining of four groups of mouse models;

[0022] Figure 3 Schematic diagram of the dewaxing steps of paraffin sections;

[0023] Figure 4 Results of intraperitoneal glucose tolerance test for four groups of mouse models;

[0024] Figure 5 Echocardiogram of four groups of mouse models;

[0025] Figure 6 Statistical results of left ventricular ejection fraction, left ventricular fractional shortening, and E / E' ratio for four groups of mouse models;

[0026] Figure 7 Statistical results of body weight and heart-to-tibia ratio for four groups of mouse models;

[0027] Figure 8 Histological detection results of four groups of mouse models. Among them, A is the representative map of heart gross (Gross), HE, WGA, Masson staining, and transmission electron microscopy (EM); B is the statistical chart of myocardial cell size in HE staining, C is the statistical chart of myocardial cell size in WGA staining, D is the statistical chart of the ratio of perivascular fibrosis area to vascular area, and E is the statistical chart of the percentage of mitochondrial cristae loss;

[0028] Figure 9 Flow chart of the multi-factor ELISA detection steps;

[0029] Figure 10 Differential gene function enrichment analysis of four groups of mouse models. Among them, A is the volcano plot of differential genes in the IMQ group and the IMQ combined with HFD+STZ model group, B is the KEGG function enrichment analysis, and C is the detection results of corresponding inflammatory indicators in plasma multi-factor ELISA; Specific implementation manner

[0030] The present invention provides a method for constructing a psoriasis-cardiovascular comorbidity mouse model, comprising the following steps:

[0031] After feeding the mice with a high-fat diet for 5-7 weeks, inject streptozotocin solution for 2-4 days; after feeding the mice with a high-fat diet for 14-16 weeks, apply imiquimod to the mice for 6-8 days to obtain a psoriasis-cardiovascular comorbidity mouse model;

[0032] Preferably, after feeding the mice with a high-fat diet for 6 weeks, streptozotocin solution is injected for 3 days; after feeding with a high-fat diet for 15 weeks, imiquimod is applied to the mice for 7 days to obtain a mouse model of psoriasis with cardiovascular comorbidity.

[0033] In the present invention, the injection is intraperitoneal injection.

[0034] In the present invention, the mice are male mice.

[0035] In the present invention, the fat calories of the high-fat diet are 50-70 kcal%, preferably 55-65 kcal%, and more preferably 60 kcal%.

[0036] In the present invention, the streptozotocin solution is prepared by mixing streptozotocin and citrate buffer.

[0037] In the present invention, the initial concentration of the citrate buffer is 0.04-0.06 mol / L, preferably 0.05 mol / L; the initial pH of the citrate buffer is 4.0-5.0, preferably 4.5.

[0038] In the present invention, the mass-volume ratio of the mixture of streptozotocin and citrate buffer is 8-12 mg:1 mL, preferably 9-11 mg:1 mL, and more preferably 10 mg:1 mL.

[0039] In the present invention, the injection amount of the streptozotocin solution is 3.2-4.8 mL / kg / d, preferably 3.6-4.4 mL / kg / d, and more preferably 4.0 mL / kg / d.

[0040] In the present invention, the application position of imiquimod is the back of the mice; the application amount of imiquimod is 60-65 mg / animal / d, preferably 62-63 mg / animal / d, and more preferably 62.5 mg / animal / d.

[0041] In the present invention, before applying imiquimod, the back of the mice needs to be depilated.

[0042] In the present invention, the initial concentration of imiquimod is 4-6%, preferably 4.5-5.5%, and more preferably 5%.

[0043] The present invention also provides a mouse model of psoriasis with cardiovascular comorbidity constructed by the above construction method.

[0044] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] After 6-week-old male C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimated to the animal house environment for two weeks, the mice were fed a high-fat diet (HFD, high-fat diet D12492, with a fat calorie of 60 kcal%). After 6 weeks of high-fat diet feeding, each mouse was intraperitoneally injected with streptozotocin (STZ) solution (streptozotocin and 0.05 mol / L citrate buffer were mixed at a mass-to-volume ratio of 10 mg:1 mL, and the pH of the citrate buffer was 4.5) at a dose of 4.0 mL / kg / d for 3 consecutive days to induce pancreatic islet cell damage. After 15 weeks of high-fat diet feeding, the back hair of the mice was depilated, and then 5% imiquimod (IMQ) was applied to the mice for 7 days at a dosage of 62.5 mg / mouse / d. Through echocardiography, it was detected that the mice had diastolic dysfunction of the heart (i.e., a significant increase in E / E'), and the levels of myocardial hypertrophy and fibrosis increased, thus obtaining a psoriasis-cardiovascular comorbidity mouse model.

[0047] Example 2

[0048] After 6-week-old male C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimated to the animal house environment for two weeks, the mice were fed a high-fat diet (HFD, high-fat diet D12492, with a fat calorie of 60 kcal%). After 7 weeks of high-fat diet feeding, each mouse was intraperitoneally injected with streptozotocin (STZ) solution (streptozotocin and 0.04 mol / L citrate buffer were mixed at a mass-to-volume ratio of 12 mg:1 mL, and the pH of the citrate buffer was 4) at a dose of 3.6 mL / kg / d for 2 consecutive days to induce pancreatic islet cell damage. After 14 weeks of high-fat diet feeding, the back hair of the mice was depilated, and then 6% imiquimod (IMQ) was applied to the mice for 6 days at a dosage of 64 mg / mouse / d. Through echocardiography, it was detected that the mice had diastolic dysfunction of the heart (i.e., a significant increase in E / E'), and the levels of myocardial hypertrophy and fibrosis increased, thus obtaining a psoriasis-cardiovascular comorbidity mouse model.

[0049] Example 3

[0050] After 6-week-old male C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimated to the animal house environment for two weeks, the mice were fed a high-fat diet (HFD, high-fat feed D12492, with a fat calorie content of 60 kcal%). After 5 weeks of high-fat diet feeding, each mouse was intraperitoneally injected with streptozotocin (STZ) solution at a dose of 4.4 mL / kg / d (streptozotocin and 0.06 mol / L citrate buffer were mixed at a mass-to-volume ratio of 8 mg:1 mL, and the pH of the citrate buffer was 5) for 4 consecutive days to induce pancreatic islet cell damage. After 16 weeks of high-fat diet feeding, the back hair of the mice was removed, and then 4% imiquimod (IMQ) was applied to the mice for 8 days at a dosage of 62 mg / mouse / d. By echocardiography, the mice had diastolic dysfunction of the heart (i.e., a significant increase in E / E'), and increased levels of myocardial hypertrophy and fibrosis, thus obtaining a mouse model of psoriasis-cardiovascular comorbidity.

[0051] Comparative Example 1

[0052] After 6-week-old male C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimated to the animal house environment for two weeks, the mice were continued to be fed a normal diet (ND). After 6 weeks of normal diet feeding, each mouse was intraperitoneally injected with 0.05 mol / L citrate buffer (pH = 4.5) at a dose of 4.0 mL / kg / d for 3 consecutive days. After 15 weeks of normal diet feeding, the back hair of the mice was removed, and then vaseline was applied to the mice for 7 days at a dosage of 62.5 mg / mouse / d to obtain a mouse model.

[0053] Comparative Example 2

[0054] Referring to Example 1, the difference from Example 1 was that 5% imiquimod was replaced with vaseline, and other steps were the same as in Example 1 to construct a mouse model.

[0055] Comparative Example 3

[0056] After 6-week-old male C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were acclimated to the animal house environment for two weeks, the mice were continued to be fed a normal diet (ND). After 6 weeks of normal diet feeding, each mouse was intraperitoneally injected with 0.05 mol / L citrate buffer (pH = 4.5) at a dose of 4.0 mL / kg / d for 3 consecutive days. After 15 weeks of normal diet feeding, the back hair of the mice was removed, and then 5% imiquimod (IMQ) was applied to the mice for 7 days at a dosage of 62.5 mg / mouse / d to obtain a mouse model.

[0057] Skin Detection in Experimental Example 1

[0058] The PASI score (Psoriasis Area and Severity Index) comprehensively scores psoriasis patients based on the severity of skin lesions (including erythema, infiltration, and scales) and the area of skin lesions. The final score is calculated through a specific formula and is often used to evaluate the severity of plaque psoriasis. It is an internationally recognized scoring standard for the severity of psoriasis skin lesions. After starting the modeling, the back skin of the mice was observed regularly every day, and their skin was photographed and recorded under the same environment and natural light. Two trained experimental personnel, without knowing the animal grouping, evaluated the severity of these three indicators on a daily basis using a 5-point scale (0-4) according to the PASI scoring standard for the psoriasis-like mouse model (shown in Table 1), based on the daily skin thickness, scales, and erythema of the back skin of the mice. The total score is the sum of the three independent scores for skin thickness, scales, and erythema. Finally, the average score of the two experimental personnel was calculated and recorded. The daily score changes were statistically analyzed to reflect the changes in the skin inflammation degree of the psoriasis-like mice (J Immunol. 2009 May 1; 182(9): 5836-45. doi: 10.4049 / jimmunol.0802999).

[0059] Table 1 PASI Scoring Standard for Psoriasis-Like Mouse Model

[0060]

[0061] According to the above method, the back skin thickness, scales, and erythema of the mouse models in the control group 1 (ND CON), control group 2 (HS CON), control group 3 (ND IMQ), and example group 1 (HSIMQ) were evaluated (the schematic diagrams of the four groups of mouse models are as shown in Figure 1 ), and the PASI score (0-12) was calculated to evaluate the severity of the skin lesions of the psoriasis-like mice. The evaluation results are as shown in Figure 2 (A and B).

[0062] The epidermis of the back and ears of the mice was subjected to Ki67 immunostaining, and the skin thickness and Ki67 immunohistochemical ratio of the epidermis of the back and ears of the mice were measured. The results are as shown in Figure 2 (C-G).

[0063] The specific steps of Ki67 immunostaining are as follows:

[0064] 1) Deparaffinization: The paraffin tissue sections were deparaffinized in the order shown in Figure 3 ;

[0065] 2) Antigen retrieval: Place the dewaxed tissue sections on a slide rack and put them into an antigen retrieval box containing 180 mL of sodium citrate antigen retrieval solution. Heat at 100 °C for 20 min for antigen retrieval. After taking out, let it cool naturally to room temperature, and then rinse the sections with PBS to remove the residual antigen retrieval solution;

[0066] 3) Inactivation of endogenous peroxidase: Place the slide rack in a staining jar containing 0.3% hydrogen peroxide (peroxidase substrate), and incubate at room temperature in the dark for 30 min;

[0067] 4) Blocking: Put the inactivated sections into a dark and humid box, use a hydrophobic immunohistochemistry pen to circle the staining area around the heart tissue, and block the tissue with 5% BSA prepared with TBS, incubate at room temperature for 1 h;

[0068] 5) Primary antibody incubation: After blocking, wash off the blocking solution with TBS, and use the primary antibody working solution against the target protein prepared with goat serum (prepared according to the concentration in the corresponding primary antibody instruction manual, generally 1:100) to completely cover the tissue, and incubate at 4 °C overnight;

[0069] 6) Secondary antibody incubation: After the primary antibody incubation is completed, wash off the primary antibody working solution with TBS, add the HRP-labeled secondary antibody from the corresponding source (dilute the secondary antibody at a concentration of 1:200, the secondary antibody diluent is a solution containing 5% BSA, and neither the primary antibody nor the secondary antibody working solution contains sodium azide), and make the secondary antibody working solution completely cover the tissue, incubate at room temperature for 1 h;

[0070] 7) DAB color development: After the secondary antibody incubation is completed, wash off the secondary antibody working solution with TBS. Then drop the DAB color development solution onto the tissue for color development. Immediately put the sections into PBS to terminate the color development after observing that the tissue in the positive control group turns brown under a white background microscope;

[0071] 8) Hematoxylin counterstaining: Drop an appropriate amount of hematoxylin staining solution on the developed sections to make the staining solution evenly cover the tissue and stain for 60 s, and then wash it with tap water;

[0072] 9) Differentiation with hydrochloric acid alcohol: Wash the sections 5 times in 0.3% HCl + 70% alcohol. After observing that the color of the sections becomes lighter, then wash it with tap water;

[0073] 10) Blueing with ammonia water: Wash the sections 5 times in 0.1% ammonia water, and wash it with tap water;

[0074] 11) Clearing and mounting: Dehydrate and clear the sections gradually in the reverse order, and finally mount them with neutral resin or non-toxic and environmentally friendly mounting agent. Figure 3 Reverse order, dehydrate and clear the sections gradually, and finally mount them with neutral resin or non-toxic and environmentally friendly mounting agent.

[0075] From Figure 2It can be seen that the PASI score of the mouse model in Example 1 group was significantly increased compared with that in Comparative Example 3 group, with epidermal thickening and obvious epidermal proliferation. It is suggested that metabolic disorders significantly exacerbate epidermal proliferation and inflammation in psoriatic mice.

[0076] Experimental Example 2

[0077] The mouse models of Comparative Example 1 group (ND CON), Comparative Example 2 group (HS CON), Comparative Example 3 group (ND IMQ) and Example 1 group (HSIMQ) were subjected to intraperitoneal glucose tolerance test, and the results are as Figure 4 shown.

[0078] Intraperitoneal glucose tolerance test (IPGTT): If the glucose tolerance decreases and the body's ability to consume glucose declines, after exogenous glucose is given, the blood glucose rises relatively rapidly and drops slowly, which reflects abnormal glucose tolerance. The specific steps are as follows:

[0079] One day before the formal experiment, change the clean bedding for the mice, remove the feed, and starve the mice for 12 h; intraperitoneally inject 20% glucose solution (ensure sterility) into the mice at an injection dose of 1 g / kg, and monitor the blood glucose at 15 min, 30 min, 60 min, 90 min, and 120 min after injection (cut off 2 mm from the tip of the mouse tail, discard the first drop, and use the second drop to detect the blood glucose).

[0080] From Figure 4 it can be seen that the glucose tolerance of Comparative Example 2 group was abnormal compared with that of Comparative Example 1 group, the glucose tolerance of Example 1 group was abnormal compared with that of Comparative Example 3 group, and there was no obvious difference between Comparative Example 2 group and Example 1 group, and between Comparative Example 1 group and Comparative Example 3 group.

[0081] Experimental Example 3

[0082] Dip a cotton swab in depilatory cream in advance to remove the hair in the precordial area of the mouse models of Comparative Example 1 group (ND CON), Comparative Example 2 group (HS CON), Comparative Example 3 group (ND IMQ) and Example 1 group (HSIMQ). Fix the mouse models in a supine position on the test bench with tape, apply a small amount of ultrasonic coupling agent on the four limbs to ensure good contact with the electrodes of the test bench; after fixing, use isoflurane for continuous gas anesthesia with the assistance of a ventilator, adjust the dose of anesthetic to keep the mouse heart rate stable at 500 - 600 bpm, apply an appropriate amount of ultrasonic coupling agent in the precordial area, and record the echocardiogram of the long axis and short axis ( Figure 5), the echocardiographic parameters of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were statistically analyzed to evaluate the cardiac systolic function of mice. The results are shown in Figure 6 ; the probe position was moved to the apex of the heart, and at the same time, the anesthetic dose was adjusted to control the heart rate of the mice at about 500 bpm. The ratio of the peak early diastolic blood flow velocity of the mitral valve to the peak early diastolic velocity of the mitral annulus (E / E' ratio) was obtained to evaluate the left ventricular diastolic function of the mice. The results are shown in Figure 6 .

[0083] From Figure 5 and Figure 6 , it can be seen that there were no significant changes in the systolic indexes reflecting the cardiac function of the mouse model, such as LVEF and LVFS, among the four groups of mice; there were no significant changes in the diastolic indexes reflecting the cardiac function of the mouse model, such as the E / E' ratio, in Comparative Example 1 and Comparative Example 3, but it increased significantly in Comparative Example 2. The diastolic index of the cardiac function of the mouse model in Example 1 group was significantly higher than that of the mouse model in Comparative Example 3 group. It can be seen that the cardiac function of the mice described in Example 1 was impaired, and the cardiac function impairment was mainly reflected in diastolic dysfunction, and the systolic function was normal. It is suggested that there is diastolic dysfunction in the cardiovascular comorbidity of psoriasis mice, and metabolic disorders exacerbate the cardiovascular comorbidity of psoriasis-like mice.

[0084] Experimental Example 4

[0085] The body weights (BW) of the mouse models described in Comparative Example 1 group (ND CON), Comparative Example 2 group (HS CON), Comparative Example 3 group (ND IMQ) and Example 1 group (HS IMQ) were measured. Then the mouse models were sacrificed, and the hearts and tibias of the mouse models were taken. The heart weights of the mouse models were weighed using a weighing instrument, and the tibia lengths of the mouse models were measured using a ruler. The heart-tibia ratio (HW / TL, heart weight / tibia length, the larger this index, the more it reflects cardiac hypertrophy) was calculated. The statistical results of the body weights and heart-tibia ratios of the mouse models in each group are shown in Figure 7 .

[0086] From Figure 7 , it can be seen that there were no significant changes in the body weights of the four groups of mice. The heart-tibia ratio of the Comparative Example 2 group was significantly higher than that of the Comparative Example 1 group, and the heart-tibia ratio of the Example 1 group was significantly higher than that of the Comparative Example 3 group.

[0087] Experimental Example 5

[0088] After sacrificing the mouse models in the negative control group 1 (ND CON), negative control group 2 (HS CON), negative control group 3 (ND IMQ), and example group 1 (HS IMQ), the heart tissues were collected and paraffin-embedded sections were prepared; then the sections were stained with hematoxylin-eosin (HE staining), wheat germ agglutinin staining (WGA staining), and Masson staining to observe the morphology of cardiomyocytes and fibrosis. The Image software was used to statistically analyze the size of cardiomyocytes and the perivascular fibrosis area, and calculate the ratio of the perivascular fibrosis area to the vessel area; the morphological and size changes of cardiomyocyte mitochondria were detected by transmission electron microscopy; the results are as Figure 8 shown.

[0089] Based on the mitochondrial electron microscopy images, combined with the image processing software (ImageJ) and morphological analysis methods, the area of mitochondrial cristae loss was calculated (the results are as Figure 8 shown), and the specific steps are as follows:

[0090] (1) Pretreatment and scale setting: Convert the electron microscopy images to a compatible format; open the images in ImageJ, click Analyze>Set Scale, and enter the known scale length (the pixel value corresponding to 1 μm marked on the electron microscopy images);

[0091] (2) Mitochondrial region segmentation: 1) Select the mitochondrial boundary: Manually circle the contour of a single mitochondrion using the Freehand Selection tool, or automatically identify the mitochondrial region through threshold segmentation (Image>Adjust>Threshold); for low-contrast images, background subtraction (Process>Subtract Background) or contrast enhancement (Enhance Contrast) can be performed first; 2) Generate ROI (region of interest): Save the selected mitochondrial region as ROI (Edit>Selection>Add to Manager) for subsequent repeated analysis;

[0092] (3) Identification and area calculation of mitochondrial cristae: 1) Distinction between cristae and matrix: Mitochondrial cristae appear as dark membranous structures in electron microscopy, and the matrix is brighter; by adjusting the threshold (Image>Adjust>Threshold), separate the cristae region (dark color) from the matrix; use the local threshold algorithm (Mean / Weighted Mean) to avoid mis-segmentation caused by global thresholds; 2) Morphological post-processing: Perform Despeckle (denoising) and Fill Holes on the binary image to ensure the continuity of the cristae structure; 3) Area calculation: Use Analyze>Measure to directly obtain the pixel area of the cristae region and convert it to the actual area (based on the scale);

[0093] (4) Data statistics and verification: 1) Calculate the proportion of cristae loss: Proportion of cristae area = Area of cristae region / Total area of mitochondria × 100%; 2) Multi-region sampling: Randomly select at least 50 mitochondria for measurement to avoid sampling bias.

[0094] Through HE staining ( Figure 8 A and Figure 8 B), it can be seen that there is no obvious hypertrophy in the cardiomyocytes of the mouse models in Comparative Example 1 group and Comparative Example 3 group, the cardiomyocytes of the mouse model in Comparative Example 2 group are more hypertrophic, and the cardiomyocytes of the mouse model in Example 1 group are significantly hypertrophied compared with those in Comparative Example 2 group. Through WGA staining ( Figure 8 A and Figure 8 C), it can be seen that the size of cardiomyocytes in the mouse model of Example 1 group is significantly increased compared with that in Comparative Example 1 group and Comparative Example 3 group. The results of Masson staining ( Figure 8 A and Figure 8 D) indicate that the degree of perivascular fibrosis in the heart of the mouse model in Example 1 group is significantly higher than that in the mouse model in Comparative Example 3 group. Electron microscopy ( Figure 8 A and Figure 8 E) shows that the mitochondria of cardiomyocytes in the mouse models of Comparative Example 1 group and Comparative Example 3 group are complete and dense, and there are no vacuoles inside the mitochondria; the mitochondria of cardiomyocytes in the mouse model of Comparative Example 2 group are fragmented and swollen, the vacuolization inside the mitochondria is obvious, the percentage of mitochondria with cristae loss is increased, the mitochondria of cardiomyocytes in the mouse model of Example 1 group are significantly fragmented and swollen, the vacuolization inside the mitochondria is aggravated, and the percentage of mitochondria with cristae loss is significantly increased, suggesting that the cardiac mitochondria morphology of psoriasis-cardiovascular comorbidity mice changes, mitochondrial function is damaged, and metabolic disorders exacerbate the cardiac function damage of psoriasis-like mice.

[0095] Experimental Example 6

[0096] Transcriptome sequencing and its data functional enrichment analysis: The original downloaded sequences obtained by sequencing on the second-generation high-throughput sequencing platform are processed to obtain high-quality sequences through processes such as removing low-quality sequences and removing adapter contamination, and all subsequent analyses are based on clean reads. The analysis process of transcriptome sequencing information is mainly divided into three parts: sequencing data quality control, data alignment analysis, and deep transcriptome analysis. Among them, sequencing data quality control includes filtering the obtained sequences, evaluating the quality of sequencing data, and calculating sequence length distribution, etc. Data alignment analysis is mainly for the sequences aligned to the genome, and classification and feature analysis are carried out in turn according to different genome annotation information, and the corresponding expression levels are calculated; then differential expression analysis is performed. In conventional transcriptome analysis, there are also alternative splicing analysis, prediction of new transcripts, and variant analysis. Deep analysis and other personalized analyses. Transcriptome sequencing and its data functional enrichment analysis were performed on the heart tissues of Comparative Example 1-3 groups and Example 1 group through the second-generation high-throughput sequencing platform. The data results of Example 1 group and Comparative Example 3 group are asFigure 10 as shown

[0097] Multifactor ELISA assay: The multi-index flow cytometry combined analysis technology was used to quantitatively analyze the concentrations of multiple targets in plasma. Antibody molecules of different substances to be detected were covalently crosslinked to specific coded microspheres, and each coded microsphere corresponded to a corresponding detection item. Different fluorescently coded microspheres of substances to be detected were mixed through a liquid-phase reaction, and then the substances to be detected were added. The generated complex reacted with the labeled fluorescein. Driven by the flowing sheath fluid, the microspheres passed through red and green lasers in a single row. The red laser was used to determine the fluorescence code of the microspheres, and the green laser was used to determine the fluorescence intensity of the reporter molecules on the microspheres, so as to achieve the purpose of rapid and accurate quantitative detection. The specific detection process is as Figure 9 shown

[0098] The multi-index flow cytometry combined analysis technology was used to detect the concentrations of IL17A, IL1β, IL1α, TNFα, IL6, IL18, and CXCL in the plasma of the mouse models in Example 1 group and Comparative Example 3 group. The results are as Figure 10 shown

[0099] Based on transcriptome sequencing and its data functional enrichment analysis ( Figure 10 A and Figure 10 B), it was found that the complement and coagulation pathway signaling pathways in the heart tissue of the Example 1 group were significantly regulated compared with the Comparative Example 3 group; the multifactor ELISA assay ( Figure 10 C) showed that IL1β in the plasma of the psoriasis-cardiovascular comorbidity mouse model was significantly increased.

[0100] As can be seen from the above examples, the present invention provides a psoriasis-cardiovascular comorbidity mouse model and a method for constructing the same. The present invention successfully constructed a new mouse model of metabolic disorder exacerbating psoriasis-cardiovascular comorbidity, laying a solid foundation for the later exploration of the mechanism of psoriasis-cardiovascular comorbidity.

[0101] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of psoriasis-cardiovascular comorbidity, characterized in that, It includes the following steps: After feeding mice with a high-fat diet for 5-7 weeks, streptozotocin solution is injected for 2-4 days; after feeding with a high-fat diet for 14-16 weeks, imiquimod is applied to the mice for 6-8 days to obtain a mouse model of psoriasis with cardiovascular comorbidity.

2. The construction method according to claim 1, characterized in that The mice are male mice.

3. The construction method according to claim 2, characterized in that, The fat calories of the high-fat diet are 50-70 kcal%.

4. The construction method according to claim 3, characterized in that The streptozotocin solution is prepared by mixing streptozotocin and citrate buffer.

5. The construction method according to claim 4, wherein The initial concentration of the citrate buffer is 0.04-0.06 mol / L; the initial pH of the citrate buffer is 4.0-5.

0.

6. The construction method according to claim 5, characterized in that The mass-volume ratio of the streptozotocin and the citrate buffer in the mixture is 8-12 mg:1 mL.

7. The construction method according to claim 6, wherein The injection volume of the streptozotocin solution is 3.2-4.8 mL / kg / d.

8. The construction method according to claim 7, characterized in that The application position of imiquimod is the back of the mice; the application amount of imiquimod is 60-65 mg / rat / d.

9. The construction method according to claim 8, characterized in that, The initial concentration of imiquimod is 4-6%.

10. A mouse model of psoriasis with cardiovascular comorbidity is constructed by the construction method described in any one of claims 1-9.

Images