Application of glabridin in preparation of medicine for treating metabolic disorder

Abstract

The invention provides an application of a glabridin (GLD) compound in preparation of a medicine for treating metabolic disorders, which is characterized in that after glabridin is injected into a Type 2 diabetes mellitus (T2DM) model mouse in an intraperitoneal cavity, the glabridin can stably reduce the fasting blood glucose level of the T2DM mouse, inhibit fat accumulation and improve insulin sensitivity, and the application of the glabridin in preparation of the medicine for treating the metabolic disorders is realized. The invention further provides related research and application of the glabridin in the aspects of glycolipid metabolism, energy metabolism and endoplasmic reticulum stress.

Description

Technical Field:

[0001] The present invention belongs to the field of medical treatment, and specifically provides the application of glabridin in the preparation of drugs for treating metabolic disorders. Background Art:

[0002] Globally, diabetes has become a public health problem that urgently needs to be solved. As a hard-hit area for diabetes in China, the number of patients has exceeded 140 million; compared with the 1990s, the number of deaths caused by diabetes has increased significantly. Among them, type 2 diabetes mellitus (T2DM) is the main type of diabetes, accounting for more than 90% of the patient population. The pathological characteristics of type 2 diabetes are the vicious cycle of insulin resistance and pancreatic islet β-cell dysfunction. The long-term hyperglycemic state will induce multi-system complications, increasing the risk of patients suffering from cardiovascular diseases. At the same time, type 2 diabetes is also the leading cause of end-stage renal disease and blindness caused by retinopathy. Although the current clinical drug system already covers nine major categories of drugs such as biguanides, sulfonylureas, and α-glucosidase inhibitors, there are still many difficulties in actual application.

[0003] Licorice, known as the "national elder" in the field of traditional Chinese medicine, is one of the most commonly used bulk medicinal materials in China. Its medicinal history was first recorded in "Shennong's Herbal Classic". Licorice has the effects of invigorating the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, relieving spasm and pain, and coordinating various medicinal herbs, and is applicable to clinical symptoms such as weakness of the spleen and stomach, physical fatigue, and excessive phlegm. Flavonoids with rich varieties and diverse structures are the important material basis for licorice to exert its medicinal effects.

[0004] Glabridin (GLD) is an isoflavone compound isolated from licorice, accounting for 0.08%-0.35% of the dry weight of licorice root. Previous studies have shown that glabridin has various pharmacological activities such as anti-inflammatory, antioxidant, and anti-tumor. Summary of the Invention:

[0005] The present invention aims to provide the application of glabridin in the preparation of drugs for treating metabolic disorders.

[0006] The present invention aims to provide the application of glabridin in the preparation of drugs for treating glucose metabolic disorders.

[0007] In the above application, the glucose metabolic disorder is a disorder of the liver glycogen synthesis pathway.

[0008] In the above application, the glucose metabolic disorder is a disorder of the liver gluconeogenesis pathway.

[0009] The present invention aims to provide the application of glabridin in the preparation of drugs for treating lipid metabolic disorders.

[0010] In the above application, the lipid metabolism disorder is a disorder of the fat generation pathway.

[0011] In the above application, the lipid metabolism disorder is a disorder of the fat decomposition pathway.

[0012] The present invention aims to provide the use of glabridin in the preparation of a drug for regulating energy metabolism disorders.

[0013] In the above application, the energy metabolism disorder is a disorder of the mitochondrial biogenesis pathway.

[0014] In the above application, the energy metabolism disorder is a disorder of the mitophagy pathway.

[0015] The present invention aims to provide the use of glabridin in the preparation of a drug for regulating endoplasmic reticulum stress.

[0016] In summary, the inventors of the present invention intend to explore the potential and feasibility of glabridin in improving metabolic disorders in type 2 diabetes by providing the above application, and systematically analyze its molecular mechanism of action, so as to lay a foundation for the development of new clinical anti-type 2 diabetes drugs. Specific embodiments:

[0017] The following further elaborates the present invention through some specific experimental examples.

[0018] Experimental Example 1 - Glabridin can effectively improve glucose metabolism disorders

[0019] Experimental method:

[0020] In vivo experiment: SPF-grade 8-week-old male C57BL / 6J mice were selected as experimental animals. After 1 week of adaptive feeding, the mice were randomly divided into a high-sugar and high-fat diet feeding group and a normal maintenance diet feeding group. After 4 weeks, the mice were fasted for 12 h. The mice in the high-sugar and high-fat diet feeding group were intraperitoneally injected with a low-dose streptozotocin solution (the injection dose was 30 mg / kg), and the mice in the normal maintenance diet feeding group were injected with an equal amount of citrate buffer solution for 5 days. After the blood glucose levels of the mice were stable, their 7-h fasting blood glucose levels were measured. If the blood glucose levels were ≥11.1 mM for 3 consecutive times, the type 2 diabetes model was considered successfully established. The successfully modeled mice were grouped, with 6 mice in each group. The grouping is as follows:

[0021] (1) Blank group: The mice in the normal maintenance diet feeding group were intraperitoneally injected with PBS buffer solution containing 0.5% sodium carboxymethylcellulose;

[0022] (2) Blank drug administration group: The mice in the normal maintenance diet feeding group were intraperitoneally injected with glabridin (20 mg / kg);

[0023] (3) Model group: The type 2 diabetes mice were intraperitoneally injected with PBS buffer solution containing 0.5% sodium carboxymethylcellulose;

[0024] (4) Low-dose administration group: The type 2 diabetic mice were intraperitoneally injected with glabridin (10 mg / kg);

[0025] (5) Medium-dose administration group: The type 2 diabetic mice were intraperitoneally injected with glabridin (20 mg / kg);

[0026] (6) High-dose administration group: The type 2 diabetic mice were intraperitoneally injected with glabridin (30 mg / kg);

[0027] (7) Positive drug group: The type 2 diabetic mice were intraperitoneally injected with metformin (MET) (200 mg / kg).

[0028] The drugs were administered once every 3 days for 21 days. During the 5th to 7th administrations, insulin tolerance test (ITT), pyruvate tolerance test (PTT), and oral glucose tolerance test (OGTT) were successively performed on the type 2 diabetic mice. After the last administration was completed, the livers of the mice were taken to prepare paraffin sections and PAS staining was performed to observe the distribution and accumulation of hepatic glycogen. The activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) were detected using an enzyme activity detection kit; the hepatic glycogen content was detected using a glycogen assay kit; the contents of glucokinase (GCK) and glycogen synthase kinase-3β (GSK-3β) were detected by ELISA.

[0029] In vitro experiments: Human liver cancer HepG2 cells were selected as the experimental cell line, and a high-glucose cell model was constructed. Different concentrations of glabridin (final concentrations of 2, 5, 10, 20 μM) and the positive drug metformin (final concentration of 10 mM) were administered to the cells in the model group, with 3 replicates in each group. After 24 h of drug treatment, an insulin solution with a final concentration of 1 μM was added. After incubation for 0.5 h, the supernatant was taken, and a glucose (GLU) test kit was used to measure the glucose content in the culture medium to evaluate the effect of glabridin treatment on glucose uptake in high-glucose model cells; a high-glucose cell model was constructed. Different concentrations of glabridin (final concentrations of 2, 5, 10, 20 μM) and the positive drug metformin (final concentration of 10 mM) were administered to the cells in the model group, with 3 replicates in each group. After 24 h of drug treatment, the cells were changed to serum-free and glucose-free DMEM medium for continued drug treatment for 18 h. Then, glucagon with a final concentration of 100 mM and sodium pyruvate solution with a final concentration of 10 mM were added. After incubation for 4 h, the supernatant was taken, and a glucose (GLU) test kit was used to measure the glucose content in the culture medium to evaluate the effect of glabridin treatment on gluconeogenesis in high-glucose model cells.

[0030] Experimental results:

[0031] As Figure 1 shown, the type 2 diabetic mice treated with glabridin showed significantly improved glucose metabolism ability in the oral glucose tolerance test (OGTT). Figure 2 The results of the insulin tolerance test (ITT) Figure 3 showed that glabridin could significantly improve the insulin sensitivity of type 2 diabetic mice. The results of the pyruvate tolerance test (PTT) Figures 4 - 5 showed that glabridin could inhibit the hepatic gluconeogenesis process in type 2 diabetic mice. Figure 6 showed that glabridin could inhibit the activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) in the liver of type 2 diabetic mice, thereby inhibiting hepatic gluconeogenesis. Figure 7 The results of liver PAS staining Figure 8 showed that after treatment with glabridin, the fibrosis and vacuolization in the liver tissue of type 2 diabetic mice were significantly improved, and the cytoplasmic staining was deepened, indicating that glabridin could increase hepatic glycogen accumulation in type 2 diabetic mice. Figure 9 The results of glycogen quantitative analysis Figures 10 - 11In vitro experimental results showed that glabridin could improve the glucose uptake ability of hyperglycemic model cells and inhibit gluconeogenesis, which was consistent with the in vivo results. The in vivo detection data of the above indicators are listed in Table 1, and the in vitro detection data are listed in Table 2.

[0032] Table 1 In vivo experimental data of Experimental Example 1 (n = 6, Mean ± SD)

[0033]

[0034] Table 2 In vitro experimental data of Experimental Example 1 (n = 3, Mean ± SD)

[0035]

[0036] In summary, it can be confirmed that glabridin can be used in the preparation of drugs for improving glucose metabolism disorders caused by type 2 diabetes: especially through one or more of the following 4 pathways:

[0037] (1) Inhibiting the protein activity of phosphoenolpyruvate carboxykinase (PEPCK);

[0038] (2) And / or inhibiting the protein activity of glucose-6-phosphatase (G6P);

[0039] (3) And / or increasing the protein level of glucokinase (GCK);

[0040] (4) And / or inhibiting the protein level of glycogen synthase kinase-3β (GSK-3β).

[0041] The above results showed that glabridin could improve glucose metabolism disorders caused by type 2 diabetes by inhibiting hepatic gluconeogenesis and promoting hepatic glycogen synthesis.

[0042] Experimental Example 2 - Glabridin can effectively improve lipid metabolism disorders

[0043] Experimental method:

[0044] In vivo experiment: The methods for mouse modeling, grouping, and drug administration were the same as those in Experimental Example 1. After the drug administration was completed, mouse blood samples were collected by enucleation of the eyeballs, serum was separated and collected, and the total cholesterol (TC) level in the serum was detected using a total cholesterol content detection kit, and the triglyceride (TG) level was detected using a triglyceride content detection kit. Mouse livers were taken, paraffin sections were prepared and subjected to HE staining and Oil Red O staining to observe the histopathological changes and lipid accumulation in the liver tissue. The triglyceride level in the mouse liver was detected using the above-mentioned kit. The transcriptional levels of the key genes for lipogenesis, sterol regulatory element binding protein 1c (SREBP1c), and the key gene for lipolysis, peroxisome proliferator-activated receptor α (PPARα), in the mouse liver were detected by real-time fluorescence quantitative RT-qPCR method.

[0045] In vitro experiment: Human hepatocarcinoma HepG2 cells were selected as the experimental cell line. A blank group, a drug administration group (the final concentration of glabridin was 2, 5, 10, 20 μM), and a positive drug group (the final concentration of metformin was 10 mM) were set up, with 3 replicates in each group. After incubation for 24 h, the total cholesterol and triglyceride levels in the cells were detected using the same kit as in the in vivo experiment, and the mRNA levels of sterol regulatory element binding protein 1c (SREBP1c) and peroxisome proliferator-activated receptor α (PPARα) were detected by real-time fluorescence quantitative RT-qPCR method.

[0046] Experimental results:

[0047] Figure 12 The results of liver HE staining showed that glabridin treatment could reduce the vacuolization of liver cells in type 2 diabetic mice and restore the normal nuclear morphology; Figure 13 The results of liver Oil Red O staining showed that after treatment with glabridin, the number of lipid droplets in the livers of type 2 diabetic mice was significantly reduced. The above results indicate that glabridin has the effect of improving hepatic steatosis. Figure 14 It was shown that glabridin could significantly reduce the total cholesterol (TC) and triglyceride (TG) levels in the serum of type 2 diabetic mice; Figure 15 It was shown that glabridin could significantly reduce the triglyceride (TG) level in the liver of type 2 diabetic mice; Figure 16 It was shown that glabridin could significantly reduce the total cholesterol (TC) and triglyceride (TG) levels in HepG2 cells. The above results indicate that glabridin can regulate lipid metabolism disorders. Figure 17It is shown that glabridin can down-regulate the transcriptional level of sterol regulatory element-binding protein 1c (SREBP1c) in the livers of type 2 diabetic mice and HepG2 cells, thereby inhibiting lipogenesis. Figure 18 It is shown that glabridin can up-regulate the transcriptional level of peroxisome proliferator-activated receptor α (PPARα) in the livers of type 2 diabetic mice and HepG2 cells, thereby promoting lipid catabolism. The in vivo detection data of each of the above indicators are listed in Table 3, and the in vitro detection data are listed in Table 4.

[0048] Table 3 In vivo experimental data of Experimental Example 2 (n = 6, Mean±SD)

[0049]

[0050] Table 4 In vitro experimental data of Experimental Example 2 (n = 3 - 6, Mean±SD)

[0051]

[0052] In summary, it can be confirmed that glabridin can be used in the preparation of drugs for improving lipid metabolism disorders caused by type 2 diabetes: in particular, it can be achieved through one or more of the following 2 pathways

[0053] (1) It can down-regulate the transcriptional level of sterol regulatory element-binding protein 1c (SREBP1c);

[0054] (2) And / or up-regulate the transcriptional level of peroxisome proliferator-activated receptor α (PPARɑ).

[0055] The above results show that glabridin can significantly reduce the levels of total cholesterol and triglycerides, effectively inhibit hepatic steatosis, and improve lipid metabolism disorders caused by type 2 diabetes. Glabridin can inhibit the expression of key genes in lipogenesis and promote lipid catabolism, thereby maintaining lipid metabolism balance.

[0056] Experimental Example 3 - Glabridin can effectively improve abnormal energy metabolism

[0057] Experimental method:

[0058] In vivo experiment: The method of mouse model establishment, grouping and administration was the same as that in Experimental Example 1. Mouse livers were taken, and the transcriptional levels of genes related to mitochondrial biogenesis and autophagy in mouse livers were detected by real-time fluorescence quantitative RT-qPCR method.

[0059] In vitro experiment: The treatment of HepG2 cells and the administration method were the same as in Experimental Example 2. The mitochondrial membrane potential (MMP) level was detected using the JC10 mitochondrial membrane potential kit, and the content of mitochondrial reactive oxygen species (ROS) was detected using the reactive oxygen species detection kit. The transcriptional levels of genes related to mitochondrial biogenesis and autophagy were detected by real-time fluorescence quantitative RT-qPCR method.

[0060] Experimental results:

[0061] Figure 19 It was shown that liquiritigenin could significantly increase the mitochondrial membrane potential (MMP) level of HepG2 cells. Figure 20 It was shown that liquiritigenin could significantly inhibit the production of mitochondrial reactive oxygen species (ROS) in HepG2 cells. It was Figures 21 - 23 known that liquiritigenin could up-regulate the transcriptional levels of sirtuin 1 (SIRT1), PPARγ co-activator 1α (PGC-1α), and mitochondrial transcription factor A (TFAM) in the livers of type 2 diabetic mice and HepG2 cells, thereby promoting mitochondrial biogenesis. It was Figure 24 and Figure 25 known that liquiritigenin could up-regulate the transcriptional levels of PTEN-induced putative kinase 1 (PINK1) and RBRE3 ubiquitin protein ligase (Parkin) in the livers of type 2 diabetic mice and HepG2 cells, thereby promoting mitochondrial autophagy. The in vivo detection data of the above indicators are listed in Table 5, and the in vitro detection data are listed in Table 6.

[0062] Table 5 In vivo experimental data of Experimental Example 3 (n = 6, Mean ± SD)

[0063]

[0064] Table 6 In vitro experimental data of Experimental Example 3 (n = 3, Mean ± SD)

[0065]

[0066] In summary, it can be confirmed that liquiritigenin can be used in the preparation of drugs for improving energy metabolism disorders caused by type 2 diabetes: especially through one or more of the following 7 pathways:

[0067] (1) It can increase the level of mitochondrial membrane potential (MMP);

[0068] (2) and / or reduce the level of mitochondrial reactive oxygen species (ROS);

[0069] (3) and / or up-regulate the transcriptional level of sirtuin 1 (SIRT1);

[0070] (4) and / or up-regulate the transcriptional level of PPARy coactivator 1α (PGC-1α);

[0071] (5) and / or up-regulate the transcriptional level of mitochondrial transcription factor A (TFAM);

[0072] (6) and / or up-regulate the transcriptional level of PTEN-induced kinase 1 (PINK1);

[0073] (7) and / or up-regulate the transcriptional level of RBRE3 ubiquitin ligase (Parkin).

[0074] The above results indicate that glabridin can improve mitochondrial function by increasing mitochondrial membrane potential and reducing the production of mitochondrial reactive oxygen species. At the transcriptional level, glabridin can promote mitochondrial biogenesis and autophagy, thereby maintaining cellular mitochondrial homeostasis.

[0075] Experimental Example 4 - Glabridin can effectively improve endoplasmic reticulum stress

[0076] Experimental method:

[0077] In vivo experiment: The method of mouse model establishment, grouping and administration was the same as that in Experimental Example 1. Mouse livers were taken, and the protein level of glucose-regulated protein 78 (GRP78) was detected by ELISA.

[0078] In vitro experiment: Human hepatocellular carcinoma HepG2 cells were selected as the experimental cell line, and an endoplasmic reticulum stress cell model was constructed. Cells in the model group were treated with different concentrations of glabridin (final concentrations of 2, 5, 10, 20 μM) and the positive drug metformin (final concentration of 10 mM), with 3 replicates in each group. After incubation for 24 h, the protein level of glucose-regulated protein 78 was detected by ELISA. The expressions of glucose-regulated protein 78 and protein kinase R-like endoplasmic reticulum kinase (PERK) were detected by WB.

[0079] Experimental results:

[0080] Figure 26 and Figure 27It was shown that glabridin could significantly reduce the protein level of glucose-regulated protein 78 (GRP78) in the liver of type 2 diabetic mice and in the HepG2 endoplasmic reticulum stress model cells. Figure 28 It was shown that glabridin could significantly reduce the phosphorylation level and total protein amount of protein kinase R-like endoplasmic reticulum kinase (PERK). The in vivo detection data of the above indicators are listed in Table 7, and the in vitro detection data are listed in Table 8.

[0081] Table 7 In vivo experimental data of Experimental Example 4 (n = 6, Mean±SD)

[0082]

[0083] Table 8 In vitro experimental data of Experimental Example 4 (n = 3, Mean±SD)

[0084]

[0085] In summary, it can be confirmed that glabridin can be used in the preparation of a drug for improving abnormal endoplasmic reticulum stress caused by type 2 diabetes: in particular, through one or more of the following two pathways:

[0086] (1) It can reduce the protein level of glucose-regulated protein 78 (GRP78);

[0087] (2) And / or inhibit the activity of protein kinase R-like endoplasmic reticulum kinase (PERK).

[0088] In all the drawings involved in the present invention, ▲ and ▲▲ in the experiments indicate P < 0.05 and P < 0.01 compared with the model group; * and ** indicate P < 0.05 and P < 0.01 compared with the blank group. Description of the Drawings:

[0089] Figure 1 Results of oral glucose tolerance test in mice

[0090] Figure 2 Results of insulin tolerance test in mice

[0091] Figure 3 Results of pyruvate tolerance test in mice

[0092] Figure 4 Results of detection of phosphoenolpyruvate carboxykinase (PEPCK) activity in mouse liver

[0093] Figure 5 Results of detection of glucose-6-phosphatase (G6P) activity in mouse liver

[0094] Figure 6 Results of PAS staining of mouse liver

[0095] Figure 7Detection Results of Glycogen Levels in Mouse Liver

[0096] Figure 8 Detection Results of Glucokinase (GCK) Levels in Mouse Liver

[0097] Figure 9 Detection Results of Glycogen Synthase Kinase-3β (GSK-3β) Levels in Mouse Liver

[0098] Figure 10 Detection Results of Glucose Uptake in HepG2 Cells under Insulin Stimulation

[0099] Figure 11 Detection Results of Gluconeogenesis in HepG2 Cells under Glucagon Stimulation

[0100] Figure 12 Results of HE Staining of Mouse Liver

[0101] Figure 13 Results of Oil Red O Staining of Mouse Liver

[0102] Figure 14 Detection Results of Serum Total Cholesterol (TC) and Triglyceride (TG) Levels in Mice Figure 15 Detection Results of Triglyceride (TG) Levels in Mouse Liver

[0103] Figure 16 Detection Results of Total Cholesterol (TC) and Triglyceride (TG) Levels in HepG2 Cells

[0104] Figure 17 Relative mRNA Levels of Sterol Regulatory Element Binding Protein 1c (SREBP1c) in Mouse Liver and HepG2 Cells

[0105] Figure 18 Relative mRNA Levels of Peroxisome Proliferator-Activated Receptor α (PPARα) in Mouse Liver and HepG2 Cells

[0106] Figure 19 Detection Results of Mitochondrial Membrane Potential (MMP) Levels in HepG2 Cells

[0107] Figure 20 Detection Results of Mitochondrial Reactive Oxygen Species (ROS) Content in HepG2 Cells

[0108] Figure 21 Relative mRNA Levels of Sirtuin 1 (SIRT1) in Mouse Liver and HepG2 Cells

[0109] Figure 22 Relative mRNA Levels of PPARγ Coactivator 1α (PGC-1α) in Mouse Liver and HepG2 Cells

[0110] Figure 23 Relative mRNA levels of mitochondrial transcription factor A (TFAM) in mouse liver and HepG2 cells

[0111] Figure 24 Relative mRNA levels of PTEN-induced kinase 1 (PINK1) in mouse liver and HepG2 cells

[0112] Figure 25 Relative mRNA levels of RBRE3 ubiquitin protein ligase (Parkin) in mouse liver and HepG2 cells

[0113] Figure 26 Detection results of glucose-regulated protein 78 (GRP78) levels in mouse liver and HepG2 cells

[0114] Figure 27 WB detection results of glucose-regulated protein 78 (GRP78) in HepG2 cells

[0115] Figure 28 WB detection results of protein kinase R-like endoplasmic reticulum kinase (PERK) in HepG2 cells

[0116] The above experimental protocol is only the preferred scheme of the present invention. It should be noted that in the above embodiments of the present invention, the descriptions of each embodiment have their own focuses. For the parts not detailed in a certain embodiment, reference can be made to the relevant descriptions of other embodiments. In addition, for those skilled in the art, without departing from the principle of the present invention, several modifications and improvements can be made, and these modifications and improvements should also be regarded as the protection scope of the present invention.

[0117] Abbreviations

[0118]

[0119]

Claims

1. Use of glabridin in the preparation of a drug for treating metabolic disorders.

2. The application according to claim 1, characterized in that The metabolic disorder is a glucose metabolism disorder.

3. The application according to claim 2, wherein The glucose metabolism disorder is a disorder of the hepatic glycogen synthesis pathway.

4. The application according to claim 2, wherein The glucose metabolism disorder is a disorder of the hepatic gluconeogenesis pathway.

5. The application according to claim 1, characterized in that The metabolic disorder is a lipid metabolism disorder.

6. The application according to claim 5, wherein The lipid metabolism disorder is a disorder of the lipogenesis pathway.

7. The application according to claim 5, characterized in that The lipid metabolism disorder is a disorder of the lipolysis pathway.

8. The application according to claim 1, wherein The metabolic disorder is an energy metabolism disorder.

9. The application according to claim 8, wherein The energy metabolism disorder is a disorder of the mitochondrial biogenesis pathway.

10. The application according to claim 8, wherein The energy metabolism disorder is a disorder of the mitophagy pathway.

11. The application according to claim 1, wherein The metabolic disorder is endoplasmic reticulum stress.

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