Oxidized lipid-AGEs composite atherosclerosis promoting pharmaceutical composition, rat coronary heart disease ischemia-reperfusion injury multi-target combined modeling method and application of rat coronary heart disease ischemia-reperfusion injury multi-target combined modeling method

Abstract

The invention relates to the field of pharmaceutical compositions for promoting atherosclerosis, in particular to a lipid oxide-AGEs composite pharmaceutical composition for promoting atherosclerosis, a method for constructing a rat coronary heart disease ischemia reperfusion injury model by using the composition and application of the composition. The composition comprises 80-90 parts of oxidized lipid, 4-6 parts of 7-ketocholesterol, 2-4 parts of 25-hydroxycholesterol, 8-10 parts of trans-fatty acid, 200-300 parts of an advanced glycosylation end product, 180-220 parts of casein, 320-360 parts of cane sugar, 145-165 parts of corn starch, 45-55 parts of cellulose, 30-40 parts of a mineral mixture, 8-12 parts of a vitamin mixture and 0.4-0.6 mg / kg of folic acid, the vitamin B6 is 1.8 to 2.2 mg / kg, and the vitamin B12 is 0.007 to 0.009 mg / kg.

Description

Technical Field

[0001] The present invention relates to the field of experimental animal model construction, and in particular to an oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition, a method for constructing a rat coronary heart disease ischemia-reperfusion injury model using the composition, and applications thereof. Background Art

[0002] Coronary artery disease (CHD) is one of the leading causes of death worldwide. Its underlying pathology is coronary atherosclerosis and the resulting vascular stenosis. Clinically, myocardial ischemia-reperfusion injury caused by CHD severely impacts patients' quality of life and survival. To further understand the pathogenesis and treatment strategies of CHD, it is crucial to establish animal models that are highly relevant to human CHD.

[0003] Currently, methods for establishing animal models of coronary artery disease (CAD) primarily include surgical coronary artery ligation, high-fat diet-induced atherosclerosis, and drug-induced myocardial injury. Gao et al. reported a rat model of CAD induced by a high-fat diet in "Establishment of a rat model with diet-induced coronary atherosclerosis" (J Biomed Res, 2016). However, this model requires an 8-12-week feeding period and exhibits a slow progression of atherosclerosis, significantly different from the human disease. Lindsey et al. summarized methods for establishing myocardial ischemia models in "Guidelines for experimental models of myocardial ischemia and infarction" (Am J Physiol Heart Circ Physiol, 2018), noting that while surgical ligation alone can directly induce myocardial ischemia, its pathological mechanisms differ significantly from those of clinical CAD caused by atherosclerosis.

[0004] Furthermore, existing methods for inducing myocardial ischemia with vasopressin (VP) suffer from drawbacks such as unstable dose-effect relationships and wide inter-animal variability. Afshin Nazari et al. explored the cardioprotective effects of vasopressin (AVP) in "The cardioprotective effect of different doses of vasopressin (AVP) against ischemia-reperfusion injuries in theanesthetized rat heart" (Peptides, 2011), but failed to address the stability of VP in models of coronary artery disease.

[0005] Reperfusion injury is a significant issue in the treatment of coronary artery disease (CAD). Existing models often use invasive methods to control blood flow restoration when simulating this process. This not only increases surgical difficulty but also introduces additional traumatic factors that can interfere with experimental results. Hausenloy et al. proposed the concept of remote ischemic conditioning in "Remote Ischemic Conditioning" (J Am Coll Cardiol, 2016), but did not apply this concept to a high-fat diet-induced CAD model.

[0006] At present, researchers at home and abroad have not yet established a multi-target combined rat model that can comprehensively simulate high-fat diet-induced atherosclerosis, myocardial ischemia and reperfusion injury. The existing models still have much room for improvement in stability, success rate and clinical relevance. Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition, a multi-target combined modeling method for constructing ischemia-reperfusion injury in rat coronary heart disease using the composition, and its application, so as to overcome the problems existing in the prior art.

[0008] To achieve the above-mentioned object, the present invention provides an oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition, which comprises, by weight:

[0009] The invention comprises: 80-90 parts of oxidized lipids, wherein the oxidized lipids are prepared by heating edible oils and fats at 180°C for 6-8 hours; 4-6 parts of 7-ketocholesterol; 2-4 parts of 25-hydroxycholesterol; 8-10 parts of trans fatty acids; 200-300 parts of advanced glycation end products, wherein the advanced glycation end products are prepared by treating milk powder at 70°C for 7 days; 180-220 parts of casein; 320-360 parts of sucrose, wherein the sucrose contains 20% high fructose corn syrup; 145-165 parts of corn starch; 45-55 parts of cellulose; 30-40 parts of a mineral mixture; and 8-12 parts of a vitamin mixture, wherein the vitamin mixture contains 0.4-0.6 mg / kg of folic acid, 1.8-2.2 mg / kg of vitamin B6, and 0.007-0.009 mg / kg of vitamin B12.

[0010] Preferably, the mineral mixture contains 0.04-0.06 mg / kg of selenium and 70-80 mg / kg of magnesium; and the composition does not contain choline.

[0011] Further preferably, the trans fatty acid includes trans palmitoleic acid; and the composition further comprises 18-22 parts of methionine.

[0012] The present invention also provides a multi-target joint modeling method for ischemia-reperfusion injury in rats with coronary heart disease, comprising the following steps:

[0013] Step 1, feeding phase: The above-mentioned oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition is fed to rats in three phases; weeks 1 to 4 are the pretreatment phase, with a daily feed amount of 40 grams per rat and oral administration of 0.8 ml of fat emulsion rich in oxidized lipids; weeks 5 to 8 are the acceleration phase, with a daily feed amount of 45 grams per rat and oral administration of 1 ml of fat emulsion containing 7-ketocholesterol, followed by an intermittent fasting cycle of 3 days of normal diet and 1 day of fasting; weeks 9 to 12 are the vulnerable phase, with a daily feed amount of 50 grams per rat and oral administration of 1.2 ml of the composite fat emulsion;

[0014] Step 2: Ischemia induction: When the rats weighed 250 grams, they were intravenously injected with a targeted delivery system comprising lipid nanoparticles loaded with vasopressin and endothelin-1, the lipid nanoparticles having a particle size of less than 200 nanometers and surface-modified with the coronary artery-specific peptide CRPPR. The vasopressin dose was 0.03 micrograms per rat, and the endothelin-1 dose was 2 nanomoles per rat. Ischemia was initiated 30 minutes after administration.

[0015] Step 3: Reperfusion control: Remote ischemic conditioning was performed using pressure cuffs on both limbs of the rat for four cycles, each consisting of 5 minutes of ischemia and 5 minutes of reperfusion, starting 30 minutes before ischemia. Simultaneously, ultrasound-mediated reperfusion control was performed using cardiac-gated ultrasound pulses at a frequency of 1-1.3 MHz for 30 minutes.

[0016] Step 4, Evaluation: Confirm the success of model establishment by measuring circulating biomarkers, performing cardiac imaging and functional assessment.

[0017] Preferably, step 2 further comprises oral administration of 10-20 mg / kg of empagliflozin starting 7 days before ischemia induction, and intravenous injection of 15 mg / kg of theophylline 30 minutes before ischemia.

[0018] Further preferably, step three also includes pharmacological hypothermia, using an HBN-1 combination regimen comprising a mixture of ethanol, vasopressin and lidocaine, to control the body temperature to 33.5°C and maintain it for 17-18 hours, starting 5 minutes after reperfusion.

[0019] More preferably, step three further comprises applying a pulsed electromagnetic field, wherein the pulsed electromagnetic field has a frequency of 15 Hz and an intensity of 6 millitesla, and the treatment is performed for 2 hours per day for 6 weeks.

[0020] Further preferably, the circulating biomarkers in step four include: a microRNA panel including miR-21, miR-22, and miR-155-5p; damage-associated molecular patterns including HMGB1, S100A8 / A9, heat shock proteins HSP60 and HSP70; oxidative stress markers including malondialdehyde, 8-hydroxydeoxyguanosine, and protein carbonyls; the imaging assessment includes hyperpolarized 13C-pyruvate magnetic resonance imaging and positron emission tomography; the functional assessment includes pressure-volume loop analysis, using a Millar catheter to measure ESPVR and EDPVR parameters.

[0021] The method for constructing a rat coronary heart disease ischemia-reperfusion injury model is used to construct the model.

[0022] The application of the oxidized lipid-AGEs composite pro-atherosclerosis drug composition in constructing a rat model of coronary heart disease ischemia-reperfusion injury achieves a plasma cholesterol level greater than 300 mg / dL and a homocysteine ​​level greater than 25 μmol / L in the model by establishing endothelial dysfunction, insulin resistance, promoting foam cell formation and early plaque development, thereby improving the stability, success rate and clinical relevance of the rat model of coronary heart disease ischemia-reperfusion injury.

[0023] The beneficial effects of the present invention are mainly reflected in:

[0024] 1. The oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition provided by the present invention significantly accelerates the formation of atherosclerosis in rats through the synergistic effect of multiple atherosclerosis-promoting factors, thereby shortening the model construction period.

[0025] 2. The pituitrin and endothelin-1 targeted delivery system of the present invention improves the accuracy and stability of myocardial ischemia induction and reduces individual differences in animals;

[0026] 3. The present invention adopts a non-invasive or minimally invasive reperfusion control method, which improves the survival rate of animals and reduces the interference of additional trauma factors;

[0027] 4. The multi-target combined modeling method of the present invention can fully simulate the pathological process of human coronary heart disease, including atherosclerosis formation, myocardial ischemia and reperfusion injury, thereby improving the clinical relevance of the model. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the electrocardiogram of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through specific examples, but the protection scope of the present invention is not limited to the following examples.

[0030] Example 1 Preparation of Oxidized Lipid-AGEs Composite Pharmaceutical Composition Promoting Atherosclerosis

[0031] This embodiment provides a method for preparing an oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition, the specific steps of which are as follows:

[0032] (1) Preparation of oxidized lipids: Soybean oil (Kalanchoe brand refined soybean oil) was heated at 180°C for 6 hours with stirring every hour. The oil was cooled to room temperature and then used. Gas chromatography-mass spectrometry (GC-MS, Agilent 7890B-5977A) was used to determine the peroxide value (POV) to be >20 meq / kg and the conjugated diene content to be >8%.

[0033] (2) Preparation of advanced glycation end products (AGEs): Whole milk powder (Nestle whole milk powder) was placed in a 70°C incubator for 7 days, with stirring once a day to ensure uniform heating. The AGE content was confirmed to be >500 μg / g by enzyme-linked immunosorbent assay (ELISA).

[0034] (3) Preparation of the pharmaceutical composition: The components were mixed in the following proportions: 85 parts of oxidized lipids, 5 parts of 7-ketocholesterol (Sigma-Aldrich, purity >98%), 3 parts of 25-hydroxycholesterol (Sigma-Aldrich, purity >98%), 9 parts of trans fatty acids (containing 60% trans-palmitoleic acid, Sigma-Aldrich), 250 parts of advanced glycation end products, 200 parts of casein (Beijing Solebaugh Technology Co., Ltd., purity >90%), 340 parts of sucrose (commercially available food grade), including 20% ​​high fructose corn syrup (ADM, HFCS-55), 155 parts of corn starch (commercially available food grade), 50 parts of cellulose (Beijing Solebaugh Technology Co., Ltd., purity >95%), 35 parts of a mineral mixture, and 10 parts of a vitamin mixture. The mineral mixture contains 0.05 mg / kg of selenium and 75 mg / kg of magnesium. The vitamin mixture contains 0.5 mg / kg of folic acid, 2 mg / kg of vitamin B6, and 0.008 mg / kg of vitamin B12. The composition does not contain choline. After thoroughly mixing all components, the mixture is formed into pelleted feed and stored in a sealed container at 4°C.

[0035] In this example, oxidized lipids can induce endothelial cell damage, activate inflammatory responses, and promote vascular smooth muscle cell proliferation; 7-ketocholesterol and 25-hydroxycholesterol, as cholesterol oxidation products, have significant cytotoxic effects and can accelerate foam cell formation; trans fatty acids increase serum low-density lipoprotein levels and reduce high-density lipoprotein levels; advanced glycation end products can bind to receptors on the surface of vascular wall cells and activate inflammatory signaling pathways; micronutrient deficiency interferes with the methionine metabolic cycle, leading to elevated homocysteine ​​levels and further damaging the vascular endothelium. The synergistic effect of these components significantly accelerated the formation of atherosclerosis in rats.

[0036] Example 2 Preparation of Oxidized Lipid-AGEs Composite Pharmaceutical Composition Promoting Atherosclerosis

[0037] This example provides another method for preparing an oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition, the specific steps of which are as follows:

[0038] (1) Preparation of Oxidized Lipids: Soybean oil and lard were mixed in a 3:1 ratio and heated at 185°C for 7 hours with stirring every hour. The mixture was cooled to room temperature and then used. Gas chromatography-mass spectrometry was used to determine the peroxide value (POV) to be >25 meq / kg and the conjugated diene content to be >10%.

[0039] (2) Preparation of advanced glycation end products (AGEs): Whole milk powder and protein powder were mixed in a 4:1 ratio and placed in a 72°C incubator for 6 days, with shaking twice daily. ELISA was used to determine the AGE content to be >550 μg / g.

[0040] (3) Preparation of the pharmaceutical composition: The components are mixed in the following proportions: 80 parts of oxidized lipids, 4 parts of 7-ketocholesterol, 2 parts of 25-hydroxycholesterol, 8 parts of trans fatty acids, 200 parts of advanced glycation end products, 180 parts of casein, 320 parts of sucrose, including 20% ​​high fructose corn syrup, 145 parts of corn starch, 45 parts of cellulose, 30 parts of a mineral mixture, 8 parts of a vitamin mixture, and 20 parts of methionine. The mineral mixture contains 0.04 mg / kg of selenium and 70 mg / kg of magnesium; the vitamin mixture contains 0.4 mg / kg of folic acid, 1.8 mg / kg of vitamin B6, and 0.007 mg / kg of vitamin B12; and the composition does not contain choline. After all the components are thoroughly mixed, the mixture is made into granular feed and sealed and stored at 4°C.

[0041] In this example, methionine supplementation further disrupts the methionine cycle, leading to a more significant increase in homocysteine ​​levels and accelerating the progression of atherosclerosis. This combination can induce significant atherosclerotic lesions in rats in a relatively short period of time.

[0042] Example 3 Preparation of Oxidized Lipid-AGEs Composite Pharmaceutical Composition Promoting Atherosclerosis

[0043] This example provides another method for preparing an oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition, the specific steps of which are as follows:

[0044] (1) Preparation of Oxidized Lipids: Palm oil was heated at 178°C for 8 hours with stirring every half hour. The oil was cooled to room temperature and then used. Gas chromatography-mass spectrometry was used to determine the peroxide value (POV) to be >30 meq / kg and the conjugated diene content to be >12%.

[0045] (2) Preparation of advanced glycation end products (AGEs): Whole milk powder was placed in a 68°C incubator for 8 days, with stirring three times daily. ELISA was used to determine if the AGE content was >600 μg / g.

[0046] (3) Preparation of the pharmaceutical composition: Mix the following components in the following proportions: 90 parts of oxidized lipids, 6 parts of 7-ketocholesterol, 4 parts of 25-hydroxycholesterol, 10 parts of trans fatty acids, 300 parts of advanced glycation end products, 220 parts of casein, 360 parts of sucrose, including 20% ​​high fructose corn syrup, 165 parts of corn starch, 55 parts of cellulose, 40 parts of a mineral mixture, 12 parts of a vitamin mixture, and 22 parts of methionine. The mineral mixture contains 0.06 mg / kg of selenium and 80 mg / kg of magnesium; the vitamin mixture contains 0.6 mg / kg of folic acid, 2.2 mg / kg of vitamin B6, and 0.009 mg / kg of vitamin B12; and the composition does not contain choline. After all components are thoroughly mixed, the mixture is made into granular feed and sealed and stored at 4°C.

[0047] In this embodiment, the component content is at the upper limit, and the content of oxidized lipids and advanced glycation end products is significantly increased, which can produce a stronger pro-atherosclerotic effect and is suitable for accelerating the modeling process. Obvious atherosclerotic lesions can be observed within 8-10 weeks.

[0048] Example 4 Multi-target joint modeling method for coronary heart disease ischemia-reperfusion injury in rats

[0049] This example provides a method for establishing a multi-target model of coronary heart disease ischemia-reperfusion injury in rats using the pharmaceutical composition described in Example 1. The specific steps are as follows:

[0050] (1) Experimental Animals: 8-week-old male Sprague-Dawley rats weighing 200 ± 20 g were used. Rats were housed in an SPF animal room at a temperature of 23 ± 2°C, a humidity of 50 ± 10%, and a 12-h light / dark cycle. Experiments were conducted after one week of acclimatization.

[0051] (2) Feeding stage: Rats were fed in stages according to the following scheme:

[0052] Weeks 1-4 (pretreatment period): 40 g / head were fed daily with the pharmaceutical composition described in Example 1, and 0.8 ml of fat emulsion rich in oxidized lipids (oxidized lipids prepared in Example 1 and vegetable oil were mixed in a ratio of 1:3) was administered orally to establish endothelial dysfunction and insulin resistance.

[0053] Weeks 5-8 (accelerated phase): The rats were fed 45 g / head daily with the pharmaceutical composition described in Example 1 and gavaged with 1 ml of fat emulsion containing 7-ketocholesterol (7-ketocholesterol concentration was 0.5 mg / ml). Intermittent fasting cycles of 3 days of normal diet and 1 day of fasting were performed to promote foam cell formation and early plaque development.

[0054] Weeks 9-12 (vulnerable period): 50 g / head of the drug composition described in Example 1 were fed daily, and 1.2 ml of compound fat emulsion (containing oxidized lipids, 7-ketocholesterol and 25-hydroxycholesterol) was gavage at the same time to promote the formation of unstable plaques.

[0055] (3) Ischemia induction: When the rats weighed 250 g, the following treatments were performed:

[0056] First, a targeted delivery system was prepared. Lipid nanoparticles were prepared using a thin-film hydration method. The starting materials included phosphatidylcholine (Avanti Polar Lipids), cholesterol (Sigma-Aldrich), and DSPE-PEG2000 (Avanti Polar Lipids), mixed in a molar ratio of 60:30:10. Pituitin (Sigma-Aldrich, purity >98%) and endothelin-1 (Abcam, purity >95%) were loaded into the nanoparticles at doses of 0.03 μg / animal and 2 nmol / animal, respectively. The coronary artery-specific peptide CRPPR (Gill Biochem, purity >98%) was covalently coupled to DSPE-PEG2000 and modified on the nanoparticle surface. Dynamic light scattering (DLS) determined the nanoparticle size to be 180 ± 15 nm. The prepared targeted delivery system was administered via tail vein injection, and ischemic treatment was initiated 30 minutes later.

[0057] (4) Reperfusion control: The following non-invasive methods are used for reperfusion control:

[0058] Remote ischemic conditioning (RIC): Starting 30 minutes before ischemia, a mouse / rat tail cuff sphygmomanometer (IITC Life Science) was used to apply pressure alternately to the hind limbs of the rats for 4 cycles, each cycle consisting of 5 minutes of ischemia (cuff pressure set to 20 mmHg above tail systolic pressure) and 5 minutes of reperfusion (pressure released).

[0059] Ultrasound-mediated reperfusion control: An ultrasound therapy device (PHYSIOSON-Basic, Physiomed) was used at a frequency of 1.1 MHz, with gated ultrasound pulses based on the electrocardiogram (ECG) for 30 minutes. To enhance the effect, a microbubble contrast agent (SonoVue, Boehringer Ingelheim) was injected intravenously at a dose of 0.1 ml / kg 10 minutes before ultrasound treatment.

[0060] (5) Evaluation: Confirm the success of the model establishment through the following methods:

[0061] Circulating biomarker measurements: Blood samples were collected before ischemia, 30 minutes after ischemia, and 2 hours, 6 hours, 24 hours, and 72 hours after reperfusion to measure the following indicators:

[0062] MicroRNA panel: including miR-21, miR-22, and miR-155-5p, detected using RT-qPCR

[0063] Damage-associated molecular patterns (DAMPs): including HMGB1, S100A8 / A9, and heat shock proteins HSP60 and HSP70, detected using ELISA

[0064] Oxidative stress markers: malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), and protein carbonyls, measured using thiobarbituric acid reactive substances (TBARS) assay, ELISA, and spectrophotometry, respectively

[0065] Cardiac imaging assessment: Hyperpolarized 13C-pyruvate magnetic resonance imaging was performed 7 days after reperfusion using a 3T MRI scanner (Siemens) and a dedicated 13C quadrature birdcage coil to measure 13C-pyruvate, 13C-lactate, and 13C-bicarbonate signals.

[0066] Functional assessment: Pressure-volume loop analysis was performed 14 days after reperfusion. The left ventricle was accessed through the carotid artery cannula using a Millar pressure catheter system (2F, Millar) to measure parameters such as the end-systolic pressure-volume relationship (ESPVR) and the end-diastolic pressure-volume relationship (EDPVR).

[0067] Example 5 Multi-target joint modeling method for coronary heart disease ischemia-reperfusion injury in rats

[0068] This example provides a method for establishing a multi-target model of coronary heart disease ischemia-reperfusion injury in rats using the pharmaceutical composition described in Example 2. The specific steps are as follows:

[0069] (1) Experimental Animals: 7-week-old male Sprague-Dawley rats weighing 190 ± 15 g were used. Rats were housed in an SPF animal room at a temperature of 22 ± 2°C, a humidity of 55 ± 5%, and a 12-h light / dark cycle. Experiments were conducted after one week of acclimatization.

[0070] (2) Feeding stage: Rats were fed in stages according to a similar scheme as in Example 4, but using the pharmaceutical composition described in Example 2.

[0071] (3) Ischemia induction: When the rats weighed 250 g, the following treatments were performed:

[0072] Seven days before induction of ischemia, patients were treated with oral empagliflozin (Selleck, purity >99%) at 15 mg / kg once daily. Thirty minutes before ischemia, theophylline (Sigma-Aldrich, purity >99%) at 15 mg / kg was administered intravenously. The targeted delivery system was then prepared and administered according to the method described in Example 4.

[0073] (4) Reperfusion control: In addition to the method in Example 4, this example also adds pharmacological hypothermia treatment:

[0074] The HBN-1 combination regimen, consisting of a mixture of ethanol (10%, 0.8 ml / kg, intravenous injection), vasopressin (Sigma-Aldrich, 0.4 IU / kg, intravenous injection), and lidocaine (Midocaine, 1 mg / kg, intravenous injection), was administered 5 minutes after reperfusion. Rectal temperature was monitored using an electronic thermometer (YSI 427, YSI), and the body temperature was controlled to 33.5°C and maintained for 17–18 hours.

[0075] (5) Evaluation: Basically the same as Example 4, but this example also adds 18F-FDG PET imaging evaluation, which is performed 10 days after reperfusion using a small animal PET / CT scanner (Inveon, Siemens) with a dose of 20 MBq. Images are collected 45 minutes after intravenous injection to evaluate myocardial metabolic activity.

[0076] Example 6 Multi-target joint modeling method for coronary heart disease ischemia-reperfusion injury in rats

[0077] This example provides a method for establishing a multi-target model of coronary heart disease ischemia-reperfusion injury in rats using the pharmaceutical composition described in Example 3. The specific steps are as follows:

[0078] (1) Experimental Animals: Eight-week-old male Wistar rats weighing 210 ± 20 g were used. Rats were housed in an SPF animal room at a temperature of 24 ± 1°C, a humidity of 50 ± 10%, and a 12-h light / dark cycle. Experiments were initiated after one week of acclimatization.

[0079] (2) Feeding stage: Rats were fed in stages according to a similar scheme as in Example 4, but the pharmaceutical composition described in Example 3 was used.

[0080] (3) Ischemia induction: When the rats weighed 250 g, the following treatments were performed:

[0081] Seven days before ischemia induction, patients were treated with oral empagliflozin (20 mg / kg) once daily. Theophylline (15 mg / kg) was administered intravenously 30 minutes before ischemia. This example also employed a photochemical thrombosis method to enhance the ischemia induction effect: Rose Bengal (Sigma-Aldrich, 10 mg / kg) was injected intravenously. Five minutes later, a wireless implantable LED device (homemade, 540 nm wavelength, 5 mW / mm²) was used to irradiate the left coronary artery region for 10 minutes. The targeted delivery system was then administered as described in Example 4.

[0082] (4) Reperfusion control: In addition to the methods in Examples 4 and 5, this example also adds pulsed electromagnetic field (PEMF) treatment:

[0083] A magnetic therapy device (YT-1000A) was used with a frequency of 15 Hz and an intensity of 6 mT. Treatment was performed for 2 hours daily starting from reperfusion and lasting for 6 weeks.

[0084] (5) Evaluation: basically the same as Example 5, but this example also adds long-term follow-up of cardiac pressure-volume loop analysis, and performs an evaluation once 1, 2, 4, and 6 weeks after reperfusion to observe the recovery of cardiac function.

[0085] Example 7 Application of the Oxidized Lipid-AGEs Composite Atherosclerosis-Promoting Pharmaceutical Composition in Establishing a Rat Model of Coronary Heart Disease Ischemia-Reperfusion Injury

[0086] This example verifies the application effect of the pharmaceutical composition described in Example 1 in constructing a rat model of coronary heart disease ischemia-reperfusion injury.

[0087] Twenty-four 8-week-old male SD rats were randomly divided into three groups, each consisting of eight rats: a model group (treated according to the method of Example 4), a sham-operated group (given the same diet but without ischemia-reperfusion treatment), and a control group (given a normal diet without any treatment). After 12 weeks of feeding, plasma biochemical parameters, electrocardiogram changes, and myocardial injury markers were measured in each group. The results are shown in Table 1.

[0088] Table 1 Comparison of main indicators of rats in each group (n=8, mean±SD)

[0089] index Model Group Sham operation group control group Plasma total cholesterol (mg / dL) 326.5±28.7* 287.3±25.4* 92.6±13.8 Plasma triglycerides (mg / dL) 186.3±21.5* 165.2±19.7* 75.4±12.3 Homocysteine ​​(μmol / L) 31.2±4.6* 28.5±3.9* 11.3±2.1 Oxidized LDL (U / L) 56.3±8.2* 48.7±7.5* 23.8±4.2 ST segment elevation (mV) 0.42±0.06* 0.12±0.03 0.08±0.02 Cardiac troponin I (ng / mL) 12.6±2.3* 0.21±0.06 0.15±0.04 Myocardial creatine kinase-MB (U / L) 235.7±31.2* 76.3±12.5 68.5±10.7

[0090] Note: Compared with the control group, *P<0.01

[0091] Results showed that after 12 weeks of feeding the pharmaceutical composition described in Example 1, both the model and sham-operated rats developed significant dyslipidemia and hyperhomocysteinemia, demonstrating successful induction of the biochemical basis of atherosclerosis. Furthermore, in the model group subjected to ischemia-reperfusion treatment, ST-segment elevation and myocardial injury markers were significantly elevated, indicating successful induction of myocardial ischemia-reperfusion injury.

[0092] Further histopathological examination of the model group rats revealed significant foam cell accumulation and lipid deposition in the coronary arteries, intimal thickening, and lumen stenosis of approximately 40-60%. Myocardial tissue also exhibited characteristic changes of ischemia-reperfusion injury, including myocardial cell edema, degeneration, necrosis, and inflammatory cell infiltration. This further demonstrates the effectiveness of the pharmaceutical composition of the present invention in establishing a rat model of coronary heart disease with ischemia-reperfusion injury.

[0093] Comparative Example 1

[0094] This comparative example aims to verify the key role of oxidized lipids in promoting atherosclerosis. The oxidized lipids in the composition of Example 1 were replaced with ordinary vegetable oil, while the other components remained unchanged, and the model was constructed according to the method of Example 4.

[0095] Sixteen eight-week-old male SD rats were randomly divided into two groups, each consisting of eight rats: an Example group (using the composition of Example 1) and a Comparative Example group (using the composition after replacing oxidized lipids). After 12 weeks of feeding, the main blood lipid indicators and the degree of atherosclerosis in each group were measured. The results are shown in Table 2.

[0096] Table 2 Comparison of main indicators between the embodiment group and the comparative example group (n=8, mean±SD)

[0097] index Example Group Comparative group Plasma total cholesterol (mg / dL) 326.5±28.7* 245.8±25.3 Plasma low-density lipoprotein (mg / dL) 198.3±22.6* 145.2±18.7 Oxidized LDL (U / L) 56.3±8.2* 32.5±6.4 Coronary artery stenosis degree (%) 53.7±7.5* 28.4±5.2 Atherosclerosis Index (AI) 4.62±0.55* 2.78±0.43 Cardiac troponin I (ng / mL) 12.6±2.3* 7.3±1.8

[0098] Note: Compared with the control group, *P<0.01

[0099] The results showed that while the control group, lacking oxidized lipids, also developed dyslipidemia and atherosclerosis, the extent was significantly lower than that of the example group. The degree of coronary artery stenosis and myocardial damage markers were also significantly lower. This confirms the key role of oxidized lipids in promoting the formation of atherosclerosis and myocardial ischemia.

[0100] Comparative Example 2

[0101] This comparative example is intended to verify the role of micronutrient deficiency in promoting hyperhomocysteinemia and atherosclerosis. The vitamin mixture in the composition of Example 1 was replaced with a vitamin mixture containing normal levels of folic acid (2.0 mg / kg), vitamin B6 (7.0 mg / kg) and vitamin B12 (0.025 mg / kg), and choline (1200 mg / kg) was added. The other components remained unchanged, and the model was constructed according to the method of Example 4.

[0102] Sixteen eight-week-old male SD rats were randomly divided into two groups, each consisting of eight rats: an Example group (using the composition of Example 1) and a Comparative Example group (using a composition modified in vitamin content). After 12 weeks of feeding, homocysteine ​​levels and the degree of atherosclerosis in each group were measured. The results are shown in Table 3.

[0103] Table 3 Comparison of main indicators between the embodiment group and the comparative example group (n=8, mean±SD)

[0104] index Example Group Comparative group Homocysteine ​​(μmol / L) 31.2±4.6* 14.5±2.8 Endothelial function (FMD, %) 5.3±1.2* 12.7±2.5 Endothelial nitric oxide synthase (eNOS) activity (U / mg protein) 0.58±0.14* 1.26±0.25 Coronary artery stenosis degree (%) 53.7±7.5* 32.1±5.8 Atherosclerotic plaque area (mm²) 0.68±0.12* 0.35±0.08 Myocardial ischemic area (%) 38.5±5.6* 25.2±4.3

[0105] Note: Compared with the control group, *P<0.01

[0106] The results showed that the control group, which had normal micronutrient levels, had significantly lower homocysteine ​​levels than the example group, significantly higher endothelial function and nitric oxide synthase activity, and significantly reduced atherosclerosis and myocardial ischemia. This confirms the important role of strategic micronutrient deficiency in promoting hyperhomocysteinemia, endothelial dysfunction, and atherosclerosis.

[0107] Comparative Example 3

[0108] This comparative example aims to verify the role of the targeted delivery system in improving the accuracy and stability of myocardial ischemia induction. The pharmaceutical composition of Example 1 was used, but vasopressin (VP) was directly injected intraperitoneally during the ischemia induction stage at a dose of 2.5 U / ml, 1 ml / mouse. The targeted delivery system was not used, and the other steps were the same as in Example 4.

[0109] Sixteen SD rats that had been fed the composition of Example 1 for 12 weeks were randomly divided into two groups, each with 8 rats: the Example group (using the targeted delivery system) and the Control group (direct injection of VP). After ischemia-reperfusion treatment, the uniformity and stability of myocardial ischemia were evaluated. The results are as follows: Figure 1 and as shown in Table 4.

[0110] Table 4 Comparison of myocardial ischemia characteristics between the example group and the comparative example group (n=8, mean±SD)

[0111] index Example Group Comparative group ECG ST segment elevation consistency (coefficient of variation, %) 12.5±2.3* 35.7±6.8 Homogeneity of myocardial ischemia area (coefficient of variation, %) 15.3±3.1* 42.6±8.5 Ischemia induction success rate (%) 100* 75 Accuracy of myocardial ischemic area (bias, %) 8.5±2.1* 26.3±5.7 Animal survival rate (%) 100* 62.5 Blood pressure fluctuation range (mmHg) 12.6±3.4* 38.5±7.2

[0112] Note: Compared with the control group, *P<0.01

[0113] The results showed that the Example group using the targeted delivery system significantly outperformed the control group using direct VP injection in terms of consistency, uniformity, and stability in inducing myocardial ischemia, with significantly higher success rates and survival rates, while also significantly reducing systemic side effects (such as blood pressure fluctuations). This confirms the key role of the targeted delivery system of the present invention in improving the accuracy and stability of inducing myocardial ischemia.

[0114] Comparative Example 4

[0115] This comparative example aims to verify the effect of non-invasive reperfusion control method in improving model survival rate and reducing additional trauma interference. Ischemia was induced using the pharmaceutical composition and targeted delivery system of Example 1, but a traditional surgical ligation / release method was used during the reperfusion control stage: after anesthetizing the rat, the chest was opened to expose the heart, and the left anterior descending coronary artery was ligated with 6-0 silk suture for 30 minutes and then released. Other steps were the same as in Example 4.

[0116] Sixteen SD rats, fed the composition of Example 1 for 12 weeks, were randomly divided into two groups of eight: the Example group (using non-invasive reperfusion control) and the Comparative Example group (using surgical ligation / release). After ischemia-reperfusion treatment, the survival rate and inflammatory response of the model were evaluated. The results are shown in Table 5.

[0117] Table 5 Comparison of model quality between the example group and the comparative example group (n=8, mean±SD)

[0118] index Example Group Comparative group Operation time (minutes) 35.2±6.3* 95.7±15.4 Operation difficulty rating (1-10 points) 3.5±0.7* 8.7±1.2 Animal survival rate (%) 100* 50 Serum inflammatory factor IL-6 (pg / ml) 125.6±25.3* 387.5±65.2 Serum inflammatory factor TNF-α (pg / ml) 45.8±10.2* 196.3±32.7 Non-cardiac tissue damage index 0.32±0.08* 3.65±0.75 Homogeneity of myocardial infarct size (coefficient of variation, %) 13.5±2.8* 38.2±7.5

[0119] Note: Compared with the control group, *P<0.01

[0120] The results showed that the non-invasive reperfusion control method significantly outperformed the control group (using surgical ligation / release) in terms of ease of use, survival rate, and inflammatory response control. It also reduced the impact of non-cardiac tissue damage on experimental results and improved the uniformity of myocardial infarction area. This demonstrates the significant value of the non-invasive reperfusion control method of the present invention in improving model quality and reducing experimental interference factors.

[0121] Example 8 Comparison of different targeted delivery systems

[0122] To optimize the performance of the targeted delivery system, this example compares three different targeted delivery systems: Type A (loaded only with vasopressin VP), Type B (loaded with a mixture of VP and endothelin-1), and Type C (same as Type B, but with the addition of the adenosine antagonist 8-phenyltheophylline). All three systems utilize the same lipid nanoparticles modified with the coronary artery-specific peptide CRPPR.

[0123] Twenty-four SD rats fed the composition of Example 1 for 12 weeks were randomly divided into three groups of eight rats each. Ischemia was induced using the Type A, Type B, and Type C targeted delivery systems, respectively. Other procedures were the same as in Example 4. The effects of the three systems were compared by evaluating the characteristics of myocardial ischemia and changes in biochemical markers. The results are shown in Table 6.

[0124] Table 6 Comparison of the effects of different targeted delivery systems (n=8, mean±SD)

[0125] index Type A system Type B system C-type system ECG ST segment elevation (mV) 0.25±0.06 0.42±0.08* 0.55±0.09*# Myocardial ischemic area (%) 22.5±4.3 38.7±5.6* 45.3±6.2*# Serum troponin I (ng / ml) 5.8±1.2 12.6±2.5* 18.3±3.1*# Ischemia induction success rate (%) 75 100* 100* Myocardial ischemia homogeneity (coefficient of variation, %) 28.3±5.2 12.5±2.8* 10.2±2.3* Myocardial lactate content (μmol / g) 3.2±0.7 6.5±1.2* 8.1±1.5*# ATP content (μmol / g) 4.5±0.8 2.3±0.5* 1.7±0.4*#

[0126] Note: Compared with type A system, *P<0.01; compared with type B system, #P<0.05

[0127] The results showed that the type B system (a mixture of VP and endothelin-1) was significantly superior to the type A system using VP alone in terms of myocardial ischemia induction and stability, while the type C system (adding an adenosine antagonist) further enhanced the ischemic effect, but the degree of improvement was smaller than that of the type B system. Considering the balance between effect and complexity, the type B system (a mixture of VP and endothelin-1) was determined to be the optimal choice.

[0128] Example 9 Pharmacodynamic application verification after model establishment

[0129] To verify the application value of the coronary heart disease ischemia-reperfusion injury model constructed by the present invention in pharmacodynamic studies, this example evaluated the protective effect of a cardioprotective agent, ATP-sensitive potassium channel opener nicorandil (Sigma-Aldrich, purity >98%), in this model.

[0130] Thirty-two rats with a coronary heart disease ischemia-reperfusion injury model established according to the method of Example 4 were randomly divided into four groups, each consisting of eight rats: a control group (administered with normal saline), a low-dose group (nicorandil 0.5 mg / kg), a medium-dose group (nicorandil 1.0 mg / kg), and a high-dose group (nicorandil 2.0 mg / kg). All drugs were administered via the tail vein 15 minutes before ischemia. The myocardial protective effect of nicorandil was compared by evaluating myocardial injury markers, cardiac function parameters, and pathological changes. The results are shown in Table 7.

[0131] Table 7 Myocardial protective effect of different doses of Nicorandil (n=8, mean±SD)

[0132] index control group Low-dose group Medium dose group High-dose group Serum troponin I (ng / ml) 12.6±2.5 8.5±1.7* 5.2±1.1*# 3.8±0.9*#△ Infarct area / risk area ratio (%) 65.3±8.6 45.2±6.5* 32.7±5.3*# 25.4±4.2*#△ Left ventricular ejection fraction (%) 40.5±5.3 52.7±6.8* 63.5±7.2*# 68.3±7.5*# Left ventricular systolic pressure (mmHg) 86.5±12.3 102.3±15.6* 115.7±16.2*# 122.5±17.3*# Myocardial cell apoptosis rate (%) 35.6±5.8 24.3±4.5* 15.8±3.2*# 10.5±2.6*#△ Oxidative stress index (MDA / SOD) 12.5±2.3 8.6±1.7* 5.3±1.2*# 4.2±1.0*# Inflammation score (0-10 points) 8.3±1.2 6.5±0.9* 4.2±0.7*# 3.5±0.6*#

[0133] Note: Compared with the control group, *P<0.01; compared with the low-dose group, #P<0.01; compared with the medium-dose group, △P<0.05

[0134] The results showed that nicorandil exhibited significant dose-dependent myocardial protection in the ischemia-reperfusion injury model of coronary heart disease established by the present invention, alleviating myocardial damage, improving cardiac function, and inhibiting oxidative stress and inflammatory responses. This result is consistent with the protective effects of nicorandil on patients with coronary heart disease in clinical studies, confirming the good clinical relevance and pharmacodynamic application value of the present invention's model.

[0135] Example 10 Model stability and repeatability evaluation

[0136] To evaluate the stability and repeatability of the coronary heart disease ischemia-reperfusion injury model constructed in the present invention, the model was constructed in three independent laboratories according to the method of Example 4, with 8 model rats in each laboratory, for a total of 24 rats, and then the consistency of the main evaluation indicators was compared.

[0137] Table 8 Comparison of the main indicators of the model established by three laboratories (n = 8 / laboratory, mean ± SD)

[0138] index Laboratory A Laboratory B Laboratory C Coefficient of variation (%) Plasma total cholesterol (mg / dL) 326.5±28.7 315.3±27.5 332.7±30.2 6.8 Homocysteine ​​(μmol / L) 31.2±4.6 29.5±4.2 32.3±4.8 8.5 Coronary artery stenosis degree (%) 53.7±7.5 50.2±7.1 55.8±8.2 9.2 ECG ST segment elevation (mV) 0.42±0.06 0.40±0.07 0.43±0.08 10.3 Myocardial infarction area (%) 38.5±5.6 36.8±5.3 39.7±6.0 8.7 Serum troponin I (ng / ml) 12.6±2.3 11.8±2.1 13.2±2.5 11.5 Survival rate (%) 100 100 87.5 - Model success rate (%) 100 87.5 100 -

[0139] The results showed that the models established by the three laboratories showed good consistency in key evaluation indicators, with coefficients of variation below 15%, and high survival and model success rates. This confirms that the coronary heart disease ischemia-reperfusion injury model constructed by this invention has good stability and reproducibility, making it suitable for promotion and application across different laboratories.

[0140] The oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition and the multi-target joint modeling method for coronary heart disease ischemia-reperfusion injury in rats provided by the present invention have the following industrial application values:

[0141] 1. Suitable for screening and evaluation of new drugs for cardiovascular diseases, especially for the development of drugs for coronary heart disease, atherosclerosis and myocardial ischemia-reperfusion injury;

[0142] 2. It can be used for basic research and preclinical evaluation of cardioprotective strategies, such as remote ischemic preconditioning and pharmacological cooling;

[0143] 3. Suitable for the study of the pathological mechanisms of coronary heart disease, providing an ideal platform for in-depth understanding of the molecular mechanisms of atherosclerosis, myocardial ischemia and reperfusion injury;

[0144] 4. It can serve as the basis for developing personalized treatment plans for cardiovascular diseases and evaluate the effects of different treatment strategies under various pathological conditions;

[0145] 5. This method can be widely adopted by pharmaceutical companies, CRO companies and cardiovascular disease research institutions, and has good promotion and application prospects.

[0146] In summary, the present invention establishes a rat model that is closer to the pathological process of human coronary heart disease through the combined action of multiple targets. While improving the model stability, success rate and clinical relevance, it also has good operability and promotion value, providing an important tool for coronary heart disease-related research and drug development.

[0147] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for promoting atherosclerosis by combining oxidized lipids and AGEs, characterized in that: Calculated by weight, including: 80-90 parts of oxidized lipids, wherein the oxidized lipids are prepared by heating edible oils and fats at 180° C. for 6-8 hours; 4-6 parts of 7-ketocholesterol; 2-4 parts of 25-hydroxycholesterol; 8-10 parts of trans fatty acids; 200-300 parts of advanced glycation end products, wherein the advanced glycation end products are prepared by treating milk powder at 70°C for 7 days; 180-220 parts of casein; 320-360 parts sucrose, including 20% ​​high fructose corn syrup; 145-165 parts corn starch; 45-55 parts of cellulose; 30-40 parts of mineral mixture; 8-12 parts of a vitamin mixture, wherein the vitamin mixture contains 0.4-0.6 mg / kg of folic acid, 1.8-2.2 mg / kg of vitamin B6, and 0.007-0.009 mg / kg of vitamin B12.

2. The oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition according to claim 1, characterized in that: The mineral mixture contains 0.04-0.06 mg / kg of selenium and 70-80 mg / kg of magnesium; and the composition does not contain choline.

3. The oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition according to claim 1, characterized in that: The trans fatty acid includes trans palmitoleic acid; and the composition further comprises 18-22 parts of methionine.

4. A multi-target combined modeling method for coronary heart disease ischemia-reperfusion injury in rats, characterized in that: The following steps are involved: Step 1, feeding stage: feeding rats with the oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition according to any one of claims 1 to 3 in three stages; weeks 1 to 4 are the pretreatment stage, with a daily feed amount of 40 grams per rat, and 0.8 ml of fat emulsion rich in oxidized lipids is administered by gavage; weeks 5 to 8 are the acceleration stage, with a daily feed amount of 45 grams per rat, and 1 ml of fat emulsion containing 7-ketocholesterol is administered by gavage, and an intermittent fasting cycle of 3 days of normal diet and 1 day of fasting is performed; weeks 9 to 12 are the vulnerable stage, with a daily feed amount of 50 grams per rat, and 1.2 ml of compound fat emulsion is administered by gavage; Step 2: Ischemia induction: When the rats weighed 250 grams, they were intravenously injected with a targeted delivery system comprising lipid nanoparticles loaded with vasopressin and endothelin-1, the lipid nanoparticles having a particle size of less than 200 nanometers and surface-modified with the coronary artery-specific peptide CRPPR. The vasopressin dose was 0.03 micrograms per rat, and the endothelin-1 dose was 2 nanomoles per rat. Ischemia was initiated 30 minutes after administration. Step 3: Reperfusion control: Remote ischemic conditioning was performed using pressure cuffs on both limbs of the rat for four cycles, each consisting of 5 minutes of ischemia and 5 minutes of reperfusion, starting 30 minutes before ischemia. Simultaneously, ultrasound-mediated reperfusion control was performed using cardiac-gated ultrasound pulses at a frequency of 1-1.3 MHz for 30 minutes. Step 4: Evaluation: Confirm the success of the model establishment by measuring circulating biomarkers, performing cardiac imaging and functional assessment.

5. The method according to claim 4, characterized in that Step 2 also includes oral administration of empagliflozin 10-20 mg / kg starting 7 days before ischemia induction and intravenous injection of theophylline 15 mg / kg 30 minutes before ischemia.

6. The method according to claim 4, characterized in that Step three also includes pharmacological hypothermia using the HBN-1 combination regimen, which includes a mixture of ethanol, vasopressin, and lidocaine to control the body temperature to 33.5°C and maintain it for 17-18 hours, starting 5 minutes after reperfusion.

7. The method according to claim 4, characterized in that Step three also includes applying a pulsed electromagnetic field with a frequency of 15 Hz and an intensity of 6 millitesla for 2 hours per day for 6 weeks.

8. The method according to claim 4, characterized in that The circulating biomarkers described in step 4 include: a microRNA panel, including miR-21, miR-22, and miR-155-5p; damage-associated molecular patterns, including HMGB1, S100A8 / A9, heat shock proteins HSP60 and HSP70; oxidative stress markers, including malondialdehyde, 8-hydroxydeoxyguanosine, and protein carbonyls; the imaging assessment includes hyperpolarized 13C-pyruvate magnetic resonance imaging and positron emission tomography; the functional assessment includes pressure-volume loop analysis, using a Millar catheter to measure ESPVR and EDPVR parameters.

9. A method for constructing a rat model of coronary heart disease ischemia-reperfusion injury, characterized in that: The model is constructed using the method described in any one of claims 4 to 8.

10. Use of the oxidized lipid-AGEs composite atherosclerosis-promoting pharmaceutical composition according to any one of claims 1 to 3 in constructing a rat model of coronary heart disease ischemia-reperfusion injury, characterized in that: By establishing endothelial dysfunction, insulin resistance, promoting foam cell formation and early plaque development, the plasma cholesterol level in the model was greater than 300 mg / dL and the homocysteine ​​level was greater than 25 μmol / L, thereby improving the stability, success rate and clinical relevance of the rat coronary heart disease ischemia-reperfusion injury model.

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