Application of 1,4-Methylimidazoleacetic acid in preparing drugs for preventing and treating obesity

Abstract

The present invention discloses the application of 1,4-Methylimidazoleacetic acid in the preparation of drugs for preventing and treating obesity, and belongs to the field of biomedical technology. The present invention verifies through animal experiments that 1,4-Methylimidazoleacetic acid can improve the glucose tolerance of obese mice induced by a high-fat diet and reduce the level of insulin resistance; and 1,4-Methylimidazoleacetic acid can significantly reduce lipid deposition and lipid droplet formation in liver tissue, and can also reduce fat mass and the expression level of obesity-related proteins, thereby improving the related symptoms of obesity.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of 1,4-Methylimidazoleacetic acid in preparing drugs for preventing and treating obesity. Background Art

[0002] Data from the WHO on March 4, 2022 showed that 39 million children in the world are overweight or obese, and this number is still increasing. The "Report on the Nutrition and Chronic Disease Status of Chinese Residents (2020)" shows that the overweight and obesity rates of children and adolescents aged 6 to 17 in my country are 11.1% and 7.9% respectively, and the overweight and obesity rates of children under 6 are 6.8% and 3.6% respectively. Childhood obesity has become a national epidemic. The "China Childhood Obesity Report" predicts that if effective intervention measures are not taken, by 2030, the detection rate of overweight and obesity in children aged 7 and above will reach 28.0%, and the number of overweight and obese people will reach 49.48 million. The occurrence of childhood obesity is jointly affected by genetic, environmental and socio-cultural factors. Among them, early life nutrition, dietary factors, physical activity and static activities are key individual factors. Childhood obesity and health risks in adulthood will also bring huge economic burdens. The causes of obesity are complex, so there is an urgent need to develop new effective intervention drugs for obesity.

[0003] Currently, surgery is the most effective method for treating obesity, but the choice of surgery has strict indications. Among the centrally acting drugs that have been developed and used for decades to control appetite, dexfenfluramine, fenfluramine and sibutramine have shown benefits for weight loss and blood sugar control. Orlistat is a pancreatic lipase inhibitor and is the first single drug approved for long-term treatment of obesity. Semaglutide injection is a new long-acting glucagon-like peptide-1 (GLP-1) analog from Novo Nordisk for blood sugar control. However, the efficacy of anti-obesity drugs is generally insufficient and their safety is questionable. The applicability of these drugs has its limitations, and the specific mechanism is still unclear. Therefore, it is very necessary and urgent to find products with few side effects, economical prices and suitable for preventing obesity in children.

[0004] 1,4-Methylimidazoleacetic acid (MIAA) is a monocarboxylic acid, a type of acetic acid, and a member of the imidazole class and monocarboxylic acids. It has the functions of a metabolite and a γ-aminobutyric acid (GABA) agonist. Studies have reported that 1,4-methylimidazoleacetic acid is the main urinary metabolite of histamine. Histamine in the brain mainly regulates the overall function of the brain. Central histaminergic nerves can regulate many functions of the hypothalamus, such as eating, chewing, drinking, neuroendocrine, and body temperature regulation. Interleukin-1β (IL-1β), hunger, and hypoglycemia caused by insulin can also stimulate histaminergic nerves. The activation of hypothalamic histaminergic nerves can stimulate the secretion of catecholamines in the adrenergic area to regulate peripheral energy metabolism. Central histaminergic nerves may be the key to activating the adrenergic system to regulate energy metabolism. However, there are no studies reporting whether supplementation with 1,4-methylimidazoleacetic acid can improve the symptoms of obesity induced by a high-fat diet. Summary of the invention

[0005] The purpose of the present invention is to provide the use of 1,4-Methylimidazoleacetic acid in the preparation of drugs or foods for preventing and treating obesity, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides application of 1,4-Methylimidazoleacetic acid in preparing medicine for preventing and treating obesity.

[0008] The present invention also provides the use of 1,4-Methylimidazoleacetic acid in preparing a drug for reducing glucose tolerance.

[0009] The present invention also provides the use of 1,4-Methylimidazoleacetic acid in preparing a medicine for reducing insulin resistance level.

[0010] The present invention also provides the use of 1,4-Methylimidazoleacetic acid in preparing a medicine for reducing liver fatty degeneration.

[0011] The present invention also provides the use of 1,4-Methylimidazoleacetic acid in preparing a medicine for reducing liver lipid deposition.

[0012] The present invention also provides the use of 1,4-Methylimidazoleacetic acid in preparing a drug for inhibiting the expression of obesity-related gene Fto.

[0013] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0014] Preferably, the dosage form of the drug includes granules, capsules, powder injections, tablets, oral liquids, pills or pastes.

[0015] Preferably, the obesity is obesity induced by a high-fat diet.

[0016] The invention also provides application of 1,4-Methylimidazoleacetic acid in preparing food for preventing and treating obesity.

[0017] The present invention discloses the following technical effects:

[0018] The present invention provides a new use of 1,4-Methylimidazoleacetic acid for preparing drugs for treating obesity induced by a high-fat diet. The present invention verifies that 1,4-Methylimidazoleacetic acid can alleviate the symptoms related to obesity induced by a high-fat diet. This effect may be achieved by affecting glucose tolerance, reducing insulin resistance levels and lipid deposition in liver tissue, and then exerting an effect through the expression level of obesity-related genes in liver tissue, thereby improving the symptoms related to obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The figure shows the effect of 1,4-Methylimidazoleacetic acid on the weight change and weight gain of obese mice induced by high-fat diet, wherein A is the weight change curve after continuous gavage of 1,4-Methylimidazoleacetic acid for 4 weeks, and B is the weight gain curve after 1,4-Methylimidazoleacetic acid for 4 weeks;

[0021] Figure 2 The effect of 1,4-Methylimidazoleacetic acid on glucose tolerance of obese mice induced by high-fat diet, wherein A is the blood glucose change curve of glucose tolerance test, and B is the area under the curve between the two groups;

[0022] Figure 3 The figure shows the effect of 1,4-Methylimidazoleacetic acid on the insulin resistance level of obese mice induced by a high-fat diet, wherein A is the blood glucose change curve of the insulin resistance test, and B is the area under the curve between the two groups;

[0023] Figure 4 The effect of 1,4-Methylimidazoleacetic acid on the weight of epididymal white fat and inguinal subcutaneous fat in high-fat diet-induced obese mice, wherein A is the weight of epididymal white fat and B is the weight of inguinal subcutaneous fat;

[0024] Figure 5 1,4-Methylimidazoleacetic acid is used to treat the pathological changes of adipose tissue and liver tissue in mice with high-fat diet-induced obesity, wherein A is the HE staining pathological picture of adipose tissue, and B is the HE staining pathological picture of liver tissue;

[0025] Figure 6 The effect of 1,4-Methylimidazoleacetic acid on the obesity-related gene Fto in the liver tissue of high-fat diet-induced obese mice;

[0026] Figure 7 The figure shows the effect of 1,4-Methylimidazoleacetic acid on the obesity-related protein Fto in the liver tissue of high-fat diet-induced obese mice, where A is the WB band graph and B is the protein quantification graph. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0033] Example 1

[0034] The preparation method of 1,4-Methylimidazoleacetic acid solution is as follows: 1,4-Methylimidazoleacetic acid is dissolved in 0.9% physiological saline at a concentration of 5 mg / ml.

[0035] Experimental animals: 20 4-week-old C57BL / 6J male mice were selected, and the room temperature was maintained at 23±1°C and the humidity was maintained at 50±60%. The light-dark cycle was 12 hours, and clean food and water were freely available for training.

[0036] Experimental methods:

[0037] High-fat group (HFD+NS) 10 rats: fed with 60% high-fat diet (calorie content 5.2 kcal / g), and given 200 μL normal saline by gavage every day;

[0038] 1,4-Methylimidazoleacetic acid gavage group (HFD+MIAA) 10 mice: fed with 60% high-fat diet (calorie content 5.2 kcal / g), gavage 200 μL 1,4-Methylimidazoleacetic acid solution every day.

[0039] The administration time was 8:00-9:00 am every day for 28 days. On the 29th day, the mice in each group fasted for 12 hours and performed a glucose tolerance test (GTT). After an interval of 3 days, the insulin resistance test (ITT) was performed. After completing the above tests, the mice in each group fasted for 12 hours and were anesthetized with Avertin (0.03 mL / 10 g). The eyeballs were removed to collect blood, and the blood was placed at room temperature for 60 minutes, then centrifuged at 12000 rpm for 5 minutes, the upper plasma was aspirated and divided, and placed in a -80°C ultra-low temperature refrigerator for testing. The collected fresh samples of liver tissue and adipose tissue were fixed with 4% paraformaldehyde and paraformaldehyde calcium, and then stained with hematoxylin and eosin (HE); the remaining liver tissue was quickly frozen in liquid nitrogen, divided into 2 mL sterile EP tubes, and stored in a -80°C refrigerator for Western Blot, qPCR and other tests.

[0040] Data are expressed as mean ± standard deviation (SD), and the differences between the two groups were evaluated by independent sample T test, with P < 0.05 indicating statistical significance. All graphs were created by GraphPadPrism 7.2.

[0041] Effects of 1.1,4-Methylimidazoleacetic acid on body weight changes and weight gain in obese mice

[0042] The body weights of the mice in the above groups were measured weekly from 8:00 to 10:00 in the morning for 12 consecutive weeks, of which the basic model period was 8 weeks and the 1,4-Methylimidazoleacetic acid gavage intervention lasted for 4 weeks.

[0043] The results are as follows Figure 1 As shown, Figure 1 The effect of 1,4-Methylimidazoleacetic acid on the weight change and weight gain of obese mice induced by high-fat diet. As can be seen from the figure, (A) is the weight change curve after continuous gavage of 1,4-Methylimidazoleacetic acid for 4 weeks; (B) is the weight gain chart after 1,4-Methylimidazoleacetic acid for 4 weeks. The results show that the weight gain of mice in the HFD+MIAA group is significantly lower than that of mice in the HFD+NS group (P<0.05). The data are expressed as mean±SD, n=10.

[0044] Effects of 1,2-1,4-Methylimidazoleacetic acid on glucose tolerance in obese mice

[0045] Glucose tolerance test (GTT): After the model cycle, fasting but not water for 12 hours at 20:00 in the evening. Fasting blood glucose was measured at 08:00 the next morning (recorded as the blood glucose value at 0 min), and then glucose (2 g / kg body weight) was injected intraperitoneally. The blood glucose values ​​at 30, 60, 90 and 120 min were tested to determine the glucose tolerance of mice and calculate the area under the curve. The formula is shown in 1-1.

[0046]

[0047] Note: In the formula, G0, G30, G60, G90, G120 are the blood glucose values ​​at 0min, 30min, 60min, 90min, 120min in the oral glucose tolerance test respectively; AUC is the area under the blood glucose curve.

[0048] The results are as follows Figure 2 As shown, Figure 2 The effect of 1,4-Methylimidazoleacetic acid on glucose tolerance in obese mice. As can be seen from the figure, (A) is the blood glucose change curve of the glucose tolerance test; (B) is the area under the curve between the two groups. The results show that the area under the curve AUC of the HFD+MIAA group of mice is significantly lower than that of the HFD+NS group of mice (P<0.05). The data are expressed as mean±SD, n=10. Figure 2 It shows that 1,4-Methylimidazoleacetic acid can improve glucose tolerance in obese mice.

[0049] Effects of 1,4-Methylimidazoleacetic acid on insulin resistance in obese mice

[0050] Insulin resistance test (ITT): After the model cycle, fasting but not water for 12 hours at 20:00 in the evening. Fasting blood glucose was measured at 08:00 the next morning (recorded as the blood glucose value at 0 min), and then insulin (0.75 U / kg body weight) was injected intraperitoneally. The blood glucose values ​​at 30, 60, 90 and 120 min were tested to determine the insulin resistance level of mice and calculate the area under the curve. The formula is shown in 1-1.

[0051] The results are as follows Figure 3 As shown, Figure 3The effect of 1,4-Methylimidazoleacetic acid on the insulin resistance level of obese mice. As can be seen from the figure, (A) is the blood glucose change curve of the insulin resistance test; (B) is the area under the curve between the two groups. The results show that the area under the curve AUC of the HFD+MIAA group of mice is significantly lower than that of the HFD+NS group of mice (P<0.05). The data are expressed as mean±SD, n=10. Figure 3 This shows that 1,4-Methylimidazoleacetic acid can reduce the level of insulin resistance.

[0052] Effects of 1,4-Methylimidazoleacetic acid on epididymal white fat and inguinal subcutaneous fat weight in obese mice

[0053] After completing the above experiments, the animal sampling began. The epididymal white fat on both sides and the inguinal subcutaneous fat were completely removed, and then weighed and the data were recorded.

[0054] The results are as follows Figure 4 As shown, Figure 4 The effect of 1,4-Methylimidazoleacetic acid on the weight of epididymal white fat and inguinal subcutaneous fat in obese mice. As can be seen from the figure, (A) the weight of epididymal white fat, the results show that the weight of epididymal white fat in the HFD+MIAA group is significantly lower than that in the HFD+NS group (P < 0.05); (B) the weight of inguinal subcutaneous fat, the results show that the weight of inguinal subcutaneous fat in the HFD+MIAA group is significantly lower than that in the HFD+NS group (P < 0.05). Data are expressed as mean ± SD, n = 10. Figure 4 It shows that 1,4-Methylimidazoleacetic acid can reduce fat accumulation in mice.

[0055] Effects of 1,5-1,4-Methylimidazoleacetic acid on adipose tissue and liver pathology in obese mice

[0056] Paraffin sections of adipose tissue and liver tissue were baked in an oven at 65°C for at least 1.5 hours to ensure that the paraffin on the tissue sections was fully melted; then, the sections were placed in xylene twice to remove the melted paraffin and dehydrated in gradient ethanol. The sections were stained with hematoxylin and eosin in sequence and then dehydrated with ethanol; after staining, the sections were clarified twice in xylene and fixed with neutral resin; the stained sections were observed under an optical microscope at magnifications of 200 and 400 times, respectively.

[0057] The results are as follows Figure 5 As shown, Figure 5 The effect of 1,4-Methylimidazoleacetic acid on the pathology of adipose tissue and liver tissue in obese mice. As can be seen from the figure, (A) HE staining pathology of adipose tissue shows that the adipocytes of mice in the HFD+NS group are larger than those in the HFD+MIAA group; (B) HE staining pathology of liver tissue shows that mice in the HFD+NS group showed obvious liver fatty degeneration and lipid deposition, and lipid accumulation vacuoles increased, while MIAA intervention effectively reduced lipid accumulation and lipid droplet formation in the liver of mice.

[0058] Effects of 1,61,4-Methylimidazoleacetic acid on obesity-related genes in liver tissue of obese mice

[0059] The liver tissues of the mice in each group collected in the above-mentioned animal experimental method were used to determine the expression level of the obesity-related gene (Fto) in the liver tissues using the Quantitative-PCR method.

[0060] The results are as follows Figure 6 As shown, Figure 6 The effect of 1,4-Methylimidazoleacetic acid on the expression level of obesity-related genes in liver tissue of obese mice. As can be seen from the figure, the expression of obesity-related genes (Fto) in liver tissue of mice in the HFD+NS group was significantly higher than that in the HFD+MIAA group. The data are expressed as mean±SD, n=3.

[0061] Effects of 1,7-1,4-Methylimidazoleacetic acid on obesity-related proteins in liver tissue of obese mice

[0062] The liver tissues of each group of mice collected in the above-mentioned animal experimental method were subjected to Western Blot immunoblotting to determine the expression levels of obesity-related proteins in the liver tissues.

[0063] The results are as follows Figure 7 As shown, Figure 7 The effect of 1,4-Methylimidazoleacetic acid on the expression level of obesity-related proteins in liver tissue of obese mice. (A) WB band graph; (B) Protein quantification graph. It can be seen from the figure that the expression of obesity-related protein (Fto) in liver tissue of mice in the HFD+NS group was significantly higher than that in the HFD+MIAA group. The data are expressed as mean±SD, n=3. The above results indicate that 1,4-Methylimidazoleacetic acid may regulate lipid metabolism and obesity-related expressions in liver tissue through a certain mechanism of action, thereby improving the related symptoms of obesity induced by a high-fat diet.

[0064] The present invention has been tested for the effect of 1,4-Methylimidazoleacetic acid on glucose tolerance and insulin resistance in obese mice, and it can be seen that 1,4-Methylimidazoleacetic acid can improve the related symptoms of obesity. At the same time, the present invention has explored the related mechanism that 1,4-Methylimidazoleacetic acid can improve obesity. Animal experiments have proved that the Fto gene is a gene related to fat metabolism and obesity, and is widely distributed in various tissues of animals. There are many single nucleotide polymorphism (SNP) sites on the Fto gene. These SNP sites will increase food intake by affecting the expression level of appetite peptides, thereby causing obesity. After 4 weeks of intragastric administration of 1,4-Methylimidazoleacetic acid nutrient solution to obese mice, the lipid deposition and lipid droplet formation in the liver tissue of the obese mice were significantly reduced, and the relative expression levels of obesity-related proteins and genes were significantly downregulated. According to the above experimental results, it is speculated that 1,4-Methylimidazoleacetic acid may reduce the expression level of obesity-related proteins and then regulate liver lipid deposition to alleviate the related symptoms of obesity induced by a high-fat diet.

[0065] In summary, the present invention verifies that 1,4-Methylimidazoleacetic acid can alleviate the related symptoms of obesity induced by a high-fat diet. This effect may be exerted by affecting glucose tolerance, reducing insulin resistance levels and lipid deposition in liver tissue, and then exerting its effect through the expression level of obesity-related genes in liver tissue.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of 1,4-Methylimidazoleacetic acid in the preparation of drugs for preventing and treating obesity.

2. The use according to claim 1, It is characterized in that The medicine also contains pharmaceutically acceptable excipients.

3. The use according to claim 2, It is characterized in that The dosage form of the drug includes granules, capsules, tablets, oral liquids, pills or ointments.

4. The use according to claim 1, It is characterized in that The obesity is obesity induced by a high-fat diet.

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