Application of tripeptide in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention discloses an application of a tripeptide in preparation of a medicine for treating non-alcoholic fatty liver disease. The tripeptide is phenylalanine-proline-isoleucine (FPI). The treatment effect of the FPI on the non-alcoholic fatty liver is determined by adopting a classical non-alcoholic fatty liver mouse model. Technicians in the field know important characteristics of glucose and lipid metabolism disorder, insulin resistance, liver lipid accumulation and liver injury in non-alcoholic fatty liver. Experimental data show that the FPI can significantly reduce the weight of a mouse with the non-alcoholic fatty liver disease, significantly improve glucose tolerance and insulin tolerance of the mouse, improve liver injury, significantly reduce liver lipid accumulation of the mouse, and significantly reduce the levels of TG, ALT and AST in serum of the mouse. Therefore, the FPI has the prospect of being developed into the medicine for treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a new use of a known compound, specifically to the application of a tripeptide in the preparation of a drug for treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic disease in which fat accumulates in the liver due to metabolic abnormalities and is not caused by excessive alcohol intake. It is usually asymptomatic in the early stage but may progress to hepatitis, cirrhosis, or even liver cancer. In addition, the probability of cardiovascular diseases (such as myocardial infarction and stroke) in NAFLD patients is significantly increased.

[0003] Currently, there is no specific drug for NAFLD, and lifestyle adjustment is the core intervention measure. Drug treatment needs to be individualized according to the type of metabolic abnormalities and the degree of liver injury in patients. However, some drugs may cause nausea, vomiting, abdominal distension, and increase the burden on the liver, and may directly damage liver cells. Long-term use of some liver-protecting drugs (such as glycyrrhizin preparations) may induce jaundice, ascites, and even acute liver failure, and lipid-lowering drugs (such as atorvastatin) may cause myositis or rhabdomyolysis.

[0004] In order to provide a drug for treating NAFLD, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of a tripeptide in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] The application of phenylalanine-proline-isoleucine in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0008] Preferably, the drug uses phenylalanine-proline-isoleucine as the active ingredient and is made into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

[0009] More preferably, the carrier or excipient is solid, liquid, or semi-solid.

[0010] More preferably, the dosage form is tablets, capsules, or injections.

[0011] Advantageous Effects:

[0012] The present invention uses a classical non-alcoholic fatty liver mouse model to determine the therapeutic effect of FPI on non-alcoholic fatty liver. Those skilled in the art know that disorders of glucose and lipid metabolism, insulin resistance, hepatic lipid accumulation, and liver injury are important characteristics of non-alcoholic fatty liver. The experimental data of the present invention show that FPI can significantly reduce the body weight of mice with non-alcoholic fatty liver, significantly improve glucose tolerance and insulin tolerance in mice, improve liver injury, significantly reduce hepatic lipid accumulation in mice, and significantly reduce the levels of TG, ALT, and AST in the serum of mice. Therefore, FPI has the prospect of being developed into a drug for the treatment of non-alcoholic fatty liver. Brief Description of the Drawings

[0013] Figure 1 Shows the changes in body weight of mice in each group;

[0014] Figure 2 Shows the OGTT curves (A) of mice in each group and the statistical results of the area under the curve (B); the ITT curves (C) of mice and the statistical results of the area under the curve (D);

[0015] Figure 3 Shows the liver tissue staining (A) of mice in each group and the statistical results of the Oil Red O staining area (B);

[0016] Figure 4 Shows the changes in TG (A), ALT (B), and AST (C) in the serum of mice in each group. Detailed Embodiments

[0017] The following specifically introduces the substantial content of the present invention in combination with embodiments, but does not limit the protection scope of the present invention thereby.

[0018] I. Experimental Materials

[0019] 1. Experimental Animals

[0020] Male C57BL / 6J mice, 7-8 weeks old, with a body weight of about 18-22 g, source: Jiangsu Jicui Yakang Biotechnology Co., Ltd. (SCXK (Su) 2023-0009).

[0021] 2. Animal Feed

[0022] Regular feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.; the high-fat feed was ResearchDiets, D12492 from the United States, with a fat content of 60%.

[0023] 3. Experimental Reagents

[0024] D-Glucose (Solarbio Life Sciences, G8150), insulin powder (Biosharp, BS901), sodium chloride injection (Double Crane Pharmaceutical), 4% PFA fixative (Biosharp, BL539A), blood glucose meter and its matching test strips (Sannuo Biochemical Co., Ltd.), triglyceride (TG) assay kit (Nanjing Jiancheng Bioengineering Institute, A110-1-1), alanine aminotransferase (ALT / GPT) test kit (Reitman-Frankel method) microplate method (Nanjing Jiancheng Bioengineering Institute, C009-2-1), aspartate aminotransferase (AST / GOT) test kit (Reitman-Frankel method) microplate method (Nanjing Jiancheng Bioengineering Institute, C009-2-1)

[0025] L-Phenylalanyl-L-prolyl-L-isoleucine (FPI, Anhui Zhuanpeptide Biotechnology Co., Ltd., purity 98%), the structure is as follows:

[0026]

[0027] II. Experimental methods

[0028] 1. Preparation of solutions

[0029] Glucose solution: Weigh glucose powder and dissolve it in pure water to a glucose gavage solution with a final concentration of 0.02 g / mL for the OGTT test.

[0030] Insulin solution: Accurately weigh 1.00 mg of insulin powder, add 1.00 mL of normal saline, vortex for 1 min, place it in an ice-water bath and sonicate for 15 min. Pipette 146 μL of the solution and add normal saline to make up to 50 mL to obtain insulin injection for the ITT test.

[0031] FPI solution: Weigh the FPI lyophilized powder and dissolve it in normal saline to a solution with a final concentration of 5 mg / mL and store it at 4°C.

[0032] 2. Establishment of non-alcoholic fatty liver mouse model and animal grouping

[0033] After 1 week of adaptive feeding, the mice were randomly grouped. The control group (Chow, n = 6) was fed with normal feed, and the model group (HFD, n = 6) and the drug administration group (FPI, n = 6) were fed with high-fat feed for 8 weeks. The mice in the FPI group were gavaged with FPI (50 mg / kg) every day, and the Chow group and the HFD group were gavaged with an equal volume of normal saline every day. During the daily drug administration, their drinking water and feeding behaviors were not restricted, and the body weights of the mice were recorded weekly.

[0034] 3. Oral Glucose Tolerance and Insulin Tolerance Tests

[0035] Oral Glucose Tolerance Test (OGTT) in mice: The OGTT was performed 6 weeks after administration. After fasting the mice in each group for 12 h, their blood glucose levels at 0 min were measured. Subsequently, a glucose solution was administered by gavage at a dose of 2 g / kg, and the blood glucose was measured at 15 min, 30 min, 60 min, 90 min, and 120 min respectively. A line graph was plotted with time on the X-axis and blood glucose on the Y-axis, and the Area Under the Curve (AUC) was calculated.

[0036] Intraperitoneal Insulin Tolerance Test (ITT) in mice: The ITT was performed 7 weeks after administration. After fasting the mice in each group for 4 h, the blood glucose level at 0 min was measured. Subsequently, each mouse was intraperitoneally injected with an insulin solution (0.75 U / kg), and the blood glucose of the mice was measured at 15 min, 30 min, 60 min, 90 min, and 120 min respectively. A line graph was plotted with time on the X-axis and blood glucose as the Y value, and the AUC was calculated.

[0037] 4. Collection and Processing of Samples at the End of Animal Experiments

[0038] After 8 weeks of administration, the mice in each group were fasted for 12 h. The blood of the mice was collected by orbital enucleation. After standing at room temperature for 4 h, it was centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was taken and stored in aliquots (-80 °C); the liver and other tissues of the mice were removed in turn, washed with normal saline, and a part was cut and fixed with 4% PFA fixative, and the rest was quickly frozen in liquid nitrogen and then transferred to a -80 °C refrigerator for frozen storage.

[0039] 5. Observation of Pathological Morphology of Liver Tissue

[0040] Take the liver samples stored in 4% PFA fixative, embed them in paraffin, and stain the embedded sections with hematoxylin-eosin (H&E). Oil Red O staining was performed on some frozen liver tissues to observe liver lipid accumulation.

[0041] 6. Detection of Biochemical Indexes in Mouse Serum

[0042] According to the instructions of the kit, the contents of triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the serum were measured.

[0043] 7. Data Analysis Methods

[0044] Statistical analysis was performed using GraphPad Prism 9.5.0 software, and the experimental data were expressed as Mean±SEM. One-way ANOVA was used to compare the differences between multiple groups of data. A P value < 0.05 was considered statistically significant (P<0.05 was marked as "*"; P<0.01 was marked as "**"; P<0.001 was marked as "***").

[0045] III. Experimental Results

[0046] 1. Improvement effect of FPI on the body weight of non-alcoholic fatty liver mice

[0047] After 8 weeks of administration, as Figure 1 can be seen, compared with the mice in the Chow group, the body weight of the mice in the HFD group was significantly increased, indicating that the HFD diet significantly increased the body weight of the mice. Compared with the HFD group, FPI significantly reduced the body weight of the mice.

[0048] 2. Improvement effect of FPI on oral glucose tolerance and insulin tolerance in non-alcoholic fatty liver mice

[0049] The OGTT and ITT of each group of mice were detected, and the results are shown in Figure 2 . Compared with the mice in the Chow group, the areas under the curves of oral glucose tolerance and insulin tolerance of the mice in the HFD group were significantly increased. After administration of FPI, the glucose tolerance and insulin tolerance of HFD mice were significantly improved.

[0050] 3. Histopathological protective effect of FPI on non-alcoholic fatty liver mice

[0051] H&E staining was performed on liver tissues ( Figure 3 A in). Compared with normal mice, a large number of vacuoles appeared in the livers of HFD mice, and obvious liver tissue damage was observed. In the mice intervened with FPI, the proportion of liver vacuoles was significantly reduced, and liver damage was improved.

[0052] Oil red O staining was performed on liver tissues ( Figure 3 A in). Compared with normal mice, a large amount of fat accumulation appeared in the hepatocytes of HFD mice. In the mice intervened with FPI, the lipid accumulation in the liver was significantly reduced ( Figure 3 B in).

[0053] 4. Histopathological protective effect of FPI on non-alcoholic fatty liver mice

[0054] According to the kit instructions, the blood lipid levels in the serum samples of mice were detected. Compared with the mice in the Chow group, the levels of TG, ALT, and AST in HFD mice were significantly increased. After administration of FPI, the levels of TG, ALT, and AST in the serum were significantly reduced.

[0055] The above-mentioned embodiments used a classical non-alcoholic fatty liver mouse model to determine the therapeutic effect of FPI on non-alcoholic fatty liver. Those skilled in the art know that disorders of glucose and lipid metabolism, insulin resistance, hepatic lipid accumulation, and liver injury are important characteristics of non-alcoholic fatty liver. The above experimental data show that FPI can significantly reduce the body weight of non-alcoholic fatty liver mice, significantly improve the glucose tolerance and insulin tolerance of mice, improve liver injury, significantly reduce hepatic lipid accumulation in mice, and significantly reduce the levels of TG, ALT, and AST in the serum of mice. Therefore, FPI has the prospect of being developed into a drug for the treatment of non-alcoholic fatty liver.

[0056] The role of the above-mentioned embodiments is to specifically introduce the substantial content of the present invention. However, those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. Use of phenylalanine-proline-isoleucine in the preparation of a medicament for treating non-alcoholic fatty liver disease.

2. The application according to claim 1, characterized in that: The medicament uses phenylalanine-proline-isoleucine as the active ingredient and is made into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

3. The application according to claim 2, wherein: The carrier or excipient is solid, liquid or semi-solid.

4. The application according to claim 2, wherein: The dosage form is tablet, capsule or injection.

Images