A method for constructing an animal model of diabetes combined with renal lipid injury

Abstract

The application belongs to the technical field of medical evaluation and detection, and particularly relates to a method for constructing an animal model of diabetes combined with kidney lipid injury. The method comprises the following steps: taking experimental animals, feeding the animals with high-fat feed for 3-5 weeks, feeding the animals with ordinary feed for 4-6 days in succession, injecting streptozotocin into the abdominal cavity of the animals, the amount of streptozotocin injected into each animal per day being 35-45 mg / Kg, and then feeding the animals with high-fat feed for 11-13 weeks, so that a disease animal of diabetes combined with kidney lipid injury is obtained. The disease animal model of diabetes combined with kidney lipid injury has high repeatability, prominent kidney lipid injury, and simple operation, and has important value and significance in basic research.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medical evaluation and detection, and particularly relates to a method for constructing an animal model of diabetes combined with kidney lipid damage. BACKGROUND

[0002] Diabetic kidney disease (DKD) is a chronic kidney disease (CKD) caused by diabetes (DM), and its pathogenesis is complex. The clinical features are persistent increase in albuminuria excretion and / or progressive decline in glomerular filtration rate (GFR), and eventually development into end-stage renal disease (ESRD).

[0003] Hyperglycemia is considered to be the main potential pathogenic factor for the progression of DKD. However, a number of large-scale studies have shown that intensive glycemic control has little effect on DKD renal endpoints, and increasing the use of hypoglycemic drugs and better glycemic control do not reduce the prevalence of DKD, but on the contrary, can lead to the worsening of DKD disease progression. Therefore, the research on pathogenesis and therapeutic drugs other than hyperglycemia is of both important practical significance and urgent clinical demand for the prevention and treatment of DKD.

[0004] Chinese patent application CN114766426A discloses a method for preparing a diabetic animal model and its application. The method comprises S1. weighing, measuring and recording the fasting blood glucose and fasting insulin value of the selected object; S2. calculating the required amount of streptozotocin according to the fasting blood glucose and body weight of the animal; S3. injecting the amount of streptozotocin obtained in step S2 into the pancreas of the animal under the guidance of endoscopic ultrasound; and S4. re-measuring the fasting blood glucose and fasting insulin value of the selected object until the diabetic index is met to obtain a diabetic animal model. The method provided by the application has the advantages of simple operation steps, easy operation, low preparation cost, strong repeatability, fast model formation, and greatly shortened preparation time of the diabetic animal model.

[0005] Disorder of lipid metabolism is a major pathological feature of diabetes, and excessive lipid metabolism disorder can also cause dysfunction of specific target organs, such as kidney. Recent studies have found that kidney lipid accumulation is a key trigger factor for the onset of DKD. Excessive lipid deposition in the kidney can damage glomeruli, tubular cells and podocytes, leading to glomerular sclerosis, reduced glomerular filtration rate, thickening of the glomerular basement membrane and loss of podocytes, ultimately leading to kidney dysfunction and inducing an increase in urinary protein. Therefore, deciphering the molecular mechanism of excessive storage of kidney lipids and reducing kidney lipid toxicity is a new strategy for the prevention and treatment of DKD. However, there is currently a lack of a unified and specific animal model of diabetic kidney lipid damage disease. SUMMARY

[0006] In order to solve the problem of lack of a unified and specific animal model of diabetic kidney lipid damage disease in the prior art, the present application provides a method for constructing an animal model of diabetic kidney lipid damage. The preparation method of the animal model of diabetic kidney lipid damage of the present application is simple in operation steps, easy to operate, low in preparation cost and high in repeatability, and improves the uniformity of the preparation of the animal model of diabetic kidney lipid damage.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0008] A method for constructing an animal model of diabetic kidney lipid damage, comprising the following steps: taking experimental animals, feeding the animals with high-fat feed for 4 weeks, then feeding the animals with ordinary feed for 4-6 consecutive days, and injecting streptozotocin into the abdominal cavity of each animal, with the amount of streptozotocin injected into each animal being 35-45 mg / Kg per day, and then feeding the animals with high-fat feed for 11-13 weeks, to obtain a disease animal model of diabetic kidney lipid damage.

[0009] Preferably, the experimental animals are TFF3 - / - animals, including mice, rats, beagles, rhesus monkeys, pigs and cows.

[0010] Preferably, the TFF3 - / - animals are animals with spontaneous intestinal damage combined with lipid metabolism disorder.

[0011] Further preferably, the TFF3 - / - animals are TFF3 - / - mice.

[0012] Still further preferably, the method comprises the following steps: taking TFF3 - / - mice, feeding the mice with high-fat feed for 4 weeks, then feeding the mice with ordinary feed for 5 consecutive days, injecting streptozotocin into the abdominal cavity of each mouse, with the amount of streptozotocin injected into each mouse being 40 mg / Kg per day, and then continuing to feed the mice with high-fat feed for 12 weeks, to obtain a disease mouse model of diabetic kidney lipid damage.

[0013] Preferably, the high-fat diet is fed in an ad libitum manner.

[0014] Preferably, during the high-fat diet feeding, TFF3 - / - The mice are allowed to drink water ad libitum.

[0015] Preferably, the high-fat diet is a high-fat diet with a fat energy ratio ≥ 60%.

[0016] Further preferably, the brand of the high-fat diet is Dyets, with a product number of HF60.

[0017] The present application also relates to the use of the animal model of diabetes combined with renal lipid injury constructed by the above method in screening drugs for treating diabetic kidney disease.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] (1) The disease animal model in the present application is a TFF3 - / - mouse induced by streptozotocin combined with a high-fat diet, which has characteristic renal lipid deposition and more significant renal lipid injury induced by drugs and diet, and can provide a relatively rapid disease animal model for researchers studying renal lipid toxicity. - / -

[0020] (2) The model constructed by the present application is rapid in modeling, prominent in renal lipid injury, good in repeatability, and simple and easy to operate. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a TFF3 immunohistochemical result graph of a WT mouse kidney, A is a TFF3 immunohistochemical result, and B is a TFF3 positive expression statistical result, ** indicates P<0.01 compared with glomerular TFF3 expression;

[0022] Figure 2 is a TFF3 immunofluorescence result graph of a WT mouse kidney, A is a TFF3 immunofluorescence result, and B is a TFF3 fluorescence intensity statistical result, ** indicates P<0.01 compared with glomerular TFF3 expression;

[0023] Figure 3 is a lipid metabolism gene protein expression result graph of a DKD mouse kidney, A is a lipid metabolism protein WB result, B is a SCAP / β-actin statistical result, C is a SREBP / β-actin statistical result, D is a TFF3 / β-actin statistical result, * indicates P<0.05 compared with WT-CT, and ** indicates P<0.01 compared with WT-CT; Note: WT-CT (wild type control), WT-DM (DKD model group).​

[0024] Figure 4 Figure 1 is a graph of the results of gene identification of TFF3 knockout mice, A is the gel electrophoresis results of mouse tail genomic PCR products of the mice bred, B is the WB results of TFF3 protein, C is the statistical results of TFF3 protein expression, ** indicates P<0.01 compared with WT; Note: WT (wild type control group), WT-TFF3 - / - (TFF3 knockout group)

[0025] Figure 5 Figure 2 is a graph of the results of the effect of TFF3 knockout on mouse glucose metabolism, A is the results of the change of body weight of mice in each group over time, B is the results of glucose tolerance test, C is the statistical results of the area under the curve of glucose tolerance, D is the results of fasting blood glucose over time, E is the results of insulin tolerance test, F is the statistical results of the area under the curve of insulin tolerance, ** indicates P<0.01 compared with WT. Note: WT (wild type control group), WT-TFF3 - / - (TFF3 knockout group), DKD-WT (wild type DKD group), DKD-TFF3 - / - (TFF3 knockout DKD group);

[0026] Figure 6 Figure 3 is a graph of the results of the effect of TFF3 knockout on mouse renal function, A is the results of 24-hour proteinuria test, B is the results of urea nitrogen test, C is the results of serum creatinine test, * indicates P<0.05 compared with WT group, ** indicates P<0.01 compared with WT group, # indicates P<0.05 compared with DKD-WT group, ## indicates P<0.01 compared with DKD-WT group; Note: WT (wild type control group), WT-TFF3 - / - (TFF3 knockout group), DKD-WT (wild type DKD group), DKD-TFF3 - / - (TFF3 knockout DKD group)

[0027] Figure 7 Figure 4 is a graph of the results of the effect of TFF3 knockout on mouse kidney pathology, A is the results of kidney pathological special staining test (HE, MASSON and PAS staining (200x, scale = 50pm)), B is the statistical results of Masson fibrosis area, C is the statistical results of mesangial matrix index, ** indicates P<0.01 compared with WT group, ## indicates P<0.01 compared with DKD-WT group; Note: WT (wild type control group), WT-TFF3 - / - (TFF3 knockout group), DKD-WT (wild type DKD group), DKD-TFF3 - / - (TFF3 knockout DKD group);

[0028] Figure 8Figure is the result of TFF3 knockout on mouse kidney lipid deposition, A is the result of kidney oil red O detection, B is the oil red O positive statistical result, ** indicates P<0.01 compared with the WT group, ## indicates P<0.01 compared with the DKD-WT group; Note: WT (wild type control group), WT-TFF3 - / - (TFF3 knockout group), DKD-WT (wild type DKD group), DKD-TFF3 - / - (TFF3 knockout DKD group).

[0029] Figure 9 Figure is the result of TFF3 knockout on mouse kidney lipid metabolism pathway, A is the result of lipid metabolism protein WB, B is the SCAP / β-actin statistical result, C is the SREBP / β-actin statistical result, D is the TFF3 / β-actin statistical result, * indicates P<0.05 compared with the WT group, ** indicates P<0.01 compared with the WT group, # indicates P<0.05 compared with the DKD-WT group, ## indicates P<0.01 compared with the DKD-WT group; Note: WT (wild type control group), WT-TFF3 - / - (TFF3 knockout group), DKD-WT (wild type DKD group), DKD-TFF3 - / - (TFF3 knockout DKD group). DETAILED DESCRIPTION

[0030] The application will be described in detail below in combination with the drawings and examples.

[0031] The application provides a method for constructing an animal model of diabetes combined with kidney lipid injury. The animals in the application include, but are not limited to, mice, rats, beagle dogs, rhesus monkeys, pigs, cows and other animals closely related to scientific experiments and human life (hereinafter taken as an example of mice).

[0032] Example 1

[0033] I. Animal preparation

[0034] SPF level 4-week-old TFF3 + / - The mice are purchased from Jiangsu Jicui Yaoke Biotechnology Co., Ltd., and the animal quality certificate number is NO.320727211100104814, and the experimental unit use license number is SYXK(Yue)2017-0125. They are raised in a SPF level environment (relative humidity 40-70%, temperature 20-26℃). They are free, drink water, and the life rhythm follows the day-night rhythm, with light-dark alternating time of 12 / 12 hours; noise <60dB. The mice are bred to homozygotes for the next experiment.

[0035] II. TFF3 - / - Genotype identification and verification of mice

[0036] TFF3 - / - Mice were raised in a SPF level environment (temperature maintained at 22-28℃, humidity maintained between 40%-60%). After the mice reached sexual maturity (8 weeks for male mice, 6 weeks for female mice), male and female mice were caged together (one male and two females) until the mice gave birth. After the newborn mice began to wean, they were identified by gender and numbered using ear tags. Then, 2-3 mm of tail tissue or nails were cut for genetic identification, following the specific steps:

[0037] (1) Cut 2 cm of mouse tail or nails into a 1.5 ml centrifuge tube;

[0038] (2) Add 110 ul of lysis solution (DirectPCR-Tail: Proteinase K = 200:1) to each centrifuge tube;

[0039] (3) Digestion: Place the centrifuge tube in a 55℃ water bath overnight for 16 hours (place the centrifuge tube on a foam floating plate to reduce contact with water and reduce contamination);

[0040] (4) Use a pipette to blow the liquid in the centrifuge tube several times to ensure that the digested tissue is fully mixed;

[0041] (5) Immediately place the metal bath at 99℃ for 10 minutes to inactivate the enzyme, then cool on ice for 10 minutes;

[0042] (6) Centrifuge at 4℃, 12000 rpm for 10 minutes, aspirate 4 ul of supernatant, and the remaining sample can be stored at -20℃;

[0043] (7) Configure the designed related system (25 ul as a system), see Table 3;

[0044] (8) Prepare agarose gel: 1.2 g of agarose is dissolved in 80 ml of 1×TAE solution, the volume can be changed according to the gel tray, wrap the conical flask with a tin foil cover and place it in a microwave oven at high heat for 3 minutes, then gently shake it with a heat-resistant glove and take out the sample. When cooled to 60℃ (about 30s), add 1×DNA Gelred (1:1000) 3 ul, rotate and shake well without generating bubbles, pour into the tray and cool and solidify;

[0045] (9) Sample loading, in 1×TAE solution, constant voltage 90v electrophoresis for about 1.5 hours;

[0046] (10) The gel scanning imaging technique is used for developing process, and the genotype is identified and analyzed according to the data obtained. The sequence of the PCR primer is shown in Table 1, the detailed information of the digestion system is shown in Table 2, and the detailed information of the amplification system is shown in Table 3. The obtained homozygotes and wild type mice are marked and bred for many times until each mouse has 12 mice for subsequent experiments.

[0047] Table 1 Primer sequence

[0048]

[0049] Table 2 Digestion system

[0050]

[0051] Table 3 Amplification system

[0052]

[0053] II. Animal grouping and model construction

[0054] The bred male mice are used for experiments, and are grouped into wild type control group (WT), TFF3 knockout control group (WT-TFF3 - / - ), wild type diabetic nephropathy group (DKD-WT) and TFF3 knockout diabetic nephropathy group (DKD-TFF3 - / - ), with 6 mice in each group.

[0055] The WT group and WT-TFF3 - / - group mice are fed with ordinary feed (Beijing Kaoshe Lihui Feed Co., Ltd., maintenance mouse feed, production batch number: 23103231), and the DKD-WT group and DKD-TFF3 - / - group mice are fed with high-fat feed (brand Dyets, item number HF60). After 4 weeks of feeding, the DKD-WT group and DKD-TFF3 - / - group mice are continuously injected with 40 mg / kg / d STZ for five days to establish a diabetic experimental model. The WT group and WT-TFF3 - / - group mice are given the same amount of intraperitoneal injection of 0.9% normal saline. The DKD-WT group and DKD-TFF3 - / - group mice are continuously fed with ordinary feed during the injection of STZ, and then fed with high-fat feed. The WT group and WT-TFF3 - / -The mice were continuously fed with normal feed. The litter was replaced every week, and the blood glucose fluctuation was recorded. If the blood glucose level reached or exceeded 11.1 mmol / L within 6 hours in the fasting state, it would be included in the study, otherwise it would be excluded. After 12 weeks of continuous feeding, the urine of the mice was collected, the 24-hour urine protein was measured, and statistical analysis was performed. When there was a significant difference between the two groups, the sample was taken.

[0056] III. Determination of physiological and biochemical indexes

[0057] After 12 weeks of feeding, the mice in each group were fasted for 12 hours, and blood was taken from the tail vein to measure fasting blood glucose, creatinine, urea nitrogen, and lipid metabolism pathway protein expression. The detection was carried out according to the commercial kit. The determination method is as follows.

[0058] 1. Dewaxing of paraffin sections:

[0059] The cut paraffin sections were placed in a 55°C oven for 30 minutes, and then sequentially treated with xylene for 10 minutes twice, anhydrous ethanol for 5 minutes twice, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and pure water for 5 minutes twice.

[0060] 2. Immunohistochemical staining:

[0061] (1) 3% hydrogen peroxide was added and incubated at room temperature for 10 minutes, and then PBS was washed for 3 times, each for 5 minutes;

[0062] (2) Antigen repair step: first, 650 mL of antigen repair solution was added to a 1L beaker, then the sections were placed in it, and then the microwave oven was used at medium-high heat for 13 minutes, and then it was naturally cooled at room temperature for 2.5 hours;

[0063] (3) Blocking: draw a circle around the tissue with a histological pen, and incubate at room temperature for 1 hour with 5% BSA;

[0064] (4) After removing the liquid on the surface of the tissue (avoiding washing, directly shaking dry), the four sides of the glass slide were carefully wiped with a paper towel;

[0065] (5) Diluted TFF3 primary antibody (1:200 ratio, 80ul / tissue) was added dropwise, and then incubated overnight at 4°C;

[0066] (6) Recover the primary antibody, and wash with PBS for 3 times, each for 5 minutes;

[0067] (7) Add secondary antibody, 80ul / tissue, and incubate at room temperature for 1 hour;

[0068] (8) Shake off the liquid on the tissue, and wash with PBS for 3 times, each for 5 minutes;

[0069] (9) Add SABC dropwise, incubate at room temperature for 1 hour, then spin dry, and immerse in PBS for 3 times, 5 minutes each time;

[0070] (10) DAB color development: add DAB solution dropwise for 6 minutes, and rinse with tap water for 1 minute;

[0071] (11) Hematoxylin staining solution for 1 minute, and rinse with tap water for 2 minutes;

[0072] (12) Rehydrate;

[0073] (13) Mounting.

[0074] 3. WB method for detecting protein expression of TFF3 in colon and kidney tissues

[0075] Take kidney and colon tissues, add appropriate amount of lysis solution and sterilized magnetic beads, homogenize for 10 minutes using a homogenizer, and centrifuge to obtain the supernatant. Then use the BCA method to determine the protein concentration of the tissue, add loading buffer (tissue solution to loading buffer ratio of 4:1), and heat denature in a 100°C metal bath instrument. Prepare 15% SDS-PAGE gel, load (80 μg per well), and electrophorese at 70V constant voltage for 1 hour, then change to 100V voltage, and end the electrophoresis at 1 cm above the green line; methanol activates PVDF membrane for 5 min, and electrically transfers at 220mA constant current for 0.5 hours, 0.5% defatted milk is placed on a 45rpm / s shaker for blocking for 1.5h, TBST is washed for 5 min (washed 3 times), primary antibody (dilution ratio of 1:1000) is incubated at 4°C overnight, the primary antibody is recovered, TBST is washed for 5 min (washed 3 times), secondary antibody R (dilution ratio of 1:3000) is incubated at room temperature for 1 hour, TBST is washed for 5 min (washed 3 times), and Bole developer is used for image exposure collection in a dark environment.

[0076] 4. Immunofluorescence method for detecting protein expression of TFF3 in kidney tissues

[0077] (1) Antigen repair step: first, add 650 mL of antigen repair solution to a 1L beaker, then put the slices into it, and then heat it in a microwave oven at medium-high heat for 13 minutes, and then let it cool naturally at room temperature for 2.5 hours;

[0078] (2) Blocking: place the slides vertically, then place one end on the blotting paper to absorb the liquid, and then use the blotting paper to wipe the tissue around to ensure that there is liquid in the tissue area. Use a histological pen to draw a circle around the tissue, and immediately add blocking solution (to prevent drying) after drawing the circle, 50ul / circle is dropped on each tissue, and it is placed at room temperature for 1 hour;

[0079] (3) Gently remove the blocking solution from the surface of the tissue (avoid washing), then carefully wipe the periphery of the slide with a paper towel; prepare the fluorescent primary antibody (1 :200) in advance, then incubate at 80ul / turn and 4°C overnight;

[0080] (4) Remove the primary antibody, add TBS for three washes, each for 5 minutes;

[0081] (5) Add the fluorescent secondary antibody (1 : 1000) dropwise, incubate at room temperature for 1 hour in the dark;

[0082] (6) Remove the secondary antibody, add TBS for three washes, each for 5 minutes;

[0083] (7) Gently shake off the liquid on the tissue (avoid washing), then wipe the periphery of the slide with a paper towel (be careful not to touch the tissue);

[0084] (8) Use DAPI anti-fluorescence quenching mounting medium for staining and mounting: remove the slice under light protection and gently wipe the bottom with an absorbent paper, then add DAPI mounting medium dropwise on the tissue (6ul per tissue), and finally cover with a cover glass;

[0085] (9) Use an Olympus microscope for photographic observation.

[0086] 5. HE staining method

[0087] (1) Dewax the paraffin section;

[0088] (2) Place the section on a slide holder, and immerse the entire section in hematoxylin staining solution for 1 minute;

[0089] (3) Take out the section together with the holder, rinse the back of the slide with running water, and if the staining is too dark, differentiate with hydrochloric acid ethanol for 2 minutes and then rinse with running water;

[0090] (4) Place the section in eosin staining solution for 3 minutes;

[0091] (5) Dehydrate and transparentize the section (xylene 2 times, each for 10 minutes; anhydrous ethanol 2 times, each for 5 minutes; 95% ethanol once, each for 5 minutes; 80% ethanol once, each for 5 minutes; pure water 2 times, each for 5 minutes);

[0092] (6) Mount with neutral resin, and observe under a microscope after the section is dried.

[0093] 6. MASSON staining method

[0094] (1) Dewax;

[0095] (2) MASSON A solution (50uL per tissue) for paraffin section, try to keep the same position during the whole process, do not move, and keep it at room temperature overnight;

[0096] (3) First, put the whole dark box in the oven at 65℃ for 30 minutes of incubation. Try to keep the movement small and smooth during the movement. Then, rinse with tap water for 30 seconds until the yellow part of the tissue fades. At the same time, put solution D and F (in the kit) in the oven at 65℃ for preheating;

[0097] (4) Mix solution B and C together in the same volume, then drop the mixed solution on the section, and rinse it with running water for 1 minute;

[0098] (5) Differentiate the section in 1% hydrochloric acid alcohol for 1 minute, the specific time depends on the differentiation effect (when the color of the cell nucleus is gray-black, and the background color is basically light gray or colorless);

[0099] (6) After slightly rinsing with tap water, remove the excess water from the section, then drop solution D in the kit on the section for 6 minutes (the tissue is bright red), if the red color is too light, you can increase the staining time in time;

[0100] (7) First, slightly filter out the excess water in the section (do not dry the section), then use solution E to drop for 1 minute (differentiation process), when the collagen fibers are differentiated to light red, stop;

[0101] (8) Remove the E solution from the section, do not rinse with water, directly use F solution for 20 seconds of staining;

[0102] (9) Gently shake off the F solution on the section, then differentiate it by continuously washing it in three cylinders of 1% glacial acetic acid, each cylinder needs 8 seconds;

[0103] (10) Put the section into three cylinders of anhydrous ethanol in succession, then dehydrate it respectively, each cylinder for 5s, 10s, and 30s;

[0104] (11) Two cylinders of n-butanol need to be dehydrated for 30 seconds and 2 minutes respectively;

[0105] (12) Two cylinders of turpentine, each for 5 minutes;

[0106] (13) Neutral resin mounting.

[0107] 7. PAS staining method

[0108] PAS staining is one of the routine staining methods in pathology, which is often used to show glycogen and other polysaccharides, the specific operation steps are as follows:

[0109] (1) Paraffin section dewaxing;

[0110] (2) Tap water rinse the back of the slide for 2.5 min, then immerse in distilled water for 2 times, 5 min each time;

[0111] (3) Drop A solution, stand at room temperature for 8 min;

[0112] (4) First rinse with tap water, then immerse in distilled water for 2 times, 5 min each time;

[0113] (5) Drop B solution at room temperature in the dark for 15 min;

[0114] (6) Rinse with tap water for 10 min;

[0115] (7) Drop C solution for 1 min, and differentiate with D solution for 4 s;

[0116] (8) Rinse with tap water for 12 min to return to blue;

[0117] (9) Gradually dehydrate, and seal with neutral balsam.

[0118] 8. Preparation of frozen section

[0119] Fresh kidney tissue was fixed in 4% paraformaldehyde solution, removed after 24 hours, and then dehydrated in 20% and 30% sucrose solution in turn, and then embedded with OCT (liquid nitrogen quick freezing). The embedded tissue was stored in a refrigerator at -80°C for later use. Embedding glue was applied to the surface of the sample holder of the freezing microtome, and the kidney was quickly placed on the surface of the sample holder and flattened with a weight to ensure that the samples were tightly bonded together. The sample was subjected to 10 μm thick trimming operation using trimming technology until it showed the maximum planar effect. The sample was adjusted to the section mode, cut to 5 μm section, and then pasted onto a glass slide, and then the section was stored in a refrigerator at -80°C for later use.

[0120] 9. Oil red O staining

[0121] (1) The cut frozen section was warmed in a normal temperature environment for 25 min;

[0122] (2) The OCT on the section was washed off with PBS 6 times;

[0123] (3) Fixed in 4% paraformaldehyde solution for 15 min;

[0124] (4) Washed with PBS 2 times, 5 min each time;

[0125] (5) Placed in 60% isopropanol solution for 10 min;

[0126] (6) Oil red O staining solution (0.5 g powder dissolved in 100 ml isopropyl alcohol, heated in a 60°C water bath, dissolved and stored at 4°C in the dark, the staining solution was prepared and used immediately) was dropped for 15 minutes;

[0127] (7) 60% isopropyl alcohol decolorization differentiation to interstitial clear, rinse 6 times;

[0128] (8) Hematoxylin staining for 1 minute;

[0129] (9) Pure water rinse 4 times;

[0130] (10) Tap water rinse for 8 minutes (observe the color during the period);

[0131] (11) Hydrochloric acid ethanol differentiation for 4s;

[0132] (12) Tap water rinse for 8 minutes;

[0133] (13) Use water-based mounting medium to mount.

[0134] 10. Glucose tolerance test

[0135] One week before the end of the experiment, oral glucose tolerance test was performed. Before the experiment, all mice were changed to fresh cages (to prevent food residue), fasted for 6 hours, and then intragastrically administered with a glucose solution (concentration 2 g / kg). The Roche blood glucose meter was used to observe and record the blood glucose fluctuations at different time periods (0 min, 15 min, 30 min, 60 min and 120 min).

[0136] 11. Insulin tolerance test

[0137] All mice were changed to fresh cages (to prevent food residue), fasted for 6 hours, and then intraperitoneally injected with insulin (concentration 1 U / kg, diluted with deionized water) at the fastest speed. The Roche blood glucose meter was used to observe and record the blood glucose fluctuations at different time periods (0 min, 15 min, 30 min, 45 min and 60 min).

[0138] 12. Statistical analysis of data

[0139] The data were analyzed using GraphPad Prism 8 software, and the measurement data of each group were represented by mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparison of the means of multiple sample groups. If the variance is equal, the LSD-T test method is used; if the variance is not equal, Dunnett’s T3 multiple comparisons test is used for analysis. P<0.05 and P<0.01 indicate that the difference between the two groups is statistically significant.

[0140] IV. Model Validation

[0141] The widely accepted standard for successful modeling of diabetic nephropathy in mice is the C57BL / 6 mouse model induced by STZ combined with a high-fat diet. However, the activity of these mice varies significantly across species, making it impossible to establish a definitive standard for assessing kidney damage. In mice, for example, positive proteinuria, positive Oil Red O staining of the kidneys, and elevated SREBP expression levels are typically used as criteria for assessing lipid-related kidney damage in diabetic nephropathy.

[0142] V. Measurement Results and Analysis

[0143] Results analysis:

[0144] (1) Expression and distribution of TFF3 in the kidneys of WT mice: TFF3 expression was significantly higher in the renal tubules than in the glomeruli.

[0145] like Figure 1 As shown, immunohistochemistry is a technique that accurately identifies intracellular antigens through a series of specific chemical reactions. In the kidneys of normal C57 mice, TFF3 is expressed in both the glomeruli and tubules. However, compared to the tubules, the expression level of TFF3 in the glomeruli is significantly reduced (P < 0.01).

[0146] like Figure 2 As shown, immunofluorescence technology mainly determines the specific location of antigenic substances in tissues or cells through the chemical reaction between antigens and antibodies. This technology has extremely high specificity, sensitivity, and rapid response characteristics. Immunofluorescence results showed that TFF3 was expressed in both the glomeruli and renal tubules of normal mouse kidneys; and compared with the glomeruli, the TFF3 content in the renal tubules was significantly increased (P < 0.01).

[0147] (2) TFF3 expression was significantly reduced in the kidneys of DKD mice.

[0148] like Figure 3 As shown, sterol regulatory element-binding protein (SREBP) plays a crucial role in regulating the biosynthesis of cholesterol, fatty acids, and triglycerides. To assess the association between lipid deposition and TFF3 and lipid-related proteins in diabetic nephropathy mice, we used Western blotting to detect the protein expression of SCAP, SREBP, and TFF3 in the kidneys. The results showed that the expression levels of SCAP and SREBP proteins in the kidneys of mice in the Model group were significantly increased (P < 0.01), while the expression level of TFF3 protein was significantly decreased (P < 0.01). This suggests that lipid deposition in the kidneys of diabetic nephropathy mice may be related to the decrease in TFF3 protein.

[0149] (3) Genotyping and validation results of TFF3 knockout mice

[0150] As shown in Figure 4 , No. 1, 3, 4, 5, 6, 7, 8, 9, 10, 11 are homozygous mice; No. 19, 20 are heterozygous mice; No. 12, 13, 14, 15, 16, 17, 18 are wild type mice. The positive tissue colon of TFF3 of wild type and homozygous mice were selected respectively for protein verification, and the results are shown: compared with wild type (WT) mice, there is no expression of TFF3 protein in the colon of knockout (KO) mice, which indicates that TFF3 knockout mice have been successfully bred.

[0151] (4) TFF3 knockout can affect glucose metabolism in mice

[0152] As shown in Figure 5 , compared with the WT group, the body weight of the WT-DKD group has significantly increased; compared with the WT-TFF3 - / - group, the body weight of the DKD-TFF3 - / - group has significantly increased. By comparing before and after the knockout of TFF3 genotype, it is found that there is no significant difference in body weight between the WT group and the WT-DKD group, the body weight of the DKD-TFF3 - / - group has increased compared with the WT-TFF3 - / - group, but there is no statistical difference. The area under the curve of glucose tolerance shows that the area under the curve of the DKD group has significantly increased compared with the WT group (P<0.01), but there is no significant difference between the DKD-TFF3 - / - group and the WT-TFF3 - / - group. The random blood glucose was measured every two weeks, and the change trend of blood glucose is shown in D of Figure 5 , compared with the WT group, the blood glucose of the WT-DKD group has significantly increased (P<0.01), and there is a difference between the WT-TFF3 - / - group and the DKD-TFF3 - / - group, but it is not significant. The area under the curve of insulin tolerance shows that the area under the curve of the DKD group has significantly increased compared with the WT group (P<0.01), but there is no significant difference between the DKD-TFF3 - / - group and the WT-TFF3 - / - group.

[0153] (5) TFF3 knockout can affect lipid deposition in the kidney of mice and aggravate the damage of renal function

[0154] As shown in Figure 6 , the results of the test of renal function show that the 24-hour urinary protein, serum creatinine and urea nitrogen detection values of the diabetic nephropathy group are significantly higher than those of the normal control group (P<0.01), which indicates that the modeling of STZ combined with high-fat diet is successful. By comparing the DKD-WT group and the DKD-TFF3 - / -In our study, we found that TFF3 gene knockout significantly increased 24-hour urinary protein, serum creatinine, and blood urea nitrogen (P < 0.01), leading to more severe renal impairment. This suggests that TFF3 plays an important protective regulatory role in DKD.

[0155] like Figure 7 As shown in the HE staining results, the WT group and WT-TFF... - / - The glomeruli in the group with normal structure and smaller volume were DKD-WT group and DKD-TFF3 group. - / - The glomeruli in this group were enlarged and adhesions occurred between the capsules. Compared with the DKD-WT group, the DKD-TFF3 group showed increased glomerular volume and adhesions between capsules. - / - The pathological changes in the WT group were more severe. In the MASSON staining results, the WT group and WT-TFF showed significantly different pathological changes. - / - The group with only a small amount of collagen fibers, while the DKD-WT group and DKD-TFF3 group... - / - The group showed significant collagen fiber proliferation (P < 0.01). Compared with the DKD-WT group, the DKD-TFF3 group showed significant collagen fiber proliferation. - / - The number of collagen fibers in the DKD group was increased (P < 0.01). In the PAS staining results, compared with the WT group, the matrix in the DKD group showed significant proliferation (P < 0.01). Compared with the DKD-WT group, the DKD-TFF3 group showed increased collagen fibers. - / - The matrix proliferation was more pronounced in the group, and the mesangial index was significantly increased (P < 0.01).

[0156] like Figure 8 As shown, Oil Red O can specifically adsorb neutral triglycerides, lipids, and lipoproteins in tissues and cells, thereby achieving the staining process of fat, and is generally used for the detection of tissue and cellular fat status. Extensive literature evidence indicates that the main sites of lipid deposition in diabetic nephropathy are the renal tubules and glomeruli. As shown in the figure, a large number of lipid droplets were generated in the kidneys of mice in the DKD-WT group, and lipid deposition was significantly increased (P < 0.01), suggesting that STZ combined with a high-fat diet induced renal lipid deposition. Compared with the DKD-WT group, DKD-TFF3... - / - Lipid deposition was more severe in the group and showed significant differences (P < 0.01).

[0157] like Figure 9 As shown in the Western blot results, compared with the WT group, the DKD-WT group showed significantly increased SRBP and SCAP expression (P < 0.01, P < 0.05), while TFF3 protein expression was significantly decreased (P < 0.01). Compared with the DKD-WT group, DKD-TFF3... - / - The expression of SRBP and SCAP was significantly increased in the group (P<0.05, P<0.01). In summary, these results indicate that TFF3 knockout promotes lipid deposition in diabetic nephropathy.

[0158] in conclusion:

[0159] (1) TFF3 is expressed in normal mouse kidney tubules and glomeruli, and is increased in diabetic nephropathy mouse kidney;

[0160] (2) TFF3 - / - Lipid deposition in the kidney can be accelerated by promoting SCAP / SREBP signaling.

[0161] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical solutions of the present application.

Claims

1. A method for constructing an animal model of diabetes combined with renal lipid injury, characterized in that, The method comprises the following steps: taking experimental animals, feeding the animals with high-fat feed for 3-5 weeks, feeding the animals with normal feed for 4-6 days continuously, injecting streptozotocin into the abdominal cavity of the animals, the amount of streptozotocin injected into each animal per day is 35-45 mg / Kg, and then feeding the animals with high-fat feed for 11-13 weeks, so that a disease animal with kidney lipid damage caused by diabetes is obtained. The experimental animal is TFF3 - / - Mouse.

2. The construction method of claim 1, wherein, The construction method comprises the following steps: taking TFF3 - / - The mice are fed with high-fat feed for 4 weeks, then fed with normal feed for 5 days continuously, and then injected with streptozotocin intraperitoneally, and the injection amount of streptozotocin per mouse per day is 40 mg / Kg, and then the mice are fed with high-fat feed for 12 weeks, so that the disease mice with kidney lipid damage are obtained.

3. The construction method of claim 1, wherein, The high-fat feed is fed in a free feeding mode.

4. The construction method of claim 1, wherein, During the high-fat diet feeding period, TFF3 - / - Mice had free access to water ad libitum.

5. The construction method of claim 1, wherein, The high-fat feed is high-fat feed with a fat energy supply ratio of greater than or equal to 60%.

6. The application of the animal model with kidney lipid damage caused by diabetes obtained by the construction method of any one of claims 1-5 in screening drugs for treating diabetic kidney disease.

Images