Application of combination of urolithin A and beta-nicotinamide mononucleotide in preparation of medicine for treating diabetes

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of urolithin A and beta-nicotinamide mononucleotide in preparation of a medicine for treating diabetes. The invention finds that when the urolithin A and the beta-nicotinamide mononucleotide are independently used, the hypoglycemic effect on diabetic mice cannot be realized, and when the urolithin A and the beta-nicotinamide mononucleotide are jointly used, the symptoms of diabetes and insulin resistance caused by high fat diet can be remarkably improved; the glucose tolerance and the insulin tolerance are improved; meanwhile, the content of low-density lipoprotein and cholesterol in a mouse body is reduced, so that the risk of thrombus occurrence is reduced; in addition, combined medication can significantly reduce liver cell lipid accumulation and improve mitochondrial morphological abnormality. The invention provides a brand new technical scheme and experimental basis for treating diabetes caused by high fat diet and relieving insulin resistance, and has important medical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the application of urolithin A combined with nicotinamide mononucleotide in the preparation of a drug for treating diabetes. BACKGROUND

[0002] Insulin resistance induced by a high-fat diet is the core inducer of type 2 diabetes (T2D), and the two form a vicious cycle of "metabolic disorder-pathological progression". T2D has become a complex and multifactorial heterogeneous disease. Although there are many drugs on the market for treating T2D, most of them may produce some side effects and drug resistance, for example, insulin can rapidly reduce blood sugar, but is easy to cause side effects such as hypoglycemia and weight gain; long-term use of metformin may appear to weaken the effect; and sulfonylurea drugs can produce side effects such as hypoglycemia, allergic reactions, and liver damage. T2D patients have complications such as neuropathy and kidney damage due to poor blood sugar control and other factors, which seriously reduces the quality of life. For patients, new drugs that are efficient, safe and convenient can improve treatment compliance, reduce the risk of hypoglycemia, delay the occurrence of serious complications such as foot ulcers and blindness, and improve the quality of life, so there is an urgent need to develop new effective, safe and convenient drugs for treating diabetes and insulin resistance. SUMMARY

[0003] In order to solve the above problems, the present application provides the application of urolithin A (UA) combined with nicotinamide mononucleotide (NMN) in the preparation of a drug for treating diabetes. The urolithin A combined with nicotinamide mononucleotide of the present application can effectively improve diabetes (especially diabetes induced by a high-fat diet) and insulin resistance, and can be used as a combined drug for preparing a drug for treating diabetes.

[0004] In order to achieve the above purpose, the present application provides the following technical solutions: The present application provides the application of urolithin A combined with nicotinamide mononucleotide in the preparation of a drug for treating diabetes.

[0005] Preferably, the mass ratio of urolithin A combined with nicotinamide mononucleotide in the drug is 2.5:300.

[0006] Preferably, the treatment of diabetes comprises one or more of the following: reducing body weight, reducing blood glucose, improving glucose tolerance test (GTT), improving insulin tolerance test (ITT), improving insulin resistance, reducing cholesterol content, reducing low-density lipoprotein (LDL) content, reducing liver vacuolization, increasing hepatocyte insulin sensitivity, reducing liver lipid accumulation, increasing mitochondrial fusion, and alleviating liver injury.

[0007] Preferably, the reduction of cholesterol content is a reduction of cholesterol content in serum.

[0008] Preferably, the reduction of low-density lipoprotein content is a reduction of low-density lipoprotein content in serum.

[0009] Preferably, the diabetes comprises diabetes and its complications caused by high-fat diet.

[0010] Preferably, the complications comprise non-alcoholic fatty liver.

[0011] The present application provides a medicine for treating diabetes, and the effective components comprise urolithin A and beta-nicotinamide mononucleotide.

[0012] Preferably, the mass ratio of urolithin A to beta-nicotinamide mononucleotide in the medicine is 2.5:300.

[0013] Preferably, the medicine further comprises a pharmaceutically acceptable excipient.

[0014] Beneficial effects: The present application provides the use of urolithin A combined with beta-nicotinamide mononucleotide in the preparation of a medicine for treating diabetes. The present application finds that urolithin A and beta-nicotinamide mononucleotide cannot achieve the effect of reducing blood glucose in diabetic mice when used alone, but the combination of the two can significantly improve the symptoms of diabetes and insulin resistance caused by high-fat diet: effectively reduce the fasting blood glucose level of diabetic mice, improve glucose tolerance and insulin tolerance; at the same time, reduce the low-density lipoprotein and cholesterol content in the body of mice, thereby reducing the risk of thrombosis; in addition, the combined use of drugs can significantly reduce liver cell lipid accumulation and improve mitochondrial morphological abnormalities. The present application provides a new technical solution and experimental basis for treating diabetes caused by high-fat diet and alleviating insulin resistance, and has important medical application value. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0016] Figure 1 Physiological and biochemical index detection results of T2D mice after NMN and UA were administered alone or in combination for 8 weeks; wherein, A is the body weight change; B is the liver weight coefficient; C is the GTT detection result; D is the ITT detection result; E is the fasting blood glucose detection result; F is the T-CHO detection result; G is the LDL detection result; H is the HDL detection result; p <0.05, p <0.01, p <0.001; Figure 2 Effects of NMN and UA administered alone or in combination on the morphology of liver and muscle of T2D mice after 8 weeks of administration; wherein, A is liver HE staining, and the white circle is vacuole; B is muscle HE staining; Figure 3 Effects of NMN and UA administered alone or in combination on the lipid accumulation and mitochondrial morphology of hepatocytes; wherein, A is the oil red O staining result of mouse liver sections, and red is lipid droplet; B is the oil red O staining detection result of Huh7 cells, and red is lipid droplet; C is the Mito tracker Red staining result of mitochondria, and red is mitochondria, showing punctate or linear; Figure 4 Results of Western Blot detection of key proteins; wherein, A is the pAMPK, Parkin1 and 4EBP2 protein expression results of Huh7 cells under the intervention of different drug groups; B is the pAMPK, Parkin1 and 4EBP2 protein expression results of liver cells of T2D mice under the intervention of different drug groups, C is the blank control group, H is the HFD group, H+N is the HFD+NMN group, H+U is the HFD+UA group, and H+N+U is the HFD+NMN+UA group. DETAILED DESCRIPTION

[0017] The application provides application of urolithin A combined with beta-nicotinamide mononucleotide in preparation of a drug for treating diabetes. As an implementation manner, the mass ratio of urolithin A combined with beta-nicotinamide mononucleotide in the drug is 2.5:300.

[0018] As an embodiment, the treating diabetes comprises one or more of reducing body weight, reducing blood glucose, improving glucose tolerance, improving insulin tolerance, improving insulin resistance, reducing cholesterol content, reducing low-density lipoprotein content, reducing liver vacuolization, increasing hepatocyte insulin sensitivity, reducing liver lipid accumulation, increasing mitochondrial fusion, and alleviating liver injury. As an embodiment, the reducing cholesterol content is reducing cholesterol content in serum. As an embodiment, the reducing low-density lipoprotein content is reducing low-density lipoprotein content in serum.

[0019] As an embodiment, the diabetes comprises diabetes caused by high-fat diet and its complications. As an embodiment, the complications comprise non-alcoholic fatty liver.

[0020] The present application finds that Urolithin A cannot achieve the effect of reducing blood glucose in diabetic mice when used alone, while the combination of Urolithin A and β-nicotinamide mononucleotide can significantly improve the symptoms of diabetes and insulin resistance caused by high-fat diet: effectively reducing the fasting blood glucose level of diabetic mice, improving glucose tolerance and insulin tolerance; at the same time, reducing the low-density lipoprotein and cholesterol content in the body of mice, thereby reducing the risk of thrombosis; in addition, the combination of drugs can significantly reduce liver cell lipid accumulation and improve mitochondrial morphological abnormalities. The present application provides a new technical solution and experimental basis for treating diabetes caused by high-fat diet and alleviating insulin resistance, and has important medical application value, and provides a candidate direction with transformation value for subsequent drug development.

[0021] Based on the above advantages, the present application provides a drug for treating diabetes, and the effective components include Urolithin A and β-nicotinamide mononucleotide. As an embodiment, the mass ratio of Urolithin A to β-nicotinamide mononucleotide in the drug is 2.5:300. As an embodiment, the drug further comprises a pharmaceutically acceptable excipient.

[0022] In order to further illustrate the present application, the application of Urolithin A combined with β-nicotinamide mononucleotide in preparing a drug for treating diabetes is described in detail below in combination with examples and drawings, but they should not be understood as limiting the scope of protection of the present application.

[0023] Example 1 1. Construction of animal model All mice were placed in SPF level animal room, temperature 22-25 ℃, humidity 40%-60%, 12 h light-dark cycle, free feeding and drinking, bedding changed 2 times per week, and the environment was disinfected regularly.

[0024] A blank control group (denoted as CTRL) and a high-fat diet-induced C57BL / 6 mouse T2D model (denoted as HFD) were set up. Six-week-old SPF C57BL / 6 male mice with a body weight of 18-22 g were adaptively fed for one week, and then fed with high-fat feed (fat accounted for 60%, purchased from Research Diets, item number D12492i) for 8 weeks to construct a T2D and insulin resistance model. The success criteria for modeling were a fasting blood glucose of greater than or equal to 7.0 mmol / L and an insulin resistance index of greater than or equal to 2.5.

[0025] 2. Drug grouping design Urolithin A (UA) and beta-nicotinamide mononucleotide (NMN) were both dissolved in 10% sulfobutyl-beta-cyclodextrin and injected intraperitoneally for 5 days per week. The model mice were randomly divided into 5 groups, 10 in each group, and the groups were as follows: The mice in the HFD group and the blank control group were both injected intraperitoneally with the same volume of 10% sulfobutyl-beta-cyclodextrin; HFD+NMN group: NMN injection dose was 300 mg / kg / d; HFD+UA group: UA injection dose was 2.5 mg / kg / d; HFD+NMN+UA group: NMN 300 mg / kg / d and UA 2.5 mg / kg / d.

[0026] 3. Physiological and biochemical index detection The mice were given drugs at a fixed time every day, and the intervention was continued for 8 weeks. The weight change of the mice was recorded every week.

[0027] Glucose tolerance test (GTT): After fasting for 14 h, each mouse was weighed and recorded, the blood glucose concentration of the mouse at fasting was detected and recorded, a 20% glucose solution was prepared with sterilized double distilled water (freshly prepared), and then injected intraperitoneally at a dose of 1 g / kg. Finally, the blood glucose of the mice was measured and recorded at 15, 30, 60, 90 and 120 min after injection, respectively.

[0028] Insulin tolerance test (ITT): After fasting for 6 h, each mouse was weighed and recorded, the blood glucose concentration of the mouse at fasting was detected and recorded. An insulin solution of 0.1 U / mL was prepared with normal saline (freshly prepared), and then injected intraperitoneally at a dose of 1 U / kg. Finally, the blood glucose of the mice was measured and recorded at 15, 30, 60, 90 and 120 min after insulin injection, respectively.

[0029] Lipid metabolism index detection: The serum of the mice was collected, and the total cholesterol (T-CHO), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were determined (Nanjing Jiancheng Biological Engineering Institute).

[0030] HE staining: Mice liver and muscle tissues were obtained, fixed, sectioned and stained to observe cell morphology.

[0031] Oil red O staining: Mice liver tissues were obtained, fixed, sectioned and stained to observe lipid droplet content.

[0032] 4. Results and analysis After 8 weeks of treatment, the body weight of mice was recorded every week, and all drug groups could reduce the body weight of HFD mice, and the combination had the best effect, which indicated that both drugs had a certain effect on weight control. Figure 1 In the middle A, after obtaining liver and other tissues, the weight was weighed and the organ coefficient was calculated, and it was found that all treatment regimens had no effect on the liver coefficient. Figure 1 In the middle B.

[0033] After 8 weeks of treatment, the GTT of mice was detected, and the results showed that the combination therapy significantly improved the GTT of mice, while the single drug had no effect on the GTT. Figure 1 In the middle C.

[0034] After 8 weeks of treatment, the ITT of mice was detected, and the results showed that the combination therapy most improved the ITT of mice, while NMN had no effect on the ITT. Figure 1 In the middle D.

[0035] After 8 weeks of treatment, the serum of mice was collected to detect the content of glucose, T-CHO, HDL and LDL, and the results showed that the combination significantly reduced T-CHO and glucose, but the single drug had no effect on T-CHO and blood glucose; The combination significantly reduced LDL, and NMN alone had no effect on LDL; all regimens had no effect on HDL. Figure 1 In the middle E-H), it can be seen that the combination of NMN and UA has a synergistic therapeutic effect on T2D and insulin resistance.

[0036] After 8 weeks of treatment, HE staining was performed on liver and muscle tissues, and the results showed that all drug regimens significantly reduced liver vacuolization, and the combination had the best effect. Figure 2 In the middle A); all drug regimens had no effect on muscle cell morphology. Figure 2 In the middle B), therefore, NMN and UA have an improvement effect on non-alcoholic fatty liver caused by high-fat diet, and have no effect on mouse muscle.

[0037] After 8 weeks of continuous treatment, the liver of mice was fixed and sectioned, and oil red O staining was performed to observe the content of lipid droplets, and the results are shown in Figure 3 In the middle A, NMN and UA can reduce liver lipid accumulation, and the combination has the best effect compared with single drug.

[0038] Example 2 Palmitic acid (PA) 300 μM was selected to induce lipid accumulation in hepatoma Huh7 cells for 24 h (denoted as PA group); at the same time, hepatoma Huh7 cells without PA induction were set as a blank control group (denoted as CTRL), and the PA group cells were divided into 4 groups, and NMN and / or UA were added for treatment for 24 h, and UA was dissolved in DMSO, and NMN was dissolved in sterile water, and the cells in each group were treated as follows: The PA group and the blank control group were both added with the same volume of DMSO; PA+NMN group: NMN treatment concentration was 0.5 mM; PA+UA group: UA treatment concentration was 10 μM; PA+NMN+UA group: NMN 0.5 mM and UA 10 μM.

[0039] After treating the cells for 24 h, oil red O staining and Mito-tracker staining were performed, and the results were consistent with those of Example 1, and the combined treatment of Huh7 cells significantly reduced lipid accumulation ( Figure 3 middle B). It was found by Mito-tracker staining that the combined use of drugs significantly increased the length of the mitochondria of Huh7 cells ( Figure 3 middle C), which may increase the fatty acid oxidation function of mitochondria. In summary, the combined use of drugs has a high efficiency in reducing liver lipid accumulation and reducing liver damage.

[0040] Western Blot was used to detect the protein expression levels of pAMPK, Parkin1 and 4EBP2 in Huh7 cells and liver cells of T2D mice in different drug groups, and GAPDH was used as an internal reference protein, and the results are shown in Figure 4 .

[0041] It was found by Western Blot that NMN can activate AMPK, but UA cannot activate AMPK; UA can increase the expression of autophagy-related proteins; the combined use of the two can activate AMPK, increase cell autophagy, and reduce the expression of 4EBP2 ( Figure 4 middle A and B). The reduction of 4EBP2 can increase the insulin sensitivity of liver cells, and therefore, the combined use of NMN and UA can increase mitochondrial fusion, reduce liver lipid accumulation, activate AMPK and reduce the expression of 4EBP2, and relieve liver damage.

[0042] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the scope of protection of the present application.

Claims

1. Application of urolithiasis A combined with β-nicotinamide mononucleotide in the preparation of drugs for treating diabetes.

2. The application according to claim 1, characterized in that, The mass ratio of urolithiasis A to β-nicotinamide mononucleotide in the drug is 2.5:

300.

3. The application according to claim 1, characterized in that, The treatment of diabetes includes one or more of the following: weight reduction, blood glucose reduction, improved glucose tolerance, improved insulin tolerance, improved insulin resistance, reduced cholesterol levels, reduced LDL cholesterol levels, reduced hepatic vacuolation, increased hepatocyte insulin sensitivity, reduced hepatic lipid accumulation, increased mitochondrial fusion, and relief of liver damage.

4. The application according to claim 3, characterized in that, The reduction of cholesterol content refers to the reduction of cholesterol content in serum.

5. The application according to claim 3, characterized in that, The reduction of low-density lipoprotein (LDL) content refers to reducing the LDL content in serum.

6. The application according to any one of claims 1-5, characterized in that, The diabetes mentioned includes diabetes caused by a high-fat diet and its complications.

7. The application according to claim 6, characterized in that, The complications include non-alcoholic fatty liver disease.

8. A drug for treating diabetes, characterized in that, The active ingredients include urolithin A and β-nicotinamide mononucleotide.

9. The medicament according to claim 8, characterized in that, The mass ratio of urolithiasis A to β-nicotinamide mononucleotide in the drug is 2.5:

300.

10. The medicament according to claim 8 or 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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