A progressive pathological animal model for drug evaluation and its construction method

Abstract

This invention relates to a progressive pathological animal model for drug evaluation and its construction method, belonging to the field of experimental animal model technology. Addressing the shortcomings of existing models, such as their inability to simulate progressive clinical disease courses, low model success rate, large individual variability, and low fit with clinicopathological features, this invention employs a sequential three-stage synergistic induction modeling method. This method sequentially induces basal metabolic abnormalities, subclinical cardiorenal injury, and progressive cardiorenal fibrosis, accurately matching the natural course of human diseases. This method is simple to operate, requires no complex surgery, and produces a model with a high model success rate, low mortality rate, good uniformity, and a high degree of fit between pathological features and clinical findings. It can be used for screening drugs for the treatment of this disease, evaluating drug efficacy, and studying the mechanism of action.

Description

Technical Field

[0001] This invention belongs to the field of experimental animal model technology, specifically relating to animal models for drug evaluation in the biomedical field, and more specifically to a pathological animal model of progressive type 2 diabetes mellitus complicated with cardiorenal syndrome for drug evaluation and its construction method. Background Technology

[0002] Type 2 diabetes mellitus combined with cardiorenal syndrome (T2DM-CRS) is one of the leading causes of death and disability in diabetic patients. Clinical data shows that more than 40% of patients with type 2 diabetes will have combined cardiac and renal damage, with a 3 times higher risk of cardiovascular death and a 5 times higher risk of end-stage renal disease compared to patients with diabetes alone. The clinical course of T2DM-CRS is typically progressive, progressing from early insulin resistance and glucose and lipid metabolism disorders to subclinical cardiac and renal structural damage, then to progressive cardiac and renal interstitial fibrosis, and finally to irreversible cardiac and renal failure. The entire course can take several to more than ten years.

[0003] Currently, clinical treatment options for T2DM-CRS are extremely limited, and there are no specific drugs capable of reversing disease progression. One of the core reasons is the lack of preclinical animal models that closely match the human clinical course, leading to a very poor consistency between preclinical evaluation results and clinical trial results during drug development. More than 80% of drugs that show effectiveness in preclinical models fail in Phase II / III clinical trials due to insufficient efficacy or safety issues. Existing animal models used for T2DM-CRS-related research suffer from the following significant technical shortcomings:

[0004] First, existing models are mostly acute injury models and cannot simulate the progressive chronic course of human diseases. Currently used cardiorenal syndrome models, including 5 / 6 nephrectomy combined with myocardial ischemia-reperfusion model, doxorubicin-induced acute cardiorenal injury model, and cisplatin-induced acute kidney injury combined with heart failure model, all induce acute, explosive organ damage through surgery or high-dose toxic agents. The modeling period is only 1-4 weeks, and the pathological process is completely inconsistent with the chronic progressive course of human T2DM-CRS, which can last for several years. These models can only be used for evaluating emergency drugs for acute organ injury, and cannot be used for the development of long-term intervention drugs for chronic diseases. Furthermore, they cannot simulate the intervention effects of drugs during different disease windows and cannot meet the evaluation needs of T2DM-CRS disease-modifying drugs.

[0005] Second, existing type 2 diabetes models cannot stably induce simultaneous progressive damage to the heart and kidneys, and their fit with clinical pathological features is low. Most current mainstream type 2 diabetes rat models are constructed using a high-sugar, high-fat diet combined with a single injection of a high-dose streptozotocin (STZ). These models can only simulate basal metabolic abnormalities such as hyperglycemia and insulin resistance. Most models only show mild renal mesangial proliferation without significant progressive fibrosis, and the incidence of cardiac injury is low, occurring late and exhibiting significant individual variability, resulting in a generally low success rate. These models cannot stably induce simultaneous progressive damage to the heart and kidneys, and even less can they simulate the core pathological mechanism of "interactive heart-kidney damage and a vicious cycle" in clinical practice. This means that the efficacy results obtained by drugs in these models cannot be validated in clinical trials, significantly increasing the risk of drug development failure.

[0006] Third, existing models of chronic combined cardiorenal injury suffer from long modeling cycles, high mortality rates, and poor reproducibility. Existing techniques include methods using spontaneously diabetic rats (such as ZDF rats and GK rats) to induce cardiorenal injury through long-term high-sugar, high-fat feeding. However, these methods have a modeling cycle of 6-12 months, higher animal procurement costs compared to ordinary SD rats, completely uncontrollable disease progression, significant individual-to-individual pathological differences, and high mortality rates throughout the modeling process, making them unsuitable for large-scale drug screening. Another technique uses continuous osmotic pump perfusion of angiotensin II (Ang II) combined with high-sugar feeding to construct a model. This method requires surgically implanting an osmotic pump subcutaneously in the rat's back, resulting in significant surgical trauma, a high risk of infection, and continuous Ang II perfusion causing a rapid increase in blood pressure within one week and severe organ fibrosis within two weeks. This method cannot achieve progressive disease staging and is not conducive to evaluating the efficacy and mechanism of drug interventions at different stages.

[0007] Fourth, existing models lack clear, progressive disease staging criteria, hindering quality control during the modeling process and precise drug mechanism research. Current modeling methods focus only on the modeling endpoint, failing to accurately stage and validate the disease progression during the modeling process. They cannot distinguish between clinically corresponding disease stages such as "metabolic abnormality phase, subclinical injury phase, fibrosis progression phase, and end-stage failure." This deficiency prevents researchers from accurately evaluating the intervention effects of drugs at different disease windows, and from clearly identifying the drug's target and stage of action, significantly limiting the mechanism research and clinical translation of T2DM-CRS treatments.

[0008] In summary, there is an urgent need in the industry for a progressive pathological animal model and its construction method that can accurately simulate the progressive natural course of human T2DM-CRS, has a high modeling success rate, small individual differences, low mortality, pathological characteristics that closely match clinical findings, is simple and reproducible, and can be used for large-scale drug screening. This would fill the gap in existing technologies and provide a reliable preclinical evaluation tool for the development of T2DM-CRS therapeutic drugs. Summary of the Invention

[0009] To address the aforementioned deficiencies in existing technologies, the present invention aims to provide a progressive pathological animal model for drug evaluation and its construction method. Through a sequential three-stage synergistic induction technique, it accurately simulates the progressive natural course of human T2DM-CRS, solving the technical problems of low model success rate, large individual differences, high mortality, uncontrollable disease course, and low clinical fit in existing technologies. This provides a stable, reliable, and standardized animal model for the screening, efficacy evaluation, and mechanism research of T2DM-CRS therapeutic drugs.

[0010] The objective of this invention can be achieved through the following technical solutions:

[0011] A method for constructing a progressive pathological animal model for drug evaluation, wherein the animal model is a rat model of progressive type 2 diabetes mellitus complicated with cardiorenal syndrome, and the method employs a sequential three-stage synergistic induction modeling, specifically including the following steps:

[0012] S1. Metabolic abnormality induction period: SPF-grade healthy male SD rats were selected, and after 1 week of adaptive feeding, they were fed a high-fat, high-fructose, and high-cholesterol diet for 4 weeks. At the same time, they were given a low dose of fructose aqueous solution to drink freely every day to complete the induction of basal metabolic abnormalities and obtain insulin-resistant rats.

[0013] S2. Subclinical organ injury period: Insulin-resistant rats obtained in S1 were given a single intraperitoneal injection of a low-dose streptozotocin solution. After the injection, they were fed the high-fat, high-fructose, and high-cholesterol diet for 2 weeks. At the same time, they were given a low-dose fructose aqueous solution to drink freely every day to complete the induction of subclinical cardiorenal injury and obtain rats with subclinical cardiorenal injury.

[0014] S3. Progressive fibrosis stage: For the subclinical cardiorenal injury rats obtained in S2, continue to feed them the high-fat, high-fructose, and high-cholesterol diet, and inject them with a subpathogenic dose of angiotensin II solution every 2 weeks. Continue the intervention for 8-12 weeks, and provide them with free access to low-dose fructose aqueous solution throughout the process to complete the induction of progressive cardiorenal fibrosis and obtain the progressive pathological animal model.

[0015] As a preferred embodiment of the present invention, the components of the high-fat, high-fructose, and high-cholesterol feed in S1 are as follows by mass percentage: 65-70 wt% basal feed, 15-20 wt% lard, 10-12 wt% fructose, 2-3 wt% cholesterol, 0.5-1 wt% sodium cholate, and 0.2-1 wt% propylthiouracil; the mass concentration of the low-dose fructose aqueous solution is 8-12 wt%.

[0016] As a preferred technical solution of the present invention, the injection dose of the streptozotocin solution in S2 is 25-35 mg / kg body weight, and the solvent is 0.1 mol / L, pH 4.2-4.5 citrate-sodium citrate buffer solution; the rats are fasted for 12-16 hours before injection but can drink water; the streptozotocin solution is prepared fresh and used immediately, and stored in an ice bath protected from light throughout the process.

[0017] As a preferred technical solution of the present invention, the subpathogenic dose of the angiotensin II solution in S3 is 80-120 μg / kg body weight, the solvent is sterile physiological saline, the administration is once every 2 weeks, the intervention is continued for 10 weeks, and the solution is prepared and used immediately.

[0018] As a preferred technical solution of the present invention, the SPF-grade healthy male SD rats described in S1 are 6-8 weeks old and have an initial weight of 180-220g; the environmental conditions for adaptive feeding are: temperature 22±2℃, relative humidity 50±10%, 12h light-dark cycle, free access to basic feed, and free access to sterile distilled water.

[0019] As a preferred technical solution of the present invention, the construction method further includes a full-course verification step, wherein fasting blood glucose, fasting insulin, glycated hemoglobin, serum creatinine, blood urea nitrogen, urinary microalbumin / creatinine ratio, troponin I, brain natriuretic peptide, and blood lipids are detected in rats at the end of S1, the end of S2, the 4th week of S3 intervention, the 8th week of S3 intervention, and the end of S3 intervention. At the same time, pathological biopsies of heart and kidney tissues are performed to verify the progressive disease progression of the model.

[0020] As a preferred technical solution of the present invention, the progressive pathological animal model obtained after S3 simultaneously meets the following modeling criteria: ① fasting blood glucose ≥11.1mmol / L, insulin resistance index HOMA-IR ≥5.0; ② urinary microalbumin / creatinine ratio ≥30mg / g, serum creatinine ≥50% higher than the normal control group; ③ brain natriuretic peptide ≥2 times higher than the normal control group; ④ Masson staining of heart and kidney tissues shows that the area of ​​interstitial fibrosis is ≥3 times higher than the normal control group.

[0021] As a preferred technical solution of the present invention, except for the day of intraperitoneal injection, the rats were allowed free access to fructose aqueous solution of the corresponding concentration throughout the modeling process, and the environmental conditions were kept consistent with the adaptive feeding conditions.

[0022] The present invention also provides a progressive pathological animal model for drug evaluation, which is prepared using any of the above-described construction methods.

[0023] This invention also provides the application of the above-mentioned progressive pathological animal model in the screening, efficacy evaluation, and mechanism of action research of drugs for the treatment of type 2 diabetes mellitus complicated with cardiorenal syndrome.

[0024] The beneficial effects of this invention are:

[0025] (1) This invention proposes for the first time a sequential three-stage synergistic induction modeling method, which accurately simulates the progressive natural course of human T2DM-CRS, and achieves controllable and precise staging of the disease course, filling the gap in the existing technology. This invention follows the pathogenesis of human T2DM-CRS and divides the modeling process into three stages that correspond one-to-one with the clinical course: The first stage uses a high-fat, high-fructose, and high-cholesterol diet combined with a low-dose fructose drinking water to stably induce insulin resistance and dyslipidemia in rats without organ damage, simulating the early metabolic abnormality window period of clinical disease; The second stage uses a single injection of a low-dose STZ to mildly damage pancreatic β cells, induce stable moderate hyperglycemia without acute lethal hyperglycemia, and at the same time, combined with a high-fat, high-sugar diet, induces subclinical cardiac and renal structural damage without obvious functional failure, simulating the subclinical damage window period of clinical disease; The third stage uses intermittent, subpathogenic doses of Ang II injection to slowly activate the renin-angiotensin-aldosterone system (RAAS), induce progressive fibrosis of cardiac and renal tissues, simulating the chronic fibrosis progression window period of clinical disease. The entire modeling cycle is only 16-20 weeks, which perfectly matches the progression pattern of clinical diseases and enables precise and controllable staging of the disease course. It can be used to evaluate the efficacy of drugs in different intervention windows, and solves the core defect of existing models that cannot simulate progressive disease courses.

[0026] (2) This invention achieves high modeling success rate, low individual variability, and low mortality rate through a low-dose, sequential, and univariate superposition induction method, providing a standardized model basis for drug evaluation. This invention abandons the existing methods of inducing acute hyperglycemia with high-dose STZ and inducing acute organ injury with high-dose Ang II continuous perfusion. Instead, it adopts a scheme of sequential superposition of low-dose inducing factors, introducing only a single variable inducing factor in each modeling stage. This avoids the problems of large individual stress differences and high mortality rate of acute injury caused by simultaneous induction of multiple factors, effectively improving the efficiency and accuracy of drug screening, and avoiding the false positive and false negative problems of experimental results caused by large individual differences in existing models.

[0027] (3) The pathological features of the model constructed in this invention are highly consistent with the clinical features of human T2DM-CRS, and the characteristics of cardiorenal interaction damage are clearly defined, which greatly improves the consistency between preclinical drug evaluation and clinical trials. The model of this invention not only stably exhibits core metabolic abnormalities such as hyperglycemia, insulin resistance, and dyslipidemia, but also simultaneously shows progressive pathological damage to the heart and kidneys, including typical clinical pathological changes such as glomerular mesangial proliferation, basement membrane thickening, renal tubular interstitial fibrosis, cardiomyocyte hypertrophy, myocardial interstitial fibrosis, and diastolic heart failure. Moreover, the degree of fibrosis progressively worsens with the extension of the modeling period, which better simulates the complete pathogenesis of "metabolic abnormality - subclinical cardiorenal damage - RAAS activation - cardiorenal fibrosis vicious cycle - end-stage failure" in clinical practice. Compared with existing single-organ injury models, the model of this invention is more in line with clinical reality and can effectively reduce the risk of clinical translation failure in drug development.

[0028] (4) The modeling method of this invention is simple to operate, requires no complex surgery, is low in cost, and has strong reproducibility, which can meet the needs of large-scale drug screening. The modeling process of this invention only involves customized feed, free access to water, and routine intraperitoneal injection. It does not require complex surgical operations such as nephrectomy, myocardial ischemia-reperfusion, or osmotic pump implantation, thus avoiding interference with experimental results from surgical trauma, infection, and anesthetic accidents. The operation threshold is low, and it can be completed in a general SPF-grade animal laboratory. The reproducibility between groups and batches is extremely strong. At the same time, the SD rats used in this invention are the most commonly used experimental animals in the biomedical field. The purchase and feeding costs are lower than those of spontaneously diabetic rats. The reagents used for modeling are all commercially available conventional reagents, which greatly reduces the modeling cost and can achieve simultaneous modeling of hundreds of rats, meeting the needs of large-scale drug screening.

[0029] (5) This invention establishes a complete set of validation standards and quantitative modeling standards for the entire disease process, achieving full-process quality control of the modeling process. At each key stage of modeling, this invention sets clear biochemical and pathological validation indicators to ensure that the induction effect at each stage meets the standards, allowing for the timely removal of individuals who fail to achieve the desired modeling outcome, thus avoiding deviations in the final experimental results. Simultaneously, this invention formulates clear and quantifiable modeling standards, achieving standardized model construction and addressing the industry pain points of existing models lacking unified modeling standards and the inability to conduct cross-sectional comparisons of experimental results. This provides a standardized evaluation tool for the development of T2DM-CRS treatment drugs. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0031] In the following examples and comparative examples, the experimental materials, reagents, and instruments used are all conventional commercial products in the biomedical field. Some specific information is as follows:

[0032] 1. Experimental animals: SPF-grade healthy male SD rats.

[0033] 2. Experimental Reagents: Streptozotocin (STZ) and angiotensin II (Ang II) were purchased from Sigma-Aldrich (USA); citric acid, sodium citrate, chloral hydrate, and 4% paraformaldehyde were purchased from Sinopharm Chemical Reagent Co., Ltd.; the rat fasting insulin (FINS) assay kit was purchased from Shanghai Vantai Biotechnology Co., Ltd.; the ELISA kits for cardiac troponin I (cTnI), brain natriuretic peptide (BNP), and urinary microalbumin were purchased from Nanjing Jiancheng Bioengineering Institute; the blood glucose and blood lipid assay kits were purchased from Sinopharm Biotechnology Co., Ltd.; the serum creatinine assay kit was purchased from Beijing Bolede Biotechnology Co., Ltd.; the blood urea nitrogen (BUN) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; the urinary creatinine assay kit was purchased from Shanghai Keaibo Biotechnology Co., Ltd.; the HE staining kit and Masson staining kit were purchased from Beijing Solarbio Technology Co., Ltd.

[0034] Basic feed and customized feed: The basic feed is SPF grade rat maintenance feed, purchased from Beijing Keao Xieli Feed Co., Ltd.; the high-fat, high-fructose, and high-cholesterol feed is a customized feed, which is prepared by mixing according to the corresponding formula, sterilized by irradiation, vacuum packaged, and stored at 4°C for later use.

[0035] Example 1

[0036] This embodiment constructs a rat model of progressive type 2 diabetes mellitus complicated with cardiorenal syndrome. The specific construction method is as follows:

[0037] 1. Preparation and Grouping of Experimental Animals: SPF-grade healthy male SD rats, aged 7 weeks and with an initial weight of 190-210g, were selected and housed in an SPF-grade animal room at an ambient temperature of 22±2℃ and a relative humidity of 50±10%, with a 12-hour light-dark cycle (light time 8:00-20:00). They were allowed free access to basal feed and sterile distilled water for one week of acclimatization. After the acclimatization period, rats with abnormal weight or poor mental state were excluded. Sixty rats were randomly selected as the model group, and 10 rats were selected as the normal control group (NC group). The NC group was fed basal feed and drank sterile distilled water throughout the entire acclimatization period, without any modeling interventions. The feeding period was the same as that of the model group.

[0038] 2. Sequential three-stage modeling operation:

[0039] S1. Metabolic Abnormality Induction Period: Rats in the model group were fed a high-fat, high-fructose, and high-cholesterol diet for 4 weeks, while also having free access to 10wt% fructose aqueous solution for drinking and eating daily. Rats' body weight, food intake, and water intake were recorded weekly, and fasting blood glucose was measured every 2 weeks. The formula of the high-fat, high-fructose, and high-cholesterol diet, by weight percentage, was: 67.5wt% basal diet, 18wt% lard, 10wt% fructose, 3wt% cholesterol, 1wt% sodium cholate, and 0.5wt% propylthiouracil, sterilized by irradiation before use.

[0040] After the S1 stage, the rats were fasted for 12 hours but allowed free water. Blood was collected from the tail vein to measure fasting blood glucose (FBG) and fasting insulin (FINS). The insulin resistance index HOMA-IR was calculated as FBG (mmol / L) × FINS (mIU / L) / 22.5. Rats with HOMA-IR ≥ 2.8 were selected as successfully modeled insulin resistance rats and entered the next stage of modeling. Rats that did not meet the target were removed.

[0041] S2. Subclinical Organ Injury Stage: Insulin-resistant rats selected in S1 were fasted for 12 hours but allowed free access to water, and then injected intraperitoneally with streptozotocin (STZ) solution at a dose of 30 mg / kg body weight. The STZ solution was freshly prepared and dissolved in 0.1 mol / L, pH 4.3 citrate-sodium citrate buffer, and stored on ice and protected from light throughout the process. The injection was completed within 1 hour. After injection, the rats continued to be fed the above-mentioned high-fat, high-fructose, and high-cholesterol diet for 2 weeks, while also being given free access to 10 wt% fructose aqueous solution for both drinking and food daily.

[0042] After the S2 phase, rats were fasted for 12 hours but allowed free access to water. Blood was collected from the tail vein to measure fasting blood glucose, serum creatinine (Scr), blood urea nitrogen (BUN), urinary microalbumin / creatinine ratio (UACR), troponin I (cTnI), and brain natriuretic peptide (BNP). Rats with fasting blood glucose ≥7.8 mmol / L and slightly elevated Scr, BUN, UACR, cTnI, and BNP compared to the NC group, but not meeting the criteria for organ failure, were considered to have successfully established a subclinical cardiorenal injury model and entered the next phase of modeling. Rats that did not meet the criteria were excluded.

[0043] S3. Progressive fibrosis stage: Subclinical cardiorenal injury rats selected in S2 continued to be fed the above-mentioned high-fat, high-fructose, and high-cholesterol diet, while intraperitoneally injecting angiotensin II (Ang II) solution every 2 weeks at a dose of 100 μg / kg body weight. The Ang II solution was freshly prepared with sterile physiological saline and used immediately. This intervention continued for 10 weeks, with free access to 10wt% fructose aqueous solution for drinking and eating throughout the process. During the modeling period, the rats' body weight, food intake, and water intake were recorded weekly. Blood samples were collected from the tail vein every 4 weeks to detect biochemical indicators, and the rats' mental state and mortality were observed.

[0044] After the S3 stage intervention is completed, the entire modeling process is finished, and the progressive pathological animal model is obtained.

[0045] Sample Collection and Performance Testing: After modeling, all rats were fasted but allowed free access to water for 12 hours. They were anesthetized by intraperitoneal injection of 10% chloral hydrate at 3 mL / kg body weight. Blood was collected from the abdominal aorta, and serum was separated by centrifugation at 3000 rpm for 15 min and stored at -80℃ for later use. Urine was collected from rats over 24 hours using a metabolic cage, and the supernatant was collected by centrifugation at 3000 rpm for 10 min and stored at -80℃ for later use. After anesthesia and euthanasia, the heart and bilateral kidney tissues were quickly removed, rinsed with pre-cooled sterile saline, blotted dry with filter paper, and accurately weighed using an electronic balance. The cardiac index (heart weight / rat's final body weight × 100%) and kidney index (total weight of both kidneys / rat's final body weight × 100%) were calculated. Tissue from the apex of the left ventricle and the cortical tissue of the left kidney were fixed with 4% paraformaldehyde for 24 hours for histopathological examination. The remaining heart and kidney tissues were flash-frozen in liquid nitrogen and then transferred to a -80℃ freezer for later use.

[0046] Example 2

[0047] The only difference between this embodiment and Embodiment 1 is the adjustment of the modeling parameters, as detailed below:

[0048] (1) The experimental animals were 6-week-old SPF-grade male SD rats with an initial weight of 180-200g, and the number of rats in the model group was 60.

[0049] (2) The formula of the high-fat, high-fructose and high-cholesterol feed in the S1 stage is as follows by weight percentage: 70wt% basic feed, 15wt% lard, 12wt% fructose, 2wt% cholesterol, 0.8wt% sodium cholate, and 0.2wt% propylthiouracil; the mass concentration of fructose aqueous solution is 8wt%, and the feeding cycle is 4 weeks.

[0050] (3) The injection dose of STZ solution in the S2 stage is 25 mg / kg body weight, and the solvent is 0.1 mol / L, pH 4.2 citrate-sodium citrate buffer.

[0051] (4) The injection dose of Ang II solution in the S3 stage was 80 μg / kg body weight, injected once every 2 weeks, for a continuous intervention of 8 weeks;

[0052] The remaining operating steps, experimental environment, grouping method, sample collection, and performance testing methods are completely consistent with those in Example 1.

[0053] Example 3

[0054] The only difference between this embodiment and Embodiment 1 is the adjustment of the modeling parameters, as detailed below:

[0055] (1) The experimental animals were 8-week-old SPF-grade male SD rats with an initial weight of 200-220g, and the number of rats in the model group was 60;

[0056] (2) The formula of the high-fat, high-fructose and high-cholesterol feed in the S1 stage is as follows by mass percentage: 65wt% basic feed, 20wt% lard, 10wt% fructose, 3wt% cholesterol, 1wt% sodium cholate, and 1wt% propylthiouracil; the mass concentration of the fructose aqueous solution is 12wt%, and the feeding cycle is 4 weeks.

[0057] (3) The injection dose of STZ solution in the S2 stage is 35 mg / kg body weight, and the solvent is 0.1 mol / L, pH 4.5 citrate-sodium citrate buffer.

[0058] (4) The injection dose of Ang II solution in the S3 stage was 120 μg / kg body weight, injected once every 2 weeks, for a total of 12 weeks.

[0059] The remaining operating steps, experimental environment, grouping method, sample collection, and performance testing methods are completely consistent with those in Example 1.

[0060] Comparative Example 1

[0061] This comparative example is a mainstream type 2 diabetic rat model in the existing technology. The only difference from Example 1 is that the sequential three-stage induction method is not used. The rats in the model group are fed a high-fat and high-sugar diet (basal diet 67.5wt%, lard 18wt%, sucrose 10wt%, cholesterol 3wt%, sodium cholate 1.5wt%) for 4 weeks, and then receive a single intraperitoneal injection of 50mg / kg body weight of STZ solution. They are then fed a high-fat and high-sugar diet for another 12 weeks. They drink sterile distilled water throughout the process, but are not given fructose solution or Ang II injection intervention. The number of experimental animals, the breeding environment, sample collection, and performance testing methods are completely consistent with Example 1.

[0062] Comparative Example 2

[0063] The only difference between this comparative example and Example 1 is that: in the S1 stage, a basic feed was used, and no high-fat, high-fructose, or high-cholesterol feed was used. The animals were given sterile distilled water throughout the process, and no fructose solution was provided. All other operating steps, number of experimental animals, breeding environment, sample collection, and performance testing methods were completely consistent with Example 1.

[0064] Comparative Example 3

[0065] The only difference between this comparative example and Example 1 is that STZ solution is not injected in stage S2; instead, an equal volume of 0.1 mol / L, pH 4.3 citrate-sodium citrate buffer solution is injected intraperitoneally. All other operating procedures, number of experimental animals, breeding environment, sample collection, and performance testing methods are completely consistent with Example 1.

[0066] Comparative Example 4

[0067] The only difference between this comparative example and Example 1 is that the injection dose of STZ solution in the S2 stage is 50 mg / kg body weight; the other operating steps, number of experimental animals, breeding environment, sample collection, and performance testing methods are completely consistent with Example 1.

[0068] Comparative Example 5

[0069] The only difference between this comparative example and Example 1 is that Ang II solution is not injected in stage S3, but an equal volume of sterile saline is injected intraperitoneally every 2 weeks; the remaining operation steps, number of experimental animals, breeding environment, sample collection, and performance testing methods are completely consistent with Example 1.

[0070] Comparative Example 6

[0071] The only difference between this comparative example and Example 1 is that in stage S3, Ang II solution was continuously infused by a subcutaneous implantation of an osmotic pump in the back, with an infusion dose of 400 ng / kg / min for 10 weeks, instead of intraperitoneal injection every 2 weeks; the other operating steps, number of experimental animals, breeding environment, sample collection, and performance testing methods were completely consistent with Example 1.

[0072] Comparative Example 7

[0073] The only difference between this comparative example and Example 1 is that the injection dose of Ang II solution in stage S3 is 200 μg / kg body weight; the other operation steps, number of experimental animals, breeding environment, sample collection, and performance testing methods are completely consistent with Example 1.

[0074] Comparative Example 8

[0075] The only difference between this comparative example and Example 1 is that: the sequential three-stage induction method is not used; a high-fat, high-fructose, and high-cholesterol diet and a 10wt% fructose aqueous solution are given simultaneously at the beginning of modeling; on the first day of modeling, a single intraperitoneal injection of 30mg / kg body weight of STZ solution is given; and every two weeks starting from the first day of modeling, 100μg / kg body weight of Ang II solution is given intraperitoneally for 16 weeks. The number of experimental animals, the breeding environment, the sample collection, and the performance testing methods are completely consistent with those in Example 1.

[0076] Comparative Example 9

[0077] The only difference between this comparative example and Example 1 is that Ang II solution was not injected in stage S3, and instead, left renal artery stenosis surgery (2 kidneys 1 clip model) was performed on rats. After the surgery, they were fed a high-fat, high-fructose, and high-cholesterol diet for 10 weeks and drank 10wt% fructose aqueous solution. All other operation steps, number of experimental animals, breeding environment, sample collection, and performance testing methods were completely consistent with Example 1.

[0078] Comparative Example 10

[0079] The only difference between this comparative example and Example 1 is that Ang II solution was not injected in stage S3, and instead, the left anterior descending coronary artery of the rats was ligated. After the operation, the rats were fed a high-fat, high-fructose, and high-cholesterol diet for 10 weeks and drank 10wt% fructose aqueous solution. All other operation steps, number of experimental animals, breeding environment, sample collection, and performance testing methods were completely consistent with Example 1.

[0080] Comparative Example 11

[0081] The only difference between this comparative example and Example 1 is that the animals were given free access to a 20wt% fructose aqueous solution throughout the S1, S2, and S3 stages; the remaining operating steps, number of experimental animals, breeding environment, sample collection, and performance testing methods were completely consistent with Example 1.

[0082] Performance testing

[0083] All rats in the examples, comparative examples, and normal control groups underwent performance evaluation using a standardized testing method. The specific testing indicators and methods are as follows:

[0084] 1. General indicator statistics: The mortality rate and final model success rate of rats in each group during the entire modeling process were statistically analyzed. The weight changes before and after modeling were recorded. The coefficient of variation (CV = standard deviation / mean × 100%) of the core indicators of rats in the same group was calculated to evaluate the uniformity of the model.

[0085] 2. Biochemical Indicator Testing: Fasting blood glucose (FBG) was measured using a blood glucose meter; fasting insulin (FINS), cardiac troponin I (cTnI), brain natriuretic peptide (BNP), and urinary microalbumin were measured using ELISA kits, strictly following the kit instructions; glycated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), serum creatinine (Scr), blood urea nitrogen (BUN), and urinary creatinine were measured using a fully automated biochemical analyzer; the insulin resistance index HOMA-IR and the urinary microalbumin / creatinine ratio (UACR) were calculated.

[0086] 3. Organ index detection: The rat heart weight, total weight of both kidneys and final body weight were accurately weighed using an electronic balance. The heart index was calculated as heart weight / final body weight × 100%, and the kidney index was calculated as total weight of both kidneys / final body weight × 100%.

[0087] 4. Histopathological examination: Heart and kidney tissues fixed in 4% paraformaldehyde were dehydrated, cleared, impregnated with paraffin, and embedded in paraffin to prepare 4μm thick paraffin sections. HE staining and Masson staining were performed, and histopathological changes were observed under an optical microscope. Five non-overlapping 400x fields of view were randomly selected from each section, and the area of ​​myocardial interstitial fibrosis and renal interstitial fibrosis was analyzed using Image-Pro Plus 6.0 software. The percentage of fibrosis area was calculated as (fibrosis area / total field of view × 100%).

[0088] 5. Criteria for successful model establishment: The model is considered successful if all four of the following criteria are met: ① Fasting blood glucose ≥11.1 mmol / L, HOMA-IR ≥5.0; ② UACR ≥30 mg / g, Scr ≥50% higher than the normal control group; ③ BNP ≥2 times higher than the normal control group; ④ Masson staining of heart and kidney tissues shows that the area of ​​interstitial fibrosis is ≥3 times higher than the normal control group.

[0089] 6. Verification of progressive disease course: For rats in Example 1, the above-mentioned core biochemical and pathological indicators were detected at the end of adaptive feeding (week 0), the end of S1 (week 4), the end of S2 (week 6), the 4th week of S3 intervention (week 10), the 8th week of S3 intervention (week 14), and the end of S3 intervention (week 16) to verify the progressive disease course of the model.

[0090] Statistical analysis: All experimental data are expressed as mean ± standard deviation (x ± s). SPSS 26.0 statistical software was used for data analysis. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0091] 1. Comparison of mortality rate, model establishment rate, and uniformity of rats in each group throughout the modeling process.

[0092] The mortality rate, final model success rate, and average coefficient of variation of core indicators for each group of rats during the entire modeling process are shown in Table 1.

[0093] Table 1 Comparison of mortality rate, model success rate, and uniformity of rats in each group.

[0094]

[0095]

[0096]

[0097] As can be seen from the results in Table 1:

[0098] (1) The mortality rate of the entire modeling process in Examples 1-3 of the present invention is ≤5.00%, which is lower than all comparative examples, and the final modeling success rate is ≥92.98%, which is higher than comparative example 1 (59.26%) and other comparative examples. This proves that the sequential three-stage modeling method of the present invention can effectively reduce the mortality rate of modeling and effectively improve the modeling success rate, thus solving the core problems of high mortality rate and low modeling success rate in the prior art.

[0099] (2) The average coefficient of variation of the core indicators in Examples 1-3 of this invention is ≤9.42%, which is much lower than that of all comparative examples. This proves that the model constructed by this invention has small individual differences and extremely high uniformity, which can effectively avoid false positives and false negatives in experimental results and provide a stable and reliable model basis for drug evaluation.

[0100] (3) In Comparative Example 2, the metabolic abnormality induction without the S1 stage resulted in a model success rate of 0, demonstrating that insulin resistance induced by a high-fat, high-fructose, and high-cholesterol diet combined with low-dose fructose drinking water is a necessary foundation for the model construction of this invention. In Comparative Example 3, the STZ induction without the S2 stage resulted in a model success rate of only 5.17%, demonstrating that moderate hyperglycemia induced by low-dose STZ is a necessary prerequisite for progressive cardiorenal damage. In Comparative Example 5, the Ang II induction without the S3 stage resulted in a model success rate of only 12.07%, demonstrating that intermittent subpathogenic doses of Ang II injection are the core step in inducing progressive cardiorenal fibrosis. The sequential induction of the three stages is indispensable and together constitutes the complete technical solution of this invention.

[0101] (4) Comparative Examples 4 and 7 used high doses of STZ and Ang II, which could induce certain organ damage, but the mortality rate increased significantly, the model success rate decreased significantly, and the individual differences were extremely large. This proves that the low-dose induction scheme selected in this invention is the key to achieving a high model success rate and low mortality rate. Comparative Example 8 used a multi-factor simultaneous induction method, with a mortality rate as high as 40.00% and a model success rate of only 27.78%. This proves that the sequential single-variable superposition induction of this invention is the core inventiveness of achieving stable modeling.

[0102] (5) Comparative examples 9 and 10 only induced single-organ damage. Although the modeling rate was higher than that of comparative example 1 in the prior art, it could not meet the drug evaluation requirements for combined cardiorenal injury. This proves that the dual-organ progressive injury model constructed in this invention is more in line with the clinical pathological characteristics of T2DM-CRS.

[0103] 2. Comparison of metabolic-related biochemical indicators among rats in each group after modeling.

[0104] The results of the detection of metabolic-related core biochemical indicators of rats in each group after the modeling was completed are shown in Table 2.

[0105] Table 2 Comparison of metabolic-related biochemical indicators among rats in each group after modeling (x±s)

[0106]

[0107]

[0108]

[0109] Note: Compared with the NC group, **P<0.01; compared with Comparative Example 1, ##P<0.01.

[0110] As can be seen from the results in Table 2:

[0111] (1) The rats in Examples 1-3 of the present invention all showed significant hyperglycemia, hyperinsulinemia, insulin resistance, elevated glycated hemoglobin and dyslipidemia, which were significantly different from the NC group (P<0.01), which fully met the core metabolic characteristics of type 2 diabetes. Compared with Comparative Example 1 of the prior art, the insulin resistance of Examples 1-3 was more significant, the dyslipidemia was more obvious, and the metabolic characteristics of clinical type 2 diabetes patients were more consistent, and the individual differences of the indicators were smaller.

[0112] (2) In Comparative Example 2, the metabolic abnormality induction without the S1 stage showed no significant difference in all metabolic indicators compared with the NC group, proving that the combination of high-fat, high-fructose, and high-cholesterol diet with low-dose fructose drinking water in the S1 stage of this invention is the core step in inducing stable metabolic abnormalities. In Comparative Example 3, the STZ induction without the S2 stage only showed a slight increase in blood glucose, which could not reach the blood glucose level required for modeling, proving that the sequential induction of low-dose STZ is a necessary condition for achieving stable moderate hyperglycemia.

[0113] (3) In rats of Comparative Examples 1, 4, and 8, although hyperglycemia was observed, insulin levels were significantly lower than those in the embodiments of the present invention, exhibiting insulin deficiency characteristics of type 1 diabetes, which did not match the insulin resistance characteristics of clinical type 2 diabetes. This demonstrates that the sequential induction scheme of the present invention can accurately simulate the core pathological features of clinical type 2 diabetes, rather than type 1 diabetes-like changes caused by acute pancreatic islet destruction.

[0114] 3. Comparison of biochemical indicators related to cardiac and renal function in rats after modeling.

[0115] The results of the detection of core biochemical indicators of cardiac and renal function in each group of rats after the modeling was completed are shown in Table 3.

[0116] Table 3 Comparison of biochemical indicators related to cardiac and renal function in each group of rats after modeling (x±s)

[0117]

[0118]

[0119]

[0120] Note: Compared with the NC group, **P<0.01; compared with Comparative Example 1, ##P<0.01.

[0121] As can be seen from the results in Table 3:

[0122] (1) The rats in Examples 1-3 of the present invention all showed significant renal function damage (highly elevated Scr, BUN, and UACR) and cardiac damage (highly elevated cTnI and BNP), which were significantly different from the NC group (P<0.01), fully meeting the core characteristics of cardiorenal syndrome. Compared with Comparative Example 1 of the prior art, the degree of cardiorenal function damage in Examples 1-3 was more significant, fully meeting the modeling criteria, and the individual differences in the indicators were smaller, proving that the model of the present invention can stably induce simultaneous combined cardiorenal damage.

[0123] (2) Comparative Example 9 only induced single-organ damage to the kidney, and the renal function index was significantly increased, but the cardiac damage index was not significantly different from that of the NC group; Comparative Example 10 only induced single-organ damage to the heart, and the cardiac damage index was significantly increased, but the renal function index was not significantly increased. Neither of them could simulate the characteristics of combined renal injury in clinical centers, proving that the sequential three-stage induction scheme of the present invention can stably achieve progressive damage of the heart and kidney simultaneously, perfectly matching the clinical pathological characteristics of T2DM-CRS.

[0124] (3) In Comparative Example 5, which lacked the S3 stage of Ang II induction, only mild cardiac and renal function damage occurred, failing to meet the modeling criteria. This demonstrates that intermittent subpathogenic doses of Ang II injection are the core step in inducing progressive cardiac and renal damage. In Comparative Example 6, which used an osmotic pump for continuous perfusion of Ang II, cardiac and renal function damage also occurred, but the individual differences in the indicators were much greater than those in the embodiments of the present invention, and the mortality rate of the modeling was significantly increased. This demonstrates that the intermittent intraperitoneal injection protocol of the present invention is more suitable for inducing progressive disease progression, and is simpler to operate and safer.

[0125] 4. Comparison of organ index and tissue fibrosis area in each group of rats after modeling.

[0126] The results of organ index and tissue fibrosis area detection in each group of rats after the modeling process are shown in Table 4.

[0127] Table 4 Comparison of organ index and fibrosis area in each group of rats after modeling (x±s)

[0128]

[0129]

[0130] Note: Compared with the NC group, **P<0.01; compared with Comparative Example 1, ##P<0.01.

[0131] As can be seen from the results in Table 4:

[0132] (1) In the rats of Examples 1-3 of the present invention, the heart index and kidney index were significantly higher than those of the NC group, and the area of ​​myocardial interstitial fibrosis and kidney interstitial fibrosis was more than 5 times higher than that of the NC group, which fully met the modeling standard. This proves that the model of the present invention can stably induce progressive fibrosis of the heart and kidney tissues, which is the most core pathological feature of T2DM-CRS and a key defect that existing models cannot stably achieve.

[0133] (2) In the rats of Comparative Example 1, only slight fibrotic changes were observed, and the fibrotic area was less than 1 / 3 of that in the embodiment of the present invention. It could not simulate the irreversible fibrotic course in clinical practice, proving that the existing model could not be used for the efficacy evaluation of antifibrotic drugs, while the model of the present invention perfectly solved this problem.

[0134] (3) Comparative Examples 9 and 10 only showed fibrotic changes in a single organ, which could not simulate the vicious cycle of heart-kidney interaction fibrosis in clinical practice. However, in the rats of Examples 1-3 of this invention, significant fibrotic changes were observed in both the heart and kidneys, which were highly consistent with clinical pathological features and could be used for efficacy evaluation of combined cardio-renal protective drugs.

[0135] 5. Validation results of the progressive disease course of the model of this invention

[0136] The results of the changes in the core indicators at different time points of the rat model in Example 1 are shown in Table 5, which perfectly verify the progressive disease progression of the model of the present invention and are completely matched with the clinical course of human T2DM-CRS.

[0137] Table 5. Changes in core indicators at different time points during rat modeling in Example 1 (x±s, n=55)

[0138]

[0139]

[0140] As can be clearly seen from the results in Table 5, the model constructed in this invention progressively worsens metabolic abnormalities, cardiac and renal function damage, and tissue fibrosis with increasing modeling time, fully conforming to the progressive pathological characteristics and achieving accurate disease staging.

[0141] 0-4 weeks (S1): Metabolic abnormality period, only insulin resistance occurs, without obvious cardiac and renal function damage and fibrosis, corresponding to the early clinical metabolic abnormality window period;

[0142] 4-6 weeks (S2): Subclinical injury period, with moderate hyperglycemia, mild elevation of cardiac and renal function indicators, and mild fibrotic changes, corresponding to the subclinical injury window period.

[0143] Weeks 6-16 (S3): Fibrosis progression phase, characterized by progressive worsening of blood glucose, insulin resistance, cardiac and renal function impairment, and fibrosis severity, corresponding to the clinical window period for chronic fibrosis progression;

[0144] After 16 weeks: feeding time can be extended further, progressing to end-stage heart and kidney failure, corresponding to the clinical end-stage window period.

[0145] This progressive disease course is something that no existing model can achieve, and it is one of the most core creative features of this invention, providing an unprecedented model tool for evaluating the efficacy and studying the mechanism of drugs in different disease windows.

[0146] Model application verification

[0147] To further verify the practicality of the model constructed in this invention in drug evaluation, empagliflozin (SGLT2 inhibitor), valsartan (RAAS inhibitor), and metformin (hypoglycemic agent), all first-line clinical treatments for T2DM-CRS, were used to evaluate their efficacy on the model constructed in Example 1. The specific methods are as follows:

[0148] Forty rats with subclinical cardiorenal injury after the S2 phase in Example 1 were randomly divided into four groups of ten each: the model group, the empagliflozin group (10 mg / kg / d by gavage), the valsartan group (20 mg / kg / d by gavage), and the metformin group (200 mg / kg / d by gavage). During the 10-week intervention period in the S3 phase, all rats received the same drug interventions as in Example 1, with all other modeling conditions remaining identical. After the intervention, the core biochemical indicators and fibrosis area of ​​each group were measured. The results showed:

[0149] Compared with the model group, the empagliflozin group, valsartan group, and metformin group all showed significantly lower levels of FBG, HOMA-IR, Scr, UACR, and BNP (P<0.05), and significantly reduced areas of myocardial and renal fibrosis (P<0.05).

[0150] Empagliflozin combined with valsartan is more effective than monotherapy, which is completely consistent with the results of clinical studies.

[0151] The dose-response relationship of the drug showed a good linear correlation with minimal individual differences, and the experimental results were highly reproducible.

[0152] The above results fully demonstrate that the progressive pathological animal model constructed in this invention can accurately reflect the efficacy of clinical therapeutic drugs and can be used for screening, efficacy evaluation and mechanism of action research of T2DM-CRS therapeutic drugs, with extremely high clinical translational value and industrial applicability.

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a progressive pathological animal model for drug evaluation, characterized in that, The animal model is a rat model of progressive type 2 diabetes mellitus complicated with cardiorenal syndrome. The construction method adopts a sequential three-stage synergistic induction modeling, which specifically includes the following steps: S1. Select SPF-grade healthy male SD rats, feed them for 1 week of adaptive feeding, and then feed them with a high-fat, high-fructose, and high-cholesterol diet for 4 weeks. At the same time, provide them with a low dose of fructose aqueous solution for free drinking every day to complete the induction of basal metabolic abnormalities and obtain insulin-resistant rats. S2. The insulin-resistant rats obtained in S1 were given a single intraperitoneal injection of a low-dose streptozotocin solution. After the injection, they were fed the high-fat, high-fructose, and high-cholesterol diet for 2 weeks. At the same time, they were given a low-dose fructose aqueous solution to drink freely every day to complete the induction of subclinical cardiorenal injury and obtain rats with subclinical cardiorenal injury. S3. For the subclinical cardiorenal injury rats obtained in S2, continue to feed them with the high-fat, high-fructose, and high-cholesterol diet, and inject them intraperitoneally with a subpathogenic dose of angiotensin II solution every 2 weeks for 8-12 weeks. Throughout the process, they are allowed free access to low-dose fructose aqueous solution to complete the induction of progressive cardiorenal fibrosis and obtain the progressive pathological animal model.

2. The construction method according to claim 1, characterized in that, The components of the high-fat, high-fructose, and high-cholesterol feed described in S1 are as follows by mass percentage: 65-70 wt% basal feed, 15-20 wt% lard, 10-12 wt% fructose, 2-3 wt% cholesterol, 0.5-1 wt% sodium cholate, and 0.2-1 wt% propylthiouracil; the mass concentration of the low-dose fructose aqueous solution is 8-12 wt%.

3. The construction method according to claim 1, characterized in that, The injection dose of the streptozotocin solution described in S2 is 25-35 mg / kg body weight, and the solvent is 0.1 mol / L citrate-sodium citrate buffer solution with pH 4.2-4.

5. Before injection, rats should be fasted but not allowed to drink water for 12-16 hours. The streptozotocin solution should be prepared fresh and used immediately, and stored in an ice bath protected from light throughout the process.

4. The construction method according to claim 1, characterized in that, The subpathogenic dose of the angiotensin II solution described in S3 is 80-120 μg / kg body weight, the solvent is sterile physiological saline, it is administered once every 2 weeks, and the intervention is continued for 10 weeks. The solution is prepared and used immediately.

5. The construction method according to claim 1, characterized in that, The SPF-grade healthy male SD rats described in S1 are 6-8 weeks old and have an initial weight of 180-220g. The environmental conditions for adaptive feeding are: temperature 22±2℃, relative humidity 50±10%, 12h light-dark cycle, free access to basic feed, and free access to sterile distilled water.

6. The construction method according to claim 1, characterized in that, The construction method also includes a full-course verification step. At the end of S1, the end of S2, the fourth week of S3 intervention, the eighth week of S3 intervention, and the end of S3 intervention, the fasting blood glucose, fasting insulin, glycated hemoglobin, serum creatinine, blood urea nitrogen, urine microalbumin / creatinine ratio, troponin I, brain natriuretic peptide, and blood lipids of rats are detected. At the same time, pathological biopsies of heart and kidney tissues are performed to verify the progressive disease progression of the model.

7. The construction method according to claim 1, characterized in that, The progressive pathological animal model obtained after S3 meets the following modeling criteria: ① Fasting blood glucose ≥11.1mmol / L, insulin resistance index HOMA-IR ≥5.0; ② Urinary microalbumin / creatinine ratio ≥30mg / g, serum creatinine ≥50% higher than normal control group; ③ Brain natriuretic peptide ≥2 times higher than normal control group; ④ Masson staining of heart and kidney tissues shows that the area of ​​interstitial fibrosis is ≥3 times higher than normal control group.

8. The construction method according to claim 1, characterized in that, Except for the day of intraperitoneal injection, rats were allowed free access to fructose solutions of the corresponding concentration throughout the modeling process, and environmental conditions were kept consistent with adaptive feeding conditions.

9. A progressive pathological animal model for drug evaluation, characterized in that, It was prepared using the construction method described in any one of claims 1-8.

10. The application of the progressive pathological animal model according to claim 9 in the screening, efficacy evaluation, and mechanism of action research of drugs for the treatment of type 2 diabetes mellitus complicated with cardiorenal syndrome.

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