A glycosylated rice residue protein peptide and its application

Abstract

The present invention belongs to the field of biochemistry technology and discloses a glycosylated rice residue protein peptide. The glycosylated rice residue protein peptide is obtained by glycosylation of a rice residue protein-derived immunoactive peptide with a monosaccharide. The amino acid sequence of the rice residue protein-derived immunoactive peptide is any one of SEQ ID NO:3 and SEQ ID NO:6, and the monosaccharide is any one of glucose and mannose. The glycosylated rice residue immunoactive peptide has superior anti-inflammatory and immunomodulatory effects and can be used as an immunomodulator or anti-inflammatory drug to regulate body functions.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and particularly relates to a glycosylated rice residue protein peptide and application thereof. Background Art

[0002] Food-derived immunoreactive peptides play an important regulatory role in both innate and adaptive immune responses, primarily by inducing or regulating cytokine and antibody production, stimulating lymphocyte proliferation, enhancing macrophage phagocytosis, natural killer cell activity, and the body's defenses against invading pathogens, and inhibiting host cell proinflammatory responses to LPS and other bacterial components. These effects are likely mediated by the binding of food-derived immunoreactive peptides to immune cell surface receptors. Rice processing in my country generates a large number of by-products annually, such as broken rice, rice bran, and rice residue. A large number of immunoreactive peptides are present in the enzymatic hydrolysis products of these by-products.

[0003] In the invention patent application previously applied for by the applicant (application number 2023107875183), a short peptide derived from rice dregs protein with excellent immunological activity was discovered and obtained through research. It has excellent immunomodulatory effects, especially excellent anti-inflammatory effects and the ability to promote antigen presentation. It can be used as an immunomodulator to regulate the body's immune function, inhibit inflammation, fight tumors and / or enhance vaccine effects. However, the inventors further discovered that in actual applications, when the active peptide derived from rice dregs protein participates in immunomodulation, it is easily hydrolyzed again by digestive enzymes after entering the body, resulting in the loss of active peptides, which affects its functional characteristics and the effect of immune activity. Summary of the Invention

[0004] The object of the present invention is to provide a glycosylated rice residue protein peptide which has excellent immunomodulatory and anti-inflammatory effects and is not easily hydrolyzed by digestive enzymes.

[0005] The above-mentioned objectives are achieved by the following technical solutions.

[0006] A first aspect of the present invention provides a glycosylated rice residue protein peptide, which is obtained by glycosylation of a rice residue protein-derived immunoactive peptide by monosaccharide; wherein the amino acid sequence of the rice residue protein-derived immunoactive peptide is any one of SEQ ID NO: 3 and SEQ ID NO: 6; and the monosaccharide is any one of glucose and mannose.

[0007] In some embodiments, the amino acid sequence of the rice residue protein-derived immunoactive peptide is shown in SEQ ID NO: 3.

[0008] In some embodiments, the monosaccharide is mannose.

[0009] The second aspect of the present invention provides a use of the glycosylated rice residue protein peptide as described above in the preparation of an immunomodulator.

[0010] In some embodiments, the immunomodulatory agent performs immunomodulation by promoting the expression of MHC II molecules and CD86 protein.

[0011] The third aspect of the present invention provides a use of the glycosylated rice residue protein peptide as described above in the preparation of a drug for inhibiting inflammation.

[0012] In some embodiments, the inhibition of inflammation is achieved by regulating the expression of IL-4, IL-10, IFN-γ and TNF-α.

[0013] The fourth aspect of the present invention provides an immunomodulator, which comprises the glycosylated rice residue protein peptide as described above.

[0014] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the glycosylated rice residue protein peptide as described above, and a pharmaceutically acceptable adjuvant.

[0015] A sixth aspect of the present invention provides a nutritional preparation composition, comprising the glycosylated rice residue protein peptide as described above, and a pharmaceutically acceptable adjuvant.

[0016] In the present invention, the rice residue protein peptide of a specific sequence is glycosylated with a specific monosaccharide to obtain an optimal glycosylated rice residue protein peptide. The glycosylated rice residue protein peptide has a digestive enzyme resistance characteristic and is not easily digested and hydrolyzed by digestive enzymes after entering the body. By analyzing the genes, signal transduction pathways, and antioxidant capacity that are closely related to the regulatory effect of the glycosylated rice residue immune active peptide in the cell, it was found that the glycosylated rice residue immune active peptide has a more excellent anti-inflammatory effect and immunomodulatory effect than conventional unglycosylated rice residue immune active peptide, and can function as an immunomodulator. This provides a further theoretical basis for the deep development of rice processing by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The values of cell viability of DC2.4 cells treated with different concentrations of RPH and MRPs are shown in Table 1. *: p < 0.05, there is a significant difference compared with the control group; **: p < 0.01, there is a very significant difference compared with the control group.

[0018] Figure 2 This is the toxicity analysis of different concentrations of RPH and MRPs on LPS-induced DC2.4 cells.

[0019] Figure 3 These are the morphological images of DC2.4 cells after different treatments.

[0020] Figure 4 This is a graph showing changes in MHCⅡ molecule expression after DC2.4 cells were treated with RPH and MRPs.

[0021] Figure 5 This figure shows the effects of different concentrations of RPH and MRPs on the secretion of cytokine IFN-γ in DC2.4 cells induced by LPS.

[0022] Figure 6 This figure shows the effects of different concentrations of RPH and MRPs on the secretion of TNF-α cytokine in LPS-induced DC2.4 cells.

[0023] Figure 7 This figure shows the effect of different concentrations of RPH and MRPs on the expression of CD86 protein in DC2.4 cells induced by LPS; *: p < 0.05, there is a significant difference compared with the control group.

[0024] Figure 8 This is a graph showing the effects of different concentrations of GI-RPH and GI-RPH-Ma on the cell viability of DC2.4 cells; *: p < 0.05, there is a significant difference compared with the control group; **: p < 0.01, there is an extremely significant difference compared with the control group.

[0025] Figure 9 This is a graph analyzing the toxicity of different concentrations of GI-RPH and GI-RPH-Ma to LPS-induced DC2.4 cells.

[0026] Figure 10 This figure shows the effects of different concentrations of GI-RPH and GI-RPH-Ma on LPS-induced IFN-γ secretion in DC2.4 cells.

[0027] Figure 11 This figure shows the effects of different concentrations of GI-RPH and GI-RPH-Ma on the secretion of cytokine TNF-α in DC2.4 cells induced by LPS.

[0028] Figure 12 It is the chemical parameters and amino acid sequence diagram of rice immune active peptides.

[0029] Figure 13 This is the hydrophilicity result diagram of rice immune active peptide; Figure 13 (A) is the hydrophilicity result diagram of OP1, where Figure 13 (B) is the hydrophilicity result diagram of GAP2. Figure 13 (C) is the hydrophilicity result diagram of PHP1. Figure 13 (D) is a graph showing the hydrophilicity of GAP1.

[0030] Figure 14(A) is the combined spatial simulation of GAP2 and MHC II molecules. Figure 14 (B) Details of the contact between GAP2 and MHC II molecules.

[0031] Figure 15 (A) is the combined spatial simulation of GAP2-Ma and MHC II molecules. Figure 15 (B) Details of the contact between GAP2-Ma and MHC II molecules.

[0032] Figure 16 The results of the effects of different concentrations of glycosylated peptides on the cell viability of DC2.4 cells; *: p < 0.05, there is a significant difference compared with the control group; **: p < 0.01, there is a very significant difference compared with the control group.

[0033] Figure 17 This is the toxicity analysis of different concentrations of glycosylated peptides on LPS-induced DC2.4 cells.

[0034] Figure 18 The figure shows the effect of different concentrations of glycosylated peptides on the secretion of cytokine IFN-γ in DC2.4 cells induced by LPS.

[0035] Figure 19 The figure shows the effect of different concentrations of glycosylated peptides on the secretion of cytokine TNF-α in DC2.4 cells induced by LPS.

[0036] Figure 20 The effect of different concentrations of glycosylated peptides on the secretion of cytokines IL-4 and IL-10 in DC2.4 cells induced by LPS.

[0037] Figure 21 The figure shows the effect of different concentrations of glycosylated peptides on the expression of CD86 protein in DC2.4 cells induced by LPS; *: p < 0.05, there is a significant difference compared with the control group. DETAILED DESCRIPTION

[0038] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0039] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0040] The meanings of the English abbreviations in the following embodiments are shown in Table 1.1.

[0041] Table 1.1 English abbreviations

[0042]

[0043]

[0044] Example 1 Preparation and characterization of glycosylated rice residue protein hydrolysate

[0045] Experimental methods

[0046] Preparation and enzymatic hydrolysis of rice residue protein

[0047] 1.1 Rice residue protein hydrolysis

[0048] Take by weighing 1g freeze-dried rice dregs protein (crude protein content 80%, Zhejiang Hecheng Biotechnology Co., Ltd.) and be disperseed in distilled water with 1:30 (g / mL), in 50 ℃ of lower stirrings 60min, guarantee the abundant hydration of albumen, regulate protein aqueous solution pH to 8 with the NaOH of 1mol / L, and in 50 ℃ water-bath, keep 15min, add trypsin (Bai Lingwei) subsequently, enzyme substrate is than 1:100, enzyme activity 1500U / mg, maintain pH8 in the enzymolysis process, prepare the protein hydrolyzate of 9% by controlling the enzymolysis time, degree of hydrolysis (degrees of hydrolysis, DH) is measured with the pH-stat method, enzymolysis is transferred into rapidly in 90 ℃ of water-baths and heat 10min after finishing, makes enzyme inactivation, after cooling, adjust pH to 7, centrifugal, get supernatant, with the solution freeze-drying, 4 ℃ of preservations, obtain rice dregs protein hydrolyzate, for further analysis.

[0049] 1.2 Preparation of Maillard glycosylated rice residue protein hydrolysate

[0050] The rice dregs protein hydrolysate (RPH) obtained above was dissolved in phosphate buffer solution at 10 mg / mL (pH 7.0). Glucose and mannose were then added to a concentration of 20 mg / mL. The solution was stirred slowly for 1 hour until completely dissolved. The mixture was heated in an 80°C water bath for 90 minutes, with RPH as a control. At the end of glycosylation, the solution was cooled in an ice bath and dialyzed using a 500 Da dialysis bag to remove salts and free sugars. Finally, the solution was lyophilized and stored at 4°C to obtain glycosylated rice dregs protein hydrolysates (MRPs), specifically rice dregs protein hydrolysate-glucose (RPH-Gl) and rice dregs protein hydrolysate-mannose (RPH-Ma), for further analysis.

[0051] 1.3 Preparation of protein hydrolysate from glycosylated rice residue by simulated gastrointestinal digestion

[0052] The sample solution was adjusted to pH 2 with 1 mol / L hydrochloric acid. Then, 11 mg of pepsin was added and incubated at 37°C in a shaker for 1 hour to simulate gastric digestion. Furthermore, the solution pH was adjusted to 7.5 with 1 mol / mL sodium hydroxide, and 11 mg of pancreatin was added. The solution was incubated in a shaker at 37°C for 2 hours. Digestion was stopped by placing the sample in a boiling water bath for 10 minutes. The sample was centrifuged at 16,000 g for 10 minutes, and the supernatant was collected. The solution was lyophilized and stored at 4°C for further analysis.

[0053] 1.4 Determination of branch node degree and browning degree

[0054] Dilute MRPs to a concentration of 10 mg / mL. Mix 200 μL of sample with 4 mL of o-phthalaldehyde reagent. Incubate in a 35°C water bath for 2 minutes, and measure the absorbance of the sample at 340 nm. A blank control group consists of 4.0 mL of OPA and 200 μL of the sample before glycosylation. Prepare the OPA reagent by dissolving 40 mg of o-phthalaldehyde in 1 mL of methanol. Add 25 mL of 0.1 mol / L borax solution, 2.5 mL of 20% SDS solution, and 100 μL of β-mercaptoethanol. Dose to 50 mL with deionized water.

[0055] Grafting degree calculation formula:

[0056] Grafting degree (%) = (A0-A1) / A0×100%

[0057] Where: A0: absorbance of blank sample at 340nm; A1: absorbance of sample at 340nm.

[0058] 1.5 Results Analysis

[0059] Determination of branch degree and browning degree

[0060] Table 1.2 Grafting degree of RPH-Gl and RPH-Ma

[0061] RPH-Gl RPH-Ma 1 57.53％ 59.21％ 2 57.30％ 59.21％ 3 58.65％ 59.21％ DG 57.83％±0.01 59.21％±0

[0062] The Maillard wet-heat method has been widely used to prepare glycosylated protein hydrolysates. The reaction between sugar carbonyl groups and protein free amino groups, and the formation of a Schiff base, results in a decrease in free amino group content. Therefore, the reduction in free amino groups indicates that sugars bind to RPHs during glycosylation. Table 1.2 shows that the DG of RPH-Gl and RPH-Ma is 57.83% and 59.21%, respectively, indicating a significant decrease in the free amino group content of RPHs under wet-heat reaction conditions. Due to the binding of glucose and mannose, the free amino group content of RPHs decreases, leading to an increase in DG. These results confirm the participation of sugars in the Maillard reaction.

[0063] Example 2 Analysis of immunological activity of glycosylated rice residue protein hydrolysate

[0064] Experimental methods

[0065] 2.1 Reagent preparation method

[0066] (1) DMEM high-glucose medium: DMEM high-glucose medium was prepared by mixing 10% FBS, 1% sodium penicillin and streptomycin sulfate (100 U / mL) with 89% DMEM liquid medium. The medium was aliquoted and stored at 4°C for later use.

[0067] (2) 1 mg / mL sample storage solution: Accurately weigh 2 mg of RPH and MPRs and fully dissolve them in 2 mL of sterile cell culture water. After aliquoting, store at -20°C in the dark until use.

[0068] 2.2 Toxicological study of glycosylated rice residue protein hydrolysate

[0069] Mouse bone marrow-derived dendritic cells DC2.4 (Hunan Fenghui) were cultured according to conventional methods.

[0070] The cytotoxicity of the peptides was evaluated in vitro by the effects of RPHs and MRPs on the proliferation of mouse bone marrow-derived dendritic cell line DC2.4. The effects of the samples on DC2.4 cell proliferation were determined using the CCK8 colorimetric assay, and the cytotoxicity of the peptides was then assessed. Pre-cultured logarithmic phase cells were seeded into 96-well plates with 5 replicates per group at a cell density of 2.0 × 10 4 / mL, 100μL cell suspension was inoculated into each well and incubated for 24h to allow the cells to adhere. The culture medium in each well was discarded, and a blank group (only DMEM complete culture medium was added), a model group (adding an equal amount of 10μg / mL LPS solution to stimulate for 24h), a sample group (adding an equal amount of 100, 200, 400, 800μg / mL sample and 10μg / mL LPS intervention for 24h) and a positive control group (adding an equal amount of 10μg / mL IgG and 10μg / mL LPS intervention for 24h) were set up. Six replicates were set for each concentration. After treating the cells, the cell viability was determined using the CCK8 method. The cell viability value was used to evaluate cytotoxicity. The cytotoxicity grading evaluation is shown in Table 2.1. The cell viability value was calculated as follows:

[0071] SI=OD1 / OD0

[0072] OD1: absorbance value of the sample group at 450 nm;

[0073] OD0: absorbance value of blank control group at 450 nm.

[0074] Table 2.1 Cell viability grading evaluation table

[0075]

[0076]

[0077] 2.3 Effects of glycosylated rice residue protein hydrolysate on MHCⅡ molecules in DC2.4 cells

[0078] The DC2.4 cells were diluted and counted at 2×10 5 Plate cells at a density of 1 mL / well onto a laser confocal microplate. Set up three replicates per group, with a volume of 1 mL per well. Incubate in a 37°C, 5% (v / v) CO2 incubator for 24 hours. After cells adhere, plate samples for control, model, and sample groups, add corresponding complete DMEM medium, and incubate for 24 hours. Aspirate the culture medium, wash three times with wash buffer, and add 1 mL of fixative solution for 10 minutes. Remove the fixative solution, wash three times with wash buffer for 3-5 minutes each, and aspirate the liquid. Manually shake several times during washes. Block with blocking buffer for 60 minutes, gently rocking on a rocker. Remove the blocking buffer and apply diluted primary antibody at 4°C for 12 hours. Remove the primary antibody, wash three-5 times with wash buffer for 3-5 minutes each, rocking gently on a rocker between washes. Remove the wash buffer, add 1 mL of diluted fluorescent-conjugated secondary antibody, and incubate in the dark for 60 minutes, rocking gently on a rocker. Recover the fluorescently labeled secondary antibody and wash the slide 3-5 times with washing buffer for 3-5 minutes each time, protecting the slide from light. Gently shake the slide on a shaker between washes. Apply a drop of the anti-fluorescence quenching mounting solution provided in the kit to the slide and cover with the coverslip containing the cells, avoiding air bubbles. Allow the cells to contact the mounting solution, and observe green fluorescence under confocal laser scanning.

[0079] 2.4 Effects of glycosylated rice residue protein hydrolysate on DC2.4 cytokine secretion

[0080] A control group, a model group, and a sample group were set up respectively, and the corresponding DMEM complete medium was added and cultured for 24 hours. The levels of cytokines CD86, IL-6, and TNF-α secreted by DC2.4 cells were determined by ELISA kits.

[0081] 2.5 Effects of simulated gastrointestinal digestion of glycosylated rice residue protein hydrolysate on DC2.4 cytokine secretion

[0082] A control group, a model group, and a sample group were set up respectively, and the corresponding DMEM complete medium was added and cultured for 24 hours. The levels of cytokines CD86, IL-6, and TNF-α secreted by DC2.4 cells were determined by ELISA kits.

[0083] 2.6 Results Analysis

[0084] 2.6.1 Toxicity analysis of glycosylated rice residue protein hydrolysate

[0085] The cell viability value is an indicator to measure the cell-killing ability of active ingredients. Detecting the effect of drugs on cell viability is a prerequisite step in cell research. Only by eliminating the interference of drugs on cell-killing can we further study the effect of drugs on cell regulatory mechanisms. The CCK8 method has become a commonly used method for detecting cytotoxicity due to its advantages such as high sensitivity and good repeatability. In order to explore the protective effects of RPH and MRPs on mouse DC2.4 cells, this example first examined the toxic effects of RPH and MRPs solutions of different concentrations on mouse DC2.4 cells. After confirming that they were non-toxic to DC2.4 cells, their protective effects on cells were further examined. The results are as follows: Figure 1 The experimental results showed that RPH and RPH-Ma showed a concentration-dependent effect on the cell viability of mouse DC2.4 cells within the concentration range of 0-800 μg / mL, with the cell viability value being greater than 75%, and had no toxic effect.

[0086] 2.6.2 Analysis of the cytotoxic effect of glycosylated rice residue protein hydrolysate on LPS-induced DC2.4 cells

[0087] Depend on Figure 2 It can be seen that compared with the control group, the cell viability of the sample group was greater than 95%, the cell viability was good, and RPH and MRPs had no cytotoxicity to DC2.4 cells induced by LPS.

[0088] 2.6.3 Effects of Glycosylated Rice Residue Protein Hydrolysate on DC2.4 Cell Morphology

[0089] like Figure 3 The results showed that when treated with 200 μg / mL RPH and MRPs, DC2.4 cells were semi-adherent, their shapes changed to spindle, star or tadpole shapes, and they had obvious proliferation. Figure 1 and Figure 2 The results analysis showed that the toxicity grades of RPH and MRPs on DC2.4 cells were both qualified, which still proved that the glycosylated rice residue protein hydrolysates had no toxic effects on cells and promoted maturation. Therefore, subsequent experimental studies can be carried out within this concentration range.

[0090] 2.6.4 Effects of glycosylated rice residue protein hydrolysate on MHCⅡ molecules in DC2.4 cells

[0091] MHC II molecules play a crucial role in the immune regulation of DC2.4 cells. MHC II is an important molecule for presenting antigens on the surface of DCs. When MHC II is stimulated externally, it forms more synaptic tails to present antigens produced outside the cell.

[0092] like Figure 4The experimental results showed that compared with the control group, MHCⅡ fluorescence increased significantly in all sample groups except the LPS and RPH-Gl groups, as well as the IgG group. Furthermore, the fluorescence intensity of RPH-Ma in the sample group was significantly greater than that in the other sample groups. Therefore, RPH-Ma can better present antigens.

[0093] 2.6.5 Effect of glycosylated rice residue protein hydrolysate on LPS-induced IFN-γ protein expression in DC2.4 cells

[0094] like Figure 5 As shown in the results, the secretion of IFN-γ increased after LPS treatment; compared with the LPS group, the secretion of cellular inflammatory factor IFN-γ decreased after RPH and MRPs treatment; among them, the IFN-γ content secreted by 200μg / mL RPH-Ma was significantly reduced, and the inhibition of IFN-γ secretion by RPH-Ma at all concentrations was more significant than that by RPH.

[0095] 2.6.6 Effect of glycosylated rice residue protein hydrolysate on the inhibition of LPS-induced TNF-α protein expression in DC2.4 cells

[0096] like Figure 6 As shown, the secretion of TNF-α increased after LPS treatment; compared with the LPS group, the secretion of the anti-inflammatory cytokine TNF-α decreased after RPH and MRPs treatment; RPH and MRPs inhibited the secretion of the pro-inflammatory cytokine TNF-α in DC2.4 cells induced by LPS.

[0097] 2.6.7 Effect of Glycosylated Rice Residue Enzyme Hydrolysate on CD86 Protein Expression in LPS-Induced DC2.4 Cells

[0098] like Figure 7 As shown in the data, after LPS treatment, the expression of CD86 did not increase significantly; compared with the LPS group, the expression of CD86 was significantly increased when treated with 200 μg / mL of RPH and RPH-Gl; 800 μg / mL of RPH-Ma was the most effective in promoting the expression of CD86 in LPS-induced DC2.4 cells, which was 26.767% higher than that of the control group.

[0099] 2.6.8 Analysis of inflammatory and anti-inflammatory factors in glycosylated proteolytic hydrolysates after gastrointestinal digestion

[0100] 2.6.8.1 Toxicity Analysis of Glycosylated Protein Hydrolysates after Gastrointestinal Digestion

[0101] like Figure 8 As shown, the experimental results showed that the cell viability values of GI-RPH and GI-RPH-Ma were greater than 75% in the concentration range of 50 to 800 μg / mL, and there was no toxic effect.

[0102] 2.6.8.2 Analysis of the toxicity of glycosylated proteolytic hydrolysates after gastrointestinal digestion on DC2.4 cells induced by LPS

[0103] like Figure 9 As shown, there was a significant difference compared with the control group, and the cell viability of 50-800 μg / mL was >95%, and the cell viability was good; GI-RPH and GI-RPH-Ma had no cytotoxicity to DC2.4 cells induced by LPS.

[0104] 2.6.8.3 Effect of Glycosylated Proteolytic Hydrolysates After Gastrointestinal Digestion on LPS-Induced IFN-γ Release in DC2.4 Cells

[0105] like Figure 10 As shown in the results, the secretion of IFN-γ increased after LPS treatment; compared with the LPS group, the secretion of inflammatory cytokine IFN-γ decreased after GI-RPH and GI-MRPs treatment; 100 μg / mL GI-RPH-Ma could significantly inhibit the secretion of inflammatory factor IFN-γ in DC2.4 cells induced by LPS compared with the control group.

[0106] 2.6.8.4 Effect of Glycosylated Proteolytic Hydrolysates After Gastrointestinal Digestion on LPS-Induced TNF-α Release in DC2.4 Cells

[0107] like Figure 11 As shown in the results, the secretion of TNF-α decreased after LPS treatment; compared with the LPS group, the secretion of the anti-inflammatory cytokine TNF-α increased after GI-RPH and GI-RPH-Ma treatment; 100 μg / mL GI-RPH-Ma could significantly inhibit the secretion of the pro-inflammatory cytokine TNF-α in DC2.4 cells induced by LPS compared with the control group.

[0108] In summary, RPH and MRPs had no toxic effects on mouse macrophage DC2.4 cells; after LPS treatment, the expression of CD86 was slightly increased; compared with the RPH group, the expression of CD86 was significantly increased when treated with 800 μg / mL RPH-Ma; MRPs promoted the secretion of cytokine IFN-γ and the expression of MHCⅡ and CD86 proteins in DC2.4 cells induced by LPS; MRPs promoted the secretion of cytokine IFN-γ in DC2.4 cells induced by LPS, and MRPs had an anti-inflammatory effect on inflammatory cells.

[0109] Example 3 Study on the immune activity mechanism of glycosylated rice residue protein peptide

[0110] 3.1 Materials

[0111] GAP2 synthetic peptide (GQLLIIPQHYAVVK), OP1 synthetic peptide (VSVVPSAAALVIK), GAP2-Ma synthetic glycopeptide (GQLLIIPQHYAVVK-Mannose), and OP1-Ma synthetic glycopeptide (VSVVPSAAALVIK-Mannose) were obtained from Cellmano Biotech Limited.

[0112] 3.2 Experimental methods

[0113] 3.2.1 Molecular structure and molecular docking screening of immunologically active peptides Glycosylated rice residue protein peptides (hereafter referred to as glycosylated peptides)

[0114] Immune peptides can bind to MHC II molecules on antigen presenting cells (APCs). This immune peptide-MHC II complex is then recognized by T cell receptors and presented to CD4+ T cells, which ultimately promote the participation of these cells in immune responses by enhancing the phagocytic function of macrophages, stimulating lymphocyte proliferation, differentiation and maturation, and secreting cytokines.

[0115] ( https: / / perkinelmerinformatics.com / products / research / chemdraw) were used to draw the two-dimensional molecular structure of the differentially hydrolyzed peptides, which were converted into *pdb suffixes using Chem3D 20.0 software. The three-dimensional structures were used as ligands for molecular docking. The crystal structure of the immune active peptide receptor MHC II molecule (PDB ID: 6BIY) was obtained from Protein Data Bank (PDB, https: / / www.rcsb.org / ). The bioactive peptides in the above hydrolyzed peptides were flexibly docked with the target protein using AutoDock Vina molecular simulation software. The amino acid sequences of the six rice residue immune active peptides that were screened out in the early stage for regulating cytokine release were NEQFQCTGTFVIR (MDP1, SEQ ID NO: 1), RGQLLIIPQHYAVVK (GAP1, SEQ ID NO: 2), GQLLIIPQHYAVVK (GAP2, SEQ ID NO: 3), IPLYQHIANLAGNK (PHP1, SEQ ID NO: 4), INTYANFRDEVLPR (GEP1, SEQ ID NO: 5), VSVVPSAAALVIK (OP1, SEQ ID NO: 6). NO:6). Since glycosylation treatment is required later, four rice residue immunoreactive peptides (GAP1, GAP2, PHP1, and OP1) with lysine as the amino acid terminus were selected for further screening. The complex structure with the highest affinity (minimum energy) was selected according to the default scoring function for analysis and visualization using PyMol 2.5 (http: / / www.pymol.org / ). The hydrogen bonds and interactions between the two-dimensional structures of the complexes were further analyzed using LigPlus (https: / / www.ebi.ac.uk / thornton-srv / software / LigPlus / ). Finally, glycosylated peptides with good immunoreactivity were screened.

[0116] 3.2.2 Reagent preparation method: The experimental method is the same as above.

[0117] 3.2.3 Cell culture: The experimental method is the same as above.

[0118] 3.2.4 Toxicity studies of glycosylated peptides

[0119] The cytotoxicity of the peptides was evaluated in vitro by their effects on the proliferation of mouse bone marrow-derived dendritic cell line DC2.4. The CCK8 colorimetric assay was used to determine the effects of the samples on DC2.4 cell proliferation and to assess the cytotoxicity of the peptides. Pre-cultured logarithmic-phase cells were seeded into 96-well plates with five replicates per group at a cell density of 2.0 × 10 4 / mL, 100μL cell suspension was inoculated into each well and incubated for 24h to allow cells to adhere. The culture medium in each well was discarded, and a blank group (only DMEM complete culture medium was added), a model group (addition of equal amount of 10μg / mL LPS solution for stimulation for 24h), a sample group (addition of equal amount of 0, 0.01, 0.1, 1, 10μg / mL sample and 10μg / mL LPS intervention for 24h) and a positive control group (addition of equal amount of 10μg / mL IgG and 10μg / mL LPS intervention for 24h) were set up. Six replicates were set for each concentration. After treatment of cells, the cell viability was determined using the CCK8 method.

[0120] 3.2.5 Effects of glycosylated peptides on cell surface characteristic molecules in DC2.4 cells

[0121] A control group, a model group, and a sample group were set up respectively, and the corresponding DMEM complete medium was added and cultured for 24 hours. The levels of cytokines IL-4, IL-10, IFN-γ, and TNF-α secreted by DC2.4 cells were determined by ELISA kits.

[0122] 3.2.6 Effects of glycosylated peptides on cytokine release in LPS-induced DC2.4 cells

[0123] A control group, a model group, and a sample group were set up respectively, and the corresponding DMEM complete medium was added and cultured for 24 hours. The levels of cytokines IL-4, IL-10, IFN-γ, and TNF-α secreted by DC2.4 cells were determined by ELISA kits.

[0124] 3.3 Results and Discussion

[0125] 3.3.1 Chemical Parameters and Amino Acid Sequences of Rice Immunoactive Peptides

[0126] like Figure 12 As shown, the four immunoreactive peptides consist of 13-14 amino acids with a molecular weight between 1200 and 1600. GAP2, OP1, and PHP1 are positively charged, while GAP1 is uncharged. Positively charged peptides interact electrostatically with negatively charged bacterial lipopolysaccharide (LPS), making positively charged peptides more beneficial for regulating immune activity.

[0127] 3.3.2 Hydrophilicity and hydrophobicity of rice immunoreactive peptides

[0128] The hydrophilicity and hydrophobicity of rice immunoreactive peptides were determined using the spiral wheel projection method to show the distribution of hydrophobic and hydrophilic amino acids. Figure 13 (A) to (D), where Figure 13 (A) is the hydrophilicity result diagram of OP1, where Figure 13 (B) is the hydrophilicity result diagram of GAP2. Figure 13(C) is the hydrophilicity result diagram of PHP1. Figure 13 (D) is a graph showing the hydrophilicity of GAP1.

[0129] In the figure, circles represent hydrophilic residues, diamonds represent hydrophobic residues, triangles represent potential negatively charged residues, and pentagons represent potential positively charged residues. GAP2, PHP1, and GAP1 showed a stronger hydrophobic effect. Based on the reference that there is a linear correlation between hydrophobicity and the ability of peptides to inhibit LPS-induced responses, hydrophobic peptides may be more conducive to anti-inflammatory effects.

[0130] 3.3.3 Docking analysis of immunoreactive peptides before and after glycosylation and MHCⅡ molecules

[0131] Table 3.1 Docking energy of immunoreactive peptides and MHCⅡ molecules before and after sugar binding

[0132]

[0133] Table 3.1 shows the docking energies of immunoreactive peptides before and after sugar binding to MHC II. While PHP1 and GAP1 have low docking energies before modification, modification does not reduce these energies and may even increase them, making their binding to MHC II less stable, thus impacting immune activity. Mannose binding to GAP2 and OP1 significantly reduces their binding energies, making their binding to MHC II more stable. Furthermore, mannose modification of the four peptides showed lower binding energies than glucose modification. Therefore, GAP2 and OP1 were selected for further study. Figure 14 (A) is the combined spatial simulation of GAP2 and MHC II molecules. Figure 14 (B) is a detailed diagram of the contact between GAP2 and MHCII molecules; Figure 15 (A) is the combined spatial simulation of GAP2-Ma and MHC II molecules. Figure 15 (B) is a detailed diagram of the contact between GAP2-Ma and MHC II molecules. Figure 14 and Figure 15 The binding energy between GAP2 and MHCII Glu55, Ser53, Asn62, Gln9, Arg71, and Thr77 is -10.9 kcal / mol. The binding energy between GAP2-Ma and MHCII Asn-82, Gln9, Arg71, Asn-69, and Asp57 is -14.3 kcal / mol. The glycomodified peptide has a lower docking energy, indicating greater stability. The binding energy between OP1 and MHCII Gln9, Asn62, and Gln70 is -9.1 kcal / mol. The binding energy between OP1-Ma and MHCII Asn82, Gln9, Asn62, and Arg76 is -12.7 kcal / mol. The glycomodified peptide has a lower docking energy, indicating greater stability.

[0134] 3.3.4 Toxicity analysis of glycosylated peptides

[0135] Depend on Figure 16 It can be seen that the experimental results show that GAP2, GAP-Ma, OP1 and OP1-Ma have a concentration-dependent effect on the cell viability of mouse presenting cells DC2.4 in the concentration range of 0.01 to 10 μg / mL, with cell viability values greater than 95% and no toxic effects.

[0136] 3.3.5 Glycosylated peptides have no toxic effect on LPS-induced DC2.4 cells

[0137] like Figure 17 It can be seen that compared with the control group, the cell viability at 0.01-10 μg / mL was greater than 95%, and the cell viability was good; GAP2, GAP-Ma, OP1 and OP1-Ma had no cytotoxicity to DC2.4 cells induced by LPS.

[0138] 3.3.6 Effect of glycosylated peptides on LPS-induced IFN-γ secretion in DC2.4 cells

[0139] Depend on Figure 18 It can be seen that the secretion of IFN-γ increased after LPS treatment; compared with the LPS group, the secretion of the proinflammatory factor IFN-γ increased after treatment with GAP2, GAP-Ma, OP1 and OP1-Ma; compared with GAP2, GAP-Ma significantly inhibited the secretion of the inflammatory factor IFN-γ; compared with OP1, OP1-Ma also significantly inhibited the secretion of the inflammatory factor IFN-γ; GAP2, GAP-Ma, OP1 and OP1-Ma can all inhibit the secretion of the inflammatory factor IFN-γ in DC2.4 cells induced by LPS, and the glycosylated peptides can inhibit the secretion of the inflammatory factor IFN-γ better than the unmodified peptides.

[0140] 3.3.7 Effect of glycosylated peptides on LPS-induced TNF-α protein expression in DC2.4 cells

[0141] Depend on Figure 19 It can be seen that the secretion of TNF-α increased after LPS treatment; compared with the LPS group, the secretion of the proinflammatory factor TNF-α increased after treatment with GAP2, GAP-Ma, OP1 and OP1-Ma; compared with GAP2, GAP-Ma significantly inhibited the secretion of the inflammatory factor TNF-α; compared with OP1, OP1-Ma could also significantly inhibit the secretion of the inflammatory factor TNF-α; GAP2, GAP-Ma, OP1 and OP1-Ma could all inhibit the secretion of the inflammatory factor TNF-α in LPS-induced DC2.4 cells, and the glycosylated peptides could inhibit the secretion of the inflammatory factor TNF-α better than the unmodified peptides.

[0142] 3.2.8 Effect of glycosylated rice residue protein hydrolysate on the expression of IL-4 and IL-10 proteins in LPS-induced DC2.4 cells

[0143] Depend on Figure 20 It can be seen that the secretion of IL-4 and IL-10 increased after LPS treatment; compared with the LPS group, the secretion of the pro-inflammatory factor TNF-α increased after treatment with GAP2, GAP-Ma, OP1 and OP1-Ma; compared with GAP2, GAP-Ma significantly promoted the secretion of anti-inflammatory factors IL-4 and IL-10; compared with OP1, OP1-Ma could also significantly promote the secretion of anti-inflammatory factors IL-4 and IL-10; GAP2, GAP-Ma, OP1 and OP1-Ma could all inhibit the secretion of inflammatory factor TNF-α in DC2.4 cells induced by LPS, and the glycosylated peptides could promote the secretion of anti-inflammatory factors IL-4 and IL-10 more than the unmodified peptides.

[0144] 3.3.9 Effect of glycosylated rice residue protein hydrolysate on CD86 protein expression in LPS-induced DC2.4 cells

[0145] Depend on Figure 21 As shown, LPS treatment increased CD86 expression. Compared with the LPS group, treatment with 10 μg / mL of GAP2-Ma peptide significantly increased CD86 expression. GAP2-Ma promoted CD86 expression in LPS-induced DC2.4 cells. DCs play a key role in the initiation and development of immune responses. Interventions in DC maturation can alter immune-mediated inflammatory responses. Therefore, regulatory DCs are potential therapeutic tools for evaluating various immune hyperreactivity diseases. LPS-stimulated imDCs regulate DCs by upregulating indoleamine 2,3-dioxygenase expression. DCs exhibit high CD11b / c expression and low CD80, CD86, and CD40 expression, and have enhanced antigen capture capacity. DC maturation is promoted by upregulating MHC II, CD80, CD86, CD40, and IL-12p70 secretion. This enhances the ability of mature DCs to stimulate T cell responses, including allogeneic T cell proliferation and activation of IFN-γ-producing CD4+ T cells.

[0146] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A glycosylated rice residue protein peptide, characterized in that: The glycosylated rice residue protein peptide is obtained by glycosylation of a rice residue protein-derived immune active peptide with a monosaccharide; wherein the amino acid sequence of the rice residue protein-derived immune active peptide is SEQ ID NO: 3; the monosaccharide is mannose; and the glycosylated rice residue protein peptide is VSVVPSAAALVIK-Mannose.

2. Use of the glycosylated rice residue protein peptide according to claim 1 in the preparation of a drug for inhibiting inflammation.

3. The use according to claim 2, characterized in that The inflammation is inhibited by regulating the expression of IL-4, IL-10, IFN-γ and TNF-α.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the glycosylated rice residue protein peptide according to claim 1 and a pharmaceutically acceptable adjuvant.

5. A nutritional preparation composition, characterized in that The nutritional preparation composition comprises the glycosylated rice residue protein peptide according to claim 1 and a pharmaceutically acceptable adjuvant.

Images