System for evaluating effect of preventing and treating obesity

Abstract

The embodiment of the invention provides an effect evaluation system for preventing and treating obesity, and relates to the technical field of analysis. The system comprises a feeding device used for containing multiple groups of obese mouse models, and the multiple groups of obese mouse models comprise experimental group mice, normal group mice and model group mice; the collecting device is used for collecting the liver tissues of the multiple groups of obese mouse models fed for the preset duration; wherein the mice in the experimental group are fed with to-be-experimented articles and high-fat feed, the mice in the model group are fed with high-fat feed, and the mice in the normal group are fed with normal feed; the measuring device is used for measuring the level of liver inflammatory factors in the liver tissue of each group of obese mouse models, and the liver inflammatory factors comprise at least one of the following items: tumor necrosis factor-alpha, interleukin-6 or interleukin-1beta; and the evaluation device is used for evaluating the obesity prevention and treatment effect of the to-be-experimented article based on the liver inflammatory factor levels of the multiple groups of obese mouse models.

Description

Technical Field

[0001] The present application relates to the field of analysis technology, and in particular to an effect evaluation system for preventing and treating obesity. Background Art

[0002] In recent years, obesity and metabolic syndrome (MS) have become a global problem, with high-fat and high-sugar diets being the primary cause. Therefore, preventing and treating obesity and MS is an urgent issue. Related technologies have disclosed the use of lipid-lowering drugs or functional food factors such as probiotics to prevent and treat obesity. However, prior to using these methods for obesity prevention and treatment, relevant experiments are generally required to evaluate their effectiveness.

[0003] When evaluating the effectiveness of a test article in preventing and treating obesity, the relevant art typically only evaluates changes in certain indicators (such as weight) caused by the test article. While evaluating based on weight change can more intuitively reflect the effectiveness of the test article in preventing and treating obesity, because obesity often causes multiple chronic diseases and may also affect the user's internal metabolic function and hormone levels, evaluating based solely on weight change cannot fully reflect the effectiveness of the test article in preventing and treating obesity. Summary of the Invention

[0004] The embodiment of the present application provides an obesity prevention and treatment effect evaluation system for evaluating the effect of a test article on the prevention and treatment of obesity based on the level of liver inflammatory factors in liver tissue.

[0005] In a first aspect, an embodiment of the present application provides an obesity prevention and treatment effect evaluation system, comprising: a feeding device for accommodating and feeding multiple groups of obese mouse models, the multiple groups of obese mouse models including experimental group mice, normal group mice and model group mice; wherein the experimental group mice are fed with an experimental article and a high-fat feed, the model group mice are fed with a high-fat feed, and the normal group mice are fed with a normal feed; a collecting device for collecting liver tissues of multiple groups of obese mouse models fed for a preset time; a measuring device for measuring the level of liver inflammatory factors in the liver tissue of each group of obese mouse models, the liver inflammatory factors including at least one of the following: tumor necrosis factor-α, interleukin-6 or interleukin-1β; an evaluation device for evaluating the effect of the experimental article in preventing and treating obesity based on the liver inflammatory factor levels of the multiple groups of obese mouse models.

[0006] The obesity prevention and treatment effect evaluation system provided in the embodiments of the present application contains and feeds multiple groups of obese mouse models through a feeding device. The experimental group mice are fed with the test article and a high-fat diet, the model group mice are fed with a high-fat diet, and the normal group mice are fed with a normal diet. It is understandable that a high-fat diet often contains a large amount of saturated fat and trans fatty acids. Long-term intake of a high-fat diet may lead to excessive body fat, obesity, and the occurrence of low-grade chronic inflammation. These fats can stimulate immune cells to produce liver inflammatory factors, such as tumor necrosis factor-α, interleukin-6, or interleukin-1β. Liver inflammatory factors can induce the production of inflammatory cytokines and trigger local tissue inflammatory reactions.

[0007] Therefore, the efficacy evaluation system provided in the embodiments of the present application uses a collection device to collect liver tissue from multiple groups of obese mouse models after being fed for a preset period of time, and then uses a measuring device to measure the levels of liver inflammatory factors in the liver tissue. The evaluation device can then more accurately determine the effect of the test article on the mouse's liver inflammation based on the levels of liver inflammatory factors in the mouse's liver tissue, and thus more accurately determine the effectiveness of the test article in preventing and treating obesity.

[0008] In combination with the first implementation method of the first aspect, the evaluation device is specifically used to determine that the test item has the effect of preventing and treating obesity when the difference in liver inflammatory factor levels between the experimental group mice and the normal group mice is smaller than the difference in liver inflammatory factor levels between the model group mice and the normal group mice.

[0009] In combination with the second implementation method of the first aspect, the system also includes: an extraction device for extracting serum from multiple groups of obese mouse models fed for a preset period of time, and performing serum lipid metabolism analysis to obtain serum analysis results for each group of obese mouse models, the serum analysis results including: triglyceride, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels; the evaluation device is also used to further evaluate the effectiveness of the experimental article in preventing and treating obesity based on the serum analysis results.

[0010] In combination with the third implementation of the first aspect, the measuring device is also used to measure the content of aspartate aminotransferase and / or alanine aminotransferase in the serum; the evaluation device is also used to evaluate the effect of the test article in preventing and treating obesity based on the content of aspartate aminotransferase and / or alanine aminotransferase.

[0011] In combination with the fourth implementation method of the first aspect, the system also includes: a cleaning device for cleaning and weighing the liver tissue and calculating the organ index of the liver tissue; an evaluation device for further evaluating the effect of the test article in preventing and treating obesity based on the organ index of the liver tissue.

[0012] In combination with the fifth implementation method of the first aspect, the collecting device is also used to collect epididymal adipose tissue from multiple groups of obese mouse models fed for a preset time; the system also includes: a cleaning device for cleaning and weighing the epididymal adipose tissue, and calculating the organ index of the epididymal adipose tissue; the evaluation device is also used to further evaluate the effect of the experimental article in preventing and treating obesity based on the organ index of the epididymal adipose tissue.

[0013] In combination with the sixth implementation method of the first aspect, the collecting device is also used to collect small intestinal tissues of multiple groups of obese mouse models fed for a preset time; the system also includes: a staining device for staining and slicing liver tissue, small intestinal tissue and epididymal fat tissue, and performing pathological analysis after scanning with an optical microscope to obtain pathological analysis results; the evaluation device is also used to further evaluate the effect of the experimental item in preventing and treating obesity based on the pathological analysis results.

[0014] In combination with the seventh implementation method of the first aspect, the staining device is specifically used to place the liver tissue, small intestinal tissue and epididymal fat tissue in the corresponding fixative for more than 24 hours; use different concentrations of ethanol to dehydrate the liver tissue, small intestinal tissue and epididymal fat tissue, embed them in paraffin and cut them into slices of preset thickness; and use hematoxylin-eosin to stain the slices.

[0015] In combination with the eighth implementation method of the first aspect, the recording device is used to record the food intake and weight changes of each group of obese mouse models within a preset time period of feeding multiple groups of obese mouse models; the evaluation device is also used to further evaluate the effect of the experimental article in preventing and treating obesity based on the food intake and weight changes.

[0016] In combination with the ninth implementation method of the first aspect, the experimental items include at least one of the following: chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and green vegetable; the multiple groups of obese mouse models also include: positive control group mice; the feeding device also includes: a positive control area for accommodating the positive control group mice; the positive control group mice are fed with high-fat feed and dietary fiber. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0018] Figure 1 A schematic diagram of an obesity prevention and treatment effect evaluation system provided in an embodiment of the present application;

[0019] Figure 2 A flow chart of a method for determining the level of liver inflammatory factors provided in an embodiment of the present application;

[0020] Figure 3 A schematic diagram of the content of tumor necrosis factor-α provided in an example of the present application;

[0021] Figure 4 A schematic diagram of interleukin-6 content provided in an embodiment of the present application;

[0022] Figure 5 A schematic diagram of interleukin-1β content provided in an embodiment of the present application;

[0023] Figure 6 A schematic diagram of triglyceride content provided in an embodiment of the present application;

[0024] Figure 7 A schematic diagram of cholesterol content provided in an embodiment of the present application;

[0025] Figure 8 A schematic diagram of low-density lipoprotein content provided in an embodiment of the present application;

[0026] Figure 9 A schematic diagram of high-density lipoprotein content provided in an embodiment of the present application;

[0027] Figure 10 A schematic diagram of the contents of aspartate aminotransferase and alanine aminotransferase is provided for the examples of the present application;

[0028] Figure 11 A flow chart of a method for obtaining serum provided in an embodiment of the present application;

[0029] Figure 12 A schematic diagram of an organ index provided in an embodiment of the present application;

[0030] Figure 13 A schematic diagram of another obesity prevention and treatment effect evaluation system provided in an embodiment of the present application;

[0031] Figure 14 A schematic diagram of an epididymal adipose tissue cell provided in an embodiment of the present application;

[0032] Figure 15 A schematic diagram of a section of small intestinal tissue cells provided in an embodiment of the present application;

[0033] Figure 16 A flow chart of a method for staining tissue cells provided in an embodiment of the present application;

[0034] Figure 17 A schematic diagram of weight change provided in an embodiment of the present application;

[0035] Figure 18 A weight gain diagram provided in an embodiment of the present application. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0037] It should be noted that the terms "first" and "second" used in the embodiments of the present invention are for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0038] In the embodiments of this application, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of this application should not be interpreted as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0039] As mentioned in the background art, in related technologies, when evaluating the effect of preventing and treating obesity, the evaluation is usually only based on the changes in some indicators (such as body weight) caused by the test article, which cannot fully reflect the effect of the test article on preventing and treating obesity.

[0040] Based on this, the embodiment of the present application provides an effect evaluation system for preventing and treating obesity. Multiple groups of obese mouse models are housed and fed by a feeding device. The experimental group mice are fed with the test article and a high-fat diet, the model group mice are fed with a high-fat diet, and the normal group mice are fed with a normal diet. It is understandable that a high-fat diet often contains a large amount of saturated fat and trans fatty acids. Long-term intake of a high-fat diet may lead to excessive body fat, obesity, and the occurrence of low-grade chronic inflammation. These fats can stimulate immune cells to produce liver inflammatory factors, such as tumor necrosis factor-α, interleukin-6, or interleukin-1β. Liver inflammatory factors can induce the production of inflammatory cytokines and trigger local tissue inflammatory reactions.

[0041] Therefore, the efficacy evaluation system provided in the embodiments of the present application uses a collection device to collect liver tissue from multiple groups of obese mouse models after being fed for a preset period of time, and then uses a measuring device to measure the levels of liver inflammatory factors in the liver tissue. The evaluation device can then more accurately determine the effect of the test article on the mouse's liver inflammation based on the levels of liver inflammatory factors in the mouse's liver tissue, and thus more accurately determine the effectiveness of the test article in preventing and treating obesity.

[0042] In some embodiments, since C57BL / 6J mice are sensitive to high-fat diets and become obese relatively quickly after high-fat diet feeding, C57BL / 6J mice are also referred to as diet-induced obesity mice (DIO). Therefore, as a feasible implementation method, the mice used in the embodiments of the present application can be obtained by feeding C57BL / 6J mice with a high-fat diet.

[0043] It should be noted that before using mice for experiments, they need to be fed for a period of time to allow them to adapt to the experimental environment and avoid being disturbed by environmental factors that may lead to a decrease in the mice's food intake.

[0044] Because male mice gain significantly more weight than female mice when fed the same high-fat diet, meaning male mice are more obese than female mice, male C57BL / 6J mice were used in the present examples for ease of experimentation.

[0045] As a feasibility implementation method, before using mice for experiments, 45 male C57BL / 6J mice (6 weeks old, weighing 18±2g) need to be raised in a sterile animal room with a temperature of 21℃~23℃, a relative humidity of 40%~70%, and a light-dark cycle of 12h for one week, and have free food and water intake to allow them to adapt to the experimental environment.

[0046] For the sake of ease of description, the following text uses the test items of chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and green vegetable as examples for explanation. However, this example does not constitute a limitation on the effect evaluation system provided by this application. In actual application, the experimenter can choose the test items according to needs.

[0047] As a feasible implementation method, four dishes including chicken breast salad, stir-fried kale, seasonal vegetable salad and mushroom and green vegetable salad need to be freeze-dried to remove moisture, prepared into vegetable powder by a wall breaker, and passed through a 60-mesh sieve for easy feeding of mice.

[0048] It is understood that for the accuracy of the experimental results, each group of mice in the examples of the present application includes 6 male C57BL / 6J mice (6 weeks old, 18 ± 2g) mice, and the content of various components in each group of mice is obtained by averaging or removing the maximum and minimum values ​​and then averaging. The examples of the present application are not limited hereto.

[0049] The following is a further introduction to the obesity prevention and treatment effect evaluation system provided in the embodiments of the present application in conjunction with the accompanying drawings.

[0050] See also Figure 1 , Figure 1 This is a schematic diagram of the obesity prevention and treatment effect evaluation system provided in the embodiment of the present application. Figure 1 As shown, the effect evaluation system includes: a feeding device 11, a collecting device 12, a measuring device 13 and an evaluation device 14.

[0051] In the embodiment of the present application, the feeding device 11 is used to accommodate and feed multiple groups of obese mouse models.

[0052] Among them, multiple groups of obese mouse models included experimental group mice, normal group mice and model group mice; the experimental group mice were fed with experimental items and high-fat feed, the model group mice were fed with high-fat feed, and the normal group mice were fed with normal feed.

[0053] In some embodiments, the feeding device may include an experimental area, a normal area, and a model area, which are used to accommodate and feed experimental group mice, normal group mice, and model group mice, respectively.

[0054] Multiple obese mice were randomly divided into groups to obtain multiple groups of obese mouse models. It should be understood that the number of groups of obese mouse models can be determined according to the type of experimental object. When a single experimental object is to be tested, the obese mice can be divided into three groups; when two experimental objects are to be tested, the obese mice can be divided into four groups. This embodiment of the present application does not limit this.

[0055] It should be understood that an experimental group refers to a randomly selected subset of experimental subjects in which mice receive some special treatment that is not given to the normal group. A normal group refers to a randomly selected subset of experimental subjects in which individuals do not receive any special treatment. A normal group is necessary because without a normal group, there is no way to determine whether the manipulation or some other variable (or combination of variables) has had an effect.

[0056] Therefore, in the embodiment of the present application, by dividing the mice into an experimental group of mice, a normal group of mice, and a model group of mice, it is possible to more accurately determine whether the experimental article has an effect on obesity in obese mice.

[0057] Among them, the mice in the experimental group were fed with the experimental articles and high-fat feed, the mice in the model group were fed with high-fat feed, and the mice in the normal group were fed with normal feed.

[0058] High-fat diets are one of the most commonly used feeds for modeling common diseases such as obesity, fatty liver disease, and hyperlipidemia. By adjusting the fat content and adding various substances such as cholesterol and sodium cholate, different modeling effects can be achieved. Feeding mice a high-fat diet closely resembles normal human life, being virtually irritating and similar to the developmental processes of related human diseases.

[0059] Therefore, the normal living conditions of humans can be simulated by feeding mice with high-fat feed, and then the experimental group mice can be fed with the test items to determine the impact of the test items on obesity in obese mice.

[0060] As a feasible implementation method, the mice in the experimental group can be fed chicken breast salad and high-fat feed, stir-fried kale and high-fat feed, seasonal vegetable salad and high-fat feed, and mushrooms and vegetables and high-fat feed.

[0061] In some embodiments, since chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and green vegetable all contain a large amount of dietary fiber, in order to determine whether dietary fiber has an impact on obesity in obese mice, multiple groups of obese mouse models can also include: positive control group mice.

[0062] The mice in the positive control group were fed dietary fiber and high-fat diet to determine whether dietary fiber had an effect on obesity in the obese mice.

[0063] As a feasible implementation method, the dietary fiber used in the embodiments of the present application was purchased from the Nestlé official flagship store on JD.com (item number: 68458377308).

[0064] As a feasible implementation method, when feeding mice with experimental items and dietary fiber, it is necessary to use an appropriate amount of physiological saline to dissolve them and gavage the mice at a certain dose.

[0065] As a feasible implementation, the feeding device may further include a positive control area for accommodating and feeding mice in the positive control group.

[0066] In the embodiment of the present application, the collecting device 12 is used to collect liver tissues of multiple groups of obese mouse models fed for a preset time.

[0067] It should be understood that obesity is a long-term disease, so it is necessary to feed multiple groups of obese mouse models for a period of time to obtain more accurate results. As a feasible implementation method, the preset period can be six weeks.

[0068] In some embodiments, to collect liver tissue from mice, the mice must first be anesthetized with ether and then slaughtered. Because the levels of various components in mice fluctuate significantly after eating, measurements may be inaccurate. Therefore, before anesthetizing with ether and slaughtering the mice, the mice must be fasted for a period of time (e.g., 12 hours) to ensure the accuracy of the content of various components.

[0069] A high-fat diet often contains a large amount of saturated fat and trans fatty acids. Long-term intake of a high-fat diet may lead to the accumulation of fat in the liver tissue. Therefore, the obesity level of the obese mouse model can be judged by the liver tissue, and then the impact of the experimental items on the obesity of the obese mice can be determined.

[0070] In the embodiment of the present application, the measuring device 13 is used to measure the level of liver inflammatory factors in the liver tissue of each group of obese mouse models.

[0071] Among them, liver inflammatory factors include at least one of the following: tumor necrosis factor-α, interleukin-6 or interleukin-1β.

[0072] Long-term consumption of a high-fat diet can trigger low-grade chronic inflammation. These fats can stimulate immune cells to produce liver inflammatory factors, such as tumor necrosis factor-α and interleukin-6, thereby triggering an inflammatory response.

[0073] Tumor necrosis factor-α is a cytokine produced by activated immune cells (such as macrophages and T lymphocytes) that plays a variety of roles in the human body. Tumor necrosis factor-α is an important mediator of inflammatory responses and can promote inflammatory responses.

[0074] Interleukin-6 transmits signals between immune cells, regulates and coordinates immune responses. It can promote the proliferation of B lymphocytes and antibody production, stimulate hepatocytes and other cells to synthesize acute phase proteins, participate in inflammatory responses, and is the primary indicator of inflammation in the body.

[0075] Interleukin-1β is a member of the interleukin family that can induce the production of inflammatory cytokines and trigger local tissue inflammatory responses.

[0076] In other words, obesity can cause an inflammatory response in liver tissue, which means that the levels of liver inflammatory factors increase. Furthermore, if the levels of liver inflammatory factors in the liver tissue of obese mice are lower, it can indicate that the treatment is more effective in preventing and treating obesity.

[0077] In some embodiments, liver inflammatory factors are small polypeptides or glycoproteins synthesized and secreted by tissue cells and are soluble antigens. A significant portion of these factors originate from tissues and cells, resulting in complex compositions. To prepare and measure liver inflammatory factor levels, tissues and cells must first be disrupted to release biomacromolecules such as proteins and nucleic acids from the tissues before measurement.

[0078] As a possible implementation, see Figure 2 When measuring the level of liver inflammatory factors in liver tissue, the measuring device performs the following steps:

[0079] S201. Add liver tissue to a homogenization medium at a weight (mg): volume (μL) ratio of 1:9, and homogenize in an ice-water bath to obtain a homogenate.

[0080] Homogenization is a process used primarily in laboratories or industrial production to thoroughly mix powdered or granular solids with liquids to form a thick mixture with uniformly suspended matter.

[0081] The homogenization medium should have an appropriate osmotic pressure to maintain normal cell morphology and function. Excessively high or low osmotic pressure may adversely affect cells. Therefore, as a feasible implementation, the homogenization medium is 0.9% normal saline.

[0082] S202. Centrifuge the homogenate and retain the supernatant.

[0083] During the homogenization process, tissue is broken down into a homogenate, and soluble antigens and other substances are dissolved in the homogenate. Centrifugation then separates these soluble antigens from the homogenate, forming a supernatant. This supernatant contains the liver inflammatory factors to be measured and can be used in subsequent experiments or production processes.

[0084] When selecting the supernatant, centrifugation is required to remove cell debris and microscopic tissue to ensure the purity and quality of the supernatant. In addition, depending on experimental or production requirements, the precipitate after centrifugation can be re-dissolved to extract other desired components.

[0085] As a feasible implementation method, S202 may specifically include: placing the homogenate in a centrifuge, centrifuging at 2500-3000 r / min for 10 min, and retaining the supernatant in the homogenate after centrifugation.

[0086] S203. Determine the levels of liver inflammatory factors in the supernatant.

[0087] As a feasibility study, the levels of tumor necrosis factor-α, interleukin-6, or interleukin-1β in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA) kits.

[0088] Enzyme-linked immunosorbent assay (ELISA), known as the gold standard for immunoassays, is an immunological assay method that involves adsorbing the supernatant onto a solid carrier such as polystyrene to conduct qualitative and quantitative immune reactions. It can more accurately measure the levels of inflammatory factors in liver tissue.

[0089] In the embodiment of the present application, the evaluation device 14 is used to evaluate the effect of the test article in preventing and treating obesity based on the levels of liver inflammatory factors in multiple groups of obese mouse models.

[0090] After obtaining the levels of liver inflammatory factors in different groups of obese mouse models, the effectiveness of the experimental items in preventing and treating obesity can be evaluated based on the levels of liver inflammatory factors.

[0091] It is understandable that since the level of liver inflammatory factors can reflect the degree of inflammation in the mouse's liver cells, that is, the severity of the mouse's disease, the level of liver inflammatory factors is positively correlated with the severity of the mouse's disease. Therefore, the lower the level of liver inflammatory factors in the mouse, the better the effect of preventing and treating chronic diseases.

[0092] As a feasible implementation method, the evaluation device is specifically used to determine that the test item has the effect of preventing and treating obesity when the difference in liver inflammatory factor levels between the experimental group mice and the normal group mice is smaller than the difference in liver inflammatory factor levels between the model group mice and the normal group mice.

[0093] For example, chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad were used as experimental items, and the experimental group mice were fed with them respectively. The liver inflammatory factor levels of each group of obese mice were finally measured as follows: Figure 3 、 Figure 4 and Figure 5 shown.

[0094] like Figure 3 As shown, the content of tumor necrosis factor-α in the liver of mice in the model group (fed only with high-fat feed) was 255.21 pg / mL, which was significantly increased by 184.63% compared with the normal group (fed only with normal feed) (P<0.05); while the content of tumor necrosis factor-α in some groups of the experimental group did not increase compared with the normal group, which inhibited the growth of tumor necrosis factor-α, and the content of tumor necrosis factor-α in some groups showed a downward trend compared with that in the model group; the content of tumor necrosis factor-α in the positive control group also increased significantly, and there was no downward trend compared with the content in the model group.

[0095] Depend on Figure 3It can be seen that chicken breast salad, stir-fried kale, seasonal vegetable salad and mushroom and greens can all inhibit the growth of tumor necrosis factor-α, and since there is no downward trend in the positive control group compared with the model group, it shows that the decrease in tumor necrosis factor-α content is not caused by dietary fiber.

[0096] like Figure 4 As shown, the interleukin-6 content in the liver of mice in the model group was 316.85 pg / mL, which was significantly increased by 130.15% compared with the normal group (P<0.05). However, the interleukin-6 content of some groups in the experimental group did not increase compared with the normal group, which inhibited the growth of interleukin-6. The interleukin-6 content of some groups showed a downward trend compared with the model group. The interleukin-6 content in the positive control group also increased significantly, and there was no downward trend compared with the model group.

[0097] Depend on Figure 4 It can be seen that chicken breast salad, stir-fried kale, seasonal vegetable salad and mushroom and greens can all inhibit the growth of interleukin-6, and since there is no downward trend in the positive control group compared with the model group, it shows that the decrease in interleukin-6 content is not caused by dietary fiber.

[0098] like Figure 5 As shown in the data, the interleukin-1β content in the liver of mice in the model group was 310.15 pg / mL, which was significantly increased by 172.75% compared with the normal feed group (P<0.05); the experimental group inhibited the growth of interleukin-1β, and the interleukin-1β content in each experimental group was lower than that in the normal group and the model group; the interleukin-1β content in the positive control group also increased significantly.

[0099] Depend on Figure 5 It can be seen that chicken breast salad, stir-fried kale, seasonal vegetable salad and mushroom and greens can all inhibit the growth of interleukin-1β, and since there is no downward trend in the positive control group compared with the model group, it shows that the decrease in interleukin-1β content is not caused by dietary fiber.

[0100] In summary, it can be seen that chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and greens can reduce the level of liver inflammatory factors in the liver tissue of mice, and dietary fiber cannot reduce the level of liver inflammatory factors. Therefore, chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and greens can have a better effect in preventing and treating obesity.

[0101] In some embodiments, a high-fat diet can lead to abnormal lipid metabolism in mice, specifically manifested as increased levels of triglycerides, total cholesterol, and low-density lipoprotein in serum, and decreased levels of high-density lipoprotein. Therefore, to further improve the accuracy of determining the effectiveness of a test article in preventing and treating obesity, the obesity prevention and treatment efficacy evaluation system provided in this application can also analyze the effectiveness of a test article in preventing and treating obesity based on mouse serum.

[0102] As a feasible implementation, the obesity prevention and treatment effectiveness evaluation system provided in this application also includes: an extraction device for extracting serum from multiple groups of obese mouse models fed for a preset period of time, performing serum lipid metabolism analysis, and obtaining serum analysis results. The evaluation device is also used to further evaluate the obesity prevention and treatment effectiveness of the test article based on the serum analysis results.

[0103] Among them, serum analysis results include: triglyceride, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels.

[0104] That is, the extraction device analyzes the serum of the mouse to measure the triglyceride, total cholesterol, high-density lipoprotein cholesterol or low-density lipoprotein cholesterol content in the serum.

[0105] Fat is an important source of energy and the foundation for building cell membranes, synthesizing hormones, vitamins, and other important substances. Poor lipid metabolism can lead to a variety of health problems. Hyperlipidemia is a primary manifestation of lipid metabolism disorders. When excessive fat intake or excessive cholesterol synthesis occurs, blood lipid levels rise. Abnormal lipid metabolism is also closely related to obesity and metabolic syndrome. When calorie intake exceeds body consumption, excess energy is stored as fat, leading to weight gain.

[0106] In other words, a high-fat diet can lead to abnormal lipid metabolism, manifested as elevated levels of triglycerides, total cholesterol, and low-density lipoprotein (LDL), and decreased levels of high-density lipoprotein (HDL). Therefore, the evaluation device can determine the effectiveness of a test article in preventing and treating obesity by analyzing the serum of multiple groups of obese mouse models.

[0107] For example, chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad were used as experimental items, and the experimental group mice were fed with them respectively. The serum analysis results of the obese mice in each group were finally measured as follows: Figure 6 、 Figure 7 、 Figure 8 as well as Figure 9 shown.

[0108] like Figure 6 As shown in Figure 2, compared with the model group mice, the triglyceride content in the serum of the mice in each experimental group was significantly reduced; Figure 7 As shown in Figure 2, compared with the model group mice, the serum cholesterol levels of some groups of mice in the experimental group were significantly reduced; Figure 8 As shown in FIG, the low-density lipoprotein content in the serum of some mice in the experimental group was significantly reduced; and Figure 9 As shown, the high-density lipoprotein content in the serum of mice in each experimental group was significantly increased.

[0109] In summary, although some of the experimental groups did not reduce serum cholesterol and low-density lipoprotein levels in mice, they all reduced triglyceride levels and increased high-density lipoprotein levels. Therefore, chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad are more effective in preventing and treating obesity.

[0110] It can be seen that by extracting the serum of mice for serum lipid metabolism analysis, the impact of the test items on the blood lipid metabolism of mice can be analyzed from the perspective of the blood lipid metabolism of mice, and then the effect of the test items on the prevention and treatment of obesity can be judged more accurately.

[0111] In some embodiments, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are key enzymes in the α-ketoglutarate-related metabolic pathway, which can promote the oxidation of fatty acids, reduce fat production, and alleviate oxidative stress and inflammatory responses. Both are enzymes present in liver cells. When the liver is damaged, they are released into the blood, causing increased enzyme levels in the blood. Therefore, the effect of the test article on the prevention and treatment of obesity can be judged by the level of AST or ALT in the blood.

[0112] As a feasible implementation method, the obesity prevention and treatment effect evaluation system provided in the embodiment of the present application, the measuring device is also used to measure the content of aspartate aminotransferase and / or alanine aminotransferase in the serum; the evaluation device is also used to evaluate the effect of the test article in preventing and treating obesity based on the content of aspartate aminotransferase and / or alanine aminotransferase.

[0113] It is understood that when liver cells are damaged by inflammation, necrosis, poisoning, etc., AST and ALT are released into the blood, increasing the serum AST and ALT levels. Therefore, in the present embodiment, the measurement device measures the serum AST and / or ALT levels, and the evaluation device can more accurately determine the damage to the mouse liver cells, thereby more accurately determining the effect of the test article on preventing and treating obesity.

[0114] For example, chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad were used as experimental items, and the experimental group mice were fed with them respectively. The final measured levels of aspartate aminotransferase and / or alanine aminotransferase in each group of obese mice were as follows: Figure 10shown.

[0115] like Figure 10 As shown, the serum AST activity of mice in the model group (fed only with a high-fat diet) was 251.12 U / L, significantly higher than that in the normal group (fed only with a normal diet) (P<0.05). The serum AST level in the experimental group was significantly lower. This suggests that chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad can reduce AST levels in mouse serum, effectively preventing and treating chronic obesity.

[0116] Please continue reading Figure 10 The serum alanine aminotransferase activity in the model group was 40.13 U / L, significantly higher than that in the normal group (P<0.05). The serum alanine aminotransferase level in the experimental group was significantly lower. This suggests that chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad can reduce serum alanine aminotransferase levels in mice, thereby effectively preventing and treating chronic obesity.

[0117] In some cases, because the concentrations of glucose, fat, and other substances in the blood of mice increase significantly after eating, subsequent measurements of serum components can be inaccurate. Therefore, before extracting serum from multiple groups of obese mouse models, the mice need to be fasted for a period of time to ensure the accuracy of the blood components.

[0118] As a possible implementation, see Figure 11 When extracting serum from multiple groups of obese mouse models, the extraction device can perform the following steps:

[0119] S1101. Fast multiple groups of obese mouse models for a preset period of time, and collect blood samples from the orbital nerve plexus.

[0120] Fasting multiple groups of obese mouse models for a preset period of time can ensure that the content of various components in the mouse serum will not change due to eating. Collecting blood samples from the orbital nerve plexus can collect a large amount of blood, which can facilitate the subsequent measurement of the content of various components in the blood.

[0121] As a feasible implementation method, the preset time may be 12 hours.

[0122] S1102. Centrifuge the blood sample at 4°C and 3000 rpm for 20 min to obtain serum.

[0123] After obtaining the blood sample, the serum is separated by centrifugation. Centrifugation is a method of separating the cellular components in the blood based on the physical properties of the supernatant of the blood to achieve the purpose of separating the serum.

[0124] In some embodiments, the organ index can be used to measure the status of the animal. Generally, the organ ratio of mice on a normal diet is relatively constant, while intervention with diet or toxic or harmful substances can cause changes in the organ index of mice.

[0125] As a feasible implementation method, the obesity prevention and treatment effect evaluation system provided in this application also includes: a cleaning device for cleaning and weighing the liver tissue and calculating the organ index of the liver tissue; an evaluation device for further evaluating the effect of the test article in preventing and treating obesity based on the organ index of the liver tissue.

[0126] As another feasible implementation method, the present application provides an effect evaluation system for preventing and treating obesity, in which the collection device is also used to collect epididymal adipose tissue from multiple groups of obese mouse models fed for a preset period of time. The system also includes: a cleaning device for cleaning and weighing the epididymal adipose tissue and calculating the organ index of the epididymal adipose tissue; and an evaluation device for further evaluating the effect of the experimental article in preventing and treating obesity based on the organ index of the epididymal adipose tissue.

[0127] That is, the effect evaluation system provided in the embodiment of the present application can determine the effect of the test article in preventing and treating obesity through the organ index of the liver tissue or epididymal fat tissue of mice.

[0128] As a feasible implementation method, the calculation formula of the organ index is as follows:

[0129]

[0130] Where m represents organ mass and M represents body weight.

[0131] For example, chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad were used as experimental items, and the experimental group mice were fed with them respectively. Finally, the organ indexes of the liver tissue and epididymal fat tissue of the obese mice in each group were measured as follows: Figure 12 shown.

[0132] The organ indexes of epididymal fat tissue and liver tissue of mice in the normal group were the lowest, while those of epididymal fat tissue and liver tissue of mice in the model group were the highest. Compared with the model group, the organ indexes of epididymal fat tissue and liver tissue of both the experimental group and the positive control group were decreased.

[0133] After the body gains weight, abnormalities will appear in the liver and fat tissue in the body. The above experiments show that the organ indexes of the epididymal fat tissue and liver tissue of the mice in the model group are higher than those in the normal group due to induced obesity, and the test articles used in the experimental group can alleviate the abnormal tissue and organ indexes caused by obesity. Therefore, it can be judged that the test articles are more effective in preventing and treating obesity.

[0134] In addition, it should be noted that during the feeding process, it was found that long-term consumption of dietary fiber would cause bloating in mice, while chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and green vegetables did not cause such a phenomenon. This indicates that experimental items such as chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and green vegetables can alleviate the abnormal organ indexes of obese mice and have no related side effects.

[0135] In some embodiments, long-term high-fat diets in mice can lead to visceral fat accumulation, causing a significant increase in the size of adipocytes. Therefore, to improve the accuracy of evaluating the effectiveness of experimental products in preventing and treating obesity, morphological changes in mouse adipocytes can be used for evaluation.

[0136] As a possible implementation, see Figure 13 The present application provides an evaluation system for preventing and treating obesity. The collecting device 12 is also used to collect small intestinal tissues of multiple groups of obese mouse models fed for a preset time. The system also includes: a staining device 15, which is used to stain and slice liver tissue, small intestinal tissue and epididymal adipose tissue, and perform pathological analysis after scanning with an optical microscope to obtain pathological analysis results; the evaluation device 14 is also used to further evaluate the effect of the experimental article on preventing and treating obesity based on the pathological analysis results.

[0137] As a feasible implementation method, the pathological analysis results provided in the embodiments of the present application may include changes in the status of tissue cells.

[0138] For example, see Figure 14 , Figure 14 This is a schematic diagram of an epididymal adipose tissue cell provided in the embodiment of the present application. Figure 14 As shown in the figure, the cells of epididymal adipose tissue of normal group mice were uniform in size and tightly arranged, while the epididymal adipocytes of experimental group, model group and positive control group showed a significant increase in volume, which indicates that feeding high-fat diet can promote the accumulation of visceral fat in mice and is the main reason for the weight gain of mice.

[0139] Moreover, compared with the model group, the tissue cell volume of some groups in the experimental group was smaller, indicating that some of the experimental items could reduce the accumulation of visceral fat in mice.

[0140] As another feasible implementation method, the pathological analysis results provided in the embodiments of the present application may include inflammatory infiltration phenomena of tissue cells.

[0141] For example, see Figure 15 , Figure 15 This is a schematic diagram of a section of small intestinal tissue cells provided in the embodiment of this application. Figure 15As shown, compared with the normal group mice, the model group mice showed a large reduction in goblet cells, villus atrophy and severe inflammatory infiltration in the small intestine (indicated by the black arrows in the figure).

[0142] Please continue reading Figure 15 Compared with the model group mice, the small intestinal damage of the experimental group mice was alleviated, the villus structure returned to normal, the number of goblet cells increased, and the inflammatory infiltration improved.

[0143] It can be seen that chicken breast salad, stir-fried kale, seasonal vegetable salad and mushroom and greens salad can improve the inflammatory infiltration phenomenon in mice and achieve better results in preventing and treating chronic obesity.

[0144] In some embodiments, see Figure 16 When staining tissue, the staining device can specifically perform the following steps:

[0145] S1601. Place the liver tissue, small intestinal tissue, and epididymal adipose tissue in the corresponding fixative and fix them for more than 24 hours.

[0146] Among them, liver tissue and small intestinal tissue can be placed in paraformaldehyde solution for more than 24 hours to denature the protein to maintain its biological structure and tissue morphology. Epididymal adipose tissue can be placed in fat fixative to denature the protein to maintain its biological structure and tissue morphology.

[0147] S1602. Dehydrate the liver tissue, small intestinal tissue, and epididymal adipose tissue using ethanol of different concentrations, embed them in paraffin, and cut them into slices of preset thickness.

[0148] Among them, the preset thickness can be 3μm, which can be set according to needs in actual use, and the embodiments of the present application do not limit this.

[0149] Liver tissue, small intestinal tissue and epididymal adipose tissue were dehydrated with different concentrations of ethanol, embedded in paraffin and cut into 3 μm thick sections.

[0150] The process of replacing water within tissues or cells with a dehydrating agent is called tissue dehydration. Both normal and diseased tissues contain varying amounts of water. Without this water, paraffin sections cannot be prepared because paraffin and water do not mix. The resulting sections will have poor light transmittance, making it difficult to clearly observe tissue structure. Therefore, removing water from tissues is crucial for sectioning.

[0151] Since ethanol can be mixed with water in any proportion, it can slowly displace water from tissues. Ethanol has a strong dehydrating effect, but it can also cause tissue hardening and brittleness. When used, it is usually used from low to high concentrations, with a concentration series of 70%, 80%, 90%, 95%, and 100%. After this series of treatments, the tissue can basically achieve the purpose of dehydration.

[0152] Paraffin sections are the most widely used method in conventional histological preparation techniques, facilitating the observation of cellular and tissue morphology. Dehydrated tissue is embedded in paraffin and cut into 3μm thick sections, resulting in paraffin sections of liver, small intestine, and epididymal adipose tissue.

[0153] S1603. Stain the sections with hematoxylin-eosin.

[0154] Hematoxylin-eosin staining is a chemical staining method, which mainly relies on the alkaline nature of hematoxylin staining solution to make the chromatin in the cell nucleus and the ribosomes in the cytoplasm appear purple-blue; eosin is an acidic dye that mainly makes the components in the cytoplasm and extracellular matrix appear red.

[0155] Hematoxylin-eosin staining can obtain clearer cell outlines, making it easier for workers to observe liver tissue, small intestine tissue, and epididymal adipose tissue under a microscope.

[0156] In some embodiments, weight change is the most intuitive observation indicator during mouse breeding. By detecting the weight of mice, it can be known whether the mice are growing normally or are overweight or obese.

[0157] Therefore, as a feasible implementation method, the obesity prevention and treatment effect evaluation system provided in the embodiment of the present application also includes: a recording device for recording the food intake and weight changes of each group of obese mouse models within a preset time period when feeding multiple groups of obese mouse models; an evaluation device for further evaluating the effect of the experimental article in preventing and treating obesity based on the food intake and weight changes.

[0158] Specifically, the mice can be fed at a rate of 2 g per day for six weeks, the weight of the obese mice can be recorded on Monday of each week, and the remaining amount of feed from the previous week can be recorded to calculate the food intake, thereby obtaining the food intake and weight changes of the mice.

[0159] It is understood that the lower the food intake and body weight of the mice, the better the experimental product's ability to prevent and treat obesity. Therefore, the effectiveness of the experimental product in preventing and treating obesity can be assessed based on the levels of liver inflammatory factors and changes in food intake and body weight.

[0160] For example, please refer to Table 1, which shows the food intake of mice (g / mouse / day).

[0161] Table 1 Food intake of mice

[0162]

[0163] As shown in Table 1, as time went on, the feed intake of the experimental group mice basically showed a downward trend and was significantly lower than that of the normal group mice and the model group mice. That is, the experimental items such as chicken breast salad, stir-fried kale, seasonal vegetable salad and mushroom and vegetable salad could change the daily food intake of mice.

[0164] The body weight changes of mice in each group were as follows Figure 17 As shown, weight gain Figure 18 As shown in the figure, mice fed a high-fat diet gained more weight, while mice fed a diet containing chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushrooms and greens showed little difference in weight change compared to mice fed a normal diet. Therefore, the chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushrooms and greens experimental items can reduce energy intake, thereby effectively inhibiting weight gain.

[0165] The above are only specific embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or replacements within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. An effect evaluation system for preventing and treating obesity, characterized in that: include: A feeding device for accommodating and feeding multiple groups of obese mouse models, wherein the multiple groups of obese mouse models include experimental group mice, normal group mice, and model group mice; wherein the experimental group mice are fed with the test article and high-fat feed, the model group mice are fed with high-fat feed, and the normal group mice are fed with normal feed; A collection device for collecting liver tissue from multiple groups of obese mouse models fed for a preset time period; An assay device is used to measure the level of liver inflammatory factors in the liver tissue of each group of obese mouse models, wherein the liver inflammatory factors include at least one of the following: tumor necrosis factor-α, interleukin-6, or interleukin-1β; The evaluation device is used to evaluate the effect of the experimental article in preventing and treating obesity based on the liver inflammatory factor levels of the multiple groups of obese mouse models.

2. The system according to claim 1, wherein: The evaluation device is specifically used to determine that the test article has the effect of preventing and treating obesity when the difference in liver inflammatory factor levels between the experimental group mice and the normal group mice is smaller than the difference in liver inflammatory factor levels between the model group mice and the normal group mice.

3. The system according to claim 1, wherein: The system further comprises: An extraction device is used to extract serum from multiple groups of obese mouse models fed for a preset time, and perform serum lipid metabolism analysis to obtain serum analysis results for each group of obese mouse models, the serum analysis results including: triglyceride, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels; The evaluation device is also used to further evaluate the effect of the test article in preventing and treating obesity based on the serum analysis results.

4. The system according to claim 3, characterized in that The measuring device is also used to measure the content of aspartate aminotransferase and / or alanine aminotransferase in serum; The evaluation device is also used to evaluate the effect of the test article in preventing and treating obesity based on the content of aspartate aminotransferase and / or alanine aminotransferase.

5. The system according to claim 1, wherein: The system also includes: a cleaning device, used for cleaning and weighing the liver tissue, and calculating the organ index of the liver tissue; The evaluation device is also used to further evaluate the effectiveness of the test article in preventing and treating obesity based on the organ index of liver tissue.

6. The system according to claim 1, wherein: The collection device is also used to collect epididymal adipose tissue from multiple groups of obese mouse models fed for a preset period of time; The system further comprises: a cleaning device for cleaning and weighing the epididymal fat tissue and calculating an organ index of the epididymal fat tissue; The evaluation device is also used to further evaluate the effectiveness of the test article in preventing and treating obesity based on the organ index of epididymal adipose tissue.

7. The system according to claim 6, characterized in that The collection device is also used to collect small intestinal tissue from multiple groups of obese mouse models fed for a preset period of time; The system also includes: a staining device for staining and slicing liver tissue, small intestinal tissue, and epididymal fat tissue, and performing pathological analysis after scanning with an optical microscope to obtain pathological analysis results; The evaluation device is also used to further evaluate the effect of the test article in preventing and treating obesity based on the results of pathological analysis.

8. The system according to claim 7, characterized in that The staining device is specifically used to place liver tissue, small intestinal tissue and epididymal fat tissue in corresponding fixatives for more than 24 hours; Liver tissue, small intestinal tissue, and epididymal adipose tissue were dehydrated with different concentrations of ethanol, embedded in paraffin, and cut into sections of preset thickness; Sections were stained with hematoxylin-eosin.

9. The system according to claim 1, wherein: The system also includes: A recording device is used to record the food intake and weight changes of each group of obese mouse models during feeding of multiple groups of obese mouse models for a preset period of time; The evaluation device is also used to further evaluate the effectiveness of the test article in preventing and treating obesity based on food intake and body weight changes.

10. The system according to claim 1, wherein: The items to be tested include at least one of the following: chicken breast salad, stir-fried kale, seasonal vegetable salad, and mushroom and vegetable salad; The multiple groups of obese mouse models also included: positive control group mice; The feeding apparatus further includes: a positive control area for accommodating mice in the positive control group; The mice in the positive control group were fed with a high-fat diet and dietary fiber.

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