Application of Alternaria spore in the preparation of drugs for the prevention and treatment of diabetes

Abstract

The present invention discloses the application of Alternariol in the preparation of diabetes prevention and treatment drugs, which belongs to the field of medical technology. The present invention first screens out the compound Alternariol through in vitro DYRK1A kinase activity experiment, and its IC 50 The value was 20μM. Then, through INS-1 cell proliferation model experiments, it was found that the compound Alternariol can promote INS-1 cell proliferation and promote insulin secretion by pancreatic islet cells. Even at very low concentrations, such as 300nmol, it can significantly promote INS-1 cell proliferation. Finally, through mouse diabetes model experiments, it was further verified that the compound Alternariol can indeed control blood sugar in T1D mice and promote pancreatic proliferation in mice, while also having no toxic effects on the mouse body. This invention has extraordinary significance for T1B diabetes with impaired pancreatic cell function.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to the application of spore sol in the preparation of medicines for preventing and treating diabetes. Background Art

[0002] Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are a highly evolutionarily conserved class of protein kinases belonging to the CMGC family of cell cycle-dependent protein kinases. In mammals, the DYRK family comprises five isoforms: 1A, 1B, 2, 3, and 4. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is the most highly expressed of these kinases, with a broad array of substrates involved in numerous physiological processes in the human body and implicated in a variety of diseases, including neurological disorders, diabetes, and cancer. Extensive evidence indicates that selective inhibition of DYRK1A can improve cognitive function in mice and enhance pancreatic β-cell proliferation in both mice and humans, suggesting that DYRK1A inhibitors hold great potential for the treatment of diabetes. Endophytic fungi are considered promising sources of novel and bioactive secondary metabolites for medical applications. However, the inhibitory effects of endophytic fungal natural products on DYRK1A activity have not been reported. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides the use of Alternariol in the preparation of diabetes prevention and treatment drugs. The present invention first screened out the secondary metabolite compound Alternariol (IC) from the fungus Alternaria alternata (Fr.) Keissl from 801 natural products isolated by the research group in the early stage through in vitro DYRK1A kinase activity experiments. 50 =20μM), and found that its inhibitory activity against DYRK1A was most significant. Subsequently, experiments in an INS-1 cell proliferation model revealed that the compound Alternariol can promote INS-1 cell proliferation and insulin secretion from pancreatic islet cells. This is particularly true even at very low concentrations, such as 300nmol. Finally, experiments in a mouse diabetes model further verified that the compound Alternariol can indeed control blood sugar in T1D mice and promote pancreatic proliferation in mice, while also being non-toxic to the mice. This invention has significant implications for the treatment of T1B diabetes characterized by impaired pancreatic cell function and may provide insights for subsequent structural optimization and future drug development.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] One of the purposes of the present invention is to provide the use of Alternariol in the preparation of diabetes prevention and treatment drugs.

[0006] Furthermore, the diabetes is islet cell function damage type diabetes.

[0007] Furthermore, the diabetes is idiopathic type 1 diabetes.

[0008] Furthermore, in the application, Alternariol can control blood sugar, promote INS-1 cell proliferation, promote pancreatic proliferation and / or promote insulin secretion.

[0009] The second purpose of the present invention is to provide the use of Alternariol in the preparation of blood sugar control drugs.

[0010] The third object of the present invention is to provide the use of the Alternariol in the preparation of INS-1 cell proliferation promoting drugs.

[0011] The fourth object of the present invention is to provide the use of Alternariol in the preparation of pancreatic proliferation promoting drugs.

[0012] The fifth object of the present invention is to provide the use of Alternariol in the preparation of insulin secretagogues.

[0013] Furthermore, the concentration of Alternariol used in the application is 0.15 μM to 40 μM.

[0014] The sixth object of the present invention is to provide a drug for preventing and treating diabetes-related diseases, which includes the Alternariol used in the application.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention first screened out the secondary metabolite compound Alternariol (IC) from the fungus Alternaria alternata (Fr.) Keissl from 801 natural products isolated by the research group in the early stage through in vitro DYRK1A kinase activity experiments. 50=20μM), and found that its inhibitory activity against DYRK1A was most significant. Subsequently, experiments in an INS-1 cell proliferation model revealed that the compound Alternariol can promote INS-1 cell proliferation and insulin secretion from pancreatic islet cells. This is particularly true even at very low concentrations, such as 300nmol. Finally, experiments in a mouse diabetes model further verified that the compound Alternariol can indeed control blood sugar in T1D mice and promote pancreatic proliferation in mice, while also being non-toxic to the mice. This invention has significant implications for the treatment of T1B diabetes characterized by impaired pancreatic cell function and may provide insights for subsequent structural optimization and future drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the DYRK1A kinase activity experiment in Example 1 of the present invention;

[0018] Figure 2 The rescreening results and structural formula of Alternariol in Example 1 of the present invention are as follows;

[0019] Figure 3 The OD values ​​of INS-1 cell viability and cell survival rates of each group in Example 2 of the present invention are shown;

[0020] Figure 4 This is the result of the effect of Alternariol on insulin secretion in INS-1 cells in Example 2 of the present invention;

[0021] Figure 5 This is the result of the effect of Alternariol on INS-1 cell proliferation in Example 2 of the present invention;

[0022] Figure 6 The results of the fasting blood glucose and oral glucose tolerance tests after two and four weeks of oral administration in Example 3 of the present invention are shown;

[0023] Figure 7 These are the HE staining results of the liver, spleen, pancreas, and kidney organs of mice in different groups in Example 3 of the present invention. DETAILED DESCRIPTION

[0024] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are intended to be within the scope of the present invention. Products, equipment, and the like used in the following examples are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0025] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0026] Example 1 In vitro DYRK1A kinase activity assay to screen for alternariol

[0027] 1 The experimental reagents and experimental instruments used in this example are shown in Tables 1 and 2.

[0028] Table 1 Experimental reagents

[0029]

[0030]

[0031] Table 2 Experimental instruments

[0032]

[0033] 2 The in vitro DYRK1A kinase activity test formula of this example is shown in Table 3.

[0034] Table 3 In vitro DYRK1A kinase activity test formula

[0035]

[0036]

[0037] 3 Follow Figure 1 The enzyme activity system was constructed according to the experimental principle shown in the figure. The absorbance change of NADH at 340nm was detected using a microplate reader (set to 30 minutes, once every minute). Among them: control is the blank group, positive drug is the 25μM harmine group, No dyrkla is the group without enzyme, and 0.625μM to 40μM are different concentrations of Alternariol. The results of the re-screening of Alternariol and its structural formula are shown in the figure. Figure 2 shown.

[0038] Experimental conclusion: Finally, the compound Alternariol (CAS No.: 641-38-3) was screened out, and its IC 50 The value is 20 μM.

[0039] Example 2 INS-1 cell proliferation model experiment

[0040] Establishment, grouping, modeling and drug administration of 1INS-1 cell proliferation model

[0041] 1.1 Cell Culture: Purchased INS-1 cells were cultured in a 37°C, 5% CO2 incubator using 1640 medium (containing 10% fetal bovine serum and 1% double-antibody). Cultured in a 37°C, 5% CO2, saturated humidity incubator. Once the cells have grown to the bottom of the culture flask, discard the medium and wash twice with PBS. Add trypsin to a final concentration of 0.25% and digest for 2 minutes. 1640 medium was then added to terminate digestion. Gently pipette and centrifuge at 1200 rpm for 5 minutes. Resuspend the cell pellet and inoculate it into a culture flask. Observe and photograph under a microscope. Once the cells have grown into a complete monolayer, they can be used for subsequent experiments.

[0042] 1.2 Preparation of working solution: Weigh a certain amount of compound Alternariol, dissolve it in DMSO, and then add PBS to make 10 -3 mol / L working solution, the volume of DMSO added is 0.5% of the working solution volume. Working solution: 10 -3 mol / L (hereinafter mol / L is represented by M) working solution was diluted 25 times with 1640 culture medium to prepare 4×10 -5 M working solution, and then serially diluted to 2 × 10 -6 M, 1×10 -6 M working solution, filter, aliquot, and store at -4°C.

[0043] 1.3 Establishment of cell proliferation model: Stably growing INS-1 cells were cultured into 1.25×10 5 Cells / mL cell suspension was then seeded in a sterile 96-well plate. Three different cell proliferation times were set, namely 24h, 36h, and 48h. 80μL of cell suspension was added to each well. After the cells were cultured for about 24 hours, the culture medium in the well was discarded. The control group was replaced with 1640 culture medium. The drug-treated group was given 10μL of different concentration gradients of 400μM, 200μM, 100μM, 50μM, 25μM, 12.5μM, 6.125μM, 3.06μM, and 1.503μM compounds, respectively, to make the final concentrations of 40μM, 20μM, 10μM, 5μM, 2.5μM, 1.25μM, 0.6125μM, 0.306μM, and 0.1503μM. The culture was then continued for 24h, 36h, and 48h.

[0044] 1.4 Detection of cell viability by CCK8 assay: After treating each group of INS-1 cells according to the method in 3.2.1, the entire volume of the culture medium was replaced with 1640 culture medium (containing 10% fetal bovine serum), 10 μL of CCK8 was added to each well, and the cells were incubated at 37°C for 3 h. The absorbance of each group of cells at 450 nm was measured by a microplate reader.

[0045] The cell viability calculation formula is shown in the following formula (1). The OD value of INS-1 cell viability and cell viability results of each group are shown in Figure 3 shown.

[0046]

[0047] Experimental conclusion: The compound Alternariol can significantly promote the proliferation of INS-1 cells even at a very low concentration of 300nmol, which is of extraordinary significance for T1B diabetes with damaged pancreatic islet cell function.

[0048] 2. Detection of changes in Insulin and Ki67 in INS-1 cells by immunofluorescence

[0049] Place sterile cell slides of appropriate size in a 6-well cell culture plate (1 slide / well), collect INS-1 cells that are in good growth condition and dilute them to a density of 1.25×10 6 A cell suspension of 100 μg / mL was then inoculated into the culture dish. After 24 hours of culture, the following experiments were performed: ① The cells were treated with the compound Alternariol (10 μM, 5 μM, and 2.5 μM) for 24 hours; ② STZ was first administered to the cells for 1 hour to stimulate injury. The supernatant was removed, and the cells were fixed with 4% paraformaldehyde for 30 minutes and then washed twice with pre-chilled PBS buffer. The cells were then treated with the following procedures:

[0050] (1) Cell disruption: First, shake the slide dry, select a location where cells are evenly distributed, and draw a circle with a histochemical pen. Add an appropriate amount of membrane disruption solution (BF0001, Wuhan Bolfu Biotechnology Co., Ltd.) into the circle to cover the cells. Incubate at room temperature for 20 min to 30 min, and wash three times with PBS (P1022, Beijing Soleibo Technology Co., Ltd.), each time for 5 min.

[0051] (2) BSA blocking: After drying slightly, 3% BSA (A8010, Beijing Soleibo Technology Co., Ltd.) was added dropwise to the circle and incubated at room temperature for 30 min.

[0052] (3) Primary antibody incubation: First remove the blocking solution, then place a drop of primary antibody on the section and place it in a humidified chamber at 4°C overnight. (Add a small amount of PBS to the humidified chamber to prevent evaporation).

[0053] (4) Adding secondary antibodies: Wash the cell slides with phosphate-buffered saline (PBS, pH 7.4) for 5 min three times. After the sections are slightly dry, add secondary antibodies of the corresponding species to the sections. Incubate at room temperature in the dark for 50 min.

[0054] (5) DAPI counterstaining of cell nuclei: The operation is the same as that in the DAPI staining kit instructions.

[0055] (6) Slide and seal the slide: After sealing, place the slide in a slice box and store it in a refrigerator at 4°C.

[0056] (7) Slide observation and photography: Place the slices under a fluorescence microscope for observation and image capture.

[0057] The results of the effects of each treatment group on insulin secretion and proliferation of INS-1 cells are shown in Figure 2. Figure 4 and Figure 5 shown.

[0058] Experimental conclusion: The compound Alternariol can promote the proliferation of INS-1 cells and the secretion of insulin by pancreatic islet cells, which is of extraordinary significance for T1B diabetes.

[0059] Example 3 Mouse diabetes model experiment

[0060] 1. Glucose tolerance test

[0061] (1) Glucose tolerance test: Oral glucose tolerance test was conducted before and one week after the intervention. Before the experiment, mice were fasted for 12 hours but not water. Then, they were gavaged with 2 g / kg glucose (dissolved in pre-prepared double-distilled water). The tails of the mice were cut and blood was collected. The blood glucose levels of the mice were measured at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after gavage.

[0062] (2) STZ (Streptozotocin) modeling method: After 1-2 weeks of adaptive feeding, mice were intraperitoneally injected with STZ (2%, freshly prepared and used). Before injection, 2% STZ solution was prepared with 0.05 mol / L citric acid (pH 4.5) and used fresh (fasting but not water for 12 hours). Indicators of successful modeling: 2 or 3 days after the injection of STZ solution, blood glucose was tested with blood glucose test strips for 3 consecutive days. If the blood glucose value was ≥8.0 mmol / L and the three more and one less symptoms appeared, the modeling was successful.

[0063] (3) An appropriate dose of Alternariol (Low drug group: 75 mg / kg; High drug group: 150 mg / kg) was administered to mice by gavage in the morning, and the daily food intake, body weight changes, and blood sugar changes of the mice were collected.

[0064] The results of fasting blood glucose and oral glucose tolerance test after two weeks and four weeks of oral administration are as follows Figure 6 shown.

[0065] 2HE staining was used to detect the pathological changes of pancreas, liver, kidney and spleen tissues

[0066] Following the glucose tolerance study described above, after the fifth week of dosing, eyeballs were removed and blood was collected for serum isolation. Mice were sacrificed by cervical dislocation. Most tissues, including liver, kidney, spleen, and pancreas, were stored in liquid nitrogen or dry ice, while a small portion was fixed in 4% paraformaldehyde. HE-stained sections were then prepared to examine pathological changes in the relevant tissues of the experimental mice. The specific procedures were as follows:

[0067] (1) Dewaxing the sections: Soak the sections in two different concentrations of xylene solutions for 10 min each, then discard the xylene and soak them in anhydrous ethanol, 90% ethanol, and 80% ethanol for 5 min each.

[0068] (2) After repeating step (1), dewax with 75% alcohol gradient for 5 minutes, and finally wash with distilled water.

[0069] (3) Staining tissue cell nuclei with hematoxylin: The sections were stained with hematoxylin for 5 minutes and then rinsed with tap water. Then, the sections were differentiated with hydrochloric acid for a few seconds, rinsed with tap water, and finally reduced to blue with ammonia.

[0070] (4) Eosin staining: The sections were dehydrated with alcohol and then stained with eosin for 5 minutes;

[0071] (5) Dehydration and sealing: Soak the paraffin sections in 95% alcohol, anhydrous ethanol, and xylene solution twice for 5 minutes each time, then discard the soaking solution. After the sections are slightly dry, add neutral gum to seal the sections.

[0072] (6) Observation and photography: The tissue on the slice was photographed using a fluorescence microscope. In the image, the cell nucleus was stained blue and the cytoplasm was stained red.

[0073] HE staining results of liver, spleen, pancreas and kidney organs of mice in different groups are shown in Figure 2. Figure 7 shown.

[0074] Experimental conclusion: Alternariol can indeed control the blood sugar of T1D mice and promote the proliferation of the mouse pancreas without any toxic effect on the mouse body.

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The use of Alternariol in the preparation of a drug for preventing and treating diabetes, characterized in that: The diabetes is idiopathic type 1 diabetes.

2. The use according to claim 1, characterized in that In the application, Alternariol can control blood sugar, promote INS-1 cell proliferation, promote pancreatic proliferation and / or promote insulin secretion.

3. The use according to any one of claims 1 to 2, characterized in that: Alternariol is used at a concentration of 0.15 in the application described. μ M~40 μ M.

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