Application of active peptides in the preparation of drugs for the prevention and treatment of ALD

Abstract

The present invention provides an application of an active peptide in the preparation of a drug for preventing and treating ALD. The amino acid sequence of the active peptide of the present invention is shown in SEQ ID NO: 1, which can not only effectively alleviate the damage of ethanol to liver cells, but also significantly inhibit the increase of alanine aminotransferase activity, lactate dehydrogenase activity and malondialdehyde content caused by ethanol, and the decrease of glutathione content, thereby playing a preventive and therapeutic role on ALD, and is suitable for preparing a drug for preventing and treating ALD, and also enriches the active peptide resource library with ALD preventive and therapeutic effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptides, and more specifically, relates to the application of active peptides in the preparation of drugs for preventing and treating ALD. Background Art

[0002] Alcoholic liver disease (ALD) is one of the common liver diseases. It is mainly the result of the interaction of multiple factors such as inflammatory response, oxidative stress, enteric endotoxin, inflammatory mediators and nutritional imbalance induced directly or indirectly by ethanol and its derivatives during metabolism. The clinical symptoms of ALD are non-specific and may be asymptomatic or accompanied by right upper abdominal distension and pain, loss of appetite, fatigue, weight loss, jaundice, etc. As the disease worsens, neuropsychiatric symptoms, spider nevi, liver palms, etc. may occur.

[0003] Active peptides have received considerable attention in the treatment of diseases such as diabetes, tumors, thrombosis, and hypertension due to their safety, non-toxicity, and suitability for targeted therapy. However, the existing active peptide resources that can be used to prevent and treat ALD are still relatively scarce. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide an active peptide with an amino acid sequence as shown in SEQ ID NO: 1, enrich the active peptide resource library with ALD prevention and treatment effects, and provide more raw material options for drugs for the prevention and treatment of ALD.

[0005] The first object of the present invention is to provide an active peptide.

[0006] The second object of the present invention is to provide the use of the above active peptide in the preparation of drugs for preventing and treating ALD.

[0007] The third object of the present invention is to provide the use of the above-mentioned active peptide-related biological materials in the preparation of drugs for preventing and treating ALD.

[0008] A fourth object of the present invention is to provide a product.

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

[0010] The present invention provides an active peptide, whose amino acid sequence is shown in SEQ ID NO:1.

[0011] The active peptide can not only effectively alleviate the damage of ethanol to liver cells, but also significantly inhibit the increase of alanine aminotransferase activity, lactate dehydrogenase activity and malondialdehyde content caused by ethanol, and the decrease of glutathione content, thereby playing a preventive and therapeutic role on ALD, and is suitable for preparing drugs for preventing and treating ALD. Therefore, the present invention also provides the use of the above-mentioned active peptides and related biological materials of the above-mentioned active peptides in the preparation of drugs for preventing and treating ALD. Among them, the related biological materials are nucleic acid molecules that can express the active peptides, or recombinant bacteria, recombinant cells, recombinant plasmids or expression cassettes containing the nucleic acid molecules.

[0012] Preferably, the prevention and treatment of ALD is to reduce the activity of alanine aminotransferase and / or lactate dehydrogenase.

[0013] Preferably, the prevention and treatment of ALD is to reduce the malondialdehyde content and / or increase the glutathione content.

[0014] Based on this, the present invention also provides a product comprising the above-mentioned active peptide, and a product comprising the related biological material of the above-mentioned active peptide, wherein the related biological material is a nucleic acid molecule capable of expressing the active peptide, or a recombinant bacterium, recombinant cell, recombinant plasmid or expression cassette containing the nucleic acid molecule.

[0015] Preferably, the product is a medicine.

[0016] More preferably, the medicine further contains excipients.

[0017] Further preferably, the dosage form of the drug is one of tablets, capsules, liquid preparations, powders or granules.

[0018] The present invention has the following beneficial effects:

[0019] The active peptide of the present invention whose amino acid sequence is shown in SEQ ID NO: 1 can not only effectively alleviate the damage of ethanol to liver cells, but also significantly inhibit the increase of alanine aminotransferase activity, lactate dehydrogenase activity and malondialdehyde content, and the decrease of glutathione content caused by ethanol, thereby playing a preventive and therapeutic effect on ALD. It is suitable for preparing drugs for preventing and treating ALD, and also enriches the active peptide resource library with ALD preventive and therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of mass spectrometry identification of active peptides.

[0021] Figure 2 This is the result of the purity identification of the active peptide.

[0022] Figure 3 The results show the cytotoxicity of active peptides to HepG2 cells.

[0023] Figure 4 Statistical results of HepG2 cell survival rate after ethanol treatment.

[0024] Figure 5 Statistical results of HepG2 cell survival rate after treatment with active peptides and / or ethanol.

[0025] Figure 6 Shows the changes in ALT and LDH enzyme activities in cell culture medium.

[0026] Figure 7 Shows the changes in MDA and GSH content in cells. DETAILED DESCRIPTION

[0027] 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.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] Example 1 Synthesis, mass spectrometry identification and purity identification of active peptides

[0030] Nanjing Peptide Valley Biotechnology Co., Ltd. was commissioned to assist in the synthesis of the active peptide (the amino acid sequence is shown in SEQ ID NO:1: SPPEF), and the active peptide was identified by mass spectrometry and purity.

[0031] 1. Mass spectrometry identification

[0032] ESI-MS mass spectrometry was used to identify the molecular weight of the active peptides.

[0033] Among them, the identification conditions of ESI-MS mass spectrum are:

[0034] Mobile phase: A is water (containing 0.1% ( v / v ) formic acid), B is acetonitrile (containing 0.1% ( v / v )formic acid);

[0035] Flow rate: 0.2 mL / min;

[0036] Running time: 1 min.

[0037] Positive ion mode;

[0038] Scan range: 0~2000 Da.

[0039] The results of mass spectrometry identification are as follows Figure 1As shown, it can be seen that the molecular ion peak m / z of the active peptide in this example is 576.36, with one charge, which is consistent with its theoretical molecular weight value, indicating that the active peptide synthesized in this example is the active peptide with an amino acid sequence as shown in SEQ ID NO: 1.

[0040] 2. Purity Identification

[0041] The purity of the active peptide was identified by HPLC.

[0042] Among them, the identification conditions of high performance liquid chromatography are:

[0043] Chromatographic column: Kromasil C18 (4.6 × 150 mm, 5 μm);

[0044] Mobile phase: A is acetonitrile (containing 0.1% ( v / v ) trifluoroacetic acid), B is water (containing 0.1% ( v / v ) trifluoroacetic acid);

[0045] 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;

[0046] Flow rate: 1 mL / min;

[0047] Detection wavelength: 214 nm;

[0048] Loading volume: 20 μL.

[0049] The results of purity identification are as follows Figure 2 As shown, it can be seen that in the active peptide synthesized in this example, the main peak accounts for the majority, only very few impurity peaks appear, and the purity is as high as 98.55%.

[0050] Example 2 Active peptides are non-toxic to cells

[0051] 1. Culture of HepG2 cells

[0052] Human hepatoma cells (HepG2 cells) were inoculated in complete culture medium (containing 1% ( v / v ) Penicillin / Streptomycin dual antibody solution and 10% ( v / v) in DMEM medium containing fetal bovine serum) and cultured in an incubator at 37 ℃, saturated humidity, and 5% CO2 until the cells basically covered the bottom of the culture bottle. Then, the waste liquid in the culture bottle was removed, the cells were washed with PBS, and digested with 0.25% trypsin-EDTA for 1 min. Two times the volume of complete culture medium was added to terminate the reaction, and the bottom of the bottle was washed with a pipette and transferred to a new centrifuge tube. Centrifuged at 1000 rpm for 5 min, the supernatant was removed, and complete culture medium was added to resuspend. The cells were subcultured at a ratio of 1:3, and the experiment was carried out after the cell growth state was stable.

[0053] 2. Detection of the toxicity of active peptides on HepG2 cells

[0054] Experimental group: HepG2 cells were cultured at 8×10 4 The cells were plated at 100 μL / well in a 96-well plate containing complete culture medium. After incubation at 37 °C for 24 h, the supernatant was discarded and 100 μL of DMEM medium containing different concentrations (12.5, 25, 50, 100 μM) of active peptides was added.

[0055] Blank group: HepG2 cells were cultured at 8×10 4 The cells were plated at 100 μL / well in a 96-well plate containing complete culture medium. After incubation at 37 °C for 24 h, the supernatant was discarded and 100 μL of DMEM culture medium was added.

[0056] Zero adjustment group: After incubating the 96-well plate containing complete medium at 37 °C for 24 h, the supernatant was discarded and 100 μL DMEM medium was added.

[0057] After the experimental group, blank group, and zero-adjustment group were cultured at 37 °C for 24 h, 10 μL CCK8 was added to each well, and the absorbance A was measured at 450 nm after culturing for 2 h. The survival rate of HepG2 cells after active peptide treatment was calculated according to the formula "cell survival rate (%) = (A experimental group-A zero-adjustment group) / (A blank group-A zero-adjustment group) × 100%". Three replicate wells were set for each group, and the results were averaged.

[0058] The results are as follows Figure 3 As shown, it can be seen that the active peptide of the present invention exhibits no toxic effects at a concentration of 12.5 to 100 μM.

[0059] Example 3 Active peptides can effectively prevent and treat ALD

[0060] 1. Ethanol concentration used in ALD cell modeling

[0061] Experimental group: HepG2 cells were cultured at 8×10 4The cells were plated at 100 μL / well in a 96-well plate containing complete culture medium. After incubation at 37 °C for 24 h, the supernatant was discarded and 100 μL of DMEM medium containing different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 M) of ethanol was added.

[0062] Blank group: HepG2 cells were cultured at 8×10 4 The cells were plated at 100 μL / well in a 96-well plate containing complete culture medium. After incubation at 37 °C for 24 h, the supernatant was discarded and 100 μL of DMEM culture medium was added.

[0063] Zero adjustment group: After incubating the 96-well plate containing complete medium at 37 °C for 24 h, the supernatant was discarded and 100 μL DMEM medium was added.

[0064] After the experimental group, blank group, and zero-adjustment group were cultured at 37 °C for 12 h, 10 μL CCK8 was added to each well, and the absorbance A was measured at 450 nm after culturing for 2 h. The survival rate of HepG2 cells after ethanol treatment was calculated according to the formula "cell survival rate (%) = (A experimental group-A zero-adjustment group) / (A blank group-A zero-adjustment group) × 100%". Three replicate wells were set for each group, and the results were averaged.

[0065] The results are as follows Figure 4 As shown. It can be seen that when the ethanol concentration is 0.1-1.2 M, the cell survival rate decreases in a dose-dependent manner, and when the ethanol concentration is 0.6 M, the cell inhibition rate is close to 50%, so 0.6 M is determined as the ethanol modeling concentration for the following experiments.

[0066] 2. The alleviating effect of active peptides on ethanol-induced cell damage

[0067] Experimental group: HepG2 cells were cultured at 8×10 4 The cells were inoculated with 100 μL of DMEM medium containing 50 μM active peptide per well at 37 °C and 100 μL of DMEM medium containing 0.6 M ethanol per well at 37 °C for 24 h. The supernatant was discarded and 100 μL of DMEM medium containing 50 μM active peptide was added. The cells were incubated at 37 °C for 24 h, the supernatant was discarded and 100 μL of DMEM medium containing 0.6 M ethanol was added.

[0068] Blank group: HepG2 cells were cultured at 8×10 4 The cells were inoculated with 100 μL of DMEM medium per well at 37 °C for 24 h, and the supernatant was discarded. 100 μL of DMEM medium was added to each well of the plate. The cells were incubated at 37 °C for 24 h, and the supernatant was discarded. 100 μL of DMEM medium was added to the plate.

[0069] Ethanol group: HepG2 cells were cultured at 8×10 4 The cells were inoculated with 100 μL of DMEM medium per well at 37 °C for 24 h, and then the supernatant was discarded and 100 μL of DMEM medium containing 0.6 M ethanol was added.

[0070] Zero adjustment group: After incubating the 96-well plate containing complete medium at 37°C for 24 h, the supernatant was discarded and 100 μL DMEM medium was added. After further incubation at 37°C for 24 h, the supernatant was discarded and 100 μL DMEM medium was added.

[0071] The experimental group, blank group, ethanol group, and zero adjustment group were cultured at 37 °C for 12 h, and 10 μL CCK8 was added to each well. After culturing for 2 h, the absorbance A was measured at 450 nm. The survival rate of HepG2 cells after active peptide and / or ethanol treatment was calculated according to the formula "cell survival rate (%) = (A experimental group-A zero adjustment group) / (A blank group-A zero adjustment group) × 100%" or "cell survival rate (%) = (A ethanol group-A zero adjustment group) / (A blank group-A zero adjustment group) × 100%". Three replicate wells were set for each group, and the results were averaged.

[0072] The results are as follows Figure 5 As shown. It can be seen that compared with the blank group, the survival rate of HepG2 cells in the ethanol group was significantly reduced; while compared with the ethanol group, the survival rate of HepG2 cells in the experimental group was significantly increased. This shows that the active peptide of the present invention can effectively alleviate the damage of ethanol to liver cells, thereby effectively preventing and treating ALD.

[0073] 3. Effects of active peptides on ALT and LDH activity

[0074] Experimental group: HepG2 cells were cultured at 3×10 5 The number of cells / well was inoculated in a 6-well plate containing complete culture medium. After culturing at 37°C for 24 h, the supernatant was discarded, and 2 mL of DMEM culture medium containing 50 μM active peptide was added. After further incubation at 37°C for 24 h, the supernatant was discarded, and 2 mL of DMEM culture medium containing 0.6 M ethanol was added.

[0075] Blank group: HepG2 cells were cultured at 3×10 5The number of cells / well was inoculated into a 6-well plate containing complete culture medium. After culturing at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium was added. After continuing to incubate at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium was added.

[0076] Ethanol group: HepG2 cells were cultured at 3×10 5 The number of cells / well was inoculated into a 6-well plate containing complete culture medium. After culturing at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium was added. After continuing to incubate at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium containing 0.6 M ethanol was added.

[0077] The three groups were cultured at 37 ℃ for 12 h, and then the cell culture fluid was collected. The enzyme activities of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in the cell culture fluid of each group were measured according to the instructions of the ALT and LDH detection kits.

[0078] The results are as follows Figure 6 As shown. It can be seen that compared with the blank group, the enzyme activities of ALT and LDH in the ethanol group were significantly increased; while compared with the ethanol group, the enzyme activities of ALT and LDH in the experimental group were significantly reduced. This shows that the active peptide of the present invention can significantly inhibit the increase of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities caused by ethanol, thereby effectively preventing and treating ALD.

[0079] IV. Effect of active peptides on MDA and GSH content

[0080] Experimental group: HepG2 cells were cultured at 3×10 5 The number of cells / well was inoculated in a 6-well plate containing complete culture medium. After culturing at 37°C for 24 h, the supernatant was discarded, and 2 mL of DMEM culture medium containing 50 μM active peptide was added. After further incubation at 37°C for 24 h, the supernatant was discarded, and 2 mL of DMEM culture medium containing 0.6 M ethanol was added.

[0081] Blank group: HepG2 cells were cultured at 3×10 5 The number of cells / well was inoculated into a 6-well plate containing complete culture medium. After culturing at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium was added. After continuing to incubate at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium was added.

[0082] Ethanol group: HepG2 cells were cultured at 3×10 5The number of cells / well was inoculated into a 6-well plate containing complete culture medium. After culturing at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium was added. After continuing to incubate at 37°C for 24 h, the supernatant was discarded and 2 mL of DMEM culture medium containing 0.6 M ethanol was added.

[0083] After the three groups were cultured at 37°C for 12 h, the cells were collected and the contents of malondialdehyde (MDA) and glutathione (GSH) in the cells of each group were determined according to the instructions of the malondialdehyde (MDA) and glutathione (GSH) detection kits.

[0084] The results are as follows Figure 7 As shown. It can be seen that compared with the blank group, the MDA content in the ethanol group increased significantly, and the GSH content decreased significantly; while compared with the ethanol group, the MDA content in the experimental group decreased significantly, and the GSH content increased significantly. This shows that the active peptide of the present invention can significantly inhibit the increase of malondialdehyde (MDA) content and the decrease of glutathione (GSH) content caused by ethanol, thereby effectively preventing and treating ALD.

[0085] 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. The use of active peptides in the preparation of drugs for preventing and treating ALD (alcoholic liver damage), characterized in that: The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

2. Application of biomaterials related to active peptides in the preparation of drugs for preventing and treating ALD (alcoholic liver damage), characterized in that: The amino acid sequence of the active peptide is shown in SEQ ID NO: 1, and the related biological material is a nucleic acid molecule capable of expressing the active peptide, or a recombinant bacterium or a recombinant plasmid containing the nucleic acid molecule.

3. Application of biomaterials related to active peptides in the preparation of drugs for preventing and treating ALD (alcoholic liver damage), characterized in that: The amino acid sequence of the active peptide is shown in SEQ ID NO: 1, and the related biological material is a recombinant cell or expression box containing a nucleic acid molecule capable of expressing the active peptide.

4. The use according to any one of claims 1 to 3, characterized in that: The ALD prevention and treatment is to reduce the activity of alanine aminotransferase and / or lactate dehydrogenase.

5. The use according to any one of claims 1 to 3, characterized in that: The ALD prevention and treatment is to reduce the malondialdehyde content and / or increase the glutathione content.

6. The use according to any one of claims 1 to 3, characterized in that: The medicine also contains auxiliary materials.

7. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is one of tablets, capsules or granules.

8. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is one of a liquid preparation or a powder.

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