Liver peptide for relieving liver damage caused by staying up late and application thereof

Abstract

The application discloses liver peptides for relieving liver injury caused by all-night work, and relates to the technical field of liver peptides.The amino acid sequence of the liver peptides is LLVW, the liver peptides can effectively relieve liver injury caused by all-night work, and thus the effect of protecting the liver is achieved.

Description

Technical Field

[0001] This invention relates to the field of liver peptide technology, and more specifically to a liver peptide that can alleviate liver damage caused by staying up late and its application. Background Technology

[0002] As a core metabolic and detoxification organ in the human body, the liver undertakes key physiological functions such as toxin removal, substance synthesis, and energy metabolism. The integrity of its function directly affects the body's health. With the accelerated pace of modern life, staying up late has become a common habit. Long-term or frequent late nights can cause continuous and multi-dimensional damage to the liver.

[0003] From a physiological perspective, the core pathways by which staying up late causes liver damage include three aspects: First, disruption of the circadian rhythm. Staying up late disrupts the body's diurnal rhythm balance, leading to abnormal expression of core circadian rhythm genes such as Clock, Bmal1, and Per2 in liver tissue. This, in turn, regulates the rhythmic synthesis and activity of downstream metabolic enzymes and antioxidant enzymes, disrupting the normal metabolic sequence of the liver. Second, oxidative stress overload leads to the accumulation of lipid peroxidation products such as malondialdehyde (MDA) and DNA oxidative damage markers such as 8-hydroxydeoxyguanosine (8-OHdG) in hepatocytes, directly attacking hepatocyte membranes, mitochondria, and genetic material, causing hepatocyte damage. In addition, staying up late also activates the NF-κB inflammatory pathway, promoting the release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), inducing hepatocyte apoptosis, inhibiting hepatocyte regeneration, and exacerbating the liver damage process.

[0004] For liver damage caused by staying up late, liver protection products on the market can be mainly divided into three categories: First, chemically synthesized drugs, such as thioproline and silymarin compounds, can improve liver cell damage to a certain extent, but they often have side effects (such as gastrointestinal discomfort and allergic reactions), and their mechanisms of action are singular, focusing on a single link of antioxidation or detoxification. They are difficult to address the multi-mechanism synergistic damage characteristics of liver damage caused by staying up late, including circadian rhythm disorder, oxidative stress, and inflammation activation, and their long-term safety is limited. Second, traditional Chinese medicine and natural product extracts, such as wolfberry extract, salvia miltiorrhiza extract, and artemisia capillaris preparations, have complex components, unstable content of effective active ingredients, and unclear mechanisms of action. They also have the problems of slow onset of action and poor targeting, and cannot quickly relieve acute liver damage after staying up late. Third, ordinary health products, such as vitamin C, vitamin E, and glutathione supplements, are mostly single nutrient supplements that can only help enhance antioxidant capacity. They lack the regulatory effect on the core damage pathways such as circadian rhythm disorder and abnormal detoxification enzyme function caused by staying up late, and their liver protection effect is limited.

[0005] Peptides are gaining increasing attention in liver protection due to their advantages such as small molecular weight, high bioavailability, few side effects, and well-defined mechanisms of action. Compared to proteins, peptides can be directly absorbed and utilized by the body, and can precisely target specific sites to regulate related physiological processes. However, currently available liver-protecting peptide products are mostly designed for chemically induced or immune-mediated liver damage, and cannot provide precise and comprehensive relief for liver damage caused by staying up late.

[0006] Therefore, developing a liver peptide that can precisely improve liver damage caused by staying up late has become an urgent need in the research and development of liver protection products. Summary of the Invention

[0007] In view of this, the present invention provides a liver peptide that can alleviate liver damage caused by staying up late. Peptide molecules have significant advantages in the field of liver protection due to their small molecular weight, good biocompatibility, easy absorption and utilization by the human body, and low side effects. Experiments have shown that the liver peptide LLVW with a specific amino acid sequence can effectively alleviate liver damage caused by staying up late.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a liver peptide that alleviates liver damage caused by staying up late, wherein the amino acid sequence of the liver peptide is LLVW.

[0009] Furthermore, the amino acid sequence of the hepatic peptide also includes a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the LLVW.

[0010] Secondly, the present invention provides a nucleic acid molecule that encodes the hepatic peptide, or is reverse complementary to the nucleotide sequence encoding the hepatic peptide.

[0011] Thirdly, the present invention provides a biomaterial containing the aforementioned nucleic acid molecules and capable of expressing the aforementioned liver peptides.

[0012] Furthermore, the biological material includes recombinant plasmids, recombinant vectors, or recombinant host cells.

[0013] Fourthly, the present invention provides the liver peptide, the nucleic acid molecule, and the biomaterial described herein for the preparation of liver peptides that alleviate liver damage caused by staying up late.

[0014] Furthermore, the method of alleviating liver damage caused by staying up late includes reducing the levels of MDA and TNF-α in the liver of sleep-deprived rats.

[0015] Furthermore, the products include food or pharmaceuticals.

[0016] Fourthly, the present invention provides a drug for relieving liver damage caused by staying up late, the drug comprising the aforementioned liver peptide.

[0017] Fifthly, the present invention provides a food for alleviating liver damage caused by staying up late, the food comprising the aforementioned liver peptide.

[0018] As shown in the above technical solution, this invention independently purifies a novel liver peptide with the amino acid sequence LLVW from natural biological raw materials. After purification, the purity reaches over 97%, exhibiting both high activity and good biocompatibility. Furthermore, the study unexpectedly discovered that this liver peptide can significantly reduce the content of MDA and inflammatory factors in liver tissue, thereby alleviating liver damage caused by staying up late. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 This is the mass spectrum of the liver peptide LLVW obtained by protease digestion. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 The amino acid sequence in this invention is LLVW (Leu-Leu-Val-Trp). The preparation steps are as follows: I. Raw material pretreatment 1. Select healthy pig liver (which has passed quarantine, is free from disease and drug residues), remove the fascia, blood vessels and connective tissue, rinse 3 times with 4℃ pre-cooled physiological saline, drain the surface water, and cut into small pieces of about 1cm³. 2. Add liver tissue to pre-cooled physiological saline at a ratio of 1:2 (mass-volume ratio, g / mL) into a high-speed tissue homogenizer and homogenize for 2 minutes under ice bath conditions to prepare a uniform liver tissue homogenate. 3. Add EDTA (ethylenediaminetetraacetic acid) to the homogenate at a final concentration of 0.05 mol / L and PMSF (phenylmethylsulfonyl fluoride) at a final concentration of 0.1 mmol / L, stir well, and let stand at 4°C for 30 min to inhibit protease activity and prevent liver peptide degradation.

[0023] II. Liver peptide extraction 1. Transfer the pretreated liver homogenate to a centrifuge tube, centrifuge at 4℃ and 8000r / min for 20min, collect the supernatant (crude extract), and discard the precipitate (tissue fragments, large molecular proteins, etc.). 2. Slowly add ammonium sulfate to the supernatant until the saturation is 60%, stir at 4°C for 3 hours to allow the impurities and proteins to precipitate fully; 3. Continue centrifuging at 4℃ and 10000r / min for 25min, collect the supernatant (containing the target hepatic peptide), and discard the impurity protein precipitate; 4. Use an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to ultrafilter the supernatant at 4°C and 0.3 MPa pressure. Collect the filtrate (to remove impurities and polymers with a molecular weight greater than 10 kDa) and retain the filtrate containing small peptides.

[0024] III. Preliminary purification (gel filtration chromatography) 1. Sephadex G-15 gel chromatography was used. The column (2.6 cm × 100 cm) was equilibrated with 0.02 mol / L, pH 7.0 phosphate-buffered saline (PBS) at a volume of 3 times the column volume. 2. Load the ultrafiltration filtrate into the chromatography column, with a loading volume of 5% of the column volume; 3. Elute with the same PBS buffer at a flow rate of 1 mL / min. Monitor the elution peak at 220 nm using a UV detector and collect the eluent in the corresponding molecular weight range (300-500 Da) (the collection range is preset according to the molecular weight of LLVW and LLVF). 4. Combine the collected eluents and freeze-dry them initially using a freeze dryer to obtain crude liver peptide powder.

[0025] IV. Purification (Reversed-phase high-performance liquid chromatography, RP-HPLC) 1. Dissolve the crude hepatin powder in 0.1% trifluoroacetic acid aqueous solution to prepare a sample solution of 10 mg / mL, and filter it through a 0.22 μm microporous membrane; 2. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with a column temperature of 30 °C; mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile; 3. Gradient elution program: 0-20 min, the volume fraction of mobile phase B increases from 10% to 30%; 20-35 min, the volume fraction of mobile phase B increases from 30% to 45%; 35-40 min, the volume fraction of mobile phase B returns to 10% to equilibrate the column. 4. Elution flow rate 1.0 mL / min, detection at 220 nm wavelength using a UV detector, and collection of the target elution peak corresponding to LLVW based on retention time (retention time determined by standard comparison: LLVW approximately 29.7 min). 5. The collected target eluents were freeze-dried to obtain high-purity liver peptide powder.

[0026] V. Purity Identification and Sequence Confirmation 1. Purity test: The purity of LLVW was repeatedly tested by HPLC and the purity was ≥96% with no obvious impurity peaks. 2. Sequence confirmation: The molecular weight was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the amino acid sequence was determined by Edman degradation method. The obtained product was confirmed to be LLVW (molecular weight 601.6 Da), which was completely consistent with the preset sequence.

[0027] Performance testing Animal selection: 100 SPF-grade SD rats, half male and half female, aged 6-8 weeks and weighing 200±20g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Adaptation feeding: One week before the experiment, 48 rats were placed in an SPF-grade animal room to adapt to the environment. The feeding conditions were: temperature 22±2℃, relative humidity 55±5%, and a basic light cycle of 12h light (06:00-18:00) / 12h darkness (18:00-06:00). The rats were allowed free access to standard rat feed and sterile drinking water. The rats' mental state, diet, water intake and defecation were observed daily. After ensuring that there were no abnormalities, the modeling process began.

[0028] An internationally recognized chronic incomplete sleep deprivation device was used to avoid interference from physical stress in the experiment. Device parameters: Customized acrylic sleep deprivation box (50cm×50cm×40cm), with 12 circular platforms, each 10cm in diameter and 8cm in height, placed inside the box. The platforms are spaced 5cm apart. The box is filled with clean water, with the water level 2cm below the surface of the platforms (to ensure that the rats will not drown when standing on the platforms, but if their muscles relax after falling asleep, they will fall into the water due to instability. After waking up, they will climb back onto the platforms on their own, thus achieving fragmented sleep deprivation). Deprivation period and duration: 36 rats were selected for modeling, and the modeling period lasted for 2 weeks. The daily sleep deprivation time was from 23:00 to 07:00 the next day (corresponding to the peak time of human staying up late, with 8 hours of sleep deprivation). During the rest of the time (07:00-23:00), the rats were moved to normal breeding cages and restored to free food and water and normal light to avoid excessive stress. Blank control group treatment: The rats in the blank control group were housed in the same size "sham deprivation box" (the platform height in the box was 15cm, the water level was 8cm from the platform surface, and the rats could sleep freely on the platform without sleep deprivation pressure). All other housing conditions and light cycles were completely consistent with the model group to exclude environmental factors.

[0029] General condition monitoring: Record the weight, food intake, and water intake of each group of rats daily, observe their mental state (such as whether they are lethargic or have reduced activity), coat luster, and defecation. If a sudden drop in weight (more than 10% of the initial weight) or obvious disease symptoms occur, remove the rats in time. Stress level control: Change the water in the deprivation chamber once a week during the modeling period to keep the water clean; polish the platform surface to avoid scratching the rat's feet; after sleep deprivation each day, gently wipe the rat's body surface dry with a dry towel to prevent it from getting cold; Model stability verification (day 21 after modeling): After modeling, three rats in the model group were randomly selected, sacrificed, and samples were collected to verify the effectiveness of the model.

[0030] After successful modeling, the rats were divided into 3 groups, and 12 rats were selected as a blank control group. The experimental grouping and administration methods are as follows: Blank control group: normal saline (oral 20ml / kg·d) -1 ); Model control group: physiological saline (oral administration 20 mg / kg / day) -1 ); Positive control group: Oral administration of thiopronine tablets 20 mg / kg·d -1 ; Experimental Group 1: Oral administration via gavage. Example 1: Liver peptide 20 mg / kg / day -1 .

[0031] Dosage regimen: Dosage begins after model establishment and continues for 2 weeks while the animal is fed a normal diet.

[0032] Sample collection and indicator detection Mice were euthanized by cervical dislocation, and liver tissue was dissected, weighed, and mixed with 9 times the volume of physiological saline to prepare a 10% liver homogenate. The homogenate was centrifuged at 3500 rpm for 10 min, and the supernatant was aliquoted. The levels of MDA and TNF-α in the liver tissue of each group of mice were measured.

[0033] The results are shown in Table 1.

[0034] Table 1. Results of in vivo experiments (SD rats) on relevant indicators (x±s, n=12) As shown in Table 1, the liver peptide in Example 1 has the effect of improving liver protection and alleviating liver damage, and the effect is significant. It is speculated that it can alleviate liver damage caused by staying up late.

[0035] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0036] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A liver peptide that alleviates liver damage caused by staying up late, characterized in that, The amino acid sequence of the hepatic peptide is LLVW.

2. The liver peptide for alleviating liver damage caused by staying up late according to claim 1, characterized in that, The amino acid sequence of the hepatic peptide also includes a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the LLVW.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the liver peptide of claim 1 or 2, or is reverse complementary to the nucleotide sequence encoding the liver peptide of claim 1 or 2.

4. A biomaterial, characterized in that, It contains the nucleic acid molecule as described in claim 3 and is capable of expressing the liver peptide as described in claim 1 or 2.

5. A biomaterial according to claim 4, characterized in that, The biomaterials include recombinant plasmids, recombinant vectors, or recombinant host cells.

6. The liver peptide according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, and the biomaterial according to claim 4 in the preparation of liver peptides that alleviate liver damage caused by staying up late.

7. The application according to claim 6, characterized in that, The proposed methods to alleviate liver damage caused by staying up late include reducing the levels of MDA and TNF-α in the livers of sleep-deprived rats.

8. The application according to claim 6, characterized in that, The products include food or medicine.

9. A drug for relieving liver damage caused by staying up late, characterized in that, The drug comprises the liver peptide as described in claim 1 or 2.

10. A food product for alleviating liver damage caused by staying up late, characterized in that, The food product includes the liver peptide as described in claim 1 or 2.

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