Application of thiadiazolidinedione GSK-3beta covalent inhibitor in preparation of medicine for treating type II diabetes

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a thiadiazolidinedione GSK-3beta covalent inhibitor in preparation of a medicine for treating type II diabetes mellitus, the thiadiazolidinedione GSK-3beta covalent inhibitor is N-(3-(4-benzyl-3, 5-dione-1, 2, 4-thiadiazole-2-yl) phenyl) acrylamide, and the structural formula of the thiadiazolidinedione GSK-3beta covalent inhibitor is shown in the description. The thiadiazolidinedione GSK-3beta covalent inhibitor is found to be capable of remarkably increasing phosphorylation of GSK-3beta of liver and pancreas in a type II diabetes body and lowering GSK-3beta protein expression quantity of the liver and the pancreas, so that the content of GSK-3beta in serum, the liver and the pancreas is reduced, the content of glucose in the serum and urine is reduced, the effect of treating diabetes is achieved, and the thiadiazolidinedione GSK-3beta covalent inhibitor can be used for preventing and treating the type II diabetes. Meanwhile, pathological changes of liver tissues and pancreatic tissues in the body of the type II diabetes mellitus can be remarkably improved, the medicine effect is good, the medicine safety is excellent, and an effective medicine and a medicine development thought are provided for treating the type II diabetes mellitus.

Description

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of a thiadiazolidinedione GSK-3β covalent inhibitor N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide in the preparation of a drug for the treatment of type II diabetes. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar. Clinically, it is divided into type 1 diabetes, type 2 diabetes, gestational diabetes and other special types of diabetes. Type 2 diabetes is a chronic disease caused by insufficient insulin secretion or ineffective use of insulin in the body, which leads to elevated blood sugar. Currently, the drugs used in clinical treatment of type 2 diabetes are mainly divided into five categories: insulin and its analogues, insulin sensitizers, insulin secretagogues, enzyme inhibitors and sodium-glucose co-transporter inhibitors. However, these drugs have adverse reactions such as severe hypoglycemia, ketoacidosis, gastrointestinal reactions, allergic reactions, abnormal liver function, metabolic and nutritional disorders, as well as reduced efficacy due to drug metabolism or unreasonable dosing regimens. Therefore, continuing to explore better ways and means of treating diabetes is of great significance for improving the quality of life of diabetic patients and prolonging their lifespan.

[0003] Glycogen synthase is a protein with different isoforms that function in tissues such as muscle and liver. Glycogen synthase exists in two forms: phosphorylated (p-GS) and unphosphorylated (GS). The unphosphorylated form is active, while phosphorylation reduces or even eliminates its activity. Phosphorylation causes a conformational change in glycogen synthase, making it difficult for it to bind to its substrate, uridine diphosphate glucose, and glycogen primers, thereby inhibiting glycogen synthesis. For example, when the body is starving or stressed, glycogen synthase becomes phosphorylated, inhibiting its activity and reducing glycogen synthesis to ensure that blood sugar levels remain within normal ranges and prioritize meeting the glucose needs of vital organs.

[0004] Glycogen synthase kinase 3 (GSK-3) is a highly evolutionarily conserved serine / threonine protein kinase. It typically exists in two isoforms, GSK-3α and GSK-3β, regulating cellular function by participating in multiple cellular signaling pathways. Abnormally elevated GSK-3β activity is highly correlated with the development of diseases such as type 2 diabetes, cancer, neurodegenerative diseases, and inflammation. GSK-3β inhibitors are key targets for drug development in diseases such as type 2 diabetes, Alzheimer's disease, and cancer.

[0005] GSK-3β expression plays a crucial role in the progression of type 2 diabetes. When insulin resistance develops, GSK-3β expression in peripheral tissues increases, accelerating the progression of type 2 diabetes. GSK-3β is normally active, but it can be phosphorylated by various signaling pathways, altering its activity. Phosphorylation of specific serine or threonine residues on the GSK-3β protein, often referred to as p-GSK-3β, inhibits GSK-3β activity.

[0006] N-(3-(4-benzyl-3,5-dione-1,2,4-thiadiazol-2-yl)phenyl)acrylamide (hereinafter referred to as GL 10a) has been shown to be a covalent GSK-3β inhibitor in Alzheimer's disease research. GL 10a's inhibition of GSK-3β kinase is non-ATP binding competitive inhibition and has high selectivity for GSK-3β. Furthermore, GL10a exhibits excellent kinase inhibitory activity and selectivity, along with a good safety profile (Yongxi Dong, Jun Lu, Shanhui Zhang, et al. Design, synthesis and bioevaluation of 1,2,4-thiadiazolidine-3,5-dione derivatives as potential GSK-3β inhibitors for the treatment of Alzheimer's disease [J], Bioorganic Chemistry, 2023, 134:106446). However, its therapeutic effect on diabetes and its application in the preparation of diabetes treatment drugs have not yet been reported.

[0007] The patent document with application number CN202311167646.4 mainly protects a compound that can inhibit the activity of GSK-3β. This compound and the compound described in the present invention have significant differences in type and structure, and this compound has not yet been used in the preparation of diabetes treatment drugs.

[0008] The patent document with application number CN201710606657.6 mainly protects a tetrahydrobenzothiophene derivative, its pharmaceutically acceptable salt, optically active form or racemate. This type of compound can inhibit the activity of GSK-3β, but there are significant differences in type and structure from the compound described in the present invention, and there has been no application of this compound in the preparation of diabetes treatment drugs.

[0009] Currently, there are no reports on the use of the thiadiazolidinedione compound GL10a for the treatment of type II diabetes. Therefore, the present invention aims to provide a thiadiazolidinedione GSK-3β covalent inhibitor compound for the preparation of a drug for the treatment of type II diabetes. Summary of the Invention

[0010] To address the problem in the prior art that there have been no reports on products and applications of the thiazolidinedione GSK-3β covalent inhibitor GL 10a for the treatment of type 2 diabetes, the present invention uses C57BL / 6J mice induced by high-fat feeding combined with streptozocin (STZ) as the subjects and provides the application of GL 10a in the preparation of drugs for the treatment of type 2 diabetes.

[0011] This is achieved specifically through the following technical solutions:

[0012] The first object of the present invention is to provide a use of a GSK-3β covalent inhibitor in the preparation of a medicament for treating type II diabetes or related diseases caused by type II diabetes, wherein the GSK-3β covalent inhibitor is a thiadiazolidinedione compound, wherein the thiadiazolidinedione compound is N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide (abbreviated as GL 10a), and its structural formula is as follows:

[0013]

[0014] The related diseases caused by type II diabetes refer to weight gain, increased blood sugar, increased urine glucose content, and pathological damage to liver or pancreatic tissue.

[0015] The second object of the present invention is to provide a composition comprising N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide and pharmaceutically acceptable excipients.

[0016] The drug for treating type II diabetes is prepared by combining the GSK-3β covalent inhibitor GL 10a with pharmaceutically acceptable excipients to prepare the drug and its dosage form, which is used for the treatment and / or prevention of type II diabetes.

[0017] The drug for treating type 2 diabetes is prepared with GL 10a as the active ingredient and supplemented with pharmaceutically acceptable excipients. The dosage forms include tablets, capsules, injections, aerosols, ointments, and granules. Acceptable excipients include one or more of fillers, disintegrants, binders, surfactants, lubricants, and carriers.

[0018] The treatment of type II diabetes refers to the treatment, prevention, alleviation and / or relief of type II diabetes.

[0019] Furthermore, the GSK-3β covalent inhibitor plays a role in treating type II diabetes by reducing the GSK-3β content and glucose content.

[0020] The reducing of GSK-3β content refers to reducing the GSK-3β content in serum, liver tissue, and pancreatic tissue.

[0021] The lowering of glucose content refers to lowering the glucose content in serum and urine.

[0022] Furthermore, the GSK-3β covalent inhibitor reduces the GSK-3β content by downregulating the expression of GSK-3β protein in liver or pancreatic tissue and increasing the phosphorylation of GSK-3β in liver or pancreatic tissue.

[0023] Furthermore, the GSK-3β covalent inhibitor reduces glucose levels by increasing liver glycogen synthesis and promoting insulin secretion.

[0024] More specifically, the N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide promotes insulin secretion by increasing the expression of PDX1 protein in the pancreas.

[0025] More specifically, the N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide increases liver glycogen synthesis by inhibiting the phosphorylation of glycogen synthase in the liver.

[0026] The drug for treating type II diabetes is a drug that reduces GSK-3β in serum, liver and pancreas.

[0027] The type II diabetes therapeutic drug is a drug that reduces the glucose content in serum and urine.

[0028] The type II diabetes treatment drug includes at least one of a drug that inhibits GSK-3β protein expression in the liver or pancreas, a drug that increases the phosphorylation of GSK-3β in the liver or pancreas, a drug that increases liver glycogen synthesis, and a drug that promotes insulin secretion.

[0029] The drug that increases hepatic glycogen synthesis is a drug that inhibits the phosphorylation of glycogen synthase in the liver.

[0030] The drug for promoting insulin secretion is a drug for increasing the expression of PDX1 protein in the pancreas.

[0031] Beneficial effects

[0032] The present invention provides the use of GL 10a in the preparation of a drug for treating type II diabetes, and its specific effects are:

[0033] 1. The GSK-3β covalent inhibitor GL 10a of the present invention has an excellent GSK-3β kinase inhibitory effect. It can not only significantly increase the phosphorylation of GSK-3β in the liver and pancreas of type II diabetic mice, but also effectively inhibit the expression of GSK-3β protein in the liver and pancreas, thereby reducing the GSK-3β levels in serum, liver, and pancreas.

[0034] 2. The GSK-3β covalent inhibitor of the present invention effectively inhibits the phosphorylation of glycogen synthase in the liver and increases the protein expression of PDX1 in the pancreas by inhibiting the activity of GSK-3β kinase, thereby effectively lowering blood glucose levels. Inhibiting the phosphorylation of glycogen synthase in the liver promotes liver glycogen synthesis, while increasing the protein expression of PDX1 in the pancreas promotes insulin secretion.

[0035] 3. The drug prepared by the GSK-3β covalent inhibitor of the present invention can improve the pathological changes in the liver and pancreatic tissues of type II diabetic mice.

[0036] In summary, the present invention has discovered a new use of GL 10a in treating, preventing, alleviating and alleviating type II diabetes, and has provided an effective drug and drug development strategy for treating type II diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Effects of GL 10a on body weight in mice treated with type 2 diabetes

[0038] Figure 2 Effects of GL 10a on fasting blood glucose levels in mice treated with type 2 diabetes

[0039] Figure 3 Effects of GL 10a on serum and urine glucose levels in mice treated with type 2 diabetes

[0040] Figure 4 Effects of GL 10a on serum and pancreatic insulin levels in mice treated with type 2 diabetes

[0041] Figure 5 Effects of GL 10a on GSK-3β levels in serum, liver and pancreas of mice treated with type 2 diabetes

[0042] Figure 6 Effects of GL 10a on hepatic glycogen content in mice treated with type 2 diabetes

[0043] Figure 7Effects of GL 10a on the expression of GSK-3β and p-GSK-3β proteins in the pancreas of mice treated with type 2 diabetes

[0044] Figure 8 Effect of GL 10a on the expression of PDX1 protein in the pancreas of mice treated with type 2 diabetes

[0045] Figure 9 Effects of GL 10a on the expression of GSK-3β and p-GSK-3β proteins in the liver of mice treated with type 2 diabetes

[0046] Figure 10 Effects of GL 10a on the expression of GS and p-GS proteins in the liver of mice treated with type 2 diabetes

[0047] Figure 11 Effects of GL 10a on liver and pancreatic pathology in mice treated with type 2 diabetes

[0048] Figure 12 The principle and pathway of GL 10a in lowering blood sugar levels DETAILED DESCRIPTION

[0049] The embodiments of the present invention are all based on the following experimental preparation:

[0050] Calculate and weigh GL 10a and corresponding excipients, and prepare them together into a drug.

[0051] The prepared drug and a control drug (in this case, metformin, a commonly used drug for treating type 2 diabetes) were used for efficacy comparison. Specifically, the prepared drug and the control drug were administered orally to successfully established diabetic mice. Various indicators of the mice were measured, and the therapeutic efficacy of the prepared drug for treating type 2 diabetes was ultimately determined based on the measured data.

[0052] Preparation of citric acid buffer solution: Weigh 200 mg of citric acid and 294 mg of sodium citrate and dissolve each in 10 mL of sterile distilled water. Mix the two solutions and adjust the pH of the mixture to 4.2-4.5 with sodium hydroxide solution and hydrochloric acid solution to obtain the citric acid buffer solution.

[0053] Preparation of STZ solution: Prepare STZ solution using the prepared citrate buffer solution. Prepare it immediately before use. Specifically, weigh 0.2401 g of STZ and dissolve it in 40 mL of citrate buffer solution to obtain a 6 mg / mL STZ solution.

[0054] Preparation of metformin solution: Weigh 0.6007 g of metformin and dissolve it in 30 mL of normal saline to obtain a 20 mg / mL metformin solution. Store at 4°C until ready to use.

[0055] Preparation of Tideglusib solution: Weigh 0.0598 g and dissolve it in 30 mL of normal saline to obtain a 2 mg / mL Tideglusib solution. Store at 4°C until ready to use.

[0056] Preparation of GL 10a high-dose solution: Weigh 0.1604 g of GL 10a and dissolve it in 80 mL of normal saline to obtain a 2 mg / mL GL 10a high-dose solution. Store at 4°C until ready to use.

[0057] Preparation of GL 10a medium-dose solution: Measure 20 mL of the above-mentioned 2 mg / mL GL 10a high-dose solution and dilute it with 20 mL of normal saline to obtain a 1 mg / mL GL 10a medium-dose solution. Store at 4°C until ready to use.

[0058] Preparation of GL 10a low-dose solution: Measure 10 mL of the above 2 mg / mL GL 10a high-dose solution and dilute it with 30 mL of normal saline to obtain a 0.5 mg / mL GL 10a low-dose solution. Store at 4°C until ready for use.

[0059] Glucose oxidase assay kit, catalog number A154-1-1, was purchased from Nanjing Jiancheng Bioengineering Institute;

[0060] Insulin detection kit, catalog number E-EL-M2614c, was purchased from Wuhan Elaruite Biotechnology Co., Ltd.;

[0061] GSK-3β detection kit, catalog number SED317Mu, was purchased from Wuhan Cloud-Clone Technology Co., Ltd.;

[0062] Glycogen assay kit, catalog number A043-1-1, was purchased from Nanjing Jiancheng Bioengineering Institute;

[0063] GSK-3β antibody (Cat. No. 22104-1-AP), p-GSK-3β antibody (Cat. No. 67558-1-Ig), PDX1 antibody (Cat. No. 20989-1-AP), and β-Tubulin antibody (10094-1-AP) were purchased from Wuhan Tri-Taiwan Biotechnology Co., Ltd.

[0064] GS antibody (catalog number T55788) and p-GS antibody (catalog number PN81022) were purchased from Abmart.

[0065] Experimental animals: SPF-grade male C57BL / 6J, weighing 20 ± 2 g, were purchased from Changsha Tianqin Biotechnology Co., Ltd. (SCXK (Xiang) 2022-0011). All experimental animals were humanely handled in accordance with the Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Laboratory Animal Ethics Committee of Guizhou Medical University.

[0066] The study randomly divided 105 healthy male C57BL / 6J mice into a control group (10 CON mice) and a modeling group (95 mice). The CON group mice were fed a standard diet, while the modeling group mice were fed a high-fat diet. Six weeks later, the modeling group mice were intraperitoneally injected with STZ (60 mg / kg), while the CON group mice were intraperitoneally injected with an equal volume of citrate buffer once daily for three days. Three days later, tail tip blood was collected to measure fasting blood glucose levels in the mice, and mice with fasting blood glucose levels ≥16.8 mmol / L were included in subsequent experiments. The mice with successful modeling were randomly divided into model group (M), metformin group (MET, 200 mg / kg), Tideglusib group (NP12, 20 mg / kg), GL 10a low-dose group (GL 10a-L, 5 mg / kg), GL 10a medium-dose group (GL 10a-M, 10 mg / kg), and GL 10a high-dose group (GL10a-H, 20 mg / kg) dosage groups, with 10 mice in each group. Each drug-dosing group was given the corresponding drug by gavage once a day for 4 weeks. During the experiment, the body weight of the mice was weighed and recorded every Friday, and the fasting blood glucose level of the mice in each group was measured using a blood glucose meter. After the end of the drug administration, the mice were placed in a metabolic cage and 24-hour urine was collected. One hour after the last drug administration, blood was collected to separate the serum. At the end of the experiment, the mice were anesthetized and killed, and the pancreas, liver, kidney, and heart were taken.

[0067] Example 1

[0068] The weight of the mice in the experiment was measured. Figure 1 As shown, after 4 weeks of administration, the body weight of mice in the M group was significantly reduced compared with the CON group (P<0.05). Compared with the M group, the body weight of mice in the MET group, NP12 group, and GL10a group at each dose was significantly increased (P<0.05, P<0.01). The results indicate that GL10a can improve the weight loss caused by type 2 diabetes. This indicates that GL10a can be used to prepare drugs for the treatment of type 2 diabetes and can effectively improve the weight loss caused by type 2 diabetes, thereby realizing the application of GL10a in the preparation of drugs to improve the weight gain caused by type 2 diabetes.

[0069] Example 2

[0070] By measuring the fasting blood glucose of mice in the experiment, the results are as follows Figure 2As shown, during the 4-week administration, the fasting blood glucose of mice in each administration group decreased compared with the M group. In particular, after 4 weeks of administration, the fasting blood glucose of mice in the M group was significantly increased compared with the CON group (P<0.01); compared with the M group, the fasting blood glucose of mice in the MET group, NP12 group, and GL 10a group were significantly reduced (P<0.05, P<0.01). The results show that GL 10a can significantly reduce the serum glucose content of mice with type 2 diabetes. This shows that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention realizes the use of GL 10a in the preparation of drugs for improving elevated blood glucose caused by type 2 diabetes.

[0071] Example 3

[0072] After the mice were anesthetized and killed, the collected blood and urine were centrifuged at 3000 r / min for 10 min at 4°C. The supernatant was taken and the glucose content in the serum and urine was detected by the glucose oxidase method according to the kit instructions.

[0073] The results are as follows Figure 3 As shown, compared with the CON group, the glucose levels in the serum and urine of mice in the M group were significantly increased (P < 0.01); compared with the M group, the glucose levels in the serum and urine of mice in the MET group, the NP12 group, and the GL 10a group were significantly decreased (P < 0.05, P < 0.01). The results indicate that GL 10a can significantly reduce the glucose levels in the serum and urine of mice with type 2 diabetes. This demonstrates that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention implements the use of GL 10a in the preparation of drugs for ameliorating elevated glucose levels in serum and urine caused by type 2 diabetes.

[0074] Example 4

[0075] Mice were anesthetized and sacrificed, and blood was collected and centrifuged at 3000 rpm for 10 minutes at 4°C. Pancreatic tissue was then obtained, weighed, and homogenized. The insulin content in serum and pancreas was determined according to the kit instructions.

[0076] like Figure 4As shown in the results, compared with the CON group, the insulin levels in the serum and pancreas of mice in the M group were significantly reduced (P < 0.01). Compared with the M group, the insulin levels in the serum and pancreas of mice in the MET group, NP12 group, GL 10a-M group, and GL 10a-H group were significantly increased (P < 0.05, P < 0.01). The results show that GL 10a can significantly increase the insulin levels in the serum and pancreas of type 2 diabetic mice and significantly improve the pancreatic islet function of type 2 diabetic mice. This shows that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention achieves the hypoglycemic effect of GL 10a by promoting insulin secretion.

[0077] Example 5

[0078] Mice were anesthetized and sacrificed, and blood was collected and centrifuged at 3000 rpm for 10 minutes at 4°C. Liver and pancreatic tissues were weighed and homogenized, and GSK-3β levels were detected according to the ELISA kit instructions.

[0079] like Figure 5 As shown, compared with the CON group, the GSK-3β levels in the serum, liver, and pancreas of mice in the M group were significantly increased (P<0.01). Compared with the M group, the GSK-3β levels in the serum, liver, and pancreas of mice in the MET group, NP12 group, GL 10a-M group, and GL 10a-H group were significantly decreased (P<0.05, P<0.01). The results show that GL 10a can significantly reduce the GSK-3β levels in the serum, liver, and pancreas of type 2 diabetic mice. This shows that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention realizes that GL 10a exerts a hypoglycemic effect by reducing the GSK-3β content in the liver or pancreas.

[0080] Example 6

[0081] After the mice in the experiment were anesthetized and killed, the liver tissues of the mice were taken out, weighed and homogenized, and the liver glycogen content was detected according to the instructions of the liver glycogen content detection kit.

[0082] like Figure 6As shown, compared with the CON group, the liver glycogen content of mice in the M group was significantly reduced (P < 0.01). Compared with the M group, the liver glycogen content of mice in the MET group, NP12 group, and GL 10a groups was significantly increased (P < 0.05, P < 0.01). The results show that GL 10a can significantly increase the liver glycogen content of type 2 diabetic mice and improve the synthesis of liver glycogen in type 2 diabetic mice. This shows that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention realizes that GL 10a exerts a hypoglycemic effect by increasing the synthesis of liver glycogen.

[0083] Example 7

[0084] In the experiment, mice were anesthetized and sacrificed, and pancreatic tissue was quickly removed and weighed. After rinsing with pre-chilled saline to remove surface blood and wiping with filter paper, the pancreatic tissue was added with RIPA cell lysis buffer and lysed in an ice-water bath for 20 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. Protein concentration was determined using the BCA assay. Proteins were electrophoresed on 10% SDS-PAGE. After transfer and blocking, GSK-3β (1:4000) and p-GSK-3β (1:3000) primary antibodies were added and incubated overnight at 4°C. The membranes were washed with TBST (3 times for 10 minutes) and then horseradish peroxidase-conjugated secondary antibodies were added. After incubation at room temperature for another 2 hours, the membranes were washed with TBST (3 times for 10 minutes) and visualized using enhanced chemiluminescence. The optical density of each band was analyzed using image analysis software. Semi-quantification was performed by comparing the optical density of the target band to the optical density of the internal reference band, and statistical analysis was performed.

[0085] like Figure 7 As shown in Figures A and B, compared with the Con group, GSK-3β protein expression in the pancreas of mice in the M group was significantly upregulated (P < 0.01), while p-GSK-3β protein expression was significantly downregulated (P < 0.01). Compared with the M group, GSK-3β protein expression in the pancreas of mice in the MET, NP12, GL10a-M, and GL10a-H groups was significantly downregulated (P < 0.05, P < 0.01), and p-GSK-3β / GSK-3β expression was significantly upregulated (P < 0.01). These results indicate that GL10a can downregulate GSK-3β protein expression and upregulate p-GSK-3β protein expression in the pancreas of type 2 diabetic mice. This demonstrates that GL10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention demonstrates that GL10a exerts its hypoglycemic effect by inhibiting GSK-3β protein expression in the liver or pancreas.

[0086] Example 8

[0087] In the experiment, mice were anesthetized and sacrificed, and pancreatic tissue was quickly removed and weighed. After rinsing with pre-chilled saline to remove surface blood and wiping with filter paper, the pancreatic tissue was added with RIPA cell lysis buffer and lysed in an ice-water bath for 20 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. Protein concentration was determined using the BCA assay. Proteins were electrophoresed on 10% SDS-PAGE, transferred to the membrane, blocked, and then incubated overnight at 4°C with PDX1 (1:1000) and the primary antibody. The membrane was then washed with TBST (10 minutes per 3 times) and a horseradish peroxidase-conjugated secondary antibody was added. The membrane was incubated at room temperature for another 2 hours, washed with TBST (10 minutes per 3 times), and visualized using enhanced chemiluminescence. The optical density of each band was analyzed using image analysis software. Semi-quantification was performed by comparing the optical density of the target band to the optical density of the internal reference band, and statistical analysis was performed.

[0088] like Figure 8 As shown in A and B, compared with the CON group, PDX1 protein expression in the pancreas of mice in the M group was significantly downregulated (P < 0.01). Compared with the M group, PDX1 protein expression in the pancreas of mice in the MET group, NP12 group, and GL 10a-H group was significantly upregulated (P < 0.01). The results indicate that GL 10a can upregulate PDX1 protein expression in the pancreas of type 2 diabetic mice. This demonstrates that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention achieves that GL 10a promotes insulin secretion by increasing PDX1 protein expression in the pancreas.

[0089] Embodiment 9

[0090] In the experiment, mice were anesthetized and sacrificed, and liver tissue was quickly removed and weighed. After rinsing with pre-chilled saline to remove surface blood and wiping with filter paper, the tissue was added with RIPA cell lysis buffer and lysed in an ice-water bath for 20 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. Protein concentration was determined using the BCA assay. Proteins were electrophoresed on 10% SDS-PAGE. After transfer and blocking, GSK-3β (1:4000) and p-GSK-3β (1:3000) primary antibodies were added and incubated overnight at 4°C. The membranes were washed with TBST (3 times for 10 minutes) and then horseradish peroxidase-conjugated secondary antibodies were added. After incubation at room temperature for another 2 hours, the membranes were washed with TBST (3 times for 10 minutes) and visualized using enhanced chemiluminescence. The optical density of each band was analyzed using image analysis software. Semi-quantification was performed by comparing the optical density of the target band to the optical density of the internal reference band, and statistical analysis was performed.

[0091] like Figure 9As shown in Figures A and B, compared with the CON group, GSK-3β protein expression in the livers of mice in the M group was significantly upregulated (P<0.01), while p-GSK-3β protein expression was significantly downregulated (P<0.01). Compared with the M group, GSK-3β protein expression in the livers of mice in the MET, NP12, GL10a-M, and GL10a-H groups was significantly downregulated (P<0.05), while p-GSK-3β protein expression was significantly upregulated (P<0.05, P<0.01). These results indicate that GL10a can downregulate GSK-3β protein expression and upregulate p-GSK-3β protein expression in the livers of type 2 diabetic mice. Combined with Example 7, the results indicate that GL10a reduces GSK-3β content by downregulating GSK-3β protein expression and increasing GSK-3β phosphorylation in liver or pancreatic tissue. This shows that GL 10a can be used to prepare drugs for treating type II diabetes. More specifically, the present invention achieves the effect of GL 10a downregulating the protein expression of GSK-3β in the liver and upregulating the protein expression of p-GSK-3β.

[0092] Example 10

[0093] In the experiment, mice were anesthetized and sacrificed, and liver tissue was quickly removed and weighed. After rinsing with pre-chilled saline to remove surface blood and wiping with filter paper, the tissue was added with RIPA cell lysis buffer and lysed in an ice-water bath for 20 minutes, followed by centrifugation at 12,000 rpm for 10 minutes. Protein concentration was determined using the BCA assay. Proteins were electrophoresed on 10% SDS-PAGE. After transfer and blocking, primary antibodies against GS (1:9000) and p-GS (1:1000) were added, and the cells were incubated overnight at 4°C. The membranes were washed with TBST (3 times for 10 minutes) and then a horseradish peroxidase-conjugated secondary antibody was added. After an additional 2 hours of incubation at room temperature, the membranes were washed with TBST (3 times for 10 minutes) and visualized using enhanced chemiluminescence. The optical density of each band was analyzed using image analysis software. Semi-quantification was performed by comparing the optical density of the target band to the optical density of the internal reference band, and statistical analysis was performed.

[0094] like Figure 10As shown in A and B, no significant changes were observed in GS protein expression in the livers of mice in each group. Compared with the CON group, p-GS protein expression in the livers of mice in the M group was significantly upregulated (P < 0.01). Compared with the M group, p-GS protein expression in the livers of mice in the MET group, NP12 group, and GL 10a dose groups was significantly downregulated (P < 0.05, P < 0.01), and the p-GS / GS values of the GL 10a-M and GL 10a-H dose groups were significantly reduced (P < 0.05, P < 0.01). The results indicate that GL 10a can downregulate p-GS protein expression in the livers of mice. This demonstrates that GL 10a can be used to prepare drugs for the treatment of type 2 diabetes. More specifically, the present invention achieves the effect of GL 10a in downregulating p-GS protein expression in the liver.

[0095] Example 11

[0096] In the experiment, mice were anesthetized and killed, and their liver and pancreatic tissues were taken, fixed in 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, and sectioned. After staining with hematoxylin-eosin, the pathological changes of the liver and pancreatic tissues were observed under an optical microscope.

[0097] like Figure 11 As shown in Figure A, the liver tissue in the CON group showed an intact capsule, intact hepatocyte structure, and clear hepatic lobule architecture. Hepatocytes were neatly arranged radially from the central vein, with regular sinusoids and neatly arranged hepatocyte cords. Compared with the CON group, the hepatocyte cords and sinusoids in the M group were irregularly arranged. Most hepatocytes were swollen and degenerated, with loosely stained and vacuolated cytoplasm. The cytoplasm was filamentous or almost transparent, and contained unstained, round, smooth-edged lipid droplets, predominantly small. Compared with the M group, liver damage in all GL10a-treated groups improved to some extent, with the GL10a-M and GL10a-H groups showing the most significant improvement. In the NP12 and MET groups, hepatocytes showed vacuolar degeneration, with vacuoles of varying sizes visible in the cytoplasm. Some hepatocytes were swollen, with filamentous or almost transparent cytoplasm, and nuclei were often suspended in the center of the cell. In the GL10a-L group, hepatocytes showed vacuolar degeneration, with a small number of vacuoles visible in the cytoplasm. In the GL 10a-M and GL 10a-H groups, the hepatocyte structure was intact, the hepatic lobule structure was clear, the hepatocytes were neatly arranged radially around the central vein, the hepatic sinusoids were regular, and the hepatocyte cords were neatly arranged.

[0098] like Figure 11As shown in Figure B, in the CON group, the acini were normal in morphology, the islets were regular in shape, the islet cells had clear borders, the cells were tightly packed, evenly distributed, and the structure was intact, with no significant pathological changes. Compared with the CON group, the islets in the M group showed disorganized arrangement, degeneration, vacuolar infiltration, and cytoplasmic lysis. Acinar cell degeneration was observed, and cytoplasmic eosinophilia was increased. Compared with the M group, pancreatic tissue damage in the MET, NP12, 10a-L, and 10a-M groups all showed some improvement, and pancreatic tissue damage in the 10a-H group was essentially restored to normal levels. Specifically, in the NP12, MET, GL 10a-L, and GL 10a-M groups, the majority of islets showed regular morphology, while a minority showed degeneration, with minor vacuoles and fibrous proliferation. The number of degenerated cells was reduced in the GL 10a-M group. In the GL 10a-H group, the islets showed basically regular morphology, clear borders, tightly packed, evenly distributed, and intact structure.

[0099] Histopathological examination results showed that GL 10a significantly improved liver and pancreatic tissue damage in type 2 diabetic mice, suggesting that GL 10a could be used to prepare drugs for the treatment of type 2 diabetes by improving liver and pancreatic tissue damage.

[0100] It should be noted that all data were statistically analyzed using SPSS 26.0 software. Data are expressed as . Multiple group comparisons were performed using one-way analysis of variance, and pairwise comparisons between groups were performed using the LSD-t test. P < 0.05 was considered statistically significant.

[0101] According to Examples 1 to 11, the experimental results above show that the GSK-3β covalent inhibitor GL10a can increase the body weight of type 2 diabetic mice, lower blood sugar, reduce the GSK-3β content in serum, liver, and pancreas, downregulate the expression of GSK-3β protein in the liver and pancreas, and upregulate the expression of p-GSK-3β protein in the liver and pancreas.

[0102] like Figure 12 As shown, GL 10a inhibits GSK-3β kinase activity, thereby reducing GSK-3β levels in serum, liver, and pancreas, and thus lowering glucose levels in serum and urine, thus playing a therapeutic role in type 2 diabetes. It lowers blood glucose levels in two ways: 1) GL 10a downregulates p-GS protein expression in mouse livers, thereby increasing glycogen synthesis; 2) GL 10a upregulates PDX1 protein expression in the mouse pancreas, thereby increasing insulin levels in serum and pancreas, and improving pathological changes in the mouse liver and pancreas.

[0103] In summary, GL10a significantly increased GSK-3β phosphorylation in the liver and pancreas of mice treated with type 2 diabetes, inhibited GSK-3β protein expression in the liver and pancreas, and reduced GSK-3β levels in serum, liver, and pancreas. This, in turn, lowered blood glucose levels through two pathways: 1) inhibiting the phosphorylation of glycogen synthase in the liver, thereby increasing liver glycogen synthesis; and 2) increasing PDX1 protein expression in the pancreas, promoting insulin secretion. Furthermore, GL10a significantly ameliorated pathological changes in the liver and pancreas of mice treated with type 2 diabetes. These results demonstrate the beneficial therapeutic effects of GL10a in the treatment of type 2 diabetes and suggest its potential application in the preparation of a drug for treating pathological damage to liver or pancreatic tissue caused by type 2 diabetes.

[0104] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Any researcher in this field may, without departing from the spirit and scope of the present invention, adopt the design parameters and contents of the above-disclosed embodiments to change and modify the research scheme of the present invention. Therefore, any simple modifications, parameter changes, and modifications made to the above-disclosed embodiments based on the research essence of the present invention without departing from the content of the present invention are within the scope of protection of the present invention.

Claims

1. Use of a thiadiazolidinedione GSK-3β covalent inhibitor in the preparation of a medicament for treating type II diabetes or related diseases caused by type II diabetes, wherein the thiadiazolidinedione GSK-3β covalent inhibitor is N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide, having the following structural formula:

2. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 1 in the preparation of a drug for treating type II diabetes, characterized in that: The drug for treating type II diabetes is prepared by using N-(3-(4-benzyl-3,5-diketo-1,2,4-thiadiazol-2-yl)phenyl)acrylamide as an active ingredient and supplemented with pharmaceutically acceptable excipients.

3. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 2 in the preparation of a drug for treating type II diabetes, characterized in that: The dosage forms of the drug for treating type II diabetes include tablets, capsules, injections, aerosols, ointments, and granules.

4. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 2 in the preparation of a drug for treating type II diabetes, characterized in that: The pharmaceutically acceptable excipients include one or more of fillers, disintegrants, binders, surfactants, lubricants, and carriers.

5. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 1 in the preparation of a drug for treating type II diabetes, characterized in that: The type II diabetes therapeutic drug is a drug for treating, preventing, alleviating and / or alleviating the symptoms of type II diabetes.

6. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 1 in the preparation of a drug for treating type II diabetes, characterized in that: The drug for treating type II diabetes is a drug for reducing the content of GSK-3β in serum, liver and pancreas.

7. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 1 in the preparation of a drug for treating type II diabetes, characterized in that: The type II diabetes therapeutic drug is a drug that reduces the glucose content in serum and urine.

8. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 1 in the preparation of a drug for treating type 2 diabetes, wherein the drug for treating type 2 diabetes comprises at least one of a drug that downregulates GSK-3β protein expression in the liver or pancreas, a drug that increases GSK-3β phosphorylation in the liver or pancreas, a drug that increases glycogen synthesis, and a drug that promotes insulin secretion.

9. Use of a thiazolidinedione GSK-3β covalent inhibitor according to claim 8 in the preparation of a drug for treating type II diabetes, wherein the drug that increases liver glycogen synthesis is a drug that inhibits the phosphorylation of glycogen synthase in the liver.

10. Use of a thiadiazolidinedione GSK-3β covalent inhibitor according to claim 8 in the preparation of a drug for treating type II diabetes, wherein the drug that promotes insulin secretion is a drug that increases PDX1 protein expression in the pancreas.

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