Anti-inflammatory polypeptide as well as preparation method and application thereof

Abstract

The invention discloses an anti-inflammatory polypeptide as well as a preparation method and application thereof. The amino acid sequence of the anti-inflammatory polypeptide is Gln-Phe-Gln-Phe (QFQF), and the anti-inflammatory polypeptide is derived from milk fat globule membrane protein and is tetrapeptide capable of being absorbed by intestinal tracts. According to the anti-inflammatory peptide, the amino acid sequence of the anti-inflammatory peptide is analyzed by LC-MS / MS, the anti-inflammatory peptide is synthesized to further verify the anti-inflammatory activity, the sequence of the anti-inflammatory peptide is determined, and the efficiency is improved; the milk fat globule membrane protein-derived anti-inflammatory polypeptide can be used for preparing functional preparations and reducing inflammation in a human body, and has a potential effect of regulating and controlling generation and development of inflammation. The polypeptide has very important significance for developing dairy products, animal feeds, health-care products, health-care foods and medicines with anti-inflammatory functions, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of active peptides, and specifically relates to an anti-inflammatory polypeptide and a preparation method and application thereof. Background Art

[0002] The structure and bioactive components of milk fat globule membrane (MFGM) play a vital role in the growth of infants and young children. These functions are mainly derived from the protein, lipid and carbohydrate components of MFGM. According to the current reports, the functional characteristics of MFGM are mainly as follows: (1) Immunomodulatory activity: BTN is one of the most abundant milk fat globule membrane proteins (MFGMPs) expressed in the lactating gland. Because it exists in immune cells such as lymphocytes, macrophages, and monocytes, it is considered to be an immune system regulator. MFGM as a complementary food can regulate metabolic abnormalities in malnourished infants, thereby improving metabolic regulation and enhancing immunity. (2) Anti-intestinal inflammatory activity: Supplementing formula milk with bovine MFGM helps reduce the incidence of acute otitis media and the utilization rate of antipyretic drugs in infants, and has an immunomodulatory effect on pneumococci. (3) Anti-tumor activity: Studies have shown that MFGM in bovine milk has anti-cancer factors that induce cell apoptosis and reduce the activity of HT29 cells. Colon cancer, breast cancer and other malignant tumors are also inhibited by the lipid and protein components of MFGM. (4) Promote cell growth and differentiation: MFGM may help prevent diseases and neonatal growth and development. Combining MFGM and lactoferrin in the diet can help infants' cognitive, GIT and respiratory health. Currently, there are few studies on the release of free fatty acids and the absorption of nutrients in the gastrointestinal digestion of breast milk fat globule emulsions, and the function of the bioactive peptides in its digestion products is still unclear. Therefore, it is valuable to study the simulated gastrointestinal digestion and absorption of breast milk fat globule emulsions.

[0003] Caco-2 cells are colon and rectal cancer cells from the human body. They spontaneously differentiate during routine in vitro culture to form polarized monolayers with brush borders and microvilli. They are monolayers with similar morphology and function to the human small intestinal epithelium and are widely used in a variety of intestinal studies, including intestinal absorption, intestinal transport, intestinal metabolism, intestinal barrier, intestinal immunity, and intestinal adhesion. Because of their extremely similar morphology and properties to those of small intestinal epithelial cells, the Caco-2 cell model is a commonly used in vitro absorption model. Based on the combination of in vitro simulated digestion and the Caco-2 cell model, the bioavailability of gastrointestinal digestion products of dairy products can be predicted, especially proteins from various dairy products. In addition, the intestinal transport model constructed using Caco-2 cells is also often used to screen peptides that can be fully digested and absorbed by the intestine.

[0004] In recent years, various diseases caused by inflammation, especially chronic inflammation, have seriously threatened human health, and traditional anti-inflammatory drugs have various toxic side effects, so finding safe and non-toxic anti-inflammatory drugs has become the key to research. As a small molecule active compound of natural origin, peptides usually have different biological activities due to different structures, such as antioxidant, antibacterial, anti-inflammatory, anti-tumor, hypoglycemic activity, etc. Among them, anti-inflammatory active peptides are a class of active peptides that can regulate the production and development of inflammation. They are safe and non-toxic and are considered to be safe alternatives to anti-inflammatory drugs. These anti-inflammatory peptides can be used to develop anti-inflammatory functional foods or become alternatives to chemical synthetic drugs. From the perspective of food safety, natural anti-inflammatory substances have unparalleled advantages over synthetic anti-inflammatory drugs and have become a research hotspot. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an intestinal absorbable anti-inflammatory polypeptide.

[0006] The technical problem that the present invention also aims to solve is to provide a method for preparing an intestinal absorbable anti-inflammatory polypeptide.

[0007] The final technical problem to be solved by the present invention is to provide an application of an anti-inflammatory polypeptide in the preparation of a drug for regulating inflammation.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides an anti-inflammatory polypeptide, the amino acid sequence of which is Gln-Phe-Gln-Phe.

[0009] The present invention also includes a method for preparing the anti-inflammatory polypeptide, which comprises obtaining the polypeptide by solid phase chemical synthesis.

[0010] The process of obtaining the anti-inflammatory polypeptide of the present invention specifically includes the following steps: digesting the raw material rich in milk fat globule membrane protein in vitro to obtain the milk fat globule membrane protein digestion solution and freeze-drying it; then absorbing it with a cell model to obtain a transport sample, enriching the polypeptide, filtering it with a water filter membrane, and performing sequence analysis using HPLC-MS / MS, and then entrusting Nanjing Jie Peptide Biotechnology Co., Ltd. to obtain the synthetic polypeptide using solid phase chemical synthesis.

[0011] The present invention also includes the use of the anti-inflammatory polypeptide or its preparation in the preparation of medicines, dairy products, animal feeds, health products or health foods for treating or preventing inflammation.

[0012] The inflammation model in the application is a model of inflammation obtained by stimulating cells with IL-1β.

[0013] Wherein, the cells include Caco-2 cells.

[0014] Wherein, the application includes down-regulating the expression level of IL-6, IL-8, TNF-α and / or MCP-1 to prevent or treat inflammation.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0016] 1. The present invention prepares polypeptides by in vitro digestion of raw materials rich in milk fat globule membrane protein and Caco-2 cell transport. Through Caco-2 cell layer transport, the complete peptide segment QFQF from gastrointestinal digestion is detected.

[0017] 2. The tetrapeptide QFQF of the present invention constructs an intestinal cell inflammation model based on interleukin-1β-stimulated Caco-2 cells, and has a significant inhibitory effect on intestinal cells.

[0018] 3. The anti-inflammatory tetrapeptide QFQF of the present invention has a good effect on regulating the generation and development of inflammation, has the potential to relieve inflammation, can be used to prepare anti-inflammatory drugs or health products for preventing inflammation-related diseases, and can also be used in combination with other health products or food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of milk fat globule release and milk fat globule membrane composition;

[0020] Figure 2 This is a process flow chart of the method for preparing the anti-inflammatory polypeptide derived from milk fat globule membrane protein of the present invention;

[0021] Figure 3 Total ion chromatogram for Caco-2 cell transport and absorption;

[0022] Figure 4 is the MS / MS spectrum of the peptide QFQF (Gln-Phe-Gln-Phe);

[0023] Figure 5 The figure shows the effect of QFQF on IL-6 mRNA expression in Caco-2 cells induced by IL-1β;

[0024] Figure 6 The figure shows the effect of QFQF on IL-8 mRNA expression in Caco-2 cells induced by IL-1β;

[0025] Figure 7 The figure shows the effect of QFQF on TNF-α mRNA expression in Caco-2 cells induced by IL-1β;

[0026] Figure 8 The figure shows the effect of QFQF on the expression of MCP-1 mRNA in Caco-2 cells induced by IL-1β;

[0027] Fig. 9 The figure shows the effect of QFQF on IL-1β mRNA expression in Caco-2 cells induced by IL-1β. DETAILED DESCRIPTION

[0028] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0029] Pepsin in the examples of the present invention was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., item number: P6322-100g. Lipase was purchased from Beijing Solebow Technology Co., Ltd., item number: L8623; double antibody and DMEM high glucose medium were purchased from Nanjing Houbai Biotechnology Co., Ltd., and serum FBS was purchased from Gibco, Australia. HBSS buffer was purchased from Hank, concentration 1×, pH 7.0-7.4.

[0030] Example 1 Preparation of Gln-Phe-Gln-Phe polypeptide

[0031] 1) Preparation of milk fat globule membrane protein hydrolysate: Use double distilled water to prepare a milk fat globule membrane protein hydrolysate with a concentration of 2% (the milk fat globule membrane protein powder used is Hilmar-7500 milk fat globule membrane protein powder, manufacturer: Hilmar Company, USA), heat it at a constant temperature of 37°C and stir it to fully dissolve it, adjust the pH to 4 with 1.0 mol / L HCl solution, place the hydrolysate in a 37°C constant temperature shaking water bath, add pepsin with a final concentration of 0.125% and lipase with a final concentration of 0.135% for hydrolysis, and continuously add 1.0 mol / L HCl solution was used to maintain the pH value of the solution at a constant value of 4, and the reaction time was 90 minutes; then the digest was adjusted to a pH value of 7 with a 1.0 mol / L NaOH solution, 0.68% potassium dihydrogen phosphate and 0.18% bile salt were added, and the reaction device was continued to be placed in a 37°C constant temperature shaking water bath, and a final concentration of 0.1% trypsin was added for hydrolysis. During the enzymatic hydrolysis, 1.0 mol / L NaOH solution was continuously added to maintain the pH value of the solution at a constant value of 7, and the reaction time was 120 minutes. After the above reaction was completed, the digested samples were collected. After the samples were collected, the enzymes were immediately inactivated in a boiling water bath for 5 minutes, cooled to room temperature, and freeze-dried to obtain the breast milk fat globule structure emulsion gastrointestinal digestion polypeptides for use.

[0032] 2) Add 20% (v / v) fetal bovine serum (FBS) to DMEM medium (purchased from Boster Biotech), and then add 1% (v / v) of a mixed double antibody consisting of 100U / mL penicillin and 100μg / mL streptomycin. The cell culture incubator provides the required environment for the growth of Caco-2 cells (purchased from Nanjing KeyGen Biotech Development Co., Ltd.), and the incubator conditions are set at 37°C and contain 5% CO2. Change the medium and pass the cells in time according to the cell growth.

[0033] 3) Caco-2 cell model simulates small intestinal absorption: cultured Caco-2 cells were inoculated into the TransWell chamber at a density of 2×10 5 cells / mL, then add 0.5mL of cell suspension to the AP side (Transwell plate chamber), add 1.5mL of DMEM culture medium containing 10% FBS (containing 1% penicillin / streptomycin double antibody) to the BL side (Tranwell plate large chamber), and culture in a 37°C, 5% CO2 incubator. Change the culture medium every other day in the first week of the culture cycle, and then change the medium every day. The culture cycle is 21 days. Dissolve the breast milk fat globule structure emulsion gastrointestinal digestion polypeptide collected in step (1) in HBSS solution to prepare a 10μg / mL active peptide solution, add it to the AP side of the transwell chamber, the volume is 0.5mL, and add 1.5mL of HBSS buffer to the BL side. Place the Transwell plate in a 37°C, 5% CO2 incubator for 4h. After the transport is completed, take out the transport sample from the BL side, enrich it, and filter it through a 0.45μm water filter to obtain the sample.

[0034] Implementation 2: Peptide sequence analysis of samples after Caco-2 transport, comprising the following steps:

[0035] The peptide structure was identified by liquid chromatography-mass spectrometry, the chromatographic column was packing: Reprosil-Pur120C18-AQ (75 μm id×250 mm, inner diameter 1.9 μm), the mobile phase A was ultrapure water containing 0.1% formic acid, the mobile phase B was an aqueous solution containing 0.1% formic acid and 80% acetonitrile, and the specific chromatographic conditions were: 0-2 min, 8% B; 2-35 min, 8-28% B; 35-55 min, 28-40% B; 55-56 min, 40-95% B; 56-66, 95% B. The mass spectrometry conditions were as follows: the full scan range of the mass spectrometry was 100-1500 m / z, the primary mass spectrometry resolution was set to 120000, AGC was Standard, and MaximumIT was 20 ms; the secondary mass spectrometry resolution was set to 15000, AGC was Standard, and MaximumIT was 22 ms, and the peptide fragmentation collision energy was set to 30. The raw data of mass spectrometry detection were generated, and the total ion current was as shown in the figure. Figure 3 shown.

[0036] Example 3 Synthesis and Verification

[0037] From the peptides screened in Example 2, a peptide QFQF was selected for solid phase synthesis (Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize the synthetic peptide using solid phase chemical synthesis, and the obtained synthetic peptide was a polypeptide solid powder with a purity of >85%). It was marked as synthetic peptide QFQF. Figure 4 This is the MS image of peptide QFQF.

[0038] Example 4 Study on the anti-inflammatory activity of the tetrapeptide QFQF

[0039] 1. Sample processing

[0040] 10 mg of QFQF polypeptide was accurately weighed using an analytical balance, and 1 mL of DMSO solution was added to obtain a mother solution with a concentration of 10 mg / mL, which was filtered through a 0.22 μm filter membrane and stored in a -80°C refrigerator for later use. When used, 10% DMEM culture medium was added to dilute it to different concentration gradients (0, 1, 10, 50 μg / mL).

[0041] 2. Construction of inflammation model

[0042] The frozen human colon cancer cells (Caco-2) were taken out of liquid nitrogen, immersed in a 37°C water bath to thaw, transferred to a sterile centrifuge tube, centrifuged and discarded the supernatant, and fresh 10% FBS DMEM culture medium (containing 1% penicillin / streptomycin double antibody) was added to evenly disperse the cells in the culture medium, and the culture bottle was placed in a 37°C, 5% CO2 incubator for 24 hours. The culture medium needs to be replaced in time when Caco-2 cells are cultured. The specific operation is as follows: the upper culture medium is aspirated and discarded, and an appropriate amount of physiological saline is added to rinse twice (about 90s each time), and then fresh culture medium is added. Caco-2 cells need to be digested before use. The specific operation is as follows: 0.25% trypsin solution is added to digest the originally attached cells (about 60s), and the real-time state of the cells is observed under a microscope until a certain cell gap appears, the digestion solution is discarded, and fresh culture medium is added and mixed evenly by blowing, and then plated for use. Caco-2 cells were treated with IL-1β inflammatory factor to establish an inflammatory model. The specific operation was as follows: cells in the logarithmic growth phase were taken and inoculated into a 6-well plate, with 3×10 cells per well. 5 Culture for 24 hours. After 24 hours, the culture medium was aspirated and replaced with serum-free DMEM culture medium for 4 hours to keep the cells in a co-culture state. Except for the blank control group, 100 μL of 0, 1, 10, and 50 μg / mL peptide solutions were added to each well in turn and cultured in a 37°C, 5% CO2 incubator for 24 hours. Afterwards, except for the blank control group, 100 μL of 1 μg / mL proinflammatory factor (interleukin-1β, 1 μg interleukin-1β dissolved in 1 mL PBS buffer) solution was added to other wells to stimulate Caco-2 cells for 24 hours. The culture medium was aspirated from the 6-well plate, and then 1 mL of PBS (concentration 1×, PH7.4) was added to the cell scraper, the supernatant was discarded after centrifugation at 5000g for 5 minutes, and the cells were stored in a -80°C refrigerator.

[0043] 3. Determination of inflammatory factors

[0044] 1) Extraction of total RNA

[0045] Take the cells stored in a -80°C refrigerator in step 2, and extract total RNA according to the RNA extraction kit of Nanjing Huapu Biotechnology Co., Ltd.

[0046] 2) Reverse transcription of RNA

[0047] According to the instructions of the reverse transcription kit (HiScript III RT Super Mix for qPCR), the reverse transcription reaction was performed with 0.98 μg template RNA reaction system in 20 μL.

[0048] 3) Primer design

[0049] The reaction primers IL-1β, IL-6, IL-8, TNF-α, and MCP-1 were synthesized by GenScript Biotech.

[0050]

[0051] 4) Real-time fluorescence quantitative PCR

[0052] According to the instructions of the real-time fluorescence quantitative kit (ChamQ Universal SYBR qPCR Master Mix), the reaction system was modified to 10 μL. Interleukin-1β (IL-1β) is one of the important pro-inflammatory cytokines that can promote the secretion of a variety of pro-inflammatory mediators. Figures 5 to 9 It can be seen that under the stimulation of IL-1β alone, the expression levels of IL-6, IL-8, TNF-α, MCP-1 and IL-1β in Caco-2 cells were higher than those in the blank group. Figure 5 The results showed that the mRNA expression level of IL-6 after IL-1β stimulation was about 2.37 times that of the blank group. The mRNA expression level of IL-6 in cells pretreated with 50μg / mLQFQF for 24h and then stimulated with IL-1β was significantly reduced (p<0.05), about 1.20 times that of the blank group, which was about half of the expression level after simple LPS stimulation. Figure 6 The results showed that the IL-8 expression of cells stimulated with IL-1β alone was about 2.28 times that of the blank group. When IL-1β was added to the cells after pretreatment with 50 μg / mL QFQF, the expression of IL-8 was significantly reduced (p<0.0001), which was about 1 / 2 of the IL-8 expression in the group stimulated with LPS alone. Figure 7 It can be seen that the expression of TNF-α mRNA in Caco-2 cells after stimulation with IL-1β alone was significantly enhanced compared with the blank group (p<0.05), which was about 1.85 times that of the blank group. In comparison, the expression of TNF-α mRNA in Caco-2 cells that had been intervened by QFQF in advance was significantly reduced after IL-1β stimulation (p<0.01). Figure 8 , 9 It was also shown that QFQF pretreatment significantly reduced the enhanced expression of MCP-1 and IL-1β mRNA in Caco-2 cells stimulated by IL-1β, indicating that QFQF has a certain immune cell regulatory effect, can inhibit the release of proinflammatory cytokines caused by IL-1β, and effectively inhibit inflammation.

[0053] In summary, the tetrapeptide QFQF of the present invention has a short amino acid sequence, a simple structure, convenient synthesis, safety and non-toxicity, and can effectively inhibit the expression of inflammatory factors and has a significant preventive and therapeutic effect on diseases caused by future microbial infections.

Claims

1. An anti-inflammatory polypeptide, characterized in that The amino acid sequence of the anti-inflammatory polypeptide is Gln-Phe-Gln-Phe.

2. The method for preparing the anti-inflammatory polypeptide according to claim 1, characterized in that: The method comprises obtaining the product by solid phase chemical synthesis.

3. Use of the anti-inflammatory polypeptide or its preparation according to claim 1 in the preparation of medicines, dairy products, animal feeds, health products or health foods for treating or preventing inflammation.

4. The use according to claim 3, characterized in that: The inflammation model used in the application is a model of inflammation obtained by stimulating cells with IL-1β.

5. The use according to claim 4, characterized in that: The cells include Caco-2 cells.

6. The use according to claim 4, characterized in that: The application includes down-regulating the expression level of IL-6, IL-8, TNF-α and / or MCP-1 to prevent or treat inflammation.

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