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

Abstract

The invention provides a preparation method of an animal model for insulin resistance and abnormal glucose metabolism caused by mild and moderate hypertriglyceridemia, which comprises the following steps: injecting 25-100mg of poloxamer 407 solution into the abdominal cavity of a mouse per kilogram, administration once every other day, continuously injecting for 12 weeks, and continuously monitoring the blood fat and blood sugar level. The preferable dosage of the composition is that 75mg of the composition is injected into each kilogram of mice. The invention also provides application of the animal model in screening and preparing drugs for treating insulin resistance and abnormal glucose metabolism caused by mild and moderate hypertriglyceridemia. According to the method, insulin resistance and abnormal glucose metabolism directly caused by mild and moderate hypertriglyceridemia are simulated, compared with a traditional high-fat diet or gene knockout model, the triglyceride level is accurately regulated and controlled through chemical intervention, factors such as obesity and inflammation and genetic manipulation risks are avoided, and high controllability and high efficiency are achieved.

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 an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia and its application. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by insulin resistance and β-cell dysfunction. Abnormal glucose metabolism, as a transitional stage from normal blood glucose to diabetes, is an important warning signal for the occurrence of diabetes, and its pathogenesis has significant heterogeneity and complexity. Mild to moderate hypertriglyceridemia (HTG) (1.7 ≤ triglyceride < 5.7 mmol / L) is not only a core manifestation of the metabolic syndrome, but also disrupts glucose metabolism 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 disorder" has important scientific significance for deeply revealing the heterogeneous mechanism of T2DM and developing targeted intervention strategies.

[0003] Table 1: TG level stratification

[0004] Currently, for animal models related to "lipotoxicity-driven glucose metabolism disorder", methods such as high-fat diet, gene editing or combined intervention are mainly used. The high-fat diet-induced model causes obesity and mild HTG through high-fat feed, but the mechanism of this model is single, mainly relying on the accumulation of free fatty acids, and it is difficult to simulate the lipid metabolism disorder dominated by HTG. Moreover, the animals in this model usually show a uniform obese phenotype and cannot accurately reflect the characteristics of non-obese diabetes in clinic. Common gene-deficient types include leptin-deficient (ob / ob) mice and leptin receptor-deficient (db / db) mice. ob / ob mice mainly develop obesity and diabetes due to leptin secretion deficiency, while db / db mice mainly develop obesity and diabetes due to leptin receptor function deficiency. Both show the characteristics of obesity and insulin resistance, and the latter mice have a higher probability of developing diabetes. In addition, there are also various gene knockout mouse models, such as IRS gene knockout, GCK gene knockout, TCF7L2 gene knockout and GLUT2 gene knockout mice. These models respectively show typical characteristics of abnormal glucose metabolism such as elevated fasting blood glucose and impaired insulin sensitivity. The diet-chemical drug-induced type uses the method of feeding mice with high-fat feed combined with injecting streptozotocin (STZ) to cause insulin resistance in the body and accelerate the destruction of pancreatic islet β-cells, simulating the pathogenesis of human T2DM.

[0005] The etiology of abnormal glucose metabolism is extremely complex. Each model has different characteristics of abnormal glucose metabolism. Different research objectives correspond to different model carriers. Clinically, there are often people who develop hypertriglyceridemia due to Lpl gene mutations, which in turn induces abnormal glucose metabolism. Such people do not show obesity characteristics. Currently, most models exhibit an obese phenotype, and there is still a lack of animal models that can simulate the insulin resistance and abnormal glucose metabolism phenomena and the disease progression caused by mild to moderate HTG.

[0006] Poloxamer 407 is a non-ionic surfactant that has 100% activity at low concentrations and is relatively non-toxic to cells. It is also a lipoprotein lipase inhibitor.

[0007] The existing animal models mainly have the following technical problems: (1) The preparation of the Lpl gene knockout mouse model requires a large amount of time and effort and is costly. In addition, the triglyceride level only increases slightly due to gene heterozygous knockout, and gene homozygous knockout easily leads to death of mice during the embryonic period or after birth.

[0008] (2) Diet induction: It is difficult to control the increase in blood lipids in the animal model induced by a high-fat diet, and it is difficult to ensure that only the triglyceride content increases during the experiment. Usually, this method is used to construct hypercholesterolemia or mixed hyperlipidemia (showing both high triglycerides and high cholesterol), which makes it controversial to construct a model of abnormal glucose metabolism caused by hypertriglyceridemia as the initiating factor. Summary of the Invention

[0009] In view of the above 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 such an 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 the lack of an animal model that can simulate the insulin resistance and abnormal glucose metabolism phenomena and the disease progression caused by HTG.

[0010] The present invention provides a method for preparing an animal model of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia. Inject a poloxamer 407 solution into the abdominal cavity of mice at a dose of 25 - 100 mg per kilogram of mice, administer the drug every other day, and continuously inject for 12 weeks, while continuously monitoring the body weight, blood lipid, and blood glucose levels.

[0011] Further, the preferred dose injected per kilogram of mice is 75 mg.

[0012] Further, prepare a physiological saline solution in which the mass-volume ratio of ultrapure water to sodium chloride is 100 mL: 0.9 g; then add poloxamer 407, dissolve it by ultrasound, and filter it with a bacterial filter for later use.

[0013] Further, before and after the preparation, detect the triglyceride, total cholesterol, fasting blood glucose, and HOMA-IR values of the mice. When the HOMA-IR is stable, the animal model is successfully constructed.

[0014] Further, the mice are male C57BL / 6 mice.

[0015] The present invention also provides the use of the animal model obtained by the above method in the preparation, screening, or preparation of drugs for treating insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia.

[0016] The optimal modeling period of the present invention is 12 weeks. Measure the body weight of the mice every week, and collect tail blood samples every 2 weeks to detect the changes in triglyceride and total cholesterol in the mouse serum. At the same time, detect the changes in fasting blood glucose and fasting insulin in the mouse serum. The present invention uses a single factor to cause mild to moderate hypertriglyceridemia in mice. In the lipid metabolism disorder, only the triglyceride level is stably elevated mildly to moderately, while the total cholesterol level does not change significantly.

[0017] The present invention uses the chemical reagent P407 to inhibit the activity of lipoprotein lipase (Lpl), thereby causing an increase in triglyceride in the whole body tissues and blood. By inhibiting Lpl in the body, mild to moderate hypertriglyceridemia is caused, which further leads to insulin resistance and abnormal glucose metabolism. The present invention observes the lipid metabolism and glucose metabolism of mice by intraperitoneal injection of different doses of P407 at different times. The optimal drug concentration and time for intraperitoneal injection of P407 to cause mild to moderate hypertriglyceridemia in mice are optimized. Different from the model that directly damages pancreatic islet β cells, the present invention uses a single factor to construct a situation of abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia, determines the time stage of insulin resistance and abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia, and can simulate the pathophysiological state of the natural occurrence and development of abnormal glucose metabolism mediated by mild to moderate hypertriglyceridemia in humans, including insulin resistance and elevated fasting blood glucose, which can be used to study the pathogenesis of abnormal glucose metabolism caused by mild to moderate hypertriglyceridemia in humans and to evaluate the therapeutic effects of related drugs.

[0018] Compared with the prior art, the technical effects of the present invention are positive and obvious. The present invention constructs a mouse model of mild to moderate hypertriglyceridemia-mediated glucose metabolism disorder caused by a single factor, which is stable, simple. By intraperitoneal injection of lipoprotein lipase inhibitor poloxamer 407 (P407), simple mild to moderate hypertriglyceridemia (HTG) is induced, successfully simulating insulin resistance and glucose metabolism disorder directly caused by mild to moderate hypertriglyceridemia (1.7 ≤ TG < 5.7 mmol / L), and for the first time clarifying the time node of HTG pathogenesis. 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 and the risks of genetic manipulation, and having both high efficiency and controllability. It provides a reliable tool for the research on the causal relationship between HTG and glucose metabolism disorder, drug target screening and exploration of early intervention timing. Moreover, the model of the present invention does not require the cultivation of transgenic animals, not only has the advantages of simple operation, low cost and high modeling success rate, but also can effectively promote the in-depth study of the pathological damage mechanism of glucose metabolism-related target organs, providing a new research platform for exploring the potential mechanisms of glucose metabolism disorder-related diseases. Brief Description of the Drawings

[0019] Figure 1 It is the flowchart for establishing the model of the mouse model of the present invention.

[0020] Figure 2 It is the external view of two groups of mice of the present invention and the trend of body weight change of mice treated with different doses.

[0021] Figure 3 It is the appearance of the sera of two groups of mice of the present invention and the trend of changes in triglyceride and total cholesterol of mice treated with different doses.

[0022] Figure 4 It is the fasting blood glucose and HOMA-IR values of mice after intraperitoneal injection of the preferred dose (75 mg / kg) of P407 of the present invention.

[0023] Figure 5 It is the HE staining map of the pancreas and the pathological score of the pancreas of mice after intraperitoneal injection of the preferred dose (75 mg / kg) of P407 of the present invention. Detailed Embodiments

[0024] Example 1

[0025] 1.1 Experimental animals and main reagents: 1.1.1 Experimental animals: Male C57BL / 6 mice at 6 weeks of age were used in the experiment, and all of them were provided by the Animal Experiment Center of Renji Hospital. In the animal house, all mice were placed in IVC cages meeting the SPF standard and were housed separately to ensure their free access to food and water. The environmental conditions in the animal house were strictly controlled at a temperature of 21 °C, a humidity of 50%-60%, and a light / dark cycle of 12 h / 12 h.

[0026] 1.1.2 Main reagents: P407 (Poloxamer 407) was purchased from MCE Company in the United States. The TG kit, TC kit, and FBG kit were all purchased from Nanjing Jiancheng Bioengineering Research Institute. The ELISA kit was purchased from Crystal Chem Inc in the United States. The blood glucose meter and blood glucose test strips were purchased from Bayer Contour. The consumables for HE staining were all purchased from Wuhan Sevier Company.

[0027] 1.2 Establishment of hypertriglyceridemia model (as Figure 1 shown): 1.2.1 Preparation of main reagents: (1) Preparation of 0.9% normal saline: Weigh 0.9 g of sodium chloride powder and dissolve it in 100 mL of ultrapure water.

[0028] (2) Preparation of Poloxamer 407 solution: Add 300 mg of Poloxamer 407 to 10 mL of 0.9% normal saline, dissolve it by ultrasonic treatment to prepare the stock solution. After filtering with a bacterial filter, calculate the dosing amount according to the body weight of the mice, aliquot and store it in a -20 °C refrigerator for later use.

[0029] 1.2.2 Grouping of animal models: (1) 6-week-old healthy wild-type C57BL / 6J male mice (48 mice). After 1 week of adaptive feeding, the mice were randomly divided into 6 groups, with 8 mice in each group: Control group: Administer the drug by intraperitoneal injection with normal saline once every other day.

[0030] 25 mg / kg P407 group (the dose injected per kg of mice is 25 mg): Administer the drug by intraperitoneal injection with 25 mg / kg P407 once every other day.

[0031] 50 mg / kg P407 group (the dose injected per kg of mice is 50 mg): Administer the drug by intraperitoneal injection with 50 mg / kg P407 once every other day.

[0032] 75 mg / kg P407 group (the dose injected per kg of mice is 75 mg): Administer the drug by intraperitoneal injection with 75 mg / kg P407 once every other day.

[0033] Group P407 at 100 mg / kg (the dosage injected per kg of mice is 100 mg): Administer by intraperitoneal injection with 100 mg / kg P407 once every other day.

[0034] Group P407 at 500 mg / kg (the dosage injected per kg of mice is 500 mg): Administer by intraperitoneal injection with 500 mg / kg P407 once every other day.

[0035] (2) Normal diet: The contents of fat, protein, and carbohydrates are maintained within the normal range.

[0036] 1.2.3 Detection of triglyceride (TG) and total cholesterol (TC): Fast the mice for 12 h overnight while allowing normal water intake. At 8:00 am the next day, cut off the distal 1 mm of the mouse's tail with scissors, gently squeeze the tail, wipe off the first drop of blood with a cotton swab, and then collect the blood. After the blood stands at room temperature for 2 h, centrifuge (4°C, 3000 r, 15 min), take the upper serum, and use TG and TC kits to detect the levels of TG and TC respectively.

[0037] 1.2.4 Detection of fasting blood glucose (FBG): Fast the mice for 12 h overnight while allowing normal water intake. At 8:00 am the next day, cut off the distal 1 mm of the mouse's tail with scissors, gently squeeze the tail, wipe off the first drop of blood with a cotton swab, and immediately detect the blood glucose value with a blood glucose test strip.

[0038] 1.2.5 Detection of fasting insulin (FINS) in mice and calculation of the HOMA-IR value (HOMA-IR is the insulin resistance index used to evaluate insulin resistance): Fast the mice for 12 h overnight while allowing normal water intake. At 8:00 am the next day, cut off the distal 1 mm of the mouse's tail with scissors, gently squeeze the tail, wipe off the first drop of blood with a cotton swab, and then collect the blood. After the blood stands at room temperature for 2 h, centrifuge (4°C, 3000 r, 15 min), take the upper serum, use an insulin ELISA kit to detect the fasting insulin level, and then calculate the HOMA-IR value. The calculation formula is: 20 × FINS (μU / mL) / (FBG (mmol / L) - 3.5).

[0039] 1.2.6 HE staining of the pancreas: At the 12th week of model establishment, anesthetize the mice with 2.5% Avertin, quickly decapitate and sacrifice them, take out the pancreatic tissue, fix it for 24 h, dehydrate it through gradient alcohol, embed it in paraffin to make paraffin blocks, cut the paraffin blocks into thin sections and attach them to glass slides, continue to dewax and hydrate, stain with hematoxylin and eosin, dehydrate and clear, seal with neutral balsam, and finally use a high-throughput digital slide scanning system for image acquisition and analysis.

[0040] 2. Experimental results: 2.1 Effects of different doses of P407 intervention on the body weight of mice As Figure 2 shown in (A, B), the body weight of mice was dynamically detected. The pictures show the body weight changes of different doses of P407 groups (including 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, and 500 mg / kg) at different time points (from week 0 to week 12). The results show that the body weight of each group of mice gradually increased over time, but there were no differences in the body weight gain among the mice in each dose group.

[0041] 2.2 Effects of different doses of P407 intervention on the contents of TG and TC in mice As Figure 3 shown, serum samples collected during the mouse modeling process. The picture ( Figure 3 A) shows that the serum of control mice is clear and transparent, while the serum of model mice is milky white. The monitoring results of serum collected from different doses of P407 (including 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, and 500 mg / kg) intervention groups at different time points (from week 0 to week 12) show that starting from week 2, the triglyceride (TG) level was stable and slightly to moderately increased ( Figure 3 B), while the cholesterol (TC) level did not change significantly ( Figure 3 C). Among all intervention groups, the TG level of mice in the 75 mg / kg P407 group showed stability and was in the range of slightly to moderately increased. Therefore, a dose of 75 mg / kg was selected as the preferred option for constructing a mild to moderate hypertriglyceridemia model of mice with glucose metabolism disorders mediated by HTG.

[0042] 2.3 Effects of 12-week intervention with the preferred dose of 75 mg / kg P407 on fasting blood glucose and insulin resistance in mice As Figure 4 shown in (A), we monitored the serum samples collected at different time points (from week 0 to week 12). The results show that at week 4, the fasting blood glucose (FBG) level of mice in the P407 administration group began to increase significantly (P < 0.05). Subsequently, during the period from week 8 to week 12, the FBG level of mice in the P407 administration group continued to increase steadily (P < 0.05). This result indicates that intraperitoneal injection of P407 can induce glucose metabolism disorders.

[0043] Then, we used the HOMA-IR value to evaluate the insulin resistance of mice. The results are as Figure 4(B)showed that the HOMA-IR value increased significantly at week 4 (P<0.05) and remained stable during weeks 8 to 12. This indicated that insulin resistance had occurred during the abnormal glucose metabolism induced by P407. Therefore, this mild to moderate hypertriglyceridemia model was characterized by HTG-mediated abnormal glucose metabolism, with insulin resistance and elevated fasting blood glucose being the prominent features of glucose metabolism disorder.

[0044] 2.4 Effect of 75 mg / kg P407 intervention for 12 weeks on pancreatic pathology in mice As Figure 5 Shown in (A, B), the HE staining results of pancreatic tissue suggested that the pancreatic tissue structure of the control group mice was intact and regular, and the acinar cells and islets maintained good morphology. The cytoplasm in the islets was pink-stained, with a relatively large number of cells, normal morphology, and deeply stained and clear cell nuclei. In contrast, after 12 weeks of intraperitoneal injection of P407, a small amount of acinar cell necrosis occurred in the exocrine part of the 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: The mice were intraperitoneally injected with poloxamer 407 solution at a dose of 25-100 mg per kg of mouse, once every other day for 12 consecutive weeks, during which body weight, blood lipid and blood glucose levels were continuously monitored.

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 dose injected per kg mouse is 75 mg.

3. 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: First, prepare a physiological saline solution, in which the mass volume ratio of ultrapure water and sodium chloride is 100 mL:0.9 g; then add Poloxamer 407, dissolve it by ultrasonication, and filter it with a bacterial filter for later use.

4. 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: Before and after the preparation, the triglyceride, total cholesterol, fasting blood glucose and HOMA-IR values ​​of the mice were tested. When the HOMA-IR was stable, the animal model was established.

5. 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.

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

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