Preparation method and application of an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia

Abstract

The present invention provides a method for preparing an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia, by injecting poloxamer 407 solution into the mouse peritoneal cavity at a dose of 25-100 mg per kilogram of mouse, administering the drug once every other day for 12 consecutive weeks, and continuously monitoring blood lipid and blood glucose levels. The preferred dose of the present invention is 75 mg per kilogram of mouse. The present invention also provides the use of the animal model in screening and preparing drugs for treating insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia. The present invention simulates insulin resistance and abnormal glucose metabolism directly caused by mild to moderate hypertriglyceridemia. Compared with traditional high-fat diets or gene knockout models, triglyceride levels are precisely regulated by chemical intervention, avoiding factors such as obesity and inflammation and the risk of genetic manipulation, and having high controllability and efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biology and relates to a method for preparing an animal model, specifically a method for preparing and applying an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by insulin resistance and β-cell dysfunction. Abnormal glucose metabolism, as the transition stage from normoglycemia to diabetes, is an important early warning sign for the development of diabetes, and its pathogenesis is remarkably heterogeneous and complex. Mild to moderate hypertriglyceridemia (HTG) (1.7 ≤ triglycerides < 5.7 mmol / L) is not only a core manifestation of metabolic syndrome but also disrupts glucose homeostasis through lipotoxic effects, highlighting the key role of lipotoxicity in the pathogenesis of diabetes. Therefore, constructing a practical, stable, and reasonable animal model of "lipotoxicity-driven glucose metabolism disorders" is of great scientific significance for further understanding the heterogeneous mechanisms of T2DM and developing targeted intervention strategies.

[0003] Table 1: TG level stratification

[0004]

[0005] Currently, animal models for studying lipotoxicity-driven glucose metabolism disorders primarily utilize high-fat diets, gene editing, or combined interventions. High-fat diet-induced models induce obesity and mild hyperlipidemia (HTG) through a high-fat diet. However, these models employ a single mechanism, primarily relying on free fatty acid accumulation, making them inadequate for mimicking HTG-driven lipid metabolism disorders. Furthermore, these models typically exhibit a uniformly obese phenotype, failing to accurately reflect clinical features of non-obese diabetes. Common genetically deficient models include leptin-deficient (ob / ob) mice and leptin receptor-deficient (db / db) mice. Ob / ob mice exhibit obesity and diabetes primarily due to a defect in leptin secretion, while db / db mice exhibit obesity and diabetes primarily due to a defect in leptin receptor function. Both exhibit features of obesity and insulin resistance, with the latter exhibiting a higher incidence of diabetes. Additionally, various knockout mouse models exist, such as IRS, GCK, TCF7L2, and GLUT2 knockout mice. These models exhibit typical features of glucose metabolism disorders, such as elevated fasting blood glucose and impaired insulin sensitivity. The diet-chemical drug-induced type uses a high-fat diet combined with injection of streptozotocin (STZ) to induce insulin resistance in the body and accelerate the destruction of pancreatic beta cells, simulating the pathogenesis of human T2DM.

[0006] The causes of abnormal glucose metabolism are extremely complex. Each model has different characteristics of abnormal glucose metabolism, and different research objectives correspond to different model carriers. Clinically, there are often people who suffer from abnormal glucose metabolism due to Lpl gene mutations, which in turn leads to hypertriglyceridemia. These people do not show characteristics of obesity. Current models mostly show obese phenotypes, and there is still a lack of animal models that can simulate the insulin resistance and abnormal glucose metabolism caused by mild to moderate HTG, as well as the disease progression.

[0007] Poloxamer 407 is a nonionic surfactant that is 100% active at low concentrations and relatively nontoxic to cells. It is also a lipoprotein lipase inhibitor.

[0008] The existing animal models have the following technical problems:

[0009] (1) The preparation of Lpl gene knockout mouse models requires a lot of time and effort, and is costly. In addition, heterozygous gene knockout only results in a mild increase in triglycerides, while homozygous gene knockout can easily lead to embryonic or postnatal death in mice.

[0010] (2) Dietary induction: It is difficult to control the increase in blood lipids in animal models induced by a high-fat diet, and it is difficult to ensure that only triglyceride levels increase during the experiment. This method is usually used to construct hypercholesterolemia or mixed hyperlipidemia (simultaneously showing high triglycerides and high cholesterol). This makes the construction of a model with hypertriglyceridemia as the initiating factor to induce abnormal glucose metabolism somewhat controversial. Summary of the Invention

[0011] In response to the above-mentioned technical problems in the prior art, the present invention provides a method for preparing and applying an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia. The method for preparing and applying the animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia aims to solve the problem in the prior art of lacking an animal model that can simulate the phenomena of insulin resistance and abnormal glucose metabolism and the course of disease caused by HTG.

[0012] The present invention provides a method for preparing an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia. The method comprises injecting a poloxamer 407 solution into the peritoneal cavity of mice at a dose of 25-100 mg per kilogram of mice, administering the drug once every other day for 12 consecutive weeks, during which body weight, blood lipid and blood glucose levels are continuously monitored.

[0013] Furthermore, the preferred injection dose is 75 mg per kilogram of mouse.

[0014] Furthermore, a physiological saline solution is first prepared, wherein the mass volume ratio of ultrapure water to sodium chloride is 100 mL:0.9 g; then poloxamer 407 is added, ultrasonically dissolved, and filtered through a bacterial filter for later use.

[0015] Furthermore, the triglyceride, total cholesterol, fasting blood glucose and HOMA-IR values of the mice were tested before and after preparation. When the HOMA-IR was stable, the animal model was established.

[0016] Furthermore, the mice are male C57BL / 6 mice.

[0017] The present invention also provides the use of the animal model obtained by the above method in preparing and screening or preparing drugs for treating insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia.

[0018] The optimal modeling period of the present invention is 12 weeks. The mice are weighed weekly and tail blood is sampled every two weeks to detect changes in triglycerides and total cholesterol in the mouse serum. Simultaneously, changes in fasting blood glucose and fasting insulin levels are also detected in the mouse serum. The present invention uses a single factor to induce mild to moderate hypertriglyceridemia in mice. Among the lipid metabolism abnormalities, only triglyceride levels remain stable with a slight to moderate increase, while total cholesterol levels do not change significantly.

[0019] This study uses the chemical reagent P407 to inhibit lipoprotein lipase (Lpl) activity, thereby elevating triglycerides in systemic tissues and blood. This Lpl inhibition leads to mild to moderate hypertriglyceridemia, which in turn causes insulin resistance and abnormal glucose metabolism. The study observed lipid and glucose metabolism in mice by intraperitoneal injection of different doses of P407 at different injection times. The optimal concentration and timing of intraperitoneal injection of P407 for producing mild to moderate hypertriglyceridemia in mice were optimized. Unlike models that directly damage pancreatic beta cells, this study uses a single factor to establish mild to moderate hypertriglyceridemia-induced glucose abnormalities. The study also identifies the time course of insulin resistance and glucose abnormalities in response to mild to moderate hypertriglyceridemia. This model can mimic the pathophysiological state of glucose abnormalities naturally occurring in humans mediated by mild to moderate hypertriglyceridemia, including insulin resistance and elevated fasting blood glucose. This study can be used to study the pathogenesis of glucose abnormalities caused by mild to moderate hypertriglyceridemia in humans and to evaluate the therapeutic efficacy of related drugs.

[0020] Compared with existing technologies, the present invention offers significant and positive technical benefits. It constructs a stable, simple, single-factor mouse model of mild-to-moderate hypertriglyceridemia-mediated glucose metabolism abnormalities. By intraperitoneally injecting the lipoprotein lipase inhibitor poloxamer 407 (P407) to induce isolated mild-to-moderate hypertriglyceridemia (HTG), the present invention successfully simulates the insulin resistance and glucose metabolism abnormalities directly caused by mild-to-moderate hypertriglyceridemia (1.7 ≤ TG < 5.7 mmol / L), and for the first time defines the time point at which HTG becomes pathogenic. Compared with traditional high-fat diet or gene knockout models, this model precisely regulates triglyceride levels through chemical intervention, avoiding confounding factors such as obesity and inflammation, as well as the risks of genetic manipulation, while being both highly effective and controllable. This provides a reliable tool for studying the causal relationship between HTG and glucose metabolism abnormalities, screening drug targets, and exploring opportunities for early intervention. Moreover, the model of the present invention does not require the cultivation of transgenic animals. It not only has the advantages of simple operation, low cost and high modeling success rate, but also can effectively promote in-depth research on the pathological damage mechanism of target organs related to glucose metabolism, and provide a new research platform for exploring the potential mechanisms of diseases related to abnormal glucose metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 is a flow chart of the modeling of the mouse model of the present invention.

[0022] Figure 2 The figures show the appearance of two groups of mice of the present invention and the weight change trend of mice treated with different doses.

[0023] Figure 3 The figures show the appearance of serum of two groups of mice of the present invention and the changing trends of triglyceride and total cholesterol in mice treated with different doses.

[0024] Figure 4 The fasting blood glucose and HOMA-IR values of mice after intraperitoneal injection of P407 at the preferred dose (75 mg / kg) of the present invention.

[0025] Figure 5 The following are the pancreatic HE staining images and pancreatic pathological scores after intraperitoneal injection of P407 at the preferred dose (75 mg / kg) of the present invention. DETAILED DESCRIPTION

[0026] Example 1

[0027] 1.1 Experimental animals and main reagents:

[0028] 1.1.1 Experimental Animals: Six-week-old male C57BL / 6 mice were used in this experiment. These mice were provided by the Animal Experimentation Center of Renji Hospital. All mice were housed in SPF-grade IVC cages in the animal room, individually housed, with free access to food and water. Environmental conditions in the animal room were strictly controlled at a temperature of 21°C, a humidity of 50%-60%, and a 12-h / 12-h light / dark cycle.

[0029] 1.1.2 Main Reagents: P407 (Poloxamer 407) was purchased from MCE (USA); TG, TC, and FBG kits were purchased from Nanjing Jiancheng Bioengineering Institute; ELSIA kits were purchased from Crystal Chem Inc. (USA); and blood glucose meters and test strips were purchased from Bioankang. HE staining consumables were purchased from Wuhan Sewell Company.

[0030] 1.2 Establishment of hypertriglyceridemia model (e.g. Figure 1 shown):

[0031] 1.2.1 Preparation of main reagents:

[0032] (1) Preparation of 0.9% normal saline: Weigh 0.9 g of sodium chloride powder and dissolve it in 100 mL of ultrapure water.

[0033] (2) Preparation of Poloxamer 407 solution: Add 300 mg of Poloxamer 407 to 10 mL of 0.9% saline, dissolve by ultrasonication to prepare the stock solution, filter with a bacterial filter, calculate the dosage according to the weight of the mouse, and store in a -20°C refrigerator for later use.

[0034] 1.2.2 Animal model groups:

[0035] (1) 6-week-old healthy wild-type C57BL / 6J male mice (48) were randomly divided into 6 groups after 1 week of adaptive feeding, with 8 mice in each group:

[0036] Control group: The animals were intraperitoneally injected with normal saline, once every other day.

[0037] 25 mg / kg P407 group (the injection dose is 25 mg per kg mouse): 25 mg / kg P407 was intraperitoneally injected once every other day.

[0038] The 50 mg / kg P407 group received 50 mg of P407 per kg of mouse through intraperitoneal injection, once every other day.

[0039] The 75 mg / kg P407 group received 75 mg of P407 per kg of mouse through intraperitoneal injection, once every other day.

[0040] The 100 mg / kg P407 group received 100 mg of P407 per kg of mouse through intraperitoneal injection, once every other day.

[0041] The 500 mg / kg P407 group received 500 mg of P407 per kg of mouse through intraperitoneal injection, once every other day.

[0042] (2) Normal feed: The content of fat, protein and carbohydrates is maintained within normal levels.

[0043] 1.2.3 Triglyceride (TG) and total cholesterol (TC) testing:

[0044] Mice were fasted for 12 hours overnight with normal water intake. The following morning at 8:00 AM, the distal 1 mm of the tail was snipped with scissors. The tail was gently squeezed, and the first drop of blood was wiped off with a cotton swab. Blood was then collected. The blood was allowed to stand at room temperature for 2 hours and then centrifuged (4°C, 3000 rpm, 15 minutes). The upper serum layer was collected and assayed for triglyceride (TG) and TC (TC) levels using TG and TC kits, respectively.

[0045] 1.2.4 Fasting blood glucose (FBG) test:

[0046] The mice were fasted for 12 hours overnight with normal water intake. The following morning at 8:00 AM, the distal 1 mm of the tail was cut with scissors. The tail was gently squeezed, and the first drop of blood was removed with a cotton swab. Blood glucose levels were then measured immediately using a blood glucose test strip.

[0047] 1.2.5 Fasting insulin (FINS) test and calculation of HOMA-IR value (HOMA-IR is the insulin resistance index used to assess insulin resistance):

[0048] Mice were fasted for 12 hours overnight with normal water intake. The following morning at 8:00 AM, the distal 1 mm of the tail was snipped with scissors. The tail was gently squeezed, and the first drop of blood was removed with a cotton swab. Blood was then collected. After standing at room temperature for 2 hours, the blood was centrifuged (4°C, 3000 rpm, 15 minutes). The supernatant serum was collected and fasting insulin levels were measured using an insulin ELISA kit. HOMA-IR values were then calculated using the formula: 20 × FINS (μU / mL) / (FBG (mmol / L) - 3.5).

[0049] 1.2.6 Pancreas HE staining:

[0050] At the 12th week of modeling, mice were anesthetized with 2.5% Avertin and quickly killed by cervical dislocation. The pancreatic tissue was removed, fixed for 24 h, dehydrated with gradient alcohol, and embedded in paraffin to make paraffin blocks. The paraffin blocks were cut into thin slices and attached to glass slides. They were further dewaxed and hydrated, stained with hematoxylin and eosin, dehydrated and transparentized, and then mounted with neutral gum. Finally, a high-throughput digital slice scanning system was used for image acquisition and analysis.

[0051] 2. Experimental results:

[0052] 2.1 Effects of different doses of P407 intervention on mouse body weight

[0053] like Figure 2 (A, B) Dynamic monitoring of mouse body weight. The images show the changes in body weight at different time points (from week 0 to week 12) in groups treated with different doses of P407 (including 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, and 500 mg / kg). The results show that the body weight of mice in each group gradually increased over time, but there was no difference in body weight gain among the mice in each dose group.

[0054] 2.2 Effects of different doses of P407 intervention on TG and TC levels in mice

[0055] like Figure 3 As shown, serum samples collected during the mouse modeling process, picture ( Figure 3 A) shows that the serum of the control mice was clear and transparent, while the serum of the model mice was milky white. The serum monitoring results of the intervention groups with different doses of P407 (including 25mg / kg, 50mg / kg, 75mg / kg, 100mg / kg and 500mg / kg) collected at different time points (from 0 to 12 weeks) showed that starting from the second week, the triglyceride (TG) level was stable and slightly increased ( Figure 3 B), while cholesterol (TC) levels did not change significantly ( Figure 3 C). Among all intervention groups, triglyceride levels in mice treated with 75 mg / kg P407 remained stable and within the mild to moderately elevated range. Therefore, the 75 mg / kg dose was selected as the preferred regimen for establishing a mild to moderate hypertriglyceridemia model in mice with HTG-mediated glucose metabolism abnormalities.

[0056] 2.3 Effects of 12-week intervention with the optimal dose of 75 mg / kg P407 on fasting blood glucose and insulin resistance in mice

[0057] like Figure 4As shown in (A), serum samples collected at different time points (from week 0 to week 12) were monitored. The results showed that fasting blood glucose (FBG) levels in mice treated with P407 began to increase significantly at week 4 (P < 0.05). Subsequently, FBG levels in mice treated with P407 continued to rise steadily from week 8 to 12 (P < 0.05). This result suggests that intraperitoneal administration of P407 can induce abnormal glucose metabolism.

[0058] Then, we used HOMA-IR value to evaluate the insulin resistance of mice. Figure 4 (B) shows that the HOMA-IR value increased significantly at week 4 (P < 0.05) and remained stable from week 8 to week 12. This indicates that insulin resistance has already occurred during the P407-induced glucose metabolism abnormalities. Therefore, this mild-to-moderate hypertriglyceridemia model is characterized by HTG-mediated glucose metabolism abnormalities, and glucose metabolism disorders are prominently characterized by insulin resistance and elevated fasting blood glucose.

[0059] 2.4 Effects of 12-week intervention with the optimal dose of 75 mg / kg P407 on pancreatic pathology in mice

[0060] like Figure 5 (A, B) HE staining of pancreatic tissue in control mice revealed intact and regular pancreatic tissue structure, with well-maintained acinar cell and islet morphology. Islets showed pink cytoplasm, numerous cells with normal morphology, and distinct dark-stained nuclei. In contrast, 12 weeks after intraperitoneal injection of P407, a small amount of acinar cell necrosis was observed in the exocrine pancreas.

Claims

1. A method for preparing an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia, characterized in that: 1) Prepare a physiological saline solution, wherein the mass volume ratio of ultrapure water to sodium chloride is 100 mL:0.9 g; 2) Prepare the poloxamer 407 solution by adding poloxamer 407 to the saline solution prepared in step 1), dissolve it by ultrasonication, and filter it through a bacterial filter. Calculate the dosage based on the weight of the mouse. 3) Mice were intraperitoneally injected with a poloxamer 407 solution at a dose of 75 mg / kg of mouse, every other day for 12 weeks. Body weight, triglycerides, total cholesterol, blood glucose, insulin, and HOMA-IR levels were continuously monitored during this period. Starting from the second week of injection, serum triglyceride levels stabilized and remained between 1.7 mmol / L and 5.7 mmol / L, and total cholesterol levels did not change significantly. From the 8th to 12th week of injection, the fasting blood glucose levels of mice continued to rise steadily. During the 8th to 12th week, after HOMA-IR stabilized, the animal model was completed.

2. The method for preparing an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia according to claim 1, characterized in that: The mice are male C57BL / 6 mice.

3. Use of the animal model obtained by the method according to claim 1 in preparing and screening drugs for treating insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia.

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