A polypeptide and its application in preparing a drug for preventing and / or treating alcoholic liver damage

Abstract

The present invention provides a polypeptide and its use in the preparation of a drug for preventing and / or treating alcoholic liver injury. The polypeptide is not only safe and has no side effects, but also can significantly reduce the content of malondialdehyde (MDA), increase the activity of superoxide dismutase (SOD), increase the content of glutathione (GSH), reduce the activity of alanine aminotransferase (ALT), reduce the activity of aspartate aminotransferase (AST), and reduce the activity of lactate dehydrogenase (LDH), and is very suitable for the preparation of a drug for preventing and / or treating alcoholic liver injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a polypeptide and its application in preparing a drug for preventing and / or treating alcoholic liver injury. Background Art

[0002] Alcoholic liver damage is a liver disease caused by long-term heavy drinking. It usually manifests as fatty liver in the early stage, and can then develop into alcoholic hepatitis, liver fibrosis and cirrhosis. The main clinical features are nausea, vomiting, jaundice, liver enlargement and tenderness, and may be complicated by liver failure and upper gastrointestinal bleeding. Severe alcoholism may induce extensive liver cell necrosis and even liver failure.

[0003] At present, the main clinical methods for treating alcoholic liver injury include quitting drinking, surgical treatment, and drug treatment. Quitting drinking is the key to treating alcoholic liver injury, but many patients usually have poor compliance due to psychological dependence and physiological reactions; surgical treatment can remove the diseased tissue, but the operation itself has risks and requires a period of recovery after surgery; drug treatment can relieve symptoms, but the main drugs currently used are polyene phosphatidylcholine, Fuzheng Huayu capsules, silybin capsules, etc., and long-term use of such drugs is prone to drug resistance and side effects. Therefore, it is urgent to find a safe and side-effect-free drug for the treatment of alcoholic liver injury.

[0004] Peptides are compounds formed by connecting multiple α-amino acids with peptide chains. They have multiple biological activities and are safe and have no side effects. However, there are still few reports of peptides with anti-alcoholic liver damage activity, which is not conducive to the development of clinical drugs. Summary of the invention

[0005] The present invention aims to overcome the deficiencies of the prior art and to provide a polypeptide as a new raw material that is safe and has no side effects and is used to prepare a drug for preventing and / or treating alcoholic liver injury.

[0006] The first object of the present invention is to provide a polypeptide.

[0007] The second objective of the present invention is to provide the use of the above polypeptide or its related biological materials in the preparation of drugs for preventing and / or treating alcoholic liver injury.

[0008] The third object of the present invention is to provide a medicine.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention finds that a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 can significantly reduce the content of malondialdehyde (MDA), increase the activity of superoxide dismutase (SOD), increase the content of glutathione (GSH), reduce the activity of alanine aminotransferase (ALT), reduce the activity of aspartate aminotransferase (AST), and reduce the activity of lactate dehydrogenase (LDH), thereby effectively preventing and / or treating alcoholic liver injury. Therefore, the present invention provides a polypeptide with an amino acid sequence as shown in SEQ ID NO:1, and the use of the above polypeptide or its related biological materials in the preparation of a drug for preventing and / or treating alcoholic liver injury, wherein the related biological material is a nucleic acid molecule capable of expressing the polypeptide, or an expression cassette, recombinant plasmid, recombinant bacteria or recombinant cell containing the nucleic acid molecule.

[0011] Preferably, the prevention and / or treatment of alcoholic liver damage is to reduce the content of malondialdehyde.

[0012] Preferably, the prevention and / or treatment of alcoholic liver damage is to enhance the activity of superoxide dismutase.

[0013] Preferably, the prevention and / or treatment of alcoholic liver damage is to increase glutathione content.

[0014] Preferably, the prevention and / or treatment of alcoholic liver injury is to reduce the activity of alanine aminotransferase.

[0015] Preferably, the prevention and / or treatment of alcoholic liver damage is to reduce the activity of aspartate aminotransferase.

[0016] Preferably, the prevention and / or treatment of alcoholic liver injury is to reduce the activity of lactate dehydrogenase.

[0017] Based on this, the present invention also provides a drug containing the above-mentioned polypeptide or its related biological material; wherein the related biological material is a nucleic acid molecule capable of expressing the polypeptide, or an expression cassette, recombinant plasmid, recombinant bacteria or recombinant cell containing the nucleic acid molecule.

[0018] Preferably, the medicine further contains excipients.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, which is not only safe and has no side effects, but also can significantly reduce the content of malondialdehyde (MDA), increase the activity of superoxide dismutase (SOD), increase the content of glutathione (GSH), reduce the activity of alanine aminotransferase (ALT), reduce the activity of aspartate aminotransferase (AST), and reduce the activity of lactate dehydrogenase (LDH), and is very suitable for preparing drugs for preventing and / or treating alcoholic liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The purity analysis results of the peptide.

[0022] Figure 2 The mass spectrometry identification results of the peptides.

[0023] Figure 3 These are the safety test results of the peptide.

[0024] Figure 4 The results show the effect of alcohol on the survival rate of HepG2 cells.

[0025] Figure 5 These are the results of in vitro activity test of the peptide against alcoholic liver damage.

[0026] Figure 6 Shows the changes in MDA content, GSH content and SOD enzyme activity.

[0027] Figure 7 Shows the changes in ALT, AST, and LDH enzyme activities. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0029] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0030] Each group of data in the examples of the present invention was measured three times, and the results were expressed as mean ± standard deviation. SPSS 27.0 was used for single factor mean comparison (ANOVA), and the LSD method was used for inter-group significance analysis.

[0031] Example 1 Peptide synthesis and identification

[0032] In this example, a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 was solid-phase synthesized (with the assistance of Nanjing Peptide Valley Biotechnology Co., Ltd.), and purity analysis and mass spectrometry identification were performed on the synthesized polypeptide. SEQ ID NO: 1: LQPPR.

[0033] 1. Purity analysis of peptides

[0034] The purity of the peptide was analyzed by high performance liquid chromatography; the analysis conditions were as follows: chromatographic column: Kromasil C18 (4.6×150 mm, 5μm); mobile phase A: acetonitrile (containing 0.1% ( v / v ) trifluoroacetic acid); mobile phase B: water (containing 0.1% ( v / v) trifluoroacetic acid); elution gradient: 0→0.01 min, 5% ( v / v )A,0.01→25 min、50%( v / v )A, 25→30 min, 90% ( v / v )A; flow rate: 1mL / min; detection wavelength: 214 nm; sample volume: 20 μL.

[0035] 2. Mass spectrometry identification of peptides

[0036] ESI-MS mass spectrometry was used to identify the peptides, and the molecular weight and theoretical value on the mass spectrum were used to determine whether they were the target products. Mass spectrometry conditions: Mobile phase A: water (containing 0.1% ( v / v ) formic acid); mobile phase B: acetonitrile (containing 0.1% ( v / v ) formic acid); flow rate: 0.2 mL / min; run time: 1 min; positive ion mode; scan range: 0-2000 Da.

[0037] The results of peptide purity analysis and mass spectrometry identification are shown in Figure 1 and Figure 2 As shown. Figure 1 It can be seen that in the polypeptide of the present invention, the main peak accounts for the majority, only a few impurity peaks appear, and the purity is as high as 95.76%. Figure 2 It can be seen that the molecular ion peak m / z of the polypeptide of the present invention is 610.4, with one charge, which is consistent with its theoretical molecular weight value. This shows that the polypeptide synthesized in this example is the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, and the purity meets the requirements of subsequent experiments.

[0038] Example 2 Peptide Safety Test

[0039] In this example, the polypeptide obtained in Example 1 was used to treat human liver cancer cells to test the safety of the polypeptide.

[0040] 1. Cultivation of human liver cancer cells

[0041] HepG2 cells (human liver cancer cells) are highly similar in morphology and metabolic function to healthy human liver cells. The status of their intracellular metabolic enzymes does not change with the increase in the number of passages. They are a commonly used cell line for establishing alcoholic liver injury models. HepG2 cells were inoculated in a medium containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin DMEM medium, cultured in an incubator at 37°C, saturated humidity, and 5% CO2 until the cells basically covered the bottom of the culture bottle, removed the waste liquid in the culture bottle, washed with PBS, digested with 0.25% trypsin-EDTA for 1 min, and added 2 times the volume of 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin DMEM medium to terminate the reaction, blow the bottom of the bottle with a pipette, transfer the liquid to a centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, and add 10% ( v / v ) fetal bovine serum and 1% ( v / v ) and resuspended in DMEM medium containing penicillin / streptomycin, and subcultured at a ratio of 1:3. Experiments were carried out after the cell growth state stabilized.

[0042] 2. Testing peptide safety

[0043] HepG2 cells were cultured at 8×10 4 Each well was inoculated with 100 μL of DMEM medium (containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin) in a 96-well plate and divided them into a control group (containing cells but no peptide) and an experimental group (containing cells but containing peptide). v / v ) fetal bovine serum and 1% ( v / v ) Penicillin / streptomycin) was set as the zero group (no cells, no polypeptide). Three replicate wells were set for each group, incubated at 37 °C for 24 h, and the supernatant was discarded. Subsequently, 100 μL of DMEM medium containing different concentrations (12.5, 25, 50, 100, 200 μM) of polypeptide was added to the experimental group, and 100 μL of DMEM medium was added to the other groups. After the three groups were cultured at 37 °C for 24 h, 10 μL of CCK8 was added to each well, and the absorbance OD was measured at 450 nm after further culture for 2 h. The cell survival rate was calculated according to the following formula:

[0044] .

[0045] The results of peptide safety testing are as follows Figure 3 As shown. Figure 3 It can be seen that the polypeptide of the present invention exhibits no toxic effect on human liver cancer cells at a concentration of 12.5 to 200 μM, indicating that the polypeptide of the present invention is safe and has no side effects.

[0046] Example 3 Testing of the Anti-alcoholic Liver Damage Activity of Peptides

[0047] 1. Determination of alcohol modeling concentration in alcoholic liver injury model

[0048] HepG2 cells were cultured at 8×10 4 Each well was inoculated with 100 μL of DMEM medium (containing 10% ( v / v ) fetal bovine serum and 1% ( v / v) penicillin / streptomycin) in a 96-well plate and divided them into a control group (containing cells and no alcohol) and an experimental group (containing cells and containing alcohol). v / v ) fetal bovine serum and 1% ( v / v ) Penicillin / streptomycin) was set as the zero group (no cells, no alcohol). Three replicate wells were set for each group. After incubation at 37 °C for 24 h, the supernatant was discarded. Subsequently, 100 μL of DMEM medium containing different concentrations of alcohol (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 M) was added to the experimental group, and 100 μL of DMEM medium was added to the other groups. After the three groups were cultured at 37 °C for 12 h, 10 μL of CCK8 was added to each well, and the absorbance OD was measured at 450 nm after further culture for 2 h. The cell survival rate was calculated according to the following formula:

[0049] .

[0050] Effect of alcohol on the survival rate of HepG2 cells Figure 4 As shown. Figure 4 It can be seen that when the alcohol concentration is 0.1-1.2 M, the cell survival rate decreases in a dose-dependent manner, and at an alcohol concentration of 0.6 M, nearly 50% of HepG2 cells are inhibited. Therefore, 0.6 M was selected as the alcohol modeling concentration in the alcoholic liver injury model.

[0051] 2. In vitro activity test of peptides against alcoholic liver damage

[0052] HepG2 cells were cultured at 8×10 4 Each well was inoculated with 100 μL of DMEM medium (containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin) in a 96-well plate and divided them into a control group (containing cells, no alcohol, no peptide), an experimental group (containing cells, containing alcohol, containing peptides) and an alcohol group (containing cells, containing alcohol, no peptides). v / v ) fetal bovine serum and 1% ( v / v) Penicillin / streptomycin) was set as the zeroing group (no cells, no alcohol, no polypeptide). Three replicate wells were set for each group. After incubation at 37 °C for 24 h, the supernatant was discarded. Subsequently, the experimental group was added with 100 μL of DMEM medium containing 50 μM polypeptide, and the other groups were added with 100 μL of DMEM medium. After the four groups were cultured at 37 °C for 24 h, the supernatant was discarded. The experimental group and the alcohol group were added with 100 μL of DMEM medium containing 0.6 M alcohol, and the control group and the zeroing group were added with 100 μL of DMEM medium. After the four groups were cultured at 37 °C for 12 h, 10 μL CCK8 was added to each well, and the absorbance OD was measured at 450 nm after continuing to culture for 2 h. The cell survival rate was calculated according to the following formula:

[0053] or

[0054] .

[0055] The results of the in vitro anti-alcoholic liver injury activity test of the peptide are as follows Figure 5 As shown. Figure 5 It can be seen that compared with the alcohol group, the survival rate of HepG2 cells in the experimental group was significantly improved, indicating that the polypeptide of the present invention can significantly improve alcohol-induced cell damage and has activity against alcoholic liver damage.

[0056] 3. Test of the effect of peptides on MDA content, GSH content and SOD enzyme activity

[0057] HepG2 cells were cultured at 3×10 5 The number of cells / well was inoculated with DMEM medium (containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin) in a 6-well plate and divided into a control group (containing cells, no alcohol, no polypeptide), an experimental group (containing cells, containing alcohol, containing polypeptide) and an alcohol group (containing cells, containing alcohol, no polypeptide), with 3 replicates in each group. After culturing at 37 °C for 24 h, the supernatant was discarded. Subsequently, 2 mL of DMEM medium containing 50 μM polypeptide was added to the experimental group, and 2 mL of DMEM medium was added to the other groups. After culturing at 37 °C for 24 h, the supernatant was discarded. 2 mL of DMEM medium containing 0.6 M alcohol was added to the experimental group and the alcohol group, respectively, and 2 mL of DMEM medium was added to the control group. After culturing at 37 °C for 12 h, the cells were collected, and the MDA content, GSH content and SOD enzyme activity in each group of cells were determined according to the instructions of the malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) detection kits.

[0058] The changes of MDA content, GSH content and SOD enzyme activity are as follows Figure 6 As shown by Figure 6 It can be seen that compared with the control group, the MDA content in the cells of the alcohol group was significantly increased, and the GSH content and SOD enzyme activity were significantly decreased; compared with the alcohol group, the MDA content in the cells of the experimental group was significantly decreased, and the GSH content and SOD enzyme activity were significantly increased, indicating that the polypeptide of the present invention can effectively prevent and / or treat alcoholic liver injury.

[0059] 4. Test of the effect of peptides on ALT, AST and LDH enzyme activity

[0060] When liver cells are damaged, the cell contents leak out, and the levels of ALT, AST, and LDH in the serum will increase significantly, and the corresponding ALT, AST, and LDH enzyme activities will also increase significantly.

[0061] HepG2 cells were cultured at 3×10 5 The number of cells / well was inoculated with DMEM medium (containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin) in a 6-well plate, divided into a control group (containing cells, no alcohol, no polypeptide), an experimental group (containing cells, containing alcohol, containing polypeptide) and an alcohol group (containing cells, containing alcohol, no polypeptide), with 3 replicates in each group. After culturing at 37 °C for 24 h, the supernatant was discarded. Subsequently, 2 mL of DMEM medium containing 50 μM polypeptide was added to the experimental group, and 2 mL of DMEM medium was added to the other groups. After culturing at 37 °C for 24 h, the supernatant was discarded. 2 mL of DMEM medium containing 0.6M alcohol was added to the experimental group and the alcohol group, respectively, and 2 mL of DMEM medium was added to the control group. After culturing at 37 °C for 12 h, the cell culture fluid was collected, and the enzyme activities of ALT, AST, and LDH in the cell culture fluid of each group were measured according to the instructions of the alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) detection kits to reflect the leakage of cell contents and the health of liver cells.

[0062] Changes in ALT, AST, and LDH enzyme activities Figure 7 As shown by Figure 7 It can be seen that compared with the control group, the enzyme activities of ALT, AST, and LDH in the alcohol group were significantly increased; and compared with the alcohol group, the enzyme activities of ALT, AST, and LDH in the experimental group were significantly decreased, indicating that the polypeptide of the present invention can effectively prevent and / or treat alcoholic liver injury.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a polypeptide or its related biological material in the preparation of a drug for preventing and / or treating alcoholic liver damage, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; the related biological material is a nucleic acid molecule capable of expressing the polypeptide, or an expression cassette, recombinant plasmid, recombinant bacteria or recombinant cell containing the nucleic acid molecule.

2. The application according to claim 1, characterized in that: The prevention and / or treatment of alcoholic liver damage is to reduce the content of malondialdehyde.

3. The application according to claim 1, characterized in that: The prevention and / or treatment of alcoholic liver damage is to enhance the activity of superoxide dismutase.

4. The use according to claim 1, characterized in that: The method for preventing and / or treating alcoholic liver damage is to increase the glutathione content.

5. The use according to claim 1, characterized in that: The prevention and / or treatment of alcoholic liver injury is to reduce the activity of alanine aminotransferase.

6. The use according to claim 1, characterized in that: The prevention and / or treatment of alcoholic liver damage is to reduce the activity of aspartate aminotransferase.

7. The use according to claim 1, characterized in that: The prevention and / or treatment of alcoholic liver injury is to reduce the activity of lactate dehydrogenase.

Images