A method of modeling diabetes mellitus atherosclerosis in a melatonin receptor 1a apolipoprotein e double knockout

Abstract

The application discloses a melatonin receptor 1A and apolipoprotein E double-knockout diabetes and atherosclerosis modeling method, relates to the technical field of experimental animal model construction, and comprises the following steps: crossing and breeding MTNR1A gene homozygous knockout mice and APOE gene homozygous knockout mice with a C57BL / 6J genetic background to obtain MTNR1A / APOE double-gene homozygous knockout mice; selecting 6-week-old male MTNR1A / APOE double-gene homozygous knockout mice, and adaptively feeding the mice for 2 weeks; after fasting for 12 hours, injecting 1 time of streptozotocin citric acid solution per day at 50 mg / kg of body weight, and continuously injecting for 5 days; detecting random blood glucose two weeks after the completion of STZ injection to confirm the modeling of diabetes; feeding high-fat feed containing 21% of fat and 0.21% of cholesterol for 12 weeks to induce the formation of diabetes and atherosclerosis vulnerable plaques; and detecting blood glucose, blood lipids and aortic and carotid pathological indexes. The application achieves the support of a high-stability and high-repeatability double-knockout modeling system for pathogenesis analysis, target verification and diabetes and AS drug screening.

Description

Technical Field

[0001] This invention relates to the field of experimental animal model construction technology, and in particular to a method for creating a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E. Background Technology

[0002] In the field of metabolic cardiovascular diseases, apolipoprotein E (APOE) gene knockout mice, due to impaired endogenous lipid clearance, can spontaneously form aortic plaques after induction with a high-fat diet, and have become an internationally recognized standard model of atherosclerosis. To further simulate the pathological characteristics of accelerated plaque vulnerability in clinical type 2 diabetes, current techniques generally employ a chemical induction strategy combining APOE single-gene knockout mice with intraperitoneal injection of streptozotocin (STZ): typically, 8-10 week old male mice are fasted and then continuously injected with a certain dose of STZ to destroy pancreatic β cells, followed by feeding with a high-fat diet containing 15%-45% fat and 0.1%-0.25% cholesterol for 8-16 weeks, thereby constructing a diabetes mellitus combined with atherosclerosis (AS). This method is relatively simple to operate, can reproduce the comorbid state of hyperglycemia and vascular lipid deposition to a certain extent, and provides a preliminary vehicle for exploring the synergistic effects of hypoglycemic and anti-AS drugs. To date, it has produced a large amount of basic medical evidence and constitutes a routine research model construction method in this field.

[0003] The aforementioned modeling protocol based on APOE single-gene knockout has gradually revealed limitations in specific dimensions during long-term experimental implementation, particularly in the evaluation of phenotypic consistency and reproducibility within the model group. Due to APOE... - / - The inherent genetic heterogeneity in mice, coupled with individual fluctuations in the degree of β-cell damage caused by STZ, results in high dispersion in existing models for maintaining blood glucose homeostasis and quantifying plaque morphology. Specifically, the intra-group coefficient of variation for random blood glucose after model establishment often falls within the range of 32%-41%, the coefficient of variation for the core area of ​​aortic sinus necrosis even exceeds 46%, and individual deviations in right carotid artery-related pathological parameters can exceed 80%. This significant data dispersion forces researchers to increase sample size to offset experimental errors, increasing both consumable and feeding costs and weakening the comparability of conclusions across batches. Although optimizing the STZ dosing rhythm or fine-tuning the feed ratio can marginally improve uniformity, existing approaches are still unable to fundamentally reduce the variability of key pathological indicators due to the compensatory limits imposed by the background of single gene deletion. The inventive concept upon which this invention is based addresses this deficiency by introducing secondary gene modification to reconstruct the metabolic-vascular interface and obtain a phenotypically convergent model of vulnerable plaques in diabetic ankylosing spondylitis. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for establishing a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E, which solves the problems of poor model stability and difficulty in reproducibility evaluation caused by the high intragroup phenotypic dispersion, large coefficient of variation of key pathological indicators and difficulty in fundamentally compressing the existing APOE single gene knockout diabetic atherosclerosis model due to gene heterogeneity and STZ damage fluctuations.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a method for establishing a diabetic arteriosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E, which includes crossbreeding MTNR1A gene homozygous knockout mice with C57BL / 6J genetic background and APOE gene homozygous knockout mice as parents, and screening to obtain MTNR1A / APOE double gene homozygous knockout mice. Six-week-old male MTNR1A / APOE double gene homozygous knockout mice were selected and acclimatized for 2 weeks. After fasting for 12 hours, administer streptozotocin citrate solution once daily at a dose of 50 mg / kg body weight for 5 consecutive days. Two weeks after STZ injection, random blood glucose levels were tested to confirm the establishment of the diabetes model. Feeding patients with a high-fat diet containing 21% fat and 0.21% cholesterol for 12 weeks induced the formation of vulnerable plaques for diabetic atherosclerosis. Detect blood glucose, blood lipids, and pathological indicators of the aorta and carotid arteries to complete model identification.

[0007] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout method for establishing a diabetic atherosclerosis model according to the present invention, the hybridization breeding specifically includes: crossing MTNR1A homozygous knockout mice with APOE homozygous knockout mice to obtain F1 generation MTNR1A + / - APOE + / - Double heterozygous mice were used, and F1 siblings were mated to obtain F2 generation. Genomic DNA from the tail tip of F2 mice was subjected to MTNR1A and APOE gene-specific PCR amplification and agarose gel electrophoresis identification to screen for MTNR1A. - / - APOE - / - Double-gene homozygous knockout mice were propagated and preserved; APOE single-gene knockout mice of the same age, sex, and background were bred simultaneously as controls.

[0008] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the adaptive feeding environment is a temperature of 22-25℃ and a humidity of 50%-60%, using a 12h light / 12h dark cycle, with mice having free access to food and water; the STZ citric acid solution is made by freshly dissolving streptozotocin in 0.1mol / L, pH 4.5 citrate buffer, while the control group is injected with an equal volume of citrate buffer.

[0009] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout method for establishing a diabetic atherosclerosis model according to the present invention, the following steps are taken: random blood glucose is measured every two weeks after STZ injection, and the diabetic model is considered successful when the random blood glucose of mice is >16.7mmol / L for three consecutive times; after 12 weeks of high-fat feeding, fasting is performed for 12 hours, and blood is taken from the orbital cavity to detect fasting blood glucose and blood lipid indicators.

[0010] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the pathological index detection specifically involves: dissecting the entire aorta from the heart to the iliac artery and performing gross Oil Red O staining; separating the heart and right common carotid artery with the aorta and performing Oil Red O staining, H&E staining, and Masson staining in sequence to determine plaque area, lipid deposition, lipid necrosis core area, fiber deposition, and fibrous cap thickness, and calculating the coefficient of variation within each group.

[0011] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the resulting model, compared with APOE single gene knockout diabetic atherosclerosis mice, has a random blood glucose group intra-group coefficient of variation of 16.15%~25.76% (single knockout is 32.16%~41.19%), a fasting blood glucose value of 22.76 mmol / L and an intra-group coefficient of variation of 19.91% (single knockout mean is 16.94 mmol / L, coefficient of variation is 30.88%).

[0012] As a preferred embodiment of the melatonin receptor 1A and apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the following parameters are specified: Aortic sinus H&E staining showed a mean plaque area ratio of 30.80% and a coefficient of variation of 9.55%, and a mean necrotic core area ratio of 21.71% and a coefficient of variation of 23.95%; right carotid artery H&E staining showed a mean plaque area ratio of 79.35% and a coefficient of variation of 21.49%, and a mean necrotic core area ratio of 24.85% and a coefficient of variation of 43.81%. All these parameters are significantly higher than those corresponding to APOE single knockout and have lower coefficients of variation.

[0013] As a preferred embodiment of the melatonin receptor 1A and apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the following features are observed: Oil Red O staining of the aortic sinus shows a mean lipid deposition area ratio of 16.98% and a coefficient of variation of 17.88% (10.96% and 23.76% for APOE single knockout); Masson staining shows a mean fibrin deposition amount of 8.05% and a coefficient of variation of 21.78%, and a mean fibrin cap thickness of 4.85 μm and a coefficient of variation of 42.48% (7.60 μm for APOE single knockout), exhibiting a vulnerable plaque phenotype with a thin fibrin cap and a large necrotic core.

[0014] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the mouse model obtained by the modeling method is used to study the mechanism of MTNR1A signaling pathway regulation of vulnerable plaque formation in diabetic patients with atherosclerosis.

[0015] As a preferred embodiment of the melatonin receptor 1A apolipoprotein E double knockout modeling method for diabetic atherosclerosis described in this invention, the mouse model obtained by the modeling method is used to screen and evaluate candidate drugs or intervention targets for hypoglycemic, anti-atherosclerotic, and stabilizing vulnerable plaques.

[0016] The beneficial effects of this invention are as follows: By using MTNR1A and APOE homozygous knockout mice with a C57BL / 6J background as parents for hybridization and PCR electrophoresis genotyping, a standardized construction of a double-gene deletion genetic background was achieved, eliminating compensatory redundancy in single-gene models and providing a host platform with excellent uniformity for metabolic-vascular interaction studies. By selecting male double knockout mice for adaptive rearing and then continuously injecting STZ intraperitoneally at a predetermined dose followed by long-term feeding with a high-fat diet, combined with time-series blood glucose monitoring and multi-stain pathological identification, the synchronous induction of diabetic hyperglycemic homeostasis and vulnerable atherosclerotic plaques was achieved, accurately replicating the clinical high-risk phenotype of thin fibrous cap and large necrotic core. The cascading effect of the above steps fundamentally compressed the intra-group dispersion of the model in terms of glucose metabolism and plaque morphology, significantly reducing the experimental sample requirements and cross-batch differences, ultimately achieving a highly stable and highly reproducible double knockout modeling system to support pathogenesis analysis, target validation, and screening of hypoglycemic and anti-AS drugs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1The double knockout model of this invention differs from the traditional APOE. - / - Comparison of intra-group variability in random blood glucose levels in single knockout models; Figure 2 This is a statistical comparison chart of the differences in fasting blood glucose between the two models mentioned above. Figure 3 The above two models show the results of whole aorta gross Oil Red O staining and the statistical comparison of lipid deposition between the groups. Figure 4 The above two models show the H&E staining results of the aortic sinus, and the statistical comparison of the differences in plaque area ratio and necrotic core area ratio within the groups. Figure 5 The above two models show the H&E staining results of the right carotid artery, and the statistical comparison of the differences in plaque area ratio and necrotic core area ratio within the groups. Figure 6 The above two models show the results of Oil Red O staining of the aortic sinus and the statistical comparison of the differences in lipid deposition between the groups. Figure 7 The above two models show the results of Masson's trichrome staining of the aortic sinus, and statistical comparison of the differences between the groups in collagen deposition and fibrous cap thickness. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Reference Figures 1-7 As one embodiment of the present invention, this embodiment provides a method for inducing diabetic atherosclerosis by double knockout of melatonin receptor 1A and apolipoprotein E, comprising the following steps: The following is in conjunction with the appendix Figure 1 The structure of the present invention will be described in detail.

[0023] Example 1:

[0024] 1.1 Laboratory Animals C57BL / 6J Background MTNR1A - / - Mice, APOE - / - The mice were all from an animal experimental research center. 1.2 Main reagents and feed STZ reagent (Sigma, catalog number: S0130), citrate buffer (self-made, 0.1 mol / L, pH 4.5), high-fat diet (21% fat, 0.21% cholesterol, Research Diet), blood glucose test kit (Nanjing Jiancheng Bioengineering Institute), blood lipid test kit (Ningbo Meikang Biotechnology Co., Ltd.). 1.3 Breeding and Grouping MTNR1A - / - Mice and APOE - / - MTTNR1A was obtained through mouse hybridization and PCR identification. - / - APOE - / - Double homozygous knockout mice: 30 eight-week-old male double knockout mice were selected as the experimental group, and 30 age-matched male APOE- / - mice were selected as the control group. They were acclimatized for 1 week.

[0025] 1.4 Construction Steps 1.4.1 STZ injection: Mice in both groups were fasted for 12 hours and then injected intraperitoneally with STZ citric acid solution at a dose of 50 mg / kg once a day for 5 consecutive days. 1.4.2 High-fat feeding: All animals were fed a high-fat diet for 12 consecutive weeks, with free access to food and water; 1.4.3 Indicator Detection: Random blood glucose was measured in mice two weeks after intraperitoneal injection of STZ, and thereafter every two weeks. After 12 weeks of high-fat diet, fasting for 12 hours was performed, and fasting blood glucose and lipid-related indicators were measured. Subsequently, the mice were sacrificed, and the entire aorta from the heart to the iliac artery was dissected and grossly stained with Oil Red O. The heart and right common carotid artery with the aorta were separated and stained with Oil Red O, H&E, and Masson staining. Indicators included plaque area, lipid deposition, lipid necrosis core area, fiber deposition, and fiber cap thickness. The within-group coefficient of variation for each indicator was calculated to complete the model phenotype and stability identification.

[0026] 1.5 Model Validation Following intraperitoneal injection of STZ (1–12 weeks), the coefficient of variation within the random blood glucose group in standard APOE- / - mice ranged from 32.16% to 41.19%, and the MTNR1A... - / - APOE - / - The coefficient of variation within the random blood glucose group in mice ranged from 16.15% to 25.76%, which was significantly lower than that of conventional APOE.- / - mice ( Figure 1 A, B).

[0027] When STZ injection combined with a high-fat diet was induced for 12 weeks, the standard APOE - / - The fasting blood glucose level in mice was 16.94 mmol / L, with an within-group coefficient of variation of 30.88%; MTNR1A - / - APOE - / - The fasting blood glucose level in mice was 22.76 mmol / L, with a coefficient of variation of 19.91% within the group. There was a significant difference in fasting blood glucose levels between the two groups (P < 0.05). Figure 2 ).

[0028] Gross Oil Red O staining results of the aorta showed that ordinary APOE - / - The mean percentage of lipid deposition area in the entire aorta of the mouse model was 12.11%, with an intragroup coefficient of variation of 40.24; MTNR1A - / - APOE - / - The mean percentage of total aortic lipid deposition area in the double knockout mouse model was 24.14%, with an intragroup coefficient of variation of 40.77%. MTNR1A - / - APOE - / - The ratio of total aortic plasma deposition area in the double knockout mouse model was significantly higher than that in the standard APOE model. - / - Mice (P<0.05) Figure 3 ).

[0029] Oil Red O staining results of the aortic sinus showed that ordinary APOE - / - The mean ratio of lipid deposition area in aortic root plaques in the mouse model was 10.96%, with an intragroup coefficient of variation of 23.76; MTNR1A - / - APOE - / - The mean lipid deposition area ratio of aortic root plaques in the double knockout mouse model was 16.98%, with an intragroup coefficient of variation of 17.88%. MTNR1A - / - APOE - / - The ratio of lipid deposition area in aortic root plaques in the double knockout mouse model was significantly higher than that in the standard APOE model. - / - Mice (P<0.01) Figure 6 ) H&E staining results of the aortic sinus showed that the mean area ratio of aortic root plaques in the standard APOE- / - mouse model was 25.82%, with an intra-group coefficient of variation of 23.35%; the mean area ratio of necrotic cores was 9.97%, with an intra-group coefficient of variation of 46.01%. MTNR1A - / - APOE - / -The mean percentage of aortic root plaque area in the double knockout mouse model was 30.80%, with an intra-group coefficient of variation of 9.55%; the mean percentage of necrotic core area was 21.71%, with an intra-group coefficient of variation of 23.95%. Figure 4 AC).

[0030] H&E staining of the right carotid artery showed that ordinary APOE - / - In the mouse model, the mean percentage of plaque area in the right carotid artery was 60.55%, with an within-group coefficient of variation of 47.14%; the mean percentage of necrotic core area was 8.52%, with an within-group coefficient of variation of 81.82%. MTNR1A - / - APOE - / - In the double knockout mouse model, the mean percentage of plaque area in the right carotid artery was 79.35%, with an intra-group coefficient of variation of 21.49%; the mean percentage of necrotic core area was 24.85%, with an intra-group coefficient of variation of 43.81%. Figure 5 AC). MTNR1A - / - APOE - / - The double knockout mouse model showed significantly larger plaque areas and lipid necrosis core areas in the aortic root and right carotid artery compared to the standard APOE model. - / - The coefficient of variation within the group was much lower than that in mice (P<0.05).

[0031] Masson staining of the aortic sinus showed that MTNR1A - / - APOE - / - The mean percentage of aortic root fibrosis area in the double knockout mouse model was 8.05%, with an intragroup coefficient of variation of 21.78; ordinary APOE... - / - The mean percentage of aortic root plaque area in the mouse model was 13.14%, with an within-group coefficient of variation of 30.95%. MTNR1A - / - APOE - / - The mean thickness of the fibrous cap of aortic root plaque in the double knockout mouse model was 4.85 μm, with an intragroup coefficient of variation of 42.48%; ordinary APOE - / - The mean thickness of the fibrous cap of the aortic root plaque in the mouse model was 7.60 μm, and the coefficient of variation within the group was 46.64%. Figure 7 AC). MTNR1A - / - APOE - / - The amount of fibrous deposition and the thickness of the fibrous cap in the aortic root of the double knockout mouse model were significantly lower than those of the standard APOE. - / - Mice (P<0.05).

[0032] The above results show that, compared to ordinary APOE - / - Single knockout mice, MTNR1A - / - APOE - / -Double knockout mice, induced by STZ combined with a high-fat diet, exhibited higher blood glucose levels and more stable glucose metabolism phenotypes. The intra-group coefficients of variation for both random and fasting blood glucose were significantly reduced, effectively improving the shortcomings of traditional models, such as large blood glucose fluctuations and poor model uniformity. Simultaneously, MTNR1A... - / - APOE - / - Double knockout mice showed significantly increased lipid deposition area, total plaque area, and lipid necrosis core area in the aortic root and carotid arteries, while exhibiting reduced plaque fiber deposition and significantly thinner fibrous caps, displaying typical pathological characteristics of vulnerable plaques. Furthermore, the intragroup coefficients of variation for all key pathological indicators in double knockout mice were significantly lower than those in APOE- / - single knockout mice, greatly reducing individual model dispersion and significantly improving the overall stability and homogeneity of the diabetic atherosclerosis model, thus better reflecting the pathological phenotype of clinical type 2 diabetes mellitus complicated by vulnerable plaques.

[0033] In summary, this invention achieves standardized construction of a double-gene deletion genetic background by using C57BL / 6J background MTNR1A and APOE homozygous knockout mice as parents for hybridization and PCR electrophoresis genotyping, eliminating compensatory redundancy in single-gene models and providing a host platform with excellent uniformity for metabolic-vascular interaction studies. By selecting male double knockout mice for adaptive rearing and then continuously injecting STZ intraperitoneally at predetermined doses followed by long-term feeding with a high-fat diet, coupled with time-series blood glucose monitoring and multi-stain pathological identification, the simultaneous induction of diabetic hyperglycemic homeostasis and vulnerable atherosclerotic plaques was achieved, accurately replicating the clinical high-risk phenotype of thin fibrous cap and large necrotic core. The cascading effect of the above steps fundamentally compresses the intra-group dispersion of the model in glucose metabolism and plaque morphology, significantly reducing experimental sample requirements and cross-batch differences, ultimately achieving a highly stable and reproducible double knockout modeling system to support pathogenesis analysis, target validation, and screening of hypoglycemic and anti-AS drugs.

[0034] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for inducing diabetic atherosclerosis by double knockout of melatonin receptor 1A and apolipoprotein E, characterized in that: include, MTNR1A gene homozygous knockout mice with C57BL / 6J genetic background and APOE gene homozygous knockout mice were crossbred to obtain MTNR1A / APOE double gene homozygous knockout mice. Six-week-old male MTNR1A / APOE double gene homozygous knockout mice were selected and acclimatized for 2 weeks. After fasting for 12 hours, administer streptozotocin citrate solution once daily at a dose of 50 mg / kg body weight for 5 consecutive days. Two weeks after STZ injection, random blood glucose levels were tested to confirm the establishment of the diabetes model. Feeding patients with a high-fat diet containing 21% fat and 0.21% cholesterol for 12 weeks induced the formation of vulnerable plaques for diabetic atherosclerosis. Detect blood glucose, blood lipids, and pathological indicators of the aorta and carotid arteries to complete model identification.

2. The method for establishing a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 1, characterized in that: The hybridization breeding specifically includes: crossing MTNR1A homozygous knockout mice with APOE homozygous knockout mice to obtain F1 generation MTNR1A mice. + / - APOE + / - Double heterozygous mice were used, and F1 siblings were mated to obtain F2 generation. Genomic DNA from the tail tip of F2 mice was subjected to MTNR1A and APOE gene-specific PCR amplification and agarose gel electrophoresis identification to screen for MTNR1A. - / - APOE - / - Double-gene homozygous knockout mice were propagated and preserved; APOE single-gene knockout mice of the same age, sex, and background were bred simultaneously as controls.

3. The method for establishing a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 2, characterized in that: The adaptive rearing environment was a temperature of 22-25℃ and a humidity of 50%-60%, using a 12h light / 12h dark cycle, with mice having free access to food and water; the STZ citric acid solution was prepared by freshly dissolving streptozotocin in 0.1mol / L, pH 4.5 citric acid buffer, while the control group was injected with an equal volume of citric acid buffer.

4. The method for inducing diabetic atherosclerosis by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 3, characterized in that: Random blood glucose was measured every two weeks after STZ injection. The diabetes model was considered successfully established when the random blood glucose level of mice was >16.7 mmol / L for three consecutive times. After 12 weeks of high-fat feeding, mice were fasted for 12 hours, and blood was collected from the orbital sinus to detect fasting blood glucose and blood lipid levels.

5. The method for inducing diabetic atherosclerosis by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 4, characterized in that: The pathological index detection specifically involves: dissecting the entire aorta from the heart to the iliac artery and performing gross Oil Red O staining; separating the heart and right common carotid artery with the aorta and performing Oil Red O staining, H&E staining, and Masson staining in sequence; measuring plaque area, lipid deposition, lipid necrosis core area, fiber deposition, and fiber cap thickness; and calculating the coefficient of variation within each group.

6. The method for establishing a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 5, characterized in that: Compared with APOE single-gene knockout diabetic atherosclerotic mice, the obtained model had a random blood glucose group with an intra-group coefficient of variation of 16.15%~25.76% (single knockout with a coefficient of variation of 32.16%~41.19%), and a fasting blood glucose level of 22.76 mmol / L with an intra-group coefficient of variation of 19.91% (single knockout with a mean of 16.94 mmol / L and a coefficient of variation of 30.88%).

7. The method for inducing diabetic atherosclerosis by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 6, characterized in that: H&E staining of the aortic sinus showed a mean plaque area ratio of 30.80% and a coefficient of variation of 9.55%, and a mean necrotic core area ratio of 21.71% and a coefficient of variation of 23.95%. H&E staining of the right carotid artery showed a mean plaque area ratio of 79.35% and a coefficient of variation of 21.49%, and a mean necrotic core area ratio of 24.85% and a coefficient of variation of 43.81%. Both were significantly higher than the corresponding indicators of APOE single knockout and had lower coefficients of variation.

8. The method for establishing a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 7, characterized in that: Oil Red O staining of the whole aorta showed a mean lipid deposition area ratio of 24.14% and a coefficient of variation of 40.77% (12.11% and 40.24% for APOE knockout alone); Oil Red O staining of the aortic sinus showed a mean lipid deposition area ratio of 16.98% and a coefficient of variation of 17.88% (10.96% and 23.76% for APOE knockout alone); Masson staining showed a mean fibrin deposition amount of 8.05% and a coefficient of variation of 21.78%, and a mean fibrin cap thickness of 4.85 μm and a coefficient of variation of 42.48% (7.60 μm for APOE knockout alone), presenting a vulnerable plaque phenotype with a thin fibrin cap and a large necrotic core.

9. The method for inducing diabetic atherosclerosis by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 8, characterized in that: The mouse model obtained by the aforementioned modeling method was used to study the mechanism of MTNR1A signaling pathway regulation in the formation of vulnerable plaques in diabetic patients with atherosclerosis.

10. The method for establishing a diabetic atherosclerosis model by double knockout of melatonin receptor 1A and apolipoprotein E as described in claim 9, characterized in that: The mouse model obtained by the aforementioned modeling method was used to screen and evaluate candidate drugs or intervention targets for hypoglycemic, anti-atherosclerotic, and stabilizing vulnerable plaques.

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