Bran coat polypeptide GR-12 with immunoregulation function and application of bran coat polypeptide GR-12

Abstract

The invention discloses a bran coat polypeptide GR-12 with an immunoregulation function and an application of the bran coat polypeptide GR-12, and the amino acid sequence of the bran coat polypeptide is Gly-Gln-Pro-Trp-Pro-Pro-Ala-Ser-Phe-Ala-Cys-Arg (GQPWPPASFACR, which is called GR-12 for short). Experiments find that the GR-12 can significantly enhance the proliferation activity and phagocytic activity of macrophages RAW264.7 and promote the macrophages RAW264.7 to secrete cytokines; meanwhile, it is found that the GR-12 can significantly enhance the immunomodulatory function of an immunosuppressive mouse induced by cyclophosphamide, specifically, the body weight, thymus index and spleen index of the immunosuppressive mouse are significantly recovered; and the secretion of cytokines and immune globulins in mouse serum is obviously increased. The bran coat polypeptide GR-12 is expected to be further researched and developed into a related medicine product with an immune regulation function, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and specifically relates to a grain bran polypeptide GR-12 with immunomodulatory function and an application thereof. Background Art

[0002] The immune system is a key system in the human body that maintains body stability and physiological balance. It can not only identify and eliminate foreign pathogens, but also monitor and eliminate damaged or abnormal cells in the body, thereby protecting us from diseases. In recent years, due to people's unhealthy lifestyles, there are also some endocrine diseases, such as diabetes, hypothyroidism, etc., which can easily cause immune disorders in the body and induce various immune-related diseases. Therefore, the research and development of pharmaceutical products that improve the body's immunity has always been a hot topic in the medical community. Plant peptides can exert biological activities that are beneficial to human health, especially in immune regulation. There have been many reports. Therefore, plant peptides can be used as an important screening source for the development of pharmaceutical products related to the prevention and treatment of immune disorders.

[0003] Millet is a traditional Chinese crop, and bran is a byproduct produced during millet processing. Bran is rich in nutrients, especially its protein content is as high as 18%, and its protein digestibility, essential amino acid composition and hypoallergenicity are significantly better than millet, and it has high potential for medical development; research has found that bran protein peptide extracts can exert biological activity of immunomodulation, such as: Yu Shujia et al. found that bran polypeptide (MBPE) can enhance the phagocytic ability of leukocytes in immunosuppressed mice, promote the development of immune organs, and increase the content of antibodies in serum hemolysin, thereby improving the body's immune ability. However, it is still unclear about the specific components of bran peptides that play an immunomodulatory function. The present invention is based on in vitro simulation of the gastrointestinal digestive system to digest and enzymatically hydrolyze bran protein extracts to obtain bran protein digestion products, and screens out a polypeptide GR-12 with immunomodulatory function, which lays a foundation for its further development of related pharmaceutical products for regulating immunity. Summary of the invention

[0004] The purpose of the present invention is to provide a grain bran polypeptide GR-12 with immunomodulatory function and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] First, the amino acid sequence and physicochemical properties of the bran polypeptide GR-12 must be determined, and it must be chemically synthesized; the immunomodulatory activity of GR-12 in vitro must be evaluated using macrophage RAW264.7 cells; and then the immunomodulatory activity of GR-12 in vivo must be evaluated by establishing a mouse immunosuppression model. The bran polypeptide GR-12 provided by the present invention is expected to be further developed into a new type of immunomodulatory health product and / or medicine.

[0007] The specific contents are:

[0008] In the first aspect, the present invention provides a grain bran polypeptide GR-12 with immunomodulatory function, wherein the amino acid sequence of the polypeptide is Gly-Gln-Pro-Trp-Pro-Pro-Ala-Ser-Phe-Ala-Cys-Arg (GQPWPPASFACR, referred to as GR-12), and its chemical structure is as follows Figure 1 .

[0009] Furthermore, the virtual software Toxin Pred and Aller TOP v.2.0 were used to predict the toxicity and allergenicity of GR-12, and the results showed that the grain bran polypeptide GR-12 was non-toxic and non-potentially allergenic.

[0010] Furthermore, the grain bran polypeptide GR-12 was obtained by artificial chemical synthesis (see Example 2), and its purity was identified to be ≥ 98%.

[0011] In the second aspect, the present invention provides an application of a grain bran polypeptide GR-12 with immunomodulatory function, wherein the grain bran polypeptide GR-12 can significantly enhance the immune activity in macrophages RAW264.7, specifically, after the addition of GR-12, the proliferation activity and phagocytic activity of macrophages RAW264.7 cells can be significantly enhanced, and the secretion of cytokines TNF-α, IL1-β, IL-6, and NO can be promoted.

[0012] Furthermore, the bran polypeptide GR-12 can significantly improve the immunity of immunosuppressed mice induced by cyclophosphamide, which is specifically manifested in that GR-12 can significantly improve the spleen and thymus indexes of immunosuppressed mice and repair tissue damage of the spleen and thymus of mice, significantly enhance the secretion of mouse serum cytokines TNF-α, IL1-β, IL-6, and NO, and promote the production of immunoglobulins IgG and IgM in mouse serum.

[0013] In a third aspect, the present invention provides a health product and / or medicine with immunomodulatory function, comprising the above-mentioned grain bran polypeptide GR-12.

[0014] Compared with the prior art, the bran polypeptide GR-12 provided by the present invention has the following advantages: significant immunomodulatory function, no toxicity and potential allergenicity; GR-12 has a small molecular weight, is easily absorbed by the body, and has high bioavailability. In summary, GR-12 can be used as an immunomodulatory active ingredient and further developed into a new type of immunomodulatory health product or medicine, with considerable application potential and broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : GR-12 chemical structure;

[0016] Figure 2 :Effects of GR-12 on the proliferation activity of macrophage RAW264.7;

[0017] Figure 3 :The effect of GR-12 on the phagocytic activity of macrophage RAW264.7;

[0018] Figure 4 :The effect of GR-12 on the release of cytokines from macrophages RAW264.7;

[0019] Figure 5 :Effects of GR-12 on body weight and organ indexes in immunosuppressed mice;

[0020] Figure 6 :Effects of GR-12 on spleen and thymus tissue morphology in immunosuppressed mice;

[0021] Figure 7 :Effects of GR-12 on serum cytokine and immunoglobulin secretion in immunosuppressed mice; DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail in combination with specific embodiments. The specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] The process, conditions, reagents, test methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no particular restrictions. The test methods without specifying specific conditions in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0025] All experimental results in the following examples are expressed as mean ± standard deviation (Mean ± SD), and the differences between the groups were compared by one-way analysis of variance (ANOVA), and the "Duncan" test was selected for significance analysis. * indicates that there is a significant difference between the blank group and each group (p < 0.05), and ** indicates that there is an extremely significant difference between the blank group and each group (p < 0.01).

[0026] Example 1: Evaluation of toxicity and potential allergenicity of GR-12

[0027] The GR-12 structure was created using Chem Draw Professional 20.0. Figure 1 As shown. ToxinPred software and Aller TOP v.2.0 software were used to predict the toxicity and potential allergenicity of GR-12. Toxin Pred software used machine learning technology support vector machine (SVM) to develop a computer simulation model, using various motifs from toxic peptides for toxicity prediction to predict the safety of protein peptides. Aller TOP v.2.0 software converts protein peptides into uniform equal-length vectors based on the automatic cross-covariance (ACC) of protein sequences. By describing the hydrophobicity, molecular size, helix formation tendency, relative abundance of amino acids and β-chain formation tendency of amino acids, the training set formed contains 2427 known allergens and 2427 non-allergens from different species. The k nearest neighbor algorithm (kNN) was used to classify protein peptides to predict potential allergenicity. The prediction results are: GR-12 is non-toxic and non-potentially allergenic, with a molecular weight of 1316.48Da.

[0028] Example 2: Chemical synthesis and purity of GR-12

[0029] The chemical synthesis of polypeptides is to obtain the target polypeptide molecule by the method of directional formation of amide bonds according to the known amino acid sequence. The present invention applies the method of solid phase polypeptide synthesis (SPPS), uses an insoluble solid phase carrier to fix the amino terminus, and directional connects amino acids to form an extension of the peptide chain to synthesize the grain bran polypeptide GR-12 (GQPWPPASFACR) (purity ≥98%).

[0030] The main process of synthesis includes the following points:

[0031] 1. Resin treatment: Take an appropriate amount of resin, swell and activate it in a reactor, then add the treated amino acid solution to react at an appropriate temperature;

[0032] 2. Removal of protecting groups: Add Pip / DMF to remove the Fmoc protecting group, and vacuum dry to remove the solvent;

[0033] 3. Washing the resin: Add DMF4 solution to the resin, wash it repeatedly several times, and vacuum dry it to remove the solvent;

[0034] 4. Resin detection: Place the resin in a test tube containing the detection reagent, place the test tube in high-temperature water for a few seconds, and observe whether the resin in the tube changes color. If there is a change, it proves that the Fmoc group has been successfully removed;

[0035] 5. Amino acid condensation: Add the amino acid solution to be connected in the reactor according to the sequence, add the coupling reagent, shake evenly to make it fully react, and vacuum dry to remove the solvent;

[0036] 6. Peptide synthesis: Repeat steps 2-5 according to the sequence until the peptide is synthesized.

[0037] 7. Polypeptide dissociation: Add the mixed dissociation solution into the polypeptide solid reactor, collect and remove a large amount of solvent, and then add anhydrous ether to the residual liquid to form a white flocculent precipitate.

[0038] 8. Peptide collection: Under a certain temperature and pressure, remove impurities by centrifugation, vacuum dry the precipitate, and freeze the obtained polypeptide powder for storage.

[0039] Example 3: In vitro evaluation of the immunomodulatory function of the grain bran polypeptide GR-12

[0040] 1. Experimental Methods

[0041] 1.1 Effect of GR-12 on the proliferation activity of macrophage RAW264.7

[0042] Mouse macrophage RAW264.7 in the logarithmic growth phase was inoculated with 100 μL of cell suspension (5,000 cells / well) in a 96-well plate and pre-cultured in an incubator for 24 h. After the cells adhered to the wall, the culture supernatant was discarded, and the non-adherent cells were washed with PBS. Different concentrations of GR-12 diluted with complete culture medium were added, and LPS (5 μg / mL) was used as a positive control. Six replicate wells were set for each group and placed at 37°C and 5% CO 2 Incubate in an incubator for 24 h; add 10 μL of CCK-8 solution to each well of the 96-well plate, incubate in the incubator for 4 h, and measure the absorbance at 450 nm using a microplate reader.

[0043]

[0044] Where: A 1 A is the absorbance measured after adding different concentrations of GR-12 or LPS and incubating for 24 hours; 2 is background PBS; A 3 The absorbance is measured after adding PBS and incubating for 24 h.

[0045] 1.2 Effect of GR-12 on the phagocytic activity of macrophages RAW264.7

[0046] The cell plating and drug addition methods were the same as those in 1.1. After culturing for 24 hours, PBS buffer was added to wash the cells and the suspended cells were removed. 100 μL of 0.1% neutral red solution was added to each well and reacted in a cell culture incubator for 3 hours. The neutral red solution was discarded and PBS buffer was added to wash the cells 3 times. 100 μL of cell lysis solution (glacial acetic acid: anhydrous ethanol = 1:1, v / v) was added to each well and allowed to stand overnight. The absorbance at 450 nm was measured using an ELISA reader.

[0047]

[0048] Where: A 1 A is the absorbance measured after adding different concentrations of GR-12 or LPS and incubating for 24 hours; 2 The absorbance is measured after adding PBS and incubating for 24 h.

[0049] 1.3 Effect of GR-12 on cytokine release from macrophages RAW264.7

[0050] The cell plating and drug addition methods were the same as those in 1.2, and the cells were cultured for 24 h. The cell culture medium was collected and centrifuged at 1000 r / min for 5 min to obtain the supernatant. According to the instructions of the kit, the cytokine detection kit was used to detect the content of TNF-α, IL1-β, IL-6, and NO in the cell culture medium.

[0051] 2. Experimental results

[0052] (1) GR-12 significantly enhances the proliferation activity of macrophage RAW264.7

[0053] like Figure 2 It can be seen that with the increase of GR-12 concentration, the proliferation activity of macrophage RAW264.7 gradually increased. When the active peptide concentration increased to 80μM, the cell growth rate slowed down. GR-12 has the function of promoting the growth of macrophage RAW264.7 cells and is not cytotoxic, so it can be used for subsequent cell experiments. And within a certain concentration range, bran active peptides can significantly promote the proliferation of macrophage RAW264.7 cells, and as the concentration increases, the effect becomes more obvious. When the GR-12 concentration is 40μM, the proliferation effect is most significant.

[0054] (2) GR-12 significantly promoted the phagocytic activity of macrophages RAW264.7

[0055] The phagocytic ability of macrophages can reflect immunity to a certain extent, such as Figure 3As shown, with the increase of GR-12 concentration, the phagocytic activity of macrophages RAW264.7 was enhanced. When the GR-12 concentration increased to 40 μM, the phagocytic activity of macrophages was significantly enhanced. Therefore, the high and low concentrations of the drug in subsequent experiments were set to 20 μM and 40 μM.

[0056] (3) GR-12 significantly promotes cytokine secretion in macrophages RAW264.7

[0057] Macrophages promote host immune defense against diseases and bacteria by increasing the production of cytokines, e.g. Figure 4 As shown, GR-12 can significantly promote the secretion of cytokines TNF-α, IL1-β, IL-6, and NO by macrophages RAW264.7 in a concentration-dependent manner, indicating that GR-12 has good immunomodulatory activity.

[0058] Example 4: In vivo evaluation of the immunomodulatory function of the grain bran polypeptide GR-12

[0059] 1. Animal source: BALB / c male mice; 6 weeks old, 18-22g

[0060] 2. Rearing conditions and feed: Ordinary level, environmental temperature conditions are 22±2℃, humidity conditions are 60±5%, indoor lighting simulates day and night, and 12-hour light and dark cycle.

[0061] 3. Experimental animal grouping: After one week of adaptive feeding, all mice were randomly divided into 4 groups (n=10), namely control group (C), model group (M), positive control group (PC) and grain bran peptide drug group (GR-12). Except for the control group, the mice in other groups were intraperitoneally injected with cyclophosphamide (Cy) at a dose of 80 mg / kg for 3 consecutive days to establish a mouse immunosuppression model. After successful modeling, the drug group was given GR-12 at a concentration of (50 mg / kg·BW) every day; the positive control group was given a positive drug levamisole hydrochloride (LH) at a concentration of (50 mg / kg·BW) every day; the control group and the model group were given an equal amount of normal saline every day. After continuous gavage for 19 days according to the dosage of each group, the mice were killed by cervical dislocation, and the internal organs, blood and intestinal contents were collected for subsequent experiments.

[0062] 4. Detection indicators:

[0063] 4.1 Effects of GR-12 on body weight and organ indexes in immunosuppressed mice

[0064] The weight of mice is one of the indicators to measure the health status of animals. During the experiment, the weight of mice was measured every three days.

[0065] The spleen and thymus are important components of immune organs and play an important role in the body's immune regulation. The organ index can also reflect the health of the body. Under normal circumstances, the ratio of each organ to body weight is relatively stable. After the construction of the immunosuppression model, the weight of the damaged organs will change, and the organ index will also change. After the experiment, the mice were killed, the thymus and spleen were removed, and the excess tissue on the surface was removed. The surface blood was absorbed with filter paper and then weighed to calculate the spleen index and thymus index.

[0066] Spleen index = spleen weight (mg) / body weight (g); thymus index = thymus weight (mg) / body weight (g).

[0067] 4.2 Effects of GR-12 on spleen and thymus tissue morphology in immunosuppressed mice

[0068] The spleen and thymus separated from mice were fixed in neutral formalin fixative to maintain the original morphological structure of the tissue, prevent endogenous lysozyme from dissolving and destroying the tissue and cells, and inhibit the growth of bacteria and fungi. After the tissue was fixed in the fixative, the fixative was discarded and the neutral formalin fixative was washed with distilled water. Then, paraffin embedding, paraffin tissue sectioning, tissue section staining, sealing and pathological section scanning were performed for section observation.

[0069] 4.3 Effects of GR-12 on serum cytokine and immunoglobulin secretion in immunosuppressed mice

[0070] Whole blood samples of mice were collected by eye sampling and put into centrifuge tubes. The samples were centrifuged at 4°C and 3000 rpm / min for 15 min. The supernatant was collected as the serum sample of mice. ELISA kits were used to detect the levels of cytokines TNF-α, IL1-β, IL-6, NO and immunoglobulins IgG and IgM in mouse serum.

[0071] 5. Experimental Results

[0072] (1) GR-12 significantly improved the body weight, spleen and thymus indexes of immunosuppressed mice

[0073] like Figure 5 As shown in the figure, after the injection of cyclophosphamide (Cy), the body weight of mice in group M decreased significantly and was significantly lower than that of normal mice, indicating that the modeling was successful; 19 days after the intragastric administration of GR-12 and positive drugs (levamisole hydrochloride, LH), the body weight of mice in group GR-12 and group PC was significantly higher than that of group M, and the body weight of mice in group GR-12 recovered very well, which was the same as that of the positive control group (PC) ( Figure 5A). Under the action of the immunosuppressant Cy, compared with the C group, the spleen index of the M group increased due to the enlarged spleen, and the thymus index of the M group decreased significantly, indicating that the cyclophosphamide-induced immunosuppressive mouse model was successful. Compared with the M group, the spleen and thymus indexes of the PC group and the GR-12 group were significantly restored, and the thymus index of the GR-12 group was better than that of the PC group ( Figure 5 B, C). This indicates that GR-12 significantly improved the body weight, spleen index, and thymus index of immunosuppressed mice.

[0074] (2) GR-12 effectively improves spleen and thymus tissue damage in immunosuppressed mice

[0075] The spleen is the main site of specific immunity in the body, mainly composed of red pulp, white pulp and marginal zone. The marginal zone is the main site for initiating immune response. Figure 6 As shown in the figure, the spleen morphology of group M was significantly enlarged compared with that of group C, while the spleen morphology of group PC and group GR-12 was restored ( Figure 6 A); HE staining results of spleen tissue showed that the marginal zone of spleen tissue in group C was obvious and regular, the boundary between red pulp and white pulp was clear, and the white pulp was deeply stained, indicating that there were a large number of lymphocytes, while the white pulp and red pulp of spleen tissue in group M were disorderly and severely deformed, the boundary between the two was unclear, and the marginal zone was almost invisible, indicating that Cy could destroy the structure of spleen tissue and lead to decreased immunity of mice. Compared with group M, the spleen tissue structure of mice in group GR-12 and group PC was significantly improved. It can be clearly seen that the boundary between red pulp and white pulp gradually became clear, the area of ​​white pulp increased, and the lymphocytes were dense. In addition, the boundary between white pulp and red pulp in group GR-12 was clearer and more regular in shape, the area of ​​white pulp was larger, and the color was darker, indicating that the number of lymphocytes increased significantly, indicating that GR-12 can effectively improve the tissue damage of spleen caused by Cy, and the recovery effect is better than that of positive drug LH ( Figure 6 B).

[0076] The thymus is an important part of the immune system. It consists of a cortex and medulla. The cortex is composed of a large number of dense lymphocytes and reticular cells, and the medulla contains a large number of reticular epithelial cells and a small number of lymphocytes. Thymic bodies can be seen in the medulla. Figure 6 As shown in the figure, the thymus volume in group M was significantly smaller than that in group C, while the thymus size in groups PC and GR-12 was restored ( Figure 6C); The results of HE staining of thymic tissue showed that the thymic tissue of group C showed a clear dividing line between the cortex and medulla, the cortex was thicker, there were a large number of closely arranged lymphocytes, and the staining was darker, while the cells of the medulla were relatively sparse and stained lighter, and thymic bodies could be seen distributed in the medulla. In the thymic tissue of group M, the cortical area became thinner and smaller, and it was difficult to find thymic bodies, indicating that Cy can destroy the structure of thymic tissue, damage immune cells, and lead to decreased immunity of mice. Compared with group M, the thymic tissue structure of mice in group GR-12 and group PC was significantly improved, the cortex was significantly thicker, and the boundary between the cortex and medulla was clear, and thymic bodies could be clearly seen. The thymic tissue of group GR-12 had a thicker cortex than that of group PC, and the boundary between the cortex and medulla was clearer, and thymic bodies were clearly visible, indicating that the number of lymphocytes increased significantly. Therefore, GR-12 showed better therapeutic effect in restoring the damage to the thymus caused by Cy ( Figure 6 D).

[0077] (3) GR-12 promotes serum cytokine and immunoglobulin secretion in immunosuppressive mice

[0078] Cytokines are soluble extracellular peptides or glycoproteins synthesized and secreted by immune cells and non-immune cells. They play a vital role in immune response and are an important indicator for measuring the body's immunity. Figure 7 As shown in the figure, compared with group C, the levels of TNF-α, IL1-β, IL-6, and NO in the serum of mice in group M were significantly reduced, indicating that cyclophosphamide can significantly reduce the immunoregulatory function of mice. Compared with group M, the levels of TNF-α, IL1-β, IL-6, and NO in the serum of mice in group GR-12 and group PC were significantly increased, and the function of GR-12 in promoting the secretion of TNF-α, IL-6, and NO in mice was better than that of the positive drug LH( Figure 7 A, B, C, D). Immunoglobulin is an important biomarker of humoral immunity. It is synthesized and secreted by B cells after they differentiate and mature into plasma cells, and participates in the humoral immunity of the whole body. Figure 7 As shown in the figure, compared with group C, the levels of IgG and IgM in the serum of mice in group M were significantly reduced; compared with group M, the levels of both immunoglobulins in the serum of mice in group GR-12 were significantly increased ( Figure 7 E, F). These results indicate that GR-12 can promote the release of serum cytokines and immunoglobulin secretion in mice and has good immunoregulatory activity.

[0079] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of protection of the invention. It should be pointed out that, for ordinary technicians in this field, the above-mentioned embodiments can also be subjected to several deformations, combinations and changes without departing from the concept of the present invention, which all belong to the protection scope of this patent.

Claims

1. A grain bran polypeptide GR-12 with immunomodulatory function, characterized in that: The amino acid sequence of the grain bran polypeptide GR-12 is Gly-Gln-Pro-Trp-Pro-Pro-Ala-Ser-Phe-Ala-Cys-Arg.

2. Use of the cereal bran polypeptide GR-12 as claimed in claim 1 in the preparation of health products or medicines with immunomodulatory function.

3. A health product with immune regulation function, characterized in that Contains the grain bran polypeptide GR-12 as claimed in claim 1.

4. A drug with immunomodulatory function, characterized in that Contains the grain bran polypeptide GR-12 as claimed in claim 1.

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