Application of dauricine in preparation of medicine for treating diabetes

Abstract

The invention discloses application of dauricine in preparation of a medicine for treating diabetes mellitus, and belongs to the technical field of biological medicine. Preliminary drug screening shows that dauricine has hypoglycemic activity. Further experiments prove that dauricine has the influence on glucose uptake of L6 cells, transfer of GLUT4 and the like. In addition, the correlation between dauricine and Ca < 2 + > is also proved. The invention provides a theoretical basis for research and development of dauricine for treating type 2 diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of dauricine in the preparation of medicines for treating diabetes. Background Art

[0002] Diabetes is a chronic disease characterized by high blood sugar, caused by absolute or relative insufficient secretion of insulin and impaired utilization. The disease is mainly divided into three types: type 1, type 2 and gestational diabetes. The cause is mainly attributed to the combined effects of genetic and environmental factors, including insufficient insulin secretion caused by islet cell dysfunction, or the body's insensitivity to insulin, or both, which prevents glucose in the blood from being effectively utilized and stored. At present, the incidence and prevalence of diabetes are on the rise worldwide.

[0003] Type 2 diabetes (T2DM) is a chronic disease caused by insufficient or inefficient use of insulin. It is common in adults and is also called adult-onset diabetes. Its main characteristic is high blood sugar. Some patients have defects in pancreatic β-cell function, insufficient insulin secretion, and inability to effectively lower blood sugar, which will cause blood sugar levels to rise; some patients have normal insulin secretion, but the body is insensitive to insulin and cannot effectively use insulin, that is, insulin resistance occurs, which will also cause blood sugar to rise.

[0004] Many early patients will show no symptoms or mild symptoms. If not intervened in time, type 2 diabetes will lead to a series of complications, such as retinopathy, foot ulcers, atherosclerosis, diabetic nephropathy, multiple neuritis, etc.

[0005] Glucose transporter 4 (GLUT4) is a very important glucose transporter. GLUT4 protein is widely distributed in skeletal muscle, myocardium, fat and other tissues, and is closely related to insulin resistance and type 2 diabetes. GLUT4 circulates inside the cell and between the cell membrane. The transport of glucose requires the translocation and activation of GLUT4 itself. Under the action of insulin, the insulin receptor on the cell surface transmits intracellular signals to the GLUT4 storage vesicles, promoting the translocation of the perinuclear GLUT4 vesicles, and then fuses to the cell membrane through three steps of tethering, anchoring and fusion, transporting extracellular glucose into the cell, thereby lowering blood sugar and improving diabetes. Therefore, new drugs for the treatment of type 2 diabetes can be screened by targeting GLUT4.

[0006] At present, the main oral hypoglycemic drugs include sulfonylureas, glinides, biguanides, thiazolidinediones and α-glucosidase inhibitors. However, long-term use of these drugs has many side effects, such as gastrointestinal discomfort and hypoglycemia. Dauricine is a diphenylisoquinoline alkaloid extracted and separated from the rhizome of the plant Dauricine (Sophora japonica), which has anti-inflammatory activity and is also a broad-spectrum antiarrhythmic drug suitable for rapid arrhythmias, especially for atrial, junctional and ventricular premature beats, and for preexcitation syndrome with ventricular tachycardia. Relevant studies have shown that some diphenylisoquinoline alkaloids have therapeutic effects on T2DM and obesity; at present, there are no reports at home and abroad on the treatment of type 2 diabetes by dauricine by promoting GLUT4 expression and transport. Summary of the invention

[0007] The purpose of the present invention is to provide the use of dauricine in the preparation of a drug for treating diabetes, so as to solve the problems existing in the above-mentioned prior art.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] One of the technical solutions of the present invention is the use of dauricine in the preparation of drugs for treating diabetes.

[0010] The second technical solution of the present invention is a drug for treating diabetes, comprising dauricine.

[0011] The third technical solution of the present invention is the use of dauricine in the preparation of drugs for promoting cellular uptake of glucose.

[0012] A fourth technical solution of the present invention is the use of dauricine in the preparation of drugs for promoting cell GLUT4 transport.

[0013] The fifth technical solution of the present invention is that dauricine is used in the preparation of a method for promoting intracellular Ca 2+ Application of drugs with elevated concentrations.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention has been screened for drugs in the early stage and it is speculated that dauricine has hypoglycemic activity. Further experiments confirmed the effects of dauricine on glucose uptake and GLUT4 transport in L6 cells. In addition, it was also confirmed that dauricine and Ca 2+ The present invention provides a theoretical basis for developing dauricine for treating type 2 diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 Dauricine promotes glucose uptake in L6 cells.

[0018] Figure 2 Dauricine promotes GLUT4 transport in L6 cells.

[0019] Figure 3 Dauricine increases intracellular Ca in L6 cells 2+ Among them, A is the Ca level in L6 cells after adding dauricine 2+ B is the fluorescence change of L6 cells after adding dauricine. 2+ level. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0026] The embodiment of the present invention provides the use of dauricine in preparing a drug for treating diabetes.

[0027] An embodiment of the present invention also provides a medicine for treating diabetes, comprising dauricine.

[0028] In some specific embodiments, a pharmaceutically acceptable excipient is also included.

[0029] In some specific embodiments, the dosage form of the drug is a solid dosage form, a liquid dosage form, a cream dosage form or an emulsion dosage form.

[0030] The embodiments of the present invention also provide the use of dauricine in the preparation of a drug for promoting the uptake of glucose by cells.

[0031] The embodiments of the present invention also provide the use of dauricine in the preparation of a drug for promoting cell GLUT4 transport.

[0032] The present invention also provides a method for preparing a method for promoting intracellular Ca 2+ Application of drugs with elevated concentrations.

[0033] Example 1

[0034] The dauricine provided in this example was purchased from the official website of Shanghai Yuanye Biotechnology Co., Ltd. Dauricine was dissolved in 3% DMSO and used for biological activity test, and the specific steps were as follows:

[0035] 1. Cellular glucose uptake experiment:

[0036] (1) When L6 cells grow to about 80% of the culture dish, L6 cells are inoculated into a 96-well plate, and complete culture medium for culturing L6 cells (45 mL DMEM medium + 5 mL fetal bovine serum FBS + 0.5 mL penicillin / streptomycin 10000 U / mL) is added, 100 μL / well. After 48 hours, complete culture medium for differentiating L6 cells (49 mL DMEM medium + 1 mL fetal bovine serum FBS + 0.5 mL penicillin / streptomycin 10000 U / mL) is added and cultured for 5 to 7 days.

[0037] (2) Serum-free α-MEM cell culture medium (50 mL of DMEM culture medium) was added to a 96-well plate and the cells were starved for 2 h. The experiment was divided into a blank control group, an insulin (100 nM) positive control group and an experimental group. Batryticatine (50 μM) was added to the experimental group and then placed in an incubator at 37°C and 5% CO2 for 30 min. The experiment was repeated three times.

[0038] (3) Measure absorbance using an ELISA reader: Take out 2 μL of the sample from each well of the plate and transfer it to a new 96-well plate. Then add 200 μL of glucose oxidase reagent to each well and immediately measure the absorbance of each well using an ELISA reader: shake for 10 s, at a wavelength of 505 nm, and use GraphPad Prism for data processing.

[0039] Figure 1 Dauricine promotes glucose uptake in L6 cells. Figure 1 As shown, dauricine significantly promoted the glucose uptake of L6 cells.

[0040] 2. Detection of GLUT4 transport by laser confocal microscopy:

[0041] In this experiment, immunofluorescence experiments were performed using the structural characteristics of GLUT4 protein in the stably expressed myc-GLUT4-mOrange L6 cell line. Since myc is the extracellular domain of the GLUT4 protein, myc-specific primary antibody and FITC-labeled secondary antibody were used to label the myc domain in the immunofluorescence experiment. Under a laser confocal microscope, GLUT4 will show two types of fluorescence, one is mOrange red fluorescence, indicating total GLUT4, and the other is green fluorescence FITC indicating GLUT4 on the cell membrane. The co-localization merge of the two indicates GLUT4 transported to the cell membrane. In this result, the two types of fluorescence can be clearly seen, indicating that GLUT4 is transported from the cell to the cell membrane under the stimulation of 50μM batricin. Therefore, monitoring myc-GLUT4–mOrange can be used to monitor the transport of GLUT4 in L6 cells. First, myc-GLUT4–mOrangeL6 was starved with serum-free culture medium for 2 hours, and then the fluorescence of myc-GLUT4–mOrange L6 cells was observed under a laser confocal microscope and the fluorescence intensity on the cell membrane was detected to detect the transport of cellular GLUT4.

[0042] Figure 2 This is a diagram showing that dauricine promotes GLUT4 transport in L6 cells. Figure 2 As shown in the figure, after adding 50 μM dauricine, the fluorescence intensity of myc-GLUT4–mOrange L6 cell membrane was significantly enhanced, indicating that dauricine can promote the transport of GLUT4 in L6 cells.

[0043] 3. Real-time detection of intracellular Ca by laser confocal microscopy 2+ level:

[0044] L6 cells were inoculated in a small dish and cultured until the cells differentiated, and then starved for 2 hours by adding serum-free α-MEM. Fluo-4AM is an acetyl methyl ester derivative of Fluo-4 and is a fluorescent dye that can penetrate the cell membrane. The fluorescence of Fluo-4AM is very weak and does not change with Ca. 2+ After entering the cell, Fluo-4AM can be cleaved by esterases in the cell to form Fluo-4, which is then retained in the cell. Fluo-4 can react with Ca 2+ Bind to Ca 2+After that, it can produce strong fluorescence. Treat the cells with PSS solution containing 2.5μM Fluo-4AM, incubate at room temperature for 20 minutes, and wash twice with PSS. Then use confocal laser scanning microscopy to detect the changes in the fluorescence intensity of Fluo4 in the cells within 5 minutes after the addition of dauricine at 488nm. After adding dauricine, take pictures every 10s, and take pictures every 60s after 1 minute, for 5 minutes. Zen 2010 software was used to analyze and record the changes in Fluo-4 fluorescence in the cytoplasm, and GraphPad Prism was used for data processing.

[0045] Figure 3 Dauricine increases intracellular Ca in L6 cells 2+ Horizontal graph, such as Figure 3 As shown, after adding 50 μM dauricine, the Ca 2+ The fluorescence intensity gradually increased in a time-dependent manner and reached a maximum value at 180 s. This indicates that dauricine can stimulate Ca 2+ Increased concentration.

[0046] In summary, the present invention has shown through preliminary drug screening that it is speculated that dauricine has hypoglycemic activity. Further experiments are intended to explore the effects of dauricine on glucose uptake and GLUT4 transport in L6 cells. In addition, the relationship between dauricine and Ca 2+ The present invention provides a theoretical basis for developing dauricine for treating type 2 diabetes.

[0047] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of dauricine in the preparation of drugs for treating diabetes.

2. A drug for treating diabetes, characterized in that: Including dauricine.

3. The drug according to claim 2, characterized in that Pharmaceutically acceptable excipients are also included.

4. The drug according to claim 3, characterized in that The dosage form of the drug is a solid dosage form, a liquid dosage form, a paste dosage form or an emulsion dosage form.

5. Application of dauricine in the preparation of drugs for promoting glucose uptake by cells.

6. Application of dauricine in the preparation of drugs for promoting cellular GLUT4 transport.

7. Dauricine is used in the preparation of Ca-promoting 2+ Application of drugs with elevated concentrations.

Images