A method for establishing a diabetes model

Abstract

The application provides a method for establishing a diabetes model, and relates to the technical field of biology, and comprises the following steps: (1) preparing a glucose solution, (2) mulberry leaf pretreatment, (3) selection of domestic silkworms, (4) domestic silkworm feeding and (5) blood glucose and body weight determination. The method can induce typical characteristics of diabetes in a short time (5-7 days), the construction method is simple, only sugar-containing mulberry leaves are given, and feeding in a low-temperature environment can be realized, the method is easy to operate, and is worth promoting.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for establishing a diabetes model. Background Technology

[0002] Diabetes mellitus (DM) is a group of metabolic diseases closely related to genetic and environmental factors, characterized by chronic hyperglycemia and insulin resistance, accompanied by a lack of insulin secretion. Its pathogenesis is highly complex and can cause a range of serious complications, such as retinopathy, kidney disease, and peripheral neuropathy. According to the latest data from the International Diabetes Federation (IDF), approximately 537 million adults (aged 20-79) worldwide had diabetes in 2021 (1 in 10 people); this number is projected to rise to 643 million by 2030 and to 783 million by 2045. During this period, the world population is estimated to grow by 20%, while the number of people with diabetes is estimated to increase by 46%. Due to the high prevalence of diabetes worldwide, extensive research is urgently needed to determine its pathogenesis and develop new antidiabetic drugs. Plasma glucose levels are regulated by systemic tissue uptake and metabolism. Therefore, evaluating antidiabetic drugs requires the use of appropriate animal models that allow for the quantitative measurement of plasma glucose levels.

[0003] Appropriate experimental animal models are essential tools for understanding pathogenesis, complications, genetic or environmental factors that increase the risk of type 2 diabetes, and for testing various therapeutic drugs. However, in the past decade or so, a range of diabetic mammals, such as mice and rats, have been used to screen antidiabetic drugs, but these models have serious limitations, such as ethical issues related to animal welfare, long modeling cycles, low success rates, and the extremely high costs of maintaining a large number of animals.

[0004] Therefore, it is urgent to establish an ideal experimental animal model of diabetes to reduce resource waste, lower modeling costs, shorten research cycles, and address animal ethics issues. Summary of the Invention

[0005] The purpose of this invention is to provide a novel method for establishing a diabetic animal model, addressing the above-mentioned technical problems.

[0006] Specifically, the present invention adopts the following technical solution:

[0007] A method for establishing a diabetes model, comprising the following steps:

[0008] (1) Preparation of glucose solution: Dissolve anhydrous glucose in ultrapure water to prepare a glucose solution with a concentration of 0.25 mg / ml;

[0009] (2) Mulberry leaf pretreatment: Spray the glucose solution prepared in step (1) evenly onto the surface of mulberry leaves, and dry the surface moisture before use.

[0010] (3) Selection of silkworms: Select fifth-instar silkworms that have been raised in an environment of 25-30℃, weigh 0.9-1.0g, and have completed the fourth molt.

[0011] (4) Silkworm rearing: The fifth instar larvae of silkworms are reared under low temperature conditions and fed with mulberry leaves treated in step (2) during the rearing period; the specific rearing conditions are: temperature of 16-20℃ and humidity of 60-70%;

[0012] (5) Blood glucose and body weight measurement: During the feeding period, the blood glucose and body weight of silkworms were measured daily. When the blood glucose > 7.0 mmol / L and the insulin action was slow or resistant, the diabetic animal model was confirmed to be successfully established.

[0013] Specifically, this involves the establishment of a type 2 diabetes model.

[0014] In this invention, further, in step (1), glucose is dissolved by water bath heating, the water bath temperature is 50-60℃, and the water bath time is 20-30min.

[0015] In this invention, further, in step (2), the glucose solution is sprayed on the back of the mulberry leaves, and after spraying, the leaves are dried until there is no dripping water. Then, the treated mulberry leaves are wrapped in plastic wrap and refrigerated at 4°C for later use.

[0016] In this invention, the silkworm variety selected in step (3) is Liangguang No. 2, and the silkworm fasts overnight during its fourth molt.

[0017] In this invention, the low-temperature conditions in step (4) are: temperature of 18°C ​​and humidity of 65%.

[0018] In this invention, the blood glucose measurement in step (5) is performed 8 hours after a meal, and the weight measurement is performed 8 hours after the first meal in the morning.

[0019] In this invention, step (5) further includes the determination of insulin, specifically the insulin content of silkworms 3 hours after meal on the sixth day of modeling (fasting for more than 12 hours before feeding).

[0020] This invention constructs an animal model of type 2 diabetes; it is more consistent with the current pathogenesis of type 2 diabetes in humans and can be used to study the pathogenesis of type 2 diabetes, find relevant targets for early diagnosis, and develop drugs and methods for the treatment and prevention of diabetes.

[0021] By adopting the above technical solution, the present invention has the following advantages:

[0022] 1. The method of this invention can induce typical characteristics of diabetes in a short period of time (5-7 days). The construction method is simple and can be achieved by feeding the animals with sugar-containing mulberry leaves in a low-temperature environment. It is easy to operate and is worth promoting.

[0023] 2. This invention first selects fifth-instar silkworm larvae that have completed their fourth molt for rearing. Then, the rearing period is controlled to be carried out in a low-temperature environment, which can double the life cycle of the silkworm. In addition, the concentration of the glucose solution required does not need to be too high, so that the silkworm’s blood sugar can rise steadily.

[0024] 3. Compared with the traditional streptozotocin-induced mouse modeling method, this invention is not only simple to operate and requires less space, but also allows for large-scale breeding and low-cost model establishment (no feed required). Furthermore, it has a high modeling rate and a short modeling cycle (only 1-2 weeks), and can be sold as a mature animal model to generate economic benefits. Attached Figure Description

[0025] Figure 1 This is a bar chart showing the average blood glucose changes in the model group and the control group;

[0026] Figure 2 This is a bar chart showing the changes in average body weight between the model group and the control group;

[0027] Figure 3 This is a bar chart comparing the 5th instar life cycle of silkworms in the normal temperature group and the low temperature group (model group).

[0028] Figure 4 This is a bar chart comparing blood glucose and insulin levels in silkworms 3 hours after a meal on the sixth day after modeling at different glucose concentrations under low temperature conditions. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0030] Example 1:

[0031] This embodiment provides a method for establishing a diabetes model, which includes the following steps:

[0032] (1) Preparation of glucose solution: Dissolve anhydrous glucose in ultrapure water and stir thoroughly to prepare a glucose solution with a concentration of 0.25 mg / ml; specifically, the glucose is dissolved in a water bath at a temperature of 50°C for 20 min; after the solution is prepared, place it in a spray bottle for later use.

[0033] (2) Mulberry leaf pretreatment: Spray the glucose solution prepared in step (1) evenly onto the mulberry leaves. The mulberry leaves should be spread evenly on the cardboard box with the back of the leaves facing up (the front is smooth and has a layer of wax, so the solution is easy to lose, while the back is rough and can retain the liquid and is not easy to lose). Hold the spray bottle and spray the glucose solution evenly 20cm above the mulberry leaves. When the mulberry leaves are slightly covered with water droplets, spread them out to dry (about half an hour until there are no obvious water droplets on the surface of the mulberry leaves). Then wrap the treated mulberry leaves with plastic wrap and refrigerate them at 4℃ for later use.

[0034] (3) Selection of silkworms: Fifth instar silkworms that are raised at 25℃, weigh 0.9g, and have completed their fourth molt are selected; the selected silkworm variety is Liangguang No. 2, and the silkworms are fasted overnight after their fourth molt.

[0035] (4) Silkworm rearing: The fifth instar larvae of the silkworms are reared under low temperature conditions and fed with mulberry leaves processed in step (2) during the rearing period. The feces at the bottom of the rearing basin should be cleaned and the absorbent filter paper should be replaced every day when feeding. The specific rearing conditions are: temperature of 16℃ and humidity of 60%.

[0036] (5) Measurement of blood glucose and weight: During the rearing period, the blood glucose of silkworms was measured daily using a Sinocare precision blood glucose analyzer, and the weight of the silkworms was recorded. The measurement method was to hold the silkworm with the thumb and forefinger of the left hand, with the silkworm's head facing the tiger's mouth, and insert the needle 0.5 cm above the first hind leg of the silkworm. The blood glucose was measured and recorded. The corresponding weight was weighed and recorded before measuring the blood glucose. The blood glucose measurement time was 8 hours after a meal, and the weight measurement time was 8 hours after the first feeding in the morning. Insulin measurement was also included, specifically the insulin content of the silkworms 3 hours after a meal on the sixth day of modeling (fasting for more than 12 hours before feeding). When the blood glucose was >7.0 mmol / L and the insulin action was slow or resistant, the type 2 diabetes animal model was confirmed to be successfully established.

[0037] Example 2:

[0038] This embodiment provides a method for establishing a diabetes model, which includes the following steps:

[0039] (1) Preparation of glucose solution: Dissolve anhydrous glucose in ultrapure water and stir thoroughly to prepare a glucose solution with a concentration of 0.25 mg / ml; specifically, the glucose is dissolved in a water bath at a temperature of 55°C for 25 min; after the solution is prepared, place it in a spray bottle for later use.

[0040] (2) Mulberry leaf pretreatment: Spray the glucose solution prepared in step (1) evenly onto the mulberry leaves. The mulberry leaves should be spread evenly on the cardboard box with the back of the leaves facing up (the front is smooth and has a layer of wax, so the solution is easy to lose, while the back is rough and can retain the liquid and is not easy to lose). Hold the spray bottle and spray the glucose solution evenly from 25cm above the mulberry leaves. When the mulberry leaves are slightly covered with water droplets, spread them out to dry (about half an hour until there are no obvious water droplets on the surface of the mulberry leaves). Then wrap the treated mulberry leaves with plastic wrap and refrigerate them at 4℃ for later use.

[0041] (3) Selection of silkworms: Fifth instar silkworms that are raised at 27℃, weigh 0.9g, and have completed their fourth molt are selected; the selected silkworm variety is Liangguang No. 2, and the silkworms are fasted overnight after their fourth molt.

[0042] (4) Silkworm rearing: The fifth instar larvae of silkworms are reared under low temperature conditions and fed with mulberry leaves processed in step (2) during the rearing period. The feces at the bottom of the rearing basin should be cleaned and the absorbent filter paper should be replaced every day when feeding. The specific rearing conditions are: temperature of 18℃ and humidity of 65%.

[0043] (5) Measurement of blood glucose and weight: During the rearing period, the blood glucose of silkworms was measured daily using a Sinocare precision blood glucose analyzer, and the weight of the silkworms was recorded. The measurement method was to hold the silkworm with the thumb and forefinger of the left hand, with the silkworm's head facing the tiger's mouth, and insert the needle 0.5 cm above the first hind leg of the silkworm. The blood glucose was measured and recorded. The corresponding weight was weighed and recorded before measuring the blood glucose. The blood glucose measurement time was 8 hours after a meal, and the weight measurement time was 8 hours after the first feeding in the morning. Insulin measurement was also included, specifically the insulin content of the silkworms 3 hours after a meal on the sixth day of modeling (fasting for more than 12 hours before feeding). When the blood glucose was >7.0 mmol / L and the insulin action was slow or resistant, the type 2 diabetes animal model was confirmed to be successfully established.

[0044] Example 3:

[0045] This embodiment provides a method for establishing a diabetes model, which includes the following steps:

[0046] (1) Preparation of glucose solution: Dissolve anhydrous glucose in ultrapure water and stir thoroughly to prepare a glucose solution with a concentration of 0.25 mg / ml; specifically, the glucose is dissolved in a water bath at a temperature of 60°C for 20-30 minutes; after the solution is prepared, place it in a spray bottle for later use.

[0047] (2) Mulberry leaf pretreatment: Spray the glucose solution prepared in step (1) evenly onto the mulberry leaves. The mulberry leaves should be spread evenly on the cardboard box with the back of the leaves facing up (the front is smooth and has a layer of wax, so the solution is easy to lose, while the back is rough and can retain the liquid and is not easy to lose). Hold the spray bottle and spray the glucose solution evenly 30cm above the mulberry leaves. When the mulberry leaves are slightly covered with water droplets, spread them out to dry (about half an hour until there are no obvious water droplets on the surface of the mulberry leaves). Then wrap the treated mulberry leaves with plastic wrap and refrigerate them at 4℃ for later use.

[0048] (3) Selection of silkworms: Fifth instar silkworms that are raised at 30℃, weigh 1.0g, and have completed their fourth molt are selected; the selected silkworm variety is Liangguang No. 2, and the silkworms are fasted overnight after their fourth molt.

[0049] (4) Silkworm rearing: The fifth instar larvae of silkworms are reared under low temperature conditions and fed with mulberry leaves processed in step (2) during the rearing period. The feces at the bottom of the rearing basin should be cleaned and the absorbent filter paper should be replaced every day when feeding. The specific rearing conditions are: temperature of 20℃ and humidity of 70%.

[0050] (5) Measurement of blood glucose and weight: During the rearing period, the blood glucose of silkworms was measured daily using a Sinocare precision blood glucose analyzer, and the weight of the silkworms was recorded. The measurement method was to hold the silkworm with the thumb and forefinger of the left hand, with the silkworm's head facing the tiger's mouth, and insert the needle 0.5 cm above the first hind leg of the silkworm. The blood glucose was measured and recorded. The corresponding weight was weighed and recorded before measuring the blood glucose. The blood glucose measurement time was 8 hours after a meal, and the weight measurement time was 8 hours after the first feeding in the morning. Insulin measurement was also included, specifically the insulin content of the silkworms 3 hours after a meal on the sixth day of modeling (fasting for more than 12 hours before feeding). When the blood glucose was >7.0 mmol / L and the insulin action was slow or resistant, the type 2 diabetes animal model was confirmed to be successfully established.

[0051] The following describes the method and effects of establishing a type 2 diabetic silkworm hypothermic animal model using the second-generation fifth-instar silkworm of Guangdong and Guangxi.

[0052] The applicant divided 300 fifth-instar larvae that had completed their fourth molt and weighed between 0.90 and 1.00 g into a model group, a normal temperature group, and a control group.

[0053] Model group: 100 animals, using the method described in Example 2 of this application to establish a diabetic animal model;

[0054] Room temperature group: 100 silkworms were raised in an environment with a room temperature (set to 28℃ for this group);

[0055] Control group: 100 birds, fed with normal mulberry leaves (without being sprayed with glucose solution);

[0056] Apart from the differences emphasized above, the room temperature group and the control group were otherwise identical to the model group.

[0057] From day one, silkworms in the low-temperature rearing control group and the model group were fed 100 grams of normal mulberry leaves and high-sugar mulberry leaves respectively every morning and evening. Blood glucose and body weight were measured and recorded 8 hours after feeding each morning for days 1-9. At the same time, the growth cycle of the 5th instar (from the start of modeling to the beginning of cocooning) of the room-temperature group and the control group was observed and recorded. The results are as follows:

[0058] First, the changes in the average blood glucose concentration of diabetic silkworms in the control group and the model group are as follows:

[0059] Table 1. Changes in mean blood glucose concentration in diabetic silkworms (n=8, x±s)

[0060]

[0061] *: The difference in mean blood glucose between the model group and the control group was statistically significant (p < 0.05). The statistical test method used was the two-sample t-test.

[0062] The above blood glucose change bar chart is as follows: Figure 1 As shown.

[0063] The changes in average body weight of diabetic silkworms in the control group and the model group are as follows:

[0064] Table 2. Changes in average body weight of diabetic silkworms (n=8, x±s)

[0065]

[0066]

[0067] *: The difference in average weight between the model group and the control group was statistically significant (p < 0.05). The statistical test method used was the two-sample t-test.

[0068] The above bar chart of weight changes is as follows Figure 2 As shown.

[0069] The results show that after modeling, the weight gain of silkworms in the control group increased significantly with the feeding time, while the weight gain in the model group was slow, and the difference between the two groups was statistically significant (P<0.05, n=8). Furthermore, the blood glucose levels of silkworms in the model group gradually increased, while those in the control group remained stable and within the normal range. The blood glucose levels in the blank group ranged from 3.0 to 7.0 mmol / L, and those in the high-glucose group ranged from 7.0 to 17 mmol / L, with statistically significant differences between the two groups (P<0.05, n=8).

[0070] like Figure 3 The figure shows a bar chart comparing the 5th instar life cycle of silkworms in the room temperature group and the low temperature group (i.e., the model group). The 5th instar life cycle of the room temperature group is 4-7 days, while that of the low temperature control group is 10-16 days.

[0071] like Figure 4 As shown in the figure, this is a bar chart comparing blood glucose and insulin levels in silkworms 3 hours after a meal on the sixth day after modeling with different glucose concentrations under low temperature conditions. In the figure, the insulin level in the 20% glucose model group was higher than that in the control group, but the blood glucose level remained at a high level, indicating that the insulin action was delayed or that insulin resistance had occurred.

[0072] In summary, silkworm glucose metabolism shares similarities with that of mammals, and while glucose levels in the hemolymph of silkworms increase with a high-sugar diet, their body weight is relatively lighter compared to normal silkworms. Furthermore, raising them under low-temperature conditions can double their 5-instar cycle. Therefore, the hypothermic diabetes model established using silkworms offers advantages in terms of economy, convenience, and animal welfare. Thus, establishing a diabetes model using silkworms can be used to evaluate antidiabetic drugs for type 1 and type 2 diabetes, and it is also important in screening traditional Chinese medicines with hypoglycemic effects that have not yet been proven effective. Therefore, we believe that this invention of establishing a diabetes model using invertebrates can provide significant value for future research into the pathogenesis of diabetes, the observation of the effects of novel treatments, and the screening of antidiabetic drugs.

[0073] The above embodiments are merely one of the preferred embodiments of the present invention, and are intended to better explain the principles and practical applications of the present invention. They should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but which still solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A method for establishing a diabetes model, characterized in that, The method includes the following steps: (1) Preparation of glucose solution: Dissolve anhydrous glucose in ultrapure water to prepare a glucose solution with a concentration of 0.25 mg / ml; (2) Mulberry leaf pretreatment: Spray the glucose solution prepared in step (1) evenly on the surface of mulberry leaves, and dry the surface moisture before use; (3) Selection of silkworms: Select fifth-instar silkworms that have been raised in an environment of 25-30℃, weigh 0.9-1.0g, and have completed the fourth molt; (4) Silkworm rearing: The fifth-instar larvae of silkworms are reared under low temperature conditions and fed with mulberry leaves processed in step (2) during the rearing period; the specific rearing conditions are: temperature of 16-20℃ and humidity of 60-70%; (5) Blood glucose and body weight measurement: During the feeding period, the blood glucose and body weight of silkworms were measured daily. When the blood glucose > 7.0 mmol / L and the insulin action was slow or resistant, the diabetic animal model was confirmed to be successfully established. The silkworms selected in step (3) are of the Liangguang No. 2 variety. The silkworms are fasted overnight after their fourth molt. The low-temperature conditions in step (4) are: temperature of 18℃ and humidity of 65%. Step (4) is carried out in a low-temperature environment, which can extend the life cycle of the silkworms by one time. Under the feeding of mulberry leaves after step (2), the high blood sugar of the silkworms can be steadily increased.

2. The method according to claim 1, characterized in that, In step (1), glucose is dissolved by heating in a water bath at a temperature of 50-60°C for 20-30 minutes.

3. The method according to claim 1, characterized in that, In step (2), the glucose solution is sprayed on the back of the mulberry leaves. After spraying, the leaves are dried until no water drips. Then, the treated mulberry leaves are wrapped in plastic wrap and refrigerated at 4°C for later use.

4. The method according to claim 1, characterized in that, The blood glucose test in step (5) is performed 8 hours after a meal, and the weight test is performed 8 hours after the first meal in the morning.

5. The method according to claim 1, characterized in that, Step (5) also includes the determination of insulin, specifically the insulin content of silkworms 3 hours after meal on the sixth day of modeling, and fasting for more than 12 hours before feeding.

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