Coctanoyl polypeptide and application thereof in preparation of medicine for promoting cold acclimatization capability of organism

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to caprylyl polypeptide and application thereof in preparation of a medicine for promoting cold acclimatization capability of an organism. The structure of the octanoyl polypeptide is shown in the specification. On one hand, the absorption and utilization degree of the HNS polypeptide is improved through capryloylation modification, on the other hand, the effect of the HNS polypeptide in promoting heat metabolism of an organism and improving the cold acclimatization function is proved, and an effective intervention medicine for promoting acclimatization metabolism is provided for health guarantee of residents in winter. .

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an octanoyl polypeptide and its use in preparing a drug for promoting the body's cold acclimatization ability. Background Art

[0002] Cold environments threaten human health and performance, causing cold injuries such as frostbite, hypothermia, immersion foot, and trench foot. They can also contribute to the development and progression of cardiovascular and cerebrovascular diseases such as hypertension and stroke. Furthermore, cold exposure can lead to varying degrees of decreased mental performance, including alertness, cognitive ability, and fine motor skills, severely impacting the lives and work of people living in cold regions. Within physiological tolerance limits, prolonged exposure (generally 4-6 weeks) to cold temperatures and repeated exposure to moderate-intensity cold can lead to a series of physiological adaptive changes. These include a gradual weakening of the cold stress response (such as shivering, skin vasoconstriction, and increased heart rate), increased metabolic heat production, and a significant improvement in cold tolerance and antifreeze capacity. This change, also known as cold acclimatization, can reduce the risk of cold injuries and related metabolic diseases. Currently, there is an urgent need to develop effective therapeutic interventions to enhance the body's cold acclimatization capacity.

[0003] Currently, methods for promoting cold acclimatization or effectively improving cold tolerance include the cold tolerance training program in standard GJB2562-96 and intervention drugs. However, in the intervention program, cold tolerance training is intensive and long-term, making it difficult to adhere to. Compared with this, intervention drugs are more convenient and feasible than cold tolerance training. Among them, intervention drugs include cold sensory channel agonists and traditional Chinese medicine compositions. Cold sensory channel agonists stimulate the body's heat metabolism by simulating cold exposure, but this type of drug is highly irritating and has large side effects, which can cause stress damage such as digestive system dysfunction. Compared with cold sensory channel agonists, traditional Chinese medicine compositions are less irritating and have almost no side effects, making them more suitable. However, this type of traditional Chinese medicine composition used to improve the body's cold acclimatization ability has the problems of slow onset and unclear drug efficacy targets. Summary of the Invention

[0004] To address the issues of slow onset and unclear target of efficacy in existing traditional Chinese medicine compositions for improving cold acclimatization, the present invention provides an octanoyl polypeptide drug that promotes cold acclimatization, with a clear target, rapid onset, strong thermogenic effect, and a weak stress response. Based on this, the present invention provides an octanoyl polypeptide and its use in the preparation of a drug for promoting cold acclimatization. To achieve the above objectives, the present invention employs the following technical solutions.

[0005] The present invention provides an octanoyl polypeptide, which is an octanoyl HNS polypeptide, and its structure is as follows: .

[0006] The octanoyl polypeptide provided by the present invention targets the RNA-binding protein HuR and, through post-transcriptional regulation, precisely increases the heat production of metabolic organs, effectively enhancing cold acclimatization. When used, the octanoyl polypeptide provided by the present invention offers the advantages of a clear target, rapid onset of action, strong heat production, and a weak stress response. This addresses the issues of slow onset and unclear target efficacy of current traditional Chinese medicine compositions used to enhance cold acclimatization.

[0007] Furthermore, the preparation method of the octanoyl polypeptide comprises the following steps: The HNS polypeptide with the amino acid sequence shown in SEQ ID NO. 1 was synthesized.

[0008] The octanoyl polypeptide is obtained by performing octanoyl modification on the N-terminal amino acid of the HNS polypeptide.

[0009] HNS is located in the hinge region of the RNA-binding protein HuR. HNS peptides can target and compete with HuR molecules, promoting their transport into the cytoplasm and exerting post-transcriptional regulatory effects. Given the poor cell penetration and short plasma half-life of peptides, the present invention synthesizes an N-terminally octanoyl-modified HNS peptide to improve its in vivo absorption and stability, and to clarify the key role of this octanoyl-modified peptide in cold acclimatization.

[0010] The present invention also provides use of the octanoyl polypeptide in preparing a drug for promoting the body's cold acclimatization ability.

[0011] Furthermore, the drug contains the octanoyl polypeptide as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0012] Furthermore, the medicine includes at least one of tablets, capsules, injections, solutions and ointments.

[0013] Furthermore, the excipient includes any one or more of corn starch, gelatin, hydroxypropyl methylcellulose magnesium stearate, polysorbate and polylactic acid.

[0014] Furthermore, the carrier includes any one or more of liposomes, exosomes, cell vesicles, nanoparticles and gels.

[0015] Furthermore, the medicine is a solution.

[0016] The solution is obtained by mixing the octanoyl polypeptide with a solvent.

[0017] The solvent is any one of physiological saline, phosphate buffer and glucose solution with a concentration of 5 g / 100 mL.

[0018] Furthermore, the concentration of the octanoyl polypeptide in the solution is 20 mg / mL to 50 mg / mL.

[0019] Furthermore, the drugs of the present invention can be administered to the body in a known manner; for example, by intravenous systemic delivery, local injection delivery, or oral delivery to the body; optionally, via intravenous, transdermal, intranasal, mucosal, oral or other delivery methods; such administration can be carried out via a single dose or multiple doses. It is understood by those skilled in the art that the actual dose to be administered in the present invention can vary greatly depending on a variety of factors, such as the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides an octanoylated polypeptide with the advantages of a clear target, rapid onset, strong thermogenic effect, and reduced stress response. This polypeptide can address the slow onset and unclear target of efficacy of current traditional Chinese medicine compositions used to enhance the body's cold acclimatization ability. The octanoylated polypeptide provided herein is an octanoylated HNS polypeptide, a further structurally improved HNS polypeptide. Compared to existing HNS polypeptides, the octanoylated polypeptide provided herein has an octanoylated domain that allows efficient entry into cell and mitochondrial membranes. Therefore, the octanoylated polypeptide provided herein has high in vivo absorption and stability, representing a significant improvement over the prior art.

[0021] 2. The present invention evaluates the key role of octanoyl peptides in cold tolerance, providing new possibilities for the development of drugs that promote cold acclimatization.

[0022] 3. On the one hand, the present invention improves the absorption and utilization of HNS polypeptide through octanoyl modification. On the other hand, it confirms its role in promoting the body's heat metabolism and improving cold acclimatization function, providing an effective acclimatization-promoting metabolic intervention drug for the health protection of residents in winter.

[0023] 4. HNS is a polypeptide molecule that promotes the cytoplasmic shuttling of the RNA-binding protein HuR, helping to facilitate HuR's translocation and localization in the cytoplasm, thereby promoting the expression of its downstream target molecules. Previous research (unpublished) has shown that HuR can bind to thermogenic and fatty acid oxidizing molecules and promote their expression and translation. Based on this finding, the present invention constructed an octanoyl-HNS polypeptide and demonstrated its ability to promote thermogenic metabolism and enhance cold acclimatization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the test results showing that the octanoyl HNS polypeptide of the present invention significantly improves the cold tolerance of mice; *P<0.05, **P<0.01, n=8 per group.

[0025] Figure 2 The octanoyl HNS polypeptide of the present invention enhances the thermal metabolism of brown fat in mice; wherein, Figure 2 Figure A shows that octanoyl HNS peptide increases brown fat epidermal temperature in mice, **P<0.01, ***P<0.001, n=8 per group; Figure 2 Figure B shows that octanoyl HNS peptide increases the oxygen consumption metabolism level of brown fat in mice, ***P < 0.001, n = 6 per group.

[0026] Figure 3 Octanoylated HNS peptide promoted the expression of thermogenic metabolic molecules in brown fat of mice; *P<0.05, **P<0.01, ***P<0.001, n=6 per group. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] The structure of the octanoyl HNS polypeptide involved in the following examples is shown below: .

[0029] The octanoyl HNS peptide was custom synthesized by Sangon Biotech (Shanghai) Co., Ltd. The octanoyl HNS peptide was desalted and had a purity of >98%.

[0030] The HNS polypeptide involved in the following examples was purchased from Sangon Biotech Co., Ltd., and its amino acid sequence is shown in SEQ ID NO.1: RRFGGPVHHQAQRFRFSPMGVDHMSGISGVNV.

[0031] The experimental animals involved in the following examples are as follows: SPF C57BL / 6J wild-type male mice, weighing 22–25 g, were provided by the Experimental Animal Center of the Fourth Military Medical University. They were housed in individual cages at approximately 25°C with a 12-hour light / dark cycle and had free access to food and water. All animal experiments were approved by the Animal Ethics Committee of the Fourth Military Medical University and conducted in strict accordance with animal ethics standards. Ethics review number: 20200453.

[0032] The mouse intervention models involved in the following examples are as follows: C57BL / 6J mice were randomly divided into a control group, an HNS peptide group, and an octanoyl HNS peptide group, with 8 mice in each group.

[0033] All three groups of mice were housed in an animal room maintained at a temperature of 24°C ± 1°C. The control group received daily intraperitoneal injections of 200 μL of saline solution. The HNS peptide group received daily intraperitoneal injections of 10 mg / kg HNS peptide solution dissolved in saline. The octanoylated HNS peptide group received daily intraperitoneal injections of 10 mg / kg octanoylated HNS peptide solution dissolved in saline.

[0034] After two weeks of intervention in the above three groups of mice, the mice's acute cold exposure tolerance, brown fat skin temperature, brown fat oxygen consumption metabolic level, and brown fat thermogenic molecular changes were tested.

[0035] Example 1: Effect of octanoyl polypeptide on acute cold exposure tolerance in mice (1) Experimental methods: 1) Peptide intervention plan: C57BL / 6J mice were randomly divided into a control group, an HNS peptide group, and an octanoylated HNS peptide group, with 8 mice in each group. The mice in the three groups were housed in an animal room at 24°C ± 1°C. The control group received daily intraperitoneal injections of 200 μL of normal saline. The HNS peptide group received daily intraperitoneal injections of a 10 mg / kg solution of HNS peptide dissolved in normal saline. The octanoylated HNS peptide group received daily intraperitoneal injections of a 10 mg / kg solution of HNS peptide dissolved in normal saline.

[0036] After two weeks of intervention, the three groups of mice were subjected to an acute cold exposure experiment at 4°C.

[0037] 2) Acute cold exposure and cold tolerance test in mice: The acute cold exposure experiment includes the following steps: Each group of mice was taken out of the experimental animal room after 3 pm on the test day and immediately placed in a 4°C low-temperature environment simulation chamber in odd-numbered individual cages without food or water. The core body temperature changes of the mice were detected and recorded every hour.

[0038] 3) Measurement of mouse core body temperature: During the cold exposure period of the mice, the rectal temperature was measured and recorded every hour by gently inserting the rectal thermometer probe 1 cm into the anorectal cavity of the mice.

[0039] (2) Results: The results are as follows Figure 1As shown: After acute cold exposure, the core body temperature of the mice in the control group gradually decreased. Compared with the control group, the core body temperature of the mice in the HNS peptide group slightly rebounded. The core body temperature of the mice in the octanoyl HNS peptide group remained significantly higher, and the difference was statistically significant compared with the control group and the HNS peptide group ( Figure 1 ).

[0040] The above results show that octanoyl HNS peptide can significantly improve the cold tolerance of mice.

[0041] Example 2: Effects of Octanoylated Peptides on the Thermogenesis of Brown Fat in Mice (1) Experimental methods: 1. Detection of brown fat skin temperature in mice: Thermal imaging was used to analyze the skin temperature of the brown fat of each group of mice. The specific steps are as follows:

[0042] 1) Equipped with an infrared camera, turn on the computer and connect it to the infrared imaging system.

[0043] 2) Mice were placed in the test environment for 30 minutes.

[0044] 3) Place the mice in a clean cage and use an infrared imaging system and infrared camera to record thermal radiation imaging data of the mice's brown fat.

[0045] 4) Using thermal imaging analysis software, select the brown fat skin area to export temperature data and calculate the average value, which is the surface temperature of the mouse brown fat.

[0046] 2. Brown adipose tissue oxygen consumption metabolism detection: 1) Using the OROBOROS O2K Cellular Energy Metabolism Analysis System, rinse the O2K instrument three times with ultrapure water, each time for 5 minutes.

[0047] 2) Add 10% (volume ratio) FBS + DMEM culture medium to the test chamber. After running for 30 minutes, perform air calibration.

[0048] 3) Prepare tissue samples by evenly chopping fresh brown adipose tissue into small pieces with scissors. Weigh 100 mg of each sample and suspend the sample in fresh culture medium containing 10% (volume ratio) FBS + DMEM. Each sample refers to the brown adipose tissue from each of the three groups of mice.

[0049] 4) Place the prepared tissue sample into the test chamber and start measurement.

[0050] 5) After the oxygen consumption rate curve stabilizes, read the value, which is the basal oxygen consumption metabolic rate per 100 mg of brown adipose tissue.

[0051] (2) Results: The results are as follows Figure 2 As shown in Figure A, compared with the control group, the local skin temperature of brown fat in mice increased after HNS peptide intervention, and the difference was statistically significant; compared with the control group and the HNS peptide intervention group, the local skin temperature of brown fat in mice was significantly increased after octanoyl HNS peptide intervention, indicating that the thermal metabolism of brown adipose tissue was significantly enhanced.

[0052] The results are as follows Figure 2 As shown in Figure B, compared with the control group, after HNS peptide intervention, the oxygen consumption metabolism of the mouse brown fat tissue was enhanced, and the difference was statistically significant; compared with the control group and the HNS peptide intervention group, after octanoyl HNS peptide intervention, the oxygen consumption metabolism level of the mouse brown fat was significantly increased, indicating that the heat metabolism of the brown fat tissue was significantly enhanced.

[0053] Example 3: Effects of octanoyl peptides on the expression levels of thermogenic metabolic molecules in brown fat of mice 1. Experimental methods: 1. RNA extraction: 1) Place fresh brown adipose tissue sample into a 1.5 mL enzyme-free EP tube pre-added with 1 mL of Trizol.

[0054] 2) Use a tissue homogenizer to fully grind the brown adipose tissue sample into a homogenate.

[0055] 3) Add 200 μL of chloroform to the brown adipose tissue sample homogenate from step 2), mix thoroughly by inversion, let stand for 5 minutes, and then place the homogenate in a pre-cooled 4°C refrigerated centrifuge and centrifuge at 12,500 rpm for 20 minutes.

[0056] 4) Transfer the supernatant to a new, labeled 1.5 mL enzyme-free EP tube, add an equal volume of isopropanol, invert thoroughly to mix, and centrifuge at 12,500 rpm for 15 minutes at 4°C.

[0057] 5) After discarding the supernatant, add 1 mL of pre-chilled 75% (volume ratio) ethanol prepared from DEPC water and anhydrous ethanol. Centrifuge at 12,500 rpm for 10 minutes at 4°C.

[0058] 6) After removing the supernatant, dry the precipitate at room temperature for 5 minutes.

[0059] 7) Use 20 μL of DEPC water to fully dissolve the RNA in the tube.

[0060] 2. RNA reverse transcription: 1) Measure RNA concentration using an ultra-micro spectrophotometer.

[0061] 2) The reverse transcription reaction system is as follows: Calculate the volume of 1 μg RNA solution based on the concentration, add 4 μL of 5× Prime Script™ RT Master Mix, and add RNase-Free dH2O to make up to 20 μL.

[0062] 3) Reaction conditions: 37°C for 15 min, 85°C for 10 s. Store the reverse-transcribed cDNA at -20°C for subsequent experiments.

[0063] 3. RT-qPCR: 1) Reagent volume: TB Green Premix E×Taq II 5 μL, ROX Reference Dye II 0.2 μL, DNA template (cDNA) 1.5 μL, DEPC water 2.5 μL, primers (upstream primer + downstream primer, 10 μM) 0.8 μL, total 10 μL.

[0064] 2) Reaction conditions: 95°C for 10 min; 95°C for 15 s, 60°C for 35 s, for 45 cycles.

[0065] Among them, the primer sequences are shown in Table 1: Table 1 Primers 2. Results result Figure 3 As shown in the results, compared with the control group, after HNS peptide intervention, the expression levels of Ucp1, Pgc1α, and Pparα in the brown adipose tissue of mice increased, and the difference was statistically significant; compared with the control group and the HNS peptide intervention group, after octanoyl HNS peptide intervention, the expression levels of Ucp1, Pgc1α, and Pparα in the brown adipose tissue of mice were significantly increased, indicating that the thermal metabolism of brown adipose tissue was significantly enhanced.

[0066] These experimental results indicate that octanoyl HNS peptide significantly promotes brown fat thermogenic metabolism and improves cold tolerance in mice. Compared to HNS peptide, octanoyl HNS peptide exhibits a more potent effect in promoting thermogenic metabolism and cold tolerance, with statistically significant differences. It is an effective cold acclimatization intervention.

[0067] The octanoyl polypeptide provided by the present invention has the advantages of a clear target, rapid onset, strong thermogenic effect, and a weak stress response. It can address the slow onset and unclear target of efficacy of current traditional Chinese medicine compositions used to improve the body's cold acclimatization ability. The octanoyl polypeptide provided in this application is an octanoyl HNS polypeptide, a polypeptide with further structural improvements based on the HNS polypeptide. Compared with existing HNS polypeptides, the octanoyl polypeptide provided by the present invention has an octanoyl domain that can efficiently enter cell membranes and mitochondrial membranes. Therefore, the octanoyl polypeptide provided by the present invention has high in vivo absorption and stability, representing a significant improvement over the prior art.

[0068] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0069] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. An octanoyl polypeptide, characterized in that The octanoyl polypeptide is an octanoyl HNS polypeptide, and its structure is shown below: 。 2. Use of the octanoyl polypeptide according to claim 1 in the preparation of a medicament for promoting the body's cold acclimatization ability.

3. The use according to claim 2, characterized in that The medicine uses the octanoyl polypeptide as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

4. The use according to claim 3, characterized in that The medicine includes at least one of tablets, capsules, injections, solutions and ointments.

5. The use according to claim 3, characterized in that The excipients include any one or more of corn starch, gelatin, hydroxypropyl methylcellulose magnesium stearate, polysorbate and polylactic acid.

6. The use according to claim 3, characterized in that The carrier includes any one or more of liposomes, exosomes, cell vesicles, nanoparticles and gels.

7. The use according to claim 3, characterized in that The medicine is a solution; The solution is prepared by mixing the octanoyl polypeptide with a solvent; The solvent is any one of physiological saline, phosphate buffer and glucose solution.

8. The use according to claim 7, characterized in that The concentration of the octanoyl polypeptide in the solution is 20 mg / mL to 50 mg / mL.

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