Improved method for establishing a postprandial hypertriglyceridemic mouse model

Abstract

This invention discloses a method for establishing an improved mouse model of postprandial hypertriglyceridemia, belonging to the field of model construction technology. The key technical points are as follows: the method includes the following steps: obtaining a predetermined number of mouse models, which are then divided into a control group and a model group; administering gavage to both the model group and the control group for 120 minutes; sampling the mice in the model group and the control group after step S2, obtaining samples from both groups; and detecting glucose and lipid-related biochemical indicators, serum SOD, and MDA levels in the samples from both groups. In this invention, a mouse model of hypertriglyceridemia simulating the postprandial dietary state in humans is established through a combination of glucose and lipid administration via gavage. This will provide a research foundation for traditional Chinese medicine research on postprandial hypertriglyceridemia and related glucose and lipid metabolic diseases, and can also provide a model reference for the evaluation of lipid-lowering and hypoglycemic drugs and the development of health products.

Description

Technical Field

[0001] This invention relates to the field of model building technology, and more specifically, to a method for establishing a modified postprandial hypertriglyceridemia mouse model. Background Technology

[0002] Recent studies have found that postprandial triglycerides are a better predictor of cardiovascular disease risk than fasting triglycerides. Traditional Chinese medicine (TCM) has a safety and efficacy advantage in treating hypertriglyceridemia, especially for transient postprandial triglyceride elevations, compared to commonly used lipid-lowering drugs such as fibrates, niacin, and statins. To better evaluate the use of TCM in the prevention and treatment of postprandial hypertriglyceridemia, relevant animal models urgently need to be developed.

[0003] Modeling methods for elevating triglycerides have been studied extensively, but they have limitations in simulating the postprandial state of humans. This invention aims to explore and establish an improved mouse model of postprandial hypertriglyceridemia to better mimic human dietary structure and habits, providing an animal model reference for research on traditional Chinese medicine in the prevention and treatment of postprandial hypertriglyceridemia and related diseases, as well as the development of related health products and drugs. Summary of the Invention

[0004] The purpose of this invention is to provide an improved method for establishing a mouse model of postprandial hypertriglyceridemia, so as to better simulate human dietary structure and habits, and to provide an animal model reference for the research on the prevention and treatment of postprandial hypertriglyceridemia and related diseases by traditional Chinese medicine, as well as the development of related health products and drugs.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a method for establishing an improved mouse model of postprandial hypertriglyceridemia, the method comprising the following steps:

[0006] S1: Obtain a preset number of mouse models and divide them into a blank group and a model group on average;

[0007] S2: The model group and the blank group were administered gavage for 120 minutes.

[0008] S3: Sample the model group mice and the blank group mice after step S2 to obtain model group samples and blank group samples.

[0009] S4: Detect glucose-lipid-related biochemical indicators, serum SOD and MDA levels in model group samples and blank group samples;

[0010] S5: Observe the liver pathology and mitochondrial morphology in hepatocytes of mice in the model group and the blank group.

[0011] S6: Statistically analyze the data obtained in steps S4 and S5 to obtain the optimal mouse model of postprandial hypertriglyceridemia.

[0012] The present invention is further configured such that the preset number of items in step S1 is 24.

[0013] The present invention is further configured such that the gavage treatment in step S2 is as follows: mice are first gavaged with 0.5 ml of edible oil and then with 0.2 ml of 30% fructose aqueous solution; the blank group is first gavaged with 0.5 ml of physiological saline and then with 0.2 ml of physiological saline.

[0014] The present invention is further configured as follows: the sampling operation in step S3 is as follows: obtain plasma from the mouse eyeball, take out the plasma and let it stand for 3-4 hours, then centrifuge at 3000 rpm and 4℃ for 10 minutes in a refrigerated centrifuge and take the supernatant.

[0015] The present invention is further configured such that the glucose and lipid-related biochemical indicators in step S4 include TG, TC, HDL-C, LDL-C, alanine aminotransferase, aspartate aminotransferase, GLU, ALP and total bile acids.

[0016] In summary, the present invention has the following beneficial effects: In the present invention, a mouse model of hypertriglyceridemia simulating the postprandial dietary state of humans is established by gavage with a combination of sugar and oil. This will provide a research basis for the study of postprandial hypertriglyceridemia and related glucose and lipid metabolism diseases in traditional Chinese medicine, and can also provide a model reference for the evaluation of lipid-lowering and hypoglycemic drugs and the development of health products. Attached Figure Description

[0017] Figure 1 This is a flowchart of a method for establishing a modified mouse model of postprandial hypertriglyceridemia in an embodiment of the present invention;

[0018] Figure 2 These are evaluation test charts of serum chylous blood, biochemical indicators, and oxidative stress indicators for each group in the embodiments of this invention; (where A: severe chylous blood phenomenon in model group mice; B: triglycerides (TG); C: total cholesterol (TC); D: high-density lipoprotein (HDL); E: low-density lipoprotein (LDL); F: alanine aminotransferase (AST); G: aspartate aminotransferase (ALT); H: blood glucose (GLU); I: alkaline phosphatase level (ALP); J: total bile acids (TBA); K: superoxide dismutase (SOD); L: malondialdehyde (MDA)).

[0019] Figure 3These are pathological sections of mice stained with HE and Oil Red O in each group according to the embodiments of the present invention: (where A: HE staining of liver tissue of each group of mice (red arrow: hepatocyte arrangement structure); B: Oil Red O staining of liver tissue of each group of mice (red arrow: lipid droplets appearing in hepatocytes); C: mitochondrial structure of hepatocytes of each group of mice (red arrow: lipid droplets appearing in hepatocytes; orange arrow: endoplasmic reticulum)).

[0020] Figure 4 The following are the biochemical indicators evaluated in each group in this embodiment of the invention: (where A: triglyceride level in each group of mice; B: total cholesterol level in each group of mice; C: high-density lipoprotein level in each group of mice; D: low-density lipoprotein level in each group of mice; each group is a blank control group, an oil group administered by gavage, a model group, and a poloxamer 407 group administered intraperitoneally). Detailed Implementation

[0021] The following is in conjunction with the appendix Figure 1-4 The present invention will be described in further detail below.

[0022] Example: A method for establishing a modified mouse model of postprandial hypertriglyceridemia, such as... Figure 1 As shown, the establishment method includes the following steps:

[0023] S1: 24 mouse models were obtained and divided into a control group and a model group, with 12 mice in each group.

[0024] It should be noted that mice need to be acclimatized for one week before obtaining the mouse model.

[0025] S2: The model group and the blank group were administered gavage for 120 minutes.

[0026] The gavage treatment was as follows: Mice were first gavaged with 0.5 ml of edible oil followed by 0.2 ml of 30% fructose solution; the control group was first gavaged with 0.5 ml of physiological saline followed by 0.2 ml of physiological saline.

[0027] It should be noted that the dosage of edible oil administered via gavage during modeling was selected based on the standard recommended in the "Chinese Dietary Guidelines (2016)" which recommends a daily intake of no more than 25-30g of edible oil. However, the average daily intake of edible oil per capita in my country is 42.1g, which is far higher than this standard. Therefore, to study the harmful effects of excessive amounts of edible oil on the body, this embodiment uses the standard value and the equivalent dose exceeding the standard as the daily gavage dose for mice. According to the formula for calculating the equivalent dose for mice (based on body weight): Equivalent dose for mice = Daily intake of humans (g) / 60kg × 13.3, 18g / (kg) (approximately 0.5ml) was selected as the gavage dose of edible oil in this embodiment.

[0028] Regarding the selection of fructose solution concentration during modeling: domestic and international studies often use a 30% fructose solution concentration for the study of high fructose-induced glucose and lipid metabolism disorders; regarding the selection of fructose solution dosage during modeling, based on the daily water requirement of mice being 4-7 ml, 5 ml / 24h, the required water volume is calculated to be 0.2 ml / h.

[0029] Therefore, in this embodiment, a mouse model of postprandial hypertriglyceridemia is to be established by gavage with a combination of 0.5 ml of edible oil and 0.2 ml of 30% fructose water.

[0030] S3: Sample the model group mice and the blank group mice after step S2 to obtain model group samples and blank group samples.

[0031] The sampling procedure is as follows: plasma is obtained from the mouse eyeball. After the plasma is removed, it is left to stand for 3-4 hours. Then, it is centrifuged at 3000 rpm and 4℃ for 10 minutes in a refrigerated centrifuge. The supernatant is collected and the condition of chylous blood in each group is observed.

[0032] S4: Use a fully automated biochemical analyzer to detect glycolipid-related biochemical indicators in the model group and blank group samples; use superoxide dismutase (Catalog No.: A001-3-2) and malondialdehyde (Catalog No.: A003-1-2) detection kits to detect serum SOD and MDA levels in the model group and blank group samples;

[0033] Glucose and lipid-related biochemical indicators include TG, TC, HDL-C, LDL-C, alanine aminotransferase (ALT), aspartine aminotransferase (AST), GLU, ALP, and total biliary acid (TBA).

[0034] S5: HE and Oil Red O staining were used to observe the liver pathology of mice in the model group and the blank group, and transmission electron microscopy was used to observe the morphology of mitochondria in hepatocytes.

[0035] The specific method for observing liver pathology is as follows: After fixing a 0.5×0.5cm liver with 4% paraformaldehyde for 24 hours, it is dehydrated by ethanol gradient, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to observe the morphology of hepatocytes; then frozen, fixed, stained, differentiated, counterstained cell nuclei, mounted, and observed the results of Oil Red O staining.

[0036] The specific method for observing the morphology of mitochondria in hepatocytes is as follows: Take a piece of liver tissue the size of a grain of rice and place it in 1 ml of electron microscopy fixative to preserve it in the dark. Before instrument sectioning, rinse it three times with 1*PBS. When preparing the sample, fix it with 2% osmium tetroxide fixative for 2 hours. Dehydrate it stepwise with acetone, soak it, embed it, solidify it and then perform ultrathin sectioning. After that, stain it with lead citrate and uranium acetate, observe it under a transmission electron microscope and take pictures.

[0037] S6: Statistically analyze the data obtained in steps S4 and S5 to obtain the optimal mouse model of postprandial hypertriglyceridemia.

[0038] It should be noted that all data were statistically processed using SPSS 20.0 and Graph PadPrism 8.0 software. For normally distributed data, the mean ± standard deviation (x ± s) was expressed as mean ± standard deviation. ANOVA analysis was used for comparisons among multiple groups, with Turkey's method used for homogeneous variances and Dunnett's T3 method used for unequal variances. Independent samples t-tests were used for comparisons between two groups, and P < 0.05 was considered statistically significant.

[0039] The specific results are as follows:

[0040] like Figure 2 As shown, the model group mice exhibited severe chylous blood in their serum;

[0041] The GLU level in the model group (6.55±0.66mmol / L) was significantly higher than that in the blank group (5.1±1.8mmol / L) (P<0.01);

[0042] The TG level in the model group (4.67±0.87mmol / L) was significantly higher than that in the blank group (1.77±0.64mmol / L) (P<0.001), and was 3-4 times higher than that in the blank group.

[0043] There was no significant difference in TC between the model group (4.87±0.59mmol / L) and the blank group (4.44±0.85mmol / L);

[0044] The HDL level in the model group (2.73±0.61 U / L) was significantly lower than that in the blank group (3.26±0.36 U / L) (P<0.05).

[0045] The LDL level in the model group (0.97±0.2 U / L) was significantly higher than that in the blank group (0.76±0.2 U / L) (P<0.05);

[0046] The ALT level in the model group (48.59±8.7 U / L) was significantly higher than that in the blank group (39.17±4.9 U / L) (P<0.01);

[0047] The AST level in the model group (155.9±29.2 U / L) was significantly higher than that in the blank group (113.1±16.84 U / L) (P<0.001);

[0048] The ALP level in the model group (157.0±17.5 U / L) was significantly higher than that in the blank group (121.7±22.4 U / L) (P<0.001);

[0049] There was no significant difference in TBA levels between the model group (1.8±0.6 μmol / L) and the blank group (2.3±0.7 μmol / L).

[0050] The serum SOD level in the model group mice (28.7±4.2 U / ml) was significantly lower than that in the blank group (34.2±4.1 U / ml) (P<0.01);

[0051] The serum MDA level in the model group mice (14.9±1.4 nmol / ml) was significantly higher than that in the blank group (12.1±2.0 nmol / ml) (P<0.01).

[0052] like Figure 3 As shown, in the pathological sections of liver tissue from mice in the blank control group, the liver lobule structure was clear and intact, the hepatocytes were tightly arranged and morphologically normal, and no fatty vacuoles, fatty degeneration, or fatty necrosis were observed in the cytoplasm. In the pathological sections of liver tissue from mice in the model group, the liver lobule structure was unclear, the hepatocytes near the central vein were disordered and morphologically incomplete, and the cytoplasm was filled with a large number of fatty vacuoles (indicated by the red arrows). Oil Red O staining of the liver showed that, compared with the blank control group, the liver tissue cells in the model group showed more orange-red staining and more fatty vacuoles (indicated by the red arrows), generally suggesting that lipid droplets, mainly composed of triglycerides, were significantly aggregated. Transmission electron microscopy showed that, compared with the blank control group, the hepatocytes of mice in the model group showed mitochondrial hypertrophy, with endoplasmic reticulum and lipid droplets accompanying the mitochondria, suggesting that mitochondria may be closely related to lipid metabolism under high glucose and high lipid conditions.

[0053] like Figure 4As shown, the TG (4.67±0.87mmol / L) levels in the model group and the TG (10.21±1.65mmol / L) levels in the P-407 intraperitoneal injection group were significantly higher than the TG (1.77±0.64mmol / L) levels in the blank group (P<0.001). The TC (3.45±0.6mmol / L) level in the simple gavage edible oil group was significantly lower than the TC (4.43±0.85mmol / L) level in the blank group (P<0.01). There were no significant differences in TC levels between the other two groups and the blank group. The HDL (2.73±0.61mmol / L) level in the model group and the simple gavage edible oil group were also significantly higher. The HDL levels in the edible oil group (2.59±0.37 mmol / L) and the intraperitoneal injection P-407 group (2.59±0.37 mmol / L) were significantly lower than those in the control group (2.24±0.41 mmol / L) (P<0.01; P<0.05; P<0.001). Conversely, the LDL levels in the model group (0.97±0.2 mmol / L) and the intraperitoneal injection P-407 group (1.07±0.21 mmol / L) were significantly higher than those in the control group (0.76±0.2 mmol / L) (P<0.05; P<0.01). Data are presented as mean ± standard deviation (x±s, n=8). *P<0.05, **P<0.01, ***P<0.01, and ****P<0.001 indicate statistically significant differences compared to the control group.

[0054] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for establishing an improved mouse model of postprandial hypertriglyceridemia, characterized in that, The establishment method includes the following steps: S1: Obtain a preset number of mouse models and divide them into a blank group and a model group on average; S2: The model group and the blank group were administered gavage for 120 minutes. S3: Sample the model group mice and the blank group mice after step S2 to obtain model group samples and blank group samples. S4: Detect glucose-lipid-related biochemical indicators, serum SOD and MDA levels in model group samples and blank group samples; S5: Observe the liver pathology and mitochondrial morphology in hepatocytes of mice in the model group and the blank group. S6: Statistically analyze the data obtained in steps S4 and S5 to obtain a mouse model of postprandial hypertriglyceridemia; The gavage treatment in step S2 is as follows: Mice were first gavaged with 0.5 ml of edible oil followed by 0.2 ml of 30% fructose solution; the control group was first gavaged with 0.5 ml of physiological saline followed by 0.2 ml of physiological saline. The glucose and lipid-related biochemical indicators mentioned in step S4 include TG, TC, HDL-C, LDL-C, alanine aminotransferase, aspartate aminotransferase, GLU, ALP, and total bile acids.

2. The method for establishing a modified mouse model of postprandial hypertriglyceridemia according to claim 1, characterized in that, The preset number of items mentioned in step S1 is 24.

3. The method for establishing a modified mouse model of postprandial hypertriglyceridemia according to claim 1, characterized in that, The sampling procedure described in step S3 is as follows: obtain plasma from the mouse eyeball, let the plasma stand for 3-4 hours, then centrifuge at 3000 rpm and 4℃ for 10 min using a refrigerated centrifuge to obtain the supernatant.

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