A rice anti-hypoxia polypeptide and its application

Abstract

The present invention belongs to the field of biomedicine, and relates to a polypeptide, in particular to a rice anti-hypoxia polypeptide and its application. The amino acid sequence of the rice anti-hypoxia polypeptide is shown in SEQ ID No.1. The polypeptide length is 36 amino acids, 1-10 (IMADTTGRIP) and 35-36 (SR) are random coils, accounting for 33.33%; 11-34 (LWLIGTVTGIAVIGLIGVFFYGSY) is an α-helix, accounting for 66.67%. The polypeptide can significantly improve the survival ability and physiological function of cells and organisms in hypoxic environments. Under hypoxic conditions, the mitochondrial function indicators (such as ATP generation) of cells treated with polypeptides in high, medium and low dose groups increased by 113%, 103% and 69% respectively, and the cell survival rate increased by 98%, 85% and 57%.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a polypeptide, in particular to a rice anti-hypoxia polypeptide and application thereof. Background Art

[0002] Hypoxia is a common problem in many physiological and pathological processes, which seriously affects human health and life activities. In order to reduce the damage caused by hypoxia, an effective measure is to inject or take anti-hypoxia or hypoxia and anti-fatigue drugs. At present, there are two main categories of anti-hypoxia or hypoxia and anti-fatigue drugs on the market. One category is Western medicine, including acetazolamide, methazolamide, dexamethasone, medroxyprogesterone, theophylline, and sumatriptan; the other category is plants and extracts with antioxidant active ingredients, including ginkgo and its extracts, horse tooth extracts, rhodiola rosea and its extracts, turnip polysaccharides, salvia miltiorrhiza and its preparations, Yunnan lily extracts of the genus Macrophylla, snow lotus and its extracts, ginsenosides, quercetin, etc. The former chemical drugs have a certain effect on alleviating the symptoms caused by hypoxia, but the side effects are large, and the drugs are highly accumulated in organs and easily induce serious immune reactions, so their actual use is greatly limited; the latter plant natural products are mostly derived from precious Chinese medicinal materials, and the extraction, separation and purification processes are complicated, resulting in high drug prices. Therefore, the best choice for developing drugs with small side effects, definite efficacy and high cost performance is to develop drugs that are both edible and medicinal and anti-hypoxic or hypoxic, and anti-fatigue. The application with publication number CN115300604A discloses princess sea anemone polypeptides and their applications for anti-stress, anti-hypoxia and anti-oxidation; the application with publication number CN114989247A discloses a yak milk residue anti-hypoxia or hypoxia, anti-fatigue peptide and its preparation method and application. The raw material of the polypeptide is yak milk residue at an altitude of 4000m on the Tibetan Plateau, which is rare; princess sea anemone and yak milk residue are both derived from animals and their by-products, and the single source is not easy to prepare in large quantities; in order to reduce the cost of anti-hypoxia polypeptides and improve the anti-hypoxia effect, this application has conducted in-depth exploration. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a rice anti-hypoxia polypeptide and application thereof.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A rice anti-hypoxia polypeptide, named: RAEP-Q0D998, and its amino acid sequence is shown in SEQ ID No. 1. In its secondary structure, the main chain skeleton of the polypeptide rises in a spiral around an axis and is stabilized by intramolecular hydrogen bonds parallel to the spiral axis. The 1st to 10th amino acids and the 35th to 36th amino acids are random coils, accounting for 33.33%; the 11th to 34th amino acids are alpha-helices, accounting for 66.67%.

[0006] The secondary structure includes α-helix 17%, β-sheet 27%, random coil 17%, and β-turn 21%.

[0007] The preparation method of the above rice anti-hypoxia polypeptide comprises the following steps:

[0008] Raw material preparation: Select high-quality rice as raw material, remove impurities, and crush it to facilitate the subsequent peptide extraction process.

[0009] Peptide extraction: Select a solid-liquid ratio of 1:10 (rice flour: 25°C water), keep stirring, adjust the pH value to 9 with 4% sodium hydroxide solution, and then use alkaline protease (addition amount relative to solid 0.3‰, enzyme activity 200000 u / g) for enzymatic hydrolysis for 2-4 hours (end point judgment is pH value does not change within 30 minutes), so that the polypeptide is released from the rice flour and dissolved in water. Subsequently, centrifuge (3000 rpm) to separate and obtain rice residue and supernatant.

[0010] Alcohol precipitation: Add 75 wt% ethanol to the supernatant to precipitate the polypeptide, then centrifuge (3000 rpm) to separate the precipitated polypeptide and discard the aqueous phase.

[0011] Purification: The above peptides are further purified by means of countercurrent filtration (countercurrent chromatography) to remove other impurities such as organic matter, sugars and lipids.

[0012] Drying: The purified rice polypeptide is subjected to freeze spray method to obtain polypeptide powder.

[0013] Circular dichroism (CD) identification of secondary structure: The secondary structure of antioxidant peptides was analyzed using a JASCO J-1500 circular dichroism spectrometer and determined by chiral scanning. In this method, the sample was a 0.2 mg / mL protein solution prepared with ultrapure water. The CD spectral data was smoothed and preprocessed using spectra manager software, saved and uploaded to the http: / / dichroweb.cryst.bbk.ac.uk website, with a wavenumber range of 190 to 240 nm, and the CONTIN algorithm was used to evaluate the relative content of the secondary structure for verification.

[0014] HPLC-MS peptide identification: First, the sample was redissolved in 50 mM NH4HCO3, and then reduced, alkylated, and desalted. The treated sample was then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain the raw file of the original mass spectrometry result. The raw file was analyzed by PEAKS Studio10.6 software, matched with the database, and then alphafoid-predicted to obtain the relevant identification results and structural information.

[0015] The application of the rice anti-hypoxia polypeptide in the preparation of anti-hypoxia drugs.

[0016] A rice anti-hypoxia polypeptide drug, wherein the amino acid sequence of the rice anti-hypoxia polypeptide is shown as SEQ ID No.1.

[0017] The present invention has the following beneficial effects:

[0018] The present application provides a rice anti-hypoxia polypeptide, which can significantly improve the survival ability and physiological function of cells and organisms in hypoxic environments. The polypeptide of the present invention has the advantages of natural source, high safety, and significant effect. Specifically, under cellular hypoxia, the survival rate of cells treated with high, medium, and low doses of polypeptides increased by 98%, 85%, and 57%, respectively, and the increase in intracellular energy (mitochondrial function indicators such as ATP generation) of hypoxic cells was 113%, 103%, and 69%, respectively. The effect is significant and can be widely used in medical, sports, aerospace and other fields. In the medical field, in animal experiments, the survival time of mice in the experimental groups treated with high, medium, and low doses of polypeptides was extended by 97.04%, 83.38%, and 31.12%, respectively, compared with the experimental group mice not gavaged; the brain tissue and liver tissue of mice recovered better than the control group, with fewer damaged cells, effectively protecting the body from hypoxic damage, and can be used to prepare drugs for the treatment of hypoxia-related diseases (such as cardiovascular diseases, cerebrovascular diseases, etc.). In the field of sports, after hypoxic mice were treated with high, medium and low doses of peptides, different doses of rice peptides could reduce the level of anaerobic glycolysis in brain tissue under hypoxic environment compared with the control group, effectively protecting the body from hypoxic damage. It can be seen that rice peptides can help athletes improve their ability to resist hypoxia and enhance their athletic performance during high-altitude training or competition. In the field of aerospace, it can provide protection for astronauts' survival in space environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be 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 paying creative work.

[0020] Figure 1 is the tertiary structure of the polypeptide, where a is the standard 3D diagram of the polypeptide; b is the 3D diagram with side chain amino acids.

[0021] Figure 2 The effect of peptides on cell survival under different hypoxia time.

[0022] Figure 3 The effect of different peptide concentrations on cell activity.

[0023] Figure 4 HE-stained sections of mouse hippocampal tissue; A: control group, B: experimental group, C: experimental group (L), D: experimental group (M), E: experimental group (H).

[0024] Figure 5 HE-stained sections of mouse liver tissue; A: control group, B: experimental group, C: experimental group (L), D: experimental group (M), E: experimental group (H).

[0025] Figure 6 This is the effect of polypeptide on the MDA content in brain tissue of normobaric hypoxic mice.

[0026] Figure 7 The effect of polypeptide on H2O2 content in brain tissue of normobaric hypoxic mice.

[0027] Figure 8 This is the effect of the polypeptide on the LDH activity in the brain tissue of normobaric hypoxic mice.

[0028] Fig. 9 This is the effect of polypeptide on ATP in brain tissue of normobaric hypoxic mice. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Cell lines: Choose cell lines that are more sensitive to hypoxia, such as cardiomyocytes.

[0032] Culture medium: Complete culture medium for H9C2 rat cardiomyocytes.

[0033] Hypoxic culture device: cell hypoxia incubator (Model PH-min). Example

[0034] The preparation method of rice anti-hypoxia polypeptide comprises the following steps:

[0035] Raw material preparation: Select high-quality rice as raw material, remove impurities, and crush it to facilitate the subsequent peptide extraction process.

[0036] Peptide extraction: Select a solid-liquid ratio of 1:10 (rice flour: 25°C water), keep stirring, add alkaline protease (0.3‰ relative to solid content, enzyme activity 200,000 u / g) for 2-4 hours (end point judgment is pH value does not change within 30 minutes), release the peptide from the rice flour and dissolve it in water. Then, centrifuge (3000rpm) to obtain rice residue and supernatant.

[0037] Alcohol precipitation: Add 75 wt% ethanol to the supernatant to precipitate the polypeptide, then centrifuge to obtain the precipitated polypeptide and discard the aqueous phase.

[0038] Purification: The above peptides are further purified by countercurrent filtration (countercurrent chromatography) to remove other impurities such as organic matter, sugars and lipids.

[0039] Drying: The purified rice polypeptide is subjected to freeze spray method to obtain polypeptide powder.

[0040] Identification:

[0041] Circular dichroism (CD) identification of secondary structure: The secondary structure of antioxidant peptides was analyzed using a JASCO J-1500 circular dichroism spectrometer and determined by chiral scanning. In this method, the sample was a 0.2 mg / mL protein solution prepared with ultrapure water. The CD spectral data was smoothed and preprocessed using spectra manager software, saved and uploaded to the http: / / dichroweb.cryst.bbk.ac.uk website, with a wavenumber range of 190 to 240 nm, and the CONTIN algorithm was used to evaluate the relative content of the secondary structure for verification.

[0042] HPLC-MS peptide identification: First, the sample was redissolved in 50 mM NH4HCO3, and then reduced, alkylated, and desalted. The treated sample was then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain the raw file of the original mass spectrometry result. The raw file was analyzed by PEAKS Studio10.6 software, matched with the database, and then alphafoid-predicted to obtain the relevant identification results and structural information.

[0043] The amino acid sequence of the obtained polypeptide is as follows: structure (primary, secondary and tertiary structure) and its characterization.

[0044] Primary structure: The amino acid sequence of RAEP-Q0D998 is shown in SEQ ID No. 1: IMADTTGRIPLWLIGTVTGIAVIGLIGVFFYGSYSR.

[0045] In the secondary structure, the main chain skeleton of the polypeptide rises in a spiral around an axis and is stabilized by intramolecular hydrogen bonds parallel to the spiral axis, of which α-helix accounts for 66.67% and Random accounts for 33.33%.

[0046] Specifically: amino acids 1-10 (IMADTTGRIP) and 35-36 (SR) are random coils, accounting for 33.33%; amino acids 11-34 (LWLIGTVTGIAVIGLIGVFFYGSY) are α-helices, accounting for 66.67%.

[0047] The tertiary structure is Figure 1 shown.

[0048] Application example 1: Cell hypoxia experiment

[0049] The polypeptides prepared in the examples were formulated into oral preparations (the concentrations of the polypeptides were 1 μg / ml, 5 μg / ml, and 10 μg / ml, respectively) and cell experiments were performed:

[0050] Experiments with different hypoxia levels: Set up multiple hypoxia gradients, such as 5%, 10%, and 15% oxygen concentration environments, and culture peptide-treated and untreated cells respectively. Detect indicators such as cell survival number, cell cycle, and expression of apoptosis-related proteins to evaluate the effect of peptides under different hypoxia severities.

[0051] In the anti-hypoxia cell experiment, the cell survival rate was calculated by MTT method:

[0052] ;

[0053] Calculation principle: Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function.

[0054] Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its absorbance value (OD value) can be measured at a specific wavelength (usually 490-570nm) using a microplate reader, which can indirectly reflect the number of living cells.

[0055] In the experiment, the OD value of the control group (normally cultured cells) was 1.2, the OD value of the experimental group without added peptide (only culture medium and detection reagent) was 0.1, and the OD value of the experimental group (cells treated with added peptide) was 0.8.

[0056] The cell viability was calculated using the MTT assay: .

[0057] 1. Peptide anti-hypoxic cell experiment

[0058] The effect of peptides on cell survival under different hypoxia times mainly studies the protective effect of peptides on cell survival under different hypoxia times.

[0059] Experimental steps:

[0060] Cell culture: cells were seeded in culture plates and cultured to an appropriate density (2×10 5 / ml).

[0061] Group processing:

[0062] Control group: cells were cultured under normal oxygen concentration (21% O2) without adding peptides.

[0063] Experimental group: cells were cultured in an oxygen-deficient environment (5% O2) without the addition of peptides.

[0064] Experimental group 1 (low-dose L): cells were cultured in a hypoxic environment (5% O2) and low-concentration L (1 μg / ml) peptide was added.

[0065] Experimental group 2 (medium dose M): cells were cultured in a hypoxic environment (5% O2) and medium concentration M (5 μg / ml) peptide was added.

[0066] Experimental group 3 (high-dose H): cells were cultured in a hypoxic environment (5% O2) and high-concentration H (10 μg / ml) peptide was added.

[0067] Hypoxia treatment: The cells in the experimental group were placed in a hypoxic incubator, and the hypoxia time was set to 6 hours, 12 hours, and 24 hours.

[0068] Cell survival assay: At each time point, the cell survival rate was detected using the MTT assay ( Figure 2 ), the survival rates of the cells in the experimental group were increased by 98%, 85% and 57% at high, medium and low doses, respectively.

[0069] 2. Effects of peptides on energy metabolism of hypoxic cells

[0070] Experimental steps:

[0071] Cell culture: cells were seeded in culture plates and cultured to an appropriate density (2×10 5 / ml).

[0072] Group processing:

[0073] Control group: cells were cultured under normal oxygen concentration (21% O2) without adding peptides.

[0074] Experimental group: cells were cultured in an oxygen-deficient environment (5% O2) without the addition of peptides.

[0075] Experimental group 1 (low-dose L): cells were cultured in a hypoxic environment (5% O2) and low-concentration L (1 μg / ml) peptide was added.

[0076] Experimental group 2 (medium dose M): cells were cultured in a hypoxic environment (5% O2) and medium concentration M (5 μg / ml) peptide was added.

[0077] Experimental group 3 (high-dose H): cells were cultured in a hypoxic environment (5% O2) and high-concentration H (10 μg / ml) peptide was added.

[0078] Hypoxia treatment: The cells in the experimental group were placed in a hypoxia incubator and treated with hypoxia for 12 hours. The energy metabolism test results were as follows: Figure 3 As shown: The intracellular ATP content was detected using an ATP detection kit. The intracellular ATP content in the high-dose group of the experimental group increased by 88% compared with the experimental group.

[0079] The intracellular ATP concentration in the experimental group increased by 113%, 103%, and 69% at high, medium, and low doses, respectively, and there was no significant difference among high, medium, and low doses ( Figure 3 ).

[0080] Application Example 2: Animal Experiment (Anti-hypoxia Animal Experiment Method)

[0081] 1. Animal Experiment Grouping and Modeling

[0082] Fifty 6-8 week old KM male mice with an initial body weight of 25-30 g were selected and purchased from Zhengzhou Huaxing Animal Center. The animals were kept in an animal room with a stable environment: temperature of 22±2℃, humidity of 50%-60%, and a light-dark cycle of 12 h:12 h. They were kept in separate cages, 4 per cage, with free access to food and water. In view of the fact that there were statistically significant differences between the high-dose group and the medium-dose group in the previous cell experiment, the medium-dose intervention was used in the animal experiment.

[0083] After one week of adaptive feeding, the experimental animals were randomly divided into 5 groups according to their body weight:

[0084] Control group: The mice were raised under normal oxygen concentration (21% O2) and no peptide was added.

[0085] Experimental group: raised in an oxygen-deficient environment (5% O2) without the addition of peptides.

[0086] Experimental group 1: raised in anoxic environment (5% O2) and supplemented with low concentration of L (1 μg / BW) peptide.

[0087] Experimental group 2: raised in an oxygen-deficient environment (5% O2) and supplemented with medium concentration M (5 μg / BW) peptide.

[0088] Experimental group 3: raised in an oxygen-deficient environment (5% O2) and supplemented with high concentration of H (10 μg / BW) peptide.

[0089] Each group contained 10 animals. During the experiment, the test samples were administered daily by gavage. The control group and the experimental group were administered with normal saline, while the other groups were administered with test solution of corresponding concentrations. During the experiment, the animals were weighed weekly. At the same time, the overall condition of the animals was observed, including food intake, coat color, mental state, and daily activities. Continuous gavage intervention lasted for 30 days. After the intervention, all animals were subjected to a normobaric hypoxia tolerance experiment to evaluate the tolerance of different treatment groups to hypoxia.

[0090] Refer to the test method of hypoxia tolerance health function. One hour after the last gavage, each mouse was placed in a ground-mouth bottle containing soda lime, and the bottle mouth was sealed with vaseline to ensure airtightness. In view of the increase in the volume of mice fed for 30 days, a 500 mL wide-mouth bottle was used, and 10 g of soda lime was placed in it. After the experiment started, the timing started immediately, and the mouse stopped breathing as the observation endpoint, and the specific time of death of each mouse due to hypoxia was recorded in detail. Once the mouse died, it was immediately dissected, and its heart, liver, spleen, lung, kidney, cecum and brain tissues were quickly removed and placed on ice to prevent tissue degradation. Subsequently, these tissues were weighed and properly stored in a -80°C refrigerator for subsequent biochemical analysis and experimental research.

[0091]

[0092] 2. Determination indicators and characterization methods

[0093] (1) Organ index

[0094] The weight of the mice was recorded before dissection. The liver tissue was taken and rinsed with pre-cooled saline. The liver tissue was absorbed with filter paper and weighed. The organ index was calculated according to formula (1).

[0095] (1)

[0096] (2) Pathological tissue section (H&E) staining: After weighing, the mouse brain tissue and liver tissue were fixed in 4% paraformaldehyde fixative, embedded in paraffin, washed with tap water, thawed and fixed with frozen sections, treated with high-definition constant staining pretreatment solution, washed with hematoxylin, differentiated with differentiation solution, washed with tap water, blued with blueing solution, rinsed with running water, stained with eosin, dehydrated and sealed, examined under a microscope, and image acquisition and analysis.

[0097] (3) Determination of protein concentration in tissue homogenate: When the experimental mice stopped breathing, the brain tissues were immediately removed and stored at -80°C. The concentrations were determined using the Nanjing Jiancheng Protein Quantification Assay Kit.

[0098] (4) Determination of lipid peroxidation-related indicators in the brain tissue of normobaric hypoxic mice: Strictly follow the operating procedures of the test kit instructions to measure lipid peroxidation-related indicators MDA and H2O2 in the brain tissue of normobaric hypoxic mice.

[0099] (5) Determination of energy metabolism-related indicators in normobaric hypoxic mice: Strictly follow the operating procedures in the test kit instructions to measure the LDH and ATP contents in the brain tissue of normobaric hypoxic mice.

[0100] (6) Statistical analysis: The values ​​are expressed as “mean ± standard deviation”. The data were analyzed using IBM SPSS 25.0 statistical software. P <0.05 was considered statistically significant, and different letters indicate statistically significant differences. Data were plotted using GraphPad Prism 8 software.

[0101] 3. Experimental Results

[0102] (1) Effects on mouse body weight and organ indexes

[0103] As shown in Table 1, statistical analysis of the initial and final body weights of mice revealed no significant differences in body weight among the groups of mice ( P >0.05), so the experimental materials had no effect on the body weight of mice.

[0104] Table 1 Effects of peptides on mouse body weight

[0105]

[0106] As can be seen from Table 2, there were no significant differences in the lung, kidney, and spleen organ indexes among the groups, but there were differences in the liver index between the control group and the other four groups ( P <0.05, but not statistically significant; cardiac index was different between the control group and other groups ( P <0.05), but it was not statistically significant.

[0107] Table 2 Effects of peptides on mouse organ indexes

[0108]

[0109] (2) Effect of peptides on the survival time of normobaric hypoxic mice

[0110] Lack of oxygen in the body is a highly stressful stimulus that affects many metabolic activities in the body, especially the body's oxidative function. Ultimately, this condition may cause dysfunction of key organs such as the heart and brain and may lead to death.

[0111] Table 3 Effect of peptides on mouse survival time

[0112]

[0113] The polypeptide of the present invention showed anti-hypoxic activity during the process of normal pressure confinement hypoxia, as shown in Table 3. Compared with the experimental group, the polypeptide was able to prolong the survival time of normal pressure confinement hypoxia mice to the maximum extent, with a significant difference ( P <0.05); and the high, medium and low dose control groups can prolong the survival time of normal pressure closed hypoxia mice, and the survival time is significantly higher than that of the experimental group ( P <0.05), and there was a dose-dependency. The survival time of mice in the high, medium, and low dose experimental groups was extended by 97.04%, 83.38%, and 31.12% compared with the mice in the non-oral gavage experimental group.

[0114] (3) Effects of peptides on pathological sections of normobaric hypoxic mice

[0115] Brain tissue sections: Figure 4 As shown, the hippocampal tissue of the experimental group mice showed normal morphological structure, the cells were arranged in an orderly and complete manner, the neurons were clearly morphological, most of them were round, the nuclei were large and round, the color was lighter, and they were centered in the cells. The boundary between the nucleus and the cytoplasm was clear and easy to see. However, after hypoxia treatment, the hippocampal tissue changed significantly, showing tissue edema and looseness. These changes were mainly cell edema, with swelling of the cell bodies of some nerve cells, while others showed a shrinkage of the nucleus. Compared with the control group and experimental group 2, the cell morphology of experimental group 3 returned to normal, and there were fewer damaged cells. Therefore, the polypeptide has a certain protective effect on the brain tissue of normobaric hypoxic mice.

[0116] Liver tissue sections: Figure 5 As shown in the figure, the liver cell structure of the mice in the control group was intact, the morphology was normal, there were no fat droplets, and there was no congestion in the liver sinusoids; the nuclei were large and round, and there was little necrosis. The boundaries between the liver cells of the mice in the experimental group were blurred, the cells showed turbid swelling, and many vacuoles were formed. Acute hypoxia caused damage to the liver tissue of the mice. The number of cells in experimental groups 1, 2, and 3 increased, the vacuoles were less, and the cell morphology was well restored. Therefore, the polypeptide has a certain protective effect on the liver cells of mice with normobaric hypoxia.

[0117] (4) Protective effect of peptides on lipid peroxidation in the brain of normobaric hypoxic mice

[0118] ① Effect of rice polypeptide on MDA content in brain tissue of normobaric hypoxic mice: MDA, as a natural product of lipid oxidation, is an important biomarker reflecting the degree of lipid peroxidation. Its increase is positively correlated with the enhancement of lipid peroxidation. Figure 6 It can be seen that compared with the control group, experimental group 1 (L), experimental group 2 (M), and experimental group 3 (H) can reduce the MDA content in the brain tissue of mice; there is no significant difference between experimental group 2 (M) and experimental group 3 (H), but there is no statistical difference; it can be seen that an appropriate dose of rice polypeptide can effectively reduce the level of MDA in the brain tissue of hypoxic mice, thereby reducing the body's lipid peroxidation and protecting the body from oxidative stress damage ( Figure 6 ).

[0119] ② Effect of rice polypeptide on H2O2 content in brain tissue of normobaric hypoxic mice: Hydrogen peroxide (H2O2) is a common byproduct of reactive oxygen metabolism. Increased H2O2 levels may mean increased production of reactive oxygen species in the body, leading to increased oxidative stress and aggravated lipid peroxidation damage. Figure 7 It can be seen that compared with the control group, the experimental group, experimental group 1 (L), experimental group 2 (M), and experimental group 3 (H) can all reduce the H2O2 content in the mouse brain tissue, and the difference in the medium dose is significant ( P <0.05), but there was no statistical difference.

[0120] (5) Effects of peptides on energy metabolism in normobaric hypoxic mice

[0121] ①Effects of polypeptides on LDH activity in brain tissue of normobaric hypoxic mice

[0122] Lactate dehydrogenase (LDH) is an enzyme related to anaerobic metabolism. Its activity increases significantly when anaerobic glycolysis is enhanced, becoming one of the important indicators of increased anaerobic glycolysis levels. Figure 8 It can be seen that compared with the control group, the experimental group, experimental group 1 (L), experimental group 2 (M), and control group 4 (H) can reduce the LDH activity in the brain tissue of mice, indicating that different doses of rice polypeptides can reduce the level of anaerobic glycolysis in brain tissue under hypoxic conditions and effectively protect the body from hypoxic damage ( Figure 8 ).

[0123] ②Effects of polypeptides on ATP in brain tissue of normobaric hypoxic mice

[0124] Depend on Fig. 9 It can be seen that both the positive control group and the experimental group increased the ATP content in the mouse brain tissue. Among them, compared with the experimental group, the experimental group 2 (L), experimental group 2 (M), and experimental group 2 (H) significantly increased the ATP content in the mouse brain tissue ( P<0.05). This result shows that rice peptides can maintain normal energy metabolism in the brain tissue of hypoxic mice.

[0125] Implementation effect analysis

[0126] The results of the normal pressure hypoxia tolerance experiment showed that different doses of rice peptides had different degrees of improvement on the hypoxia tolerance of mice; the experimental group could significantly prolong the survival time of hypoxic mice ( P <0.05). The slice results show that the peptide has a certain protective effect on the hippocampal tissue cells and liver cells of normobaric hypoxic mice. Compared with the experimental group, the normobaric hypoxic mice were able to reduce the MDA content in their brain tissue after intervention with high-dose and medium-dose peptides. Different doses of peptides can significantly reduce the H2O2 content in the brain tissue of mice, and the high-dose effect is particularly significant ( P <0.05). At the same time, all doses of peptides can reduce the LDH activity in mouse brain tissue ( P <0.05, can increase the ATP content in mouse brain tissue ( P <0.05). Therefore, the peptide has a significant antioxidant protective effect on mouse brain tissue. The experimental results show that rice peptide has good anti-hypoxia activity and can effectively protect the mouse brain from damage in a closed hypoxic environment at normal pressure, thereby reducing the damage of oxidative stress to the body.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rice anti-hypoxia polypeptide, the amino acid sequence of which is shown in SEQ ID No.

1.

2. The rice anti-hypoxia polypeptide according to claim 1, wherein the polypeptide main chain skeleton in its secondary structure rises in a spiral shape around an axis and is stabilized by intramolecular hydrogen bonds parallel to the spiral axis.

3. The rice anti-hypoxia polypeptide according to claim 2, characterized in that: The 1st to 10th amino acids and the 35th to 36th amino acids of the rice anti-hypoxia polypeptide are random coils, accounting for 33.33%; the 11th to 34th amino acids are α-helices, accounting for 66.67%.

4. Use of the rice anti-hypoxia polypeptide according to any one of claims 1 to 3 in the preparation of anti-hypoxia drugs.

5. The use of the rice anti-hypoxia polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for treating hypoxia-related diseases, characterized in that: The hypoxia-related disease is a cardiovascular disease or a cerebrovascular disease.

6. A medicine comprising the rice anti-hypoxia polypeptide according to any one of claims 1 to 3.

Images