A blood protein peptide composition for improving body shape and posture symptoms

Abstract

The application relates to a blood protein peptide composition for improving body shape and body posture symptoms, and belongs to the field of bioactive peptides. The blood protein peptide composition disclosed by the application comprises an LMN polypeptide and an LTL polypeptide; the amino acid sequence of the LMN polypeptide is Leu-Met-Asn; and the amino acid sequence of the LTL polypeptide is Leu-Thr-Leu. The blood protein peptide composition prepared by using the technical scheme of the application can improve the body shape and body posture symptoms caused by lymphedema, and good economic benefits and social benefits are exhibited.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioactive peptides, in particular to a blood protein peptide composition for improving body shape and posture symptoms and application thereof. BACKGROUND

[0002] China has abundant blood resources, but they have not been truly utilized, resulting in a large amount of resource waste. Blood contains abundant proteins, and improving the utilization rate of proteins is the key to the current blood research and utilization. Blood protein peptides are fragments with biological activity, have unique biological activity beyond basic nutritional needs, have small molecular weight, are easy to absorb, and are simple to prepare, and thus are attracting more and more attention.

[0003] As a key network of human immune defense and fluid balance, the lymphatic system bears the core functions of tissue fluid recovery, pathogen clearance and macromolecular substance transport. When lymphatic vessels or lymph nodes are blocked due to infection (such as filariasis), tumor compression, surgical damage (such as axillary lymph node dissection for breast cancer), or fibrosis after radiotherapy, lymphatic reflux disorder is caused, thereby affecting body shape and posture, for example, limb contour deformity, skin barrier damage and metabolic dysfunction. However, the blood protein peptide has not been explored in improving the body shape and posture symptoms caused by lymphatic edema.

[0004] Therefore, there is an urgent need in the art to develop a blood protein peptide composition for improving body shape and posture symptoms. SUMMARY

[0005] In a first aspect, the present application provides a blood protein peptide composition, comprising an LMN polypeptide and an LTL polypeptide; the amino acid sequence of the LMN polypeptide is Leu-Met-Asn; the amino acid sequence of the LTL polypeptide is Leu-Thr-Leu.

[0006] Optionally, the mass ratio of the LMN polypeptide to the LTL polypeptide is 1:1.

[0007] Optionally, the blood protein peptide composition further comprises human pluripotent stem cell exosomes.

[0008] Optionally, the preparation method of the human pluripotent stem cell exosomes is as follows: human induced pluripotent stem cells are cultured in a basic medium containing SB431542, Dorsomorphin and Noggin; and the culture supernatant is centrifuged to obtain high-purity exosomes.

[0009] Optionally, the blood protein peptide composition further comprises a pharmaceutically acceptable carrier.

[0010] Optionally, the carrier is one or more of mannitol and phosphate buffer.

[0011] In a second aspect, the present application provides a use of the above-mentioned blood protein peptide composition in the preparation of a drug for improving the body shape and posture symptoms associated with lymphedema.

[0012] In summary, the present application includes at least one of the following beneficial technical effects:

[0013] 1) Human pluripotent stem cell exosomes and blood protein peptides synergistically significantly improve the body shape and posture disorders of lymphedema: As shown in Example 5, after administration of composition B containing human pluripotent stem cell exosomes for 3 weeks, the tail volume of the lymphedema model mice was significantly reduced compared with the model control group, and the therapeutic effect was also significantly improved compared with the polypeptide group (composition A), effectively reversing the limb contour deformity.

[0014] 2) Precise regulation of inflammatory microenvironment to restore lymphatic system homeostasis;

[0015] 3) The present application first realizes the application breakthrough of blood protein peptides in the field of lymphatic system repair, provides a new solution for the lack of lymphedema drugs in clinical practice, reduces the cost of raw materials, and has significant economic and social benefits. DETAILED DESCRIPTION

[0016] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below in conjunction with the following specific examples, but it should not be understood as limiting the scope of the present application.

[0017] Fresh human whole blood was added to an anticoagulant, sterile water was added in a mass ratio of 1:1 (blood:water), and ultrasonic assisted hemolysis was performed. After the cells were broken, centrifugation was performed for 15 min to remove the cell wall, and human blood protein solution was obtained.

[0018] Alkaline protease was added according to the mass of human blood protein, the addition amount was 10000 U / g, and enzyme hydrolysis was performed at 50°C for 4h; the enzyme activity was inactivated by boiling, and the pH value was adjusted to 7.0; trypsin was added according to the mass of bovine blood protein, the addition amount was 5000 U / g, and enzyme hydrolysis was performed at 37°C for 2h. The enzyme activity was inactivated by boiling, and the pH value was adjusted to 7.0, thereby obtaining human blood protein hydrolysate. Centrifugation was performed at 10000 r / min for 30 min to obtain the supernatant, which was first filtered through a 0.45 μm filter membrane, and the filtrate was collected; the filtrate was separated by a 3 kDa ultrafiltration membrane, and the polypeptide component with a molecular weight of <3 kDa was collected for LC-MS / MS liquid analysis. LC-MS / MS analysis and database search comparison were performed, and finally two polypeptides, LMN polypeptide and LTL polypeptide (sequence: Leu-Thr-Leu) were selected for artificial synthesis.

[0019] The LTL peptide segment and the WVV peptide segment were synthesized by solid-phase synthesis using an automatic peptide synthesizer (synthesized by Jinshui Biotech Co., Ltd.), purified by high-performance liquid chromatography, and identified by mass spectrometry and amino acid sequence analysis to confirm the sequence and purity of the synthesized peptides, and finally obtain a peptide segment powder with a purity of more than 95%.

[0020] A layer of embryonic stem cell matrix glue was laid in a culture bottle, and human induced pluripotent stem cells were cultured in hPSC XF medium containing 10 μM Rock inhibitor. The medium was replaced daily, and the cells were maintained at 37°C in a 5% CO2 environment. When the cell density reached 80%, the cells were passaged. The passaged human induced pluripotent stem cells were cultured in DMEM / F-12 medium containing 10 μM SB431542, 1 μM Dorsomorphin, and 50 ng / mLNoggin (containing 0.5 vol% GlutaMAX-I additive and 1 vol% N-2 additive) for 7 days. The culture supernatant was centrifuged at 300 x g for 10 minutes at 4°C to remove cells, and then centrifuged at 2000 g for 10 minutes to remove debris; centrifuged at 10000 g for 30 minutes, and then filtered with a 0.22 μm filter membrane to remove apoptotic bodies; centrifuged at 100000 g for 2 hours, and then resuspended in PBS and centrifuged again to obtain high-purity exosomes.

[0021] The blood protein peptide composition is prepared by the following steps.

[0022] The LMN polypeptide and the LTL polypeptide were dissolved in phosphate buffer (pH 7.2) and stirred at 35°C and 1000 r / min for 30 min until completely dissolved; mannitol was then added and mixed uniformly; the solution was filtered with a 0.22 μm polyether sulfone filter membrane to remove bacteria to obtain the blood protein peptide composition A, wherein the content of the LMN polypeptide is 5%, and the content of the LTL polypeptide is 5%.

[0023] The blood protein peptide composition is prepared by the following steps.

[0024] The LMN polypeptide, the LTL polypeptide, and the exosomes of Example 2 were dissolved in phosphate buffer (pH 7.2) and stirred at 35°C and 1000 r / min for 30 min until completely dissolved; mannitol was then added and mixed uniformly; the solution was filtered with a 0.22 μm polyether sulfone filter membrane to remove bacteria to obtain the blood protein peptide composition B, wherein the content of the LMN polypeptide is 5%, the content of the LTL polypeptide is 5%, and the content of the human pluripotent stem cell exosomes is 3%.

[0025] After 4 days of adaptive feeding, 40 rats were divided into 4 groups, named blank control group, model control group, blood protein peptide composition A group and blood protein peptide composition B group, 10 rats in each group. The rat tail lymphedema model was established by ligating the lymphatic vessels of the hind limbs of rats (SD rats, male, 200g) to induce edema.

[0026] Intraperitoneal administration of 0.5 mg / kg blood protein peptide composition A, 0.5 mg / kg blood protein peptide composition B, and normal saline (blank control group / model control group) every three days. The administration lasted for 3 weeks.

[0027] From the incision site, the tail circumference was measured every 1 cm, and the digital volume was calculated accordingly. The results are shown in Table 1. From the results in Table 1, it can be seen that the lymphedema model was successful, as shown by a significant increase in tail volume in each group compared to the blank control group at week 0 (p<0.001); the volume of the blood protein peptide composition A group was reduced by 35% (p<0.01 vs model group), indicating that the basic polypeptide composition has anti-edema activity; the volume of the blood protein peptide composition B group was significantly reduced by 47% (p<0.01 vs model group), which was more effective than the polypeptide group (A group) alone, confirming the synergistic effect of exosomes and polypeptides.

[0028] Table 1

[0029]

[0030] At the end of the animal experiment, blood was taken and centrifuged (4°C, 3000 r / min) for 15 min after standing at 4°C for 6 h. The serum was taken and the content of inflammatory factors IL-6, IL-10, and TNF-α in the serum was detected according to the kit instructions. All data are expressed as mean ± SD, and statistical analysis was performed by SPSS 26 software. The results are shown in Table 2. From the results in Table 1, it can be seen that the blood protein peptide composition A group and the blood protein peptide composition B group can significantly reduce the concentrations of TNF-α, IL-6, and IL-10 in lymphedema rats.

[0031] Table 2

[0032]

[0033] Those skilled in the art will further appreciate that the application can be practiced in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the disclosure has disclosed only exemplary embodiments, it should be understood that other variations are considered within the scope of the application. Therefore, the application is not limited to the specific embodiments described in detail herein. Rather, the scope and content of the application should be indicated by reference to the appended claims.

Claims

1. A hemoprotein peptide composition, characterized in that, It is composed of an LMN polypeptide and an LTL polypeptide; the amino acid sequence of the LMN polypeptide is Leu-Met-Asn; and the amino acid sequence of the LTL polypeptide is Leu-Thr-Leu.

2. The hemoprotein peptide composition according to claim 1, characterized in that, The mass ratio of the LMN peptide to the LTL peptide is 1:

1.

3. The use of the hemoprotein peptide composition according to any one of claims 1-2 in the preparation of a medicament for improving lymphedema.

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