A bioactive polypeptide and its application

Abstract

The present invention relates to the field of biotechnology. The present invention relates to a bioactive polypeptide, which has the effect of protecting the liver. Specifically, the present invention provides a bioactive polypeptide molecule PGPIP derived from A2β-casein in cow's milk, and its amino acid sequence is Pro-Gly-Pro-Ile-Pro. The present invention has confirmed through in vitro and in vivo experiments that it can effectively reduce liver lipid accumulation and hepatocyte damage, providing a feasible direction for the treatment of liver diseases, especially the prevention and treatment of alcoholic fatty liver; this bioactive polypeptide also has great application potential in the fields of food and health products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a bioactive polypeptide, which has the function of protecting the liver and promoting liver health. Background Art

[0002] Bioactive polypeptides are a class of peptide compounds with special physiological effects on organisms. Generally formed by the different permutations and combinations of 20 natural amino acids, their functions are also determined by the composition sequence of amino acids. The differences between peptides with different permutation methods and different lengths are huge. Often, a difference of one amino acid leads to completely different physiological activities of the polypeptide. Bioactive polypeptides have a low molecular weight, a relatively simple structure, weak antigenicity, low adverse reactions, and are easily absorbed by the human body. With the in-depth study of polypeptide molecules, the functions of a large number of bioactive peptides in aspects such as antibacterial, antioxidant, anti-tumor, immune regulation, nerve information transmission regulation, blood pressure reduction, cholesterol reduction, and trace element transportation have been discovered. They have broad development prospects in the research fields of medicine, food, etc.

[0003] As an important aspect of human social activities, a large number of people drink alcohol. However, long-term excessive drinking has also led to a series of social and health problems. Alcoholic liver disease (ALD) is a series of liver lesions caused by long-term excessive drinking, including alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis and cirrhosis, and even liver cancer. Alcoholic fatty liver disease is a classification of alcoholic liver disease. If a patient has a long-term drinking history, generally more than 5 years, and has no special symptoms, most patients will show some distending pain in the upper right abdomen, loss of appetite, fatigue, and weight loss clinically. If long-term alcoholic fatty liver is not intervened, it is very likely to deteriorate further and lead to more serious health problems. At present, the US Food and Drug Administration has not approved any drugs for the treatment of alcoholic fatty liver.

[0004] Previous research by the present inventors found that the polypeptide molecule PGPIPN derived from A2 β-casein in cow's milk has biological functions such as antioxidant, antihypertensive, and anti-tumor effects. PGPIPN can inhibit the invasion and metastasis of ovarian cancer cells and enhance the sensitivity of ovarian cancer cells to cisplatin; PGPIPN can affect the transformation of T lymphocytes and the secretion of macrophages, thereby improving immunity; PGPIPN can improve the antioxidant capacity of liver cells, scavenge free radicals generated by excessive alcohol intake, regulate lipid metabolism, and reduce the apoptosis of liver cells, thus protecting liver cells and can be used for the prevention and treatment of acute alcoholic fatty liver. CN202210008755.0 uses multiple proteases to hydrolyze bovine colostrum, screens and obtains a bioactive peptide with a brand-new amino acid structure, which can scavenge oxygen free radicals, promote the proliferation of immune cells including lymphocytes, inhibit the growth and reproduction of harmful microorganisms, induce the secretion of immune factors in the body, and improve the immune capacity of the body. CN201510671040.3 discloses a liver-protecting food containing oligopeptides, and the oligopeptides are derived from soybean protein, polysaccharide peptide of Coriolus versicolor, and goat milk protein. However, there is still a lack of discovery of more and more effective polypeptides with biological and physiological activities. Summary of the Invention

[0005] Based on the need to develop more bioactive polypeptides with physiological and biochemical activities, the present inventors have conducted a large number of research and test screenings and unexpectedly found that the polypeptide molecule PGPIP (Pro-Gly-Pro-Ile-Pro) of A2 β-casein in cow's milk also has biological activity and has an unexpected therapeutic effect in the prevention and treatment of alcoholic fatty liver. Based on this, the technical solution of the present invention is proposed. Specifically:

[0006] The present invention provides a bioactive polypeptide, and the amino acid sequence of the polypeptide molecule is Pro-Gly-Pro-Ile-Pro.

[0007] The present invention provides the application of the active polypeptide in the preparation of a drug for assisting in improving the health of an organism. The active polypeptide is PGPIP, and its amino acid sequence is Pro-Gly-Pro-Ile-Pro. Based on the various effects of the polypeptide of the present invention, other health-promoting substances can also be added to the drug for combined use. Preferably, the substance is a prebiotic and / or a probiotic.

[0008] In one aspect of the present invention, the present invention provides the application of the active polypeptide in the preparation of a drug for preventing / treating liver diseases. More preferably, the liver disease is fatty liver, and more preferably, the liver disease is alcoholic fatty liver. The dosage of the active polypeptide is 0.1-10 mg / kg body weight, preferably 1-5 mg / kg body weight, and more preferably 2.0 mg / kg body weight.

[0009] In one aspect of the present invention, the pharmaceutical composition of the present invention further contains pharmaceutically acceptable excipients. The pharmaceutical composition of the present invention can be prepared into various dosage forms according to the usage scenarios, convenience, and efficacy, such as injections, tablets, capsules, lyophilized powders, nanoparticle preparations, etc.

[0010] In one aspect of the present invention, the present invention provides a pharmaceutical composition containing a bioactive peptide, and the amino acid sequence of the polypeptide molecule is Pro-Gly-Pro-Ile-Pro. Beneficial effects

[0011] The present invention provides a bioactive polypeptide molecule PGPIP derived from A2 β-casein in cow's milk. In vitro and in vivo experiments have confirmed that it can effectively reduce lipid accumulation in the liver and hepatocyte damage, providing a feasible direction for the treatment of liver diseases, especially the prevention and treatment of alcoholic fatty liver. Description of the drawings

[0012] Figure 1 : Cytotoxicity experiment diagram of the bioactive polypeptide PGPIP of the present invention. Compared with the alcohol group: *P<0.05, **P<0.01; compared with the control group: #P<0.05, ##P<0.01.

[0013] Figure 2 : Oil red O staining lipid droplet diagram of liver tissue sections of different groups.

[0014] Figure 3 : HE staining detection of hepatocyte damage in different liver tissue sections.

[0015] Figure 4 : Analysis diagram of the bioactive polypeptide PGPIP of the present invention through the SwissADME network pharmacology system.

[0016] Figure 5 : Mouse primary hepatocytes and human normal hepatocyte L-02 cell diagrams.

[0017] Figure 6 : Analysis diagram of the molecular mechanism involved in the liver protection of the bioactive polypeptide PGPIP. Detailed implementation manners

[0018] The present invention will be further described below in conjunction with specific embodiments. Embodiment

[0019] Preparation of the bioactive polypeptide PGPIP pentapeptide: Entrust Shanghai Bioengineering Company for chemical synthesis of the polypeptide. Embodiment

[0020] Cytotoxicity experiment of the bioactive polypeptide PGPIP in vitro culture

[0021] The viability of human normal hepatocyte line L-02 (HL-7702) was determined using the WST-1 method. Cell culture experiments were divided into a blank control group, an alcohol group, a PGPIP polypeptide group, and an alcohol and PGPIP polypeptide combined group. The cells were cultured using DMEM medium containing 100 U / ml penicillin and 100 μg / ml streptomycin, 10% fetal bovine serum, and cultured in a 37 °C incubator containing 5% CO2. The stimulation concentration of human L-02 cells in the alcohol group was 85.63 mmol / L, the stimulation concentration of the PGPIP pentapeptide was 15 μmol / L, and the stimulation time lasted for 48 hours. The absorbance values of each group were measured at 450 nm. Cell viability (%) = (A450nm absorbance value of the experimental group / A450nm absorbance value of the control group) × 100%. Statistical analysis was performed using SPSS 13.0 statistical software for data processing. The results were expressed as mean ± standard deviation, and one-way ANOVA was used for comparison among multiple groups. See Figure 1 , alcohol treatment had obvious toxicity to human normal hepatocyte line, and the cell viability decreased significantly. However, the PGPIP pentapeptide had no obvious toxicity, and the PGPIP pentapeptide had a good protective effect on the decrease in cell viability of human normal hepatocytes caused by alcohol stimulation. Example

[0022] Protective effect of bioactive polypeptide PGPIP on alcoholic liver

[0023] Establishment of the NIAAA alcoholic liver disease animal model (according to the method of the National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health, USA) and the method of polypeptide administration: 24 female C57BL / 6 mice aged 10 - 12 weeks with a body weight of 19 grams were purchased from the Experimental Animal Center of Anhui Medical University. The alcohol-free control Lieber-DeCarli diet and the Lieber-DeCarli diet containing alcohol were both purchased from Trofi Feed Technology Co., Ltd. in Nantong, Jiangsu. The mice were divided into three groups: a blank control group, an alcohol group, and a PGPIP polypeptide group, with 8 mice in each group. Except for the gavage experiment, the mice could freely eat the fed diet. To make the mice adapt to the alcohol diet, all mice were pre-fed for five days. Starting from the first day, except for the control group fed with the alcohol-free Lieber-DeCarli diet containing isocaloric maltodextrin, the other two groups were fed with the Lieber-DeCarli diet containing 1% (v / v) alcohol concentration, and the polypeptide group was gavaged with PGPIP pentapeptide at a concentration of 2.0 mg / kg body weight of the mice. After that, the alcohol concentration was increased by 1% (v / v) every day for five days. On the fifth day, the alcohol group and the polypeptide group were fed with the Lieber-DeCarli diet containing 5% (v / v) alcohol, and during this period, the polypeptide group continued to be gavaged with pentapeptide to the mice. After pre-feeding for five days, the Lieber-DeCarli diet containing 5% (v / v) alcohol concentration was continuously fed for a total of 10 days, and during this period, the polypeptide group continued to be gavaged with pentapeptide to the mice. After 10 days, after the polypeptide group was gavaged with pentapeptide to the mice 30 minutes earlier in the morning, the alcohol group and the polypeptide group were simultaneously gavaged with alcohol at a concentration of 5 g / kg body weight of the mice, and the control group was gavaged with isocaloric maltodextrin. 9 hours later, all the mice were sacrificed, and liver and serum samples were collected respectively for testing and analyzing the protective effect of the bioactive polypeptide PGPIP on the liver.

[0024] Oil Red O staining was used to analyze the lipid accumulation in the liver, see Figure 2 It can be seen that there was no obvious lipid droplet accumulation in the liver of the control group, and there was a large amount of lipid accumulation in the liver of the model group (red indicates lipid droplets, and blue indicates liver cell nuclei), and there were some relatively large lipid droplets, indicating that an alcoholic fatty liver model had been successfully established. The number of lipid droplets in the liver of the polypeptide group was significantly reduced compared with that of the model group and there was no obvious difference compared with the control group, suggesting that PGPIP had a good effect on reducing lipid accumulation in the liver. HE staining was used to analyze the liver injury situation, see Figure 3It can be seen that the hepatocytes in the control group were radially arranged around the central vein, and the hepatic lobule structure was clear. The hepatocytes anastomosed with each other to form a network, and there were sinusoids and blood sinuses between the meshes. In the model group, the reticular structure of the liver hepatocytes was disordered, there were large lipid vacuoles in many hepatocytes, the hepatic nuclei were squeezed to one side by the vacuoles, and ballooning degeneration of the hepatocytes occurred. The boundaries between hepatocytes were blurred, and infiltration of immune cells could also be observed. Compared with the alcohol group, no similar damage was observed in the liver of the polypeptide group, and there was no obvious difference compared with the control group. Scale bar = 20 μm. Example

[0025] 4.1 Pharmacological analysis of the active polypeptide PGPIP by SwissADME

[0026] The network pharmacology analysis of the PGPIP pentapeptide was completed using the SwissADME software established by the Swiss Institute of Bioinformatics (https: / / www.expasy.org / resources / swissadme). First, the molecular structural formula of PGPIP was drawn using ChemDraw software from CambridgeSoft Corporation in the United States, then converted into the SMILES format, and relevant information was obtained using the SwissADME software. Refer to Figure 4 It can be seen that the relevant parameters of the analysis results are divided into aspects such as physicochemical properties, lipophilicity, water solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties. The molecular weight of PGPIP is 479.57 Daltons, it is soluble in water, does not inhibit CYP enzymes related to drug metabolism, can pass the Lipinski's rule of five for drug-likeness, and has the potential for development as an oral drug.

[0027] 4.2 Analysis of the mechanism of action of the active polypeptide PGPIP in protecting the liver

[0028] The analysis of the mechanism of action of the PGPIP pentapeptide was carried out through experiments on primary mouse hepatocytes isolated and cultured in vitro and the human normal hepatocyte L-02 cell line. Glutathione (GSH) and the PGPIPN hexapeptide were used as controls for the PGPIP pentapeptide. First, primary hepatocytes were isolated from the livers of C57BL / 6 female mice by two-step collagenase perfusion method, and at the same time, the cryopreserved human normal hepatocyte L-02 cell line was revived and co-cultured in a cell culture incubator. Refer to Figure 5 It can be seen that compared with the cultured human normal hepatocyte line L-02, most primary mouse hepatocytes showed a binuclear morphology (indicated by black arrows), the cells were polygonal, and the cell nuclei were clearly visible. The cell boundaries were relatively obvious. From the culture map of primary mouse hepatocytes, the cell purity was greater than 90%. The human normal hepatocyte line L-02 showed obvious epithelial-like cell characteristics. Scale bar = 20 μm.

[0029] The cultured primary mouse hepatocytes were divided into five groups, namely the control group, the alcohol group, the GSH group, the PGPIPN hexapeptide group, and the PGPIP pentapeptide group. The stimulation concentration of the alcohol group was 100 mM. In the GSH group, the PGPIPN hexapeptide group, and the PGPIP pentapeptide group, in addition to being stimulated with alcohol, they were respectively stimulated with the same concentration of GSH, PGPIPN hexapeptide, and PGPIP pentapeptide, all at 15 μM, and the stimulation time lasted for 16 hours. The cells were collected and lysed, and the expression of related lipid metabolism-related genes was analyzed by Western blotting. The quantification of protein expression levels and relative protein phosphorylation levels was analyzed using Image Pro Plus software from Media Cybernetics, USA. Refer to Figure 6 As can be seen from A, B, and E in Figure 6 , PGPIP restored the decrease in the phosphorylation level of serine 79 of acetyl-CoA carboxylase (ACC) induced by alcohol. The induction of PGPIP also significantly increased the expression of peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α). In addition, PGPIP had no obvious effect on the gene expression of fatty acid synthase (FASN) and peroxisome proliferator-activated receptor α (PPARα). To analyze the effect of different concentrations of PGPIP pentapeptide on gene expression, the cultured human L-02 cells were divided into six groups, namely the control group, the alcohol group, the GSH group, the low-concentration PGPIP pentapeptide group, the medium-concentration PGPIP pentapeptide group, and the high-concentration PGPIP pentapeptide group. The stimulation concentration of the alcohol group was 85.63 mM. In the GSH group and the low, medium, and high-concentration PGPIP pentapeptide groups, in addition to being stimulated with alcohol, they were respectively stimulated with 15 μM of GSH, 0.15 μM, 1.5 μM, and 15 μM of PGPIP pentapeptide, and the stimulation time lasted for 16 hours. Refer to

[0030] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope determined by the claims submitted for the present invention.

Claims

1. Use of an active polypeptide in the preparation of a medicament for assisting in improving the health of an organism, characterized in that, The amino acid sequence of the active polypeptide molecule is Pro-Gly-Pro-Ile-Pro; the improvement of the health of the organism is the improvement of alcoholic fatty liver.

2. The application according to claim 1, characterized in that, The medicine also contains other substances that promote health.

3. Use of an active polypeptide in the preparation of a medicament for preventing / treating liver diseases, characterized in that, The amino acid sequence of the active polypeptide molecule is Pro-Gly-Pro-Ile-Pro; the liver disease is alcoholic fatty liver.

4. The application according to claim 3, wherein The dosage of the active polypeptide is 0.1-10 mg / kg body weight.

5. The application according to claim 4, characterized in that, The medicine also contains pharmaceutically acceptable excipients.

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