A polypeptide and uses thereof

Abstract

This invention discloses a polypeptide and its applications, relating to the field of biomedical technology. The present invention discloses a polypeptide with a novel amino acid structure, the amino acid sequence of which is shown in SEQ ID NO.1. The polypeptide provided by this invention can significantly reduce the paralysis rate of transgenic CL4176 nematodes and has significant anti-Alzheimer's disease activity. Furthermore, the polypeptide provided by this invention has a significant inhibitory effect on IgE secretion by U266 cells, and this polypeptide can achieve an anti-allergic effect by inhibiting IgE production. The polypeptide provided by this invention shows promise as a new drug for treating Alzheimer's disease or allergies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide and its application in the treatment of Alzheimer's disease or allergy drugs. Background Technology

[0002] Alzheimer's disease (AD), the leading cause of death among neurodegenerative diseases, involves complex mechanisms such as abnormal deposition of β-amyloid (Aβ) to form plaques, hyperphosphorylation of tau protein leading to neurofibrillary tangles, and neurotransmitter system disorders. According to data from the World Health Organization in 2024, the number of AD patients worldwide has exceeded 70 million, and continues to grow at a rate of one new case every 3 seconds. Patients not only face the physical challenges of memory loss, language impairment, and overall cognitive decline, but also often experience psychiatric symptoms such as depression and anxiety, eventually progressing to complete dependence on others for daily living and requiring 24 / 7 professional care. Currently approved AD treatments, such as the cholinesterase inhibitor donepezil and the NMDA receptor antagonist memantine, can only temporarily alleviate symptoms by regulating neurotransmitter levels and are insufficient to curb disease progression, placing enormous pressure on patients' families and societal healthcare resources.

[0003] Food allergy is a type I hypersensitivity reaction mediated by immunoglobulin E (IgE). Its main mechanism involves the binding of IgE antibodies to specific food proteins (such as peanuts, milk, and eggs), inducing the release of inflammatory mediators such as histamine from mast cells and basophils. This leads to symptoms affecting multiple systems, including the skin, respiratory tract, and digestive tract, and in severe cases, can cause anaphylactic shock and even death. In recent years, the global incidence of allergic diseases has increased, particularly among children, with a significant rise in food allergies, becoming a major threat to public health. This disease not only limits patients' diet and quality of life but also imposes long-term psychological burdens and social costs. Current clinical treatment mainly relies on strict avoidance of allergens, supplemented by antihistamines and corticosteroids to relieve acute symptoms, and emergency use of adrenaline. However, these methods only temporarily control the reaction and cannot interrupt the IgE-mediated immune response; long-term use may also cause side effects such as drug resistance and metabolic disorders. Although desensitization strategies such as oral immunotherapy can gradually improve tolerance, their efficacy is unstable, treatment time is long, and there is a risk of severe allergic reactions. Therefore, the development of new drugs that inhibit IgE responses and precisely regulate immune responses is key to breaking through the bottleneck in the treatment of food allergies. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention designs and synthesizes a novel polypeptide that, through its unique amino acid sequence, simultaneously targets both AD pathology and allergic immune regulation mechanisms, providing a new strategy for the treatment of these two diseases.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a polypeptide having the amino acid sequence TFSHAEGT (SEQ ID NO. 1). In a second aspect, the present invention provides the use of the above-mentioned polypeptide in the preparation of a drug for treating Alzheimer's disease.

[0009] Thirdly, the present invention provides the application of the above-mentioned polypeptide in the preparation of drugs for treating allergies.

[0010] Specifically, the above-mentioned drugs use the polypeptide as the active ingredient and are formulated into pharmaceutically acceptable dosage forms using pharmaceutically acceptable carrier excipients; the dosage forms include tablets, capsules, powders, injections, oral liquids, powders, and lyophilized powders.

[0011] (III) Beneficial Effects

[0012] 1. This invention discloses a polypeptide with a novel amino acid structure, the amino acid sequence of which is shown in SEQ ID NO. 1. The polypeptide provided by this invention has no adverse effects on cell activity or nematode survival rate, and is safe and has no toxic side effects.

[0013] 2. The polypeptide provided by this invention can effectively inhibit the occurrence of paralysis in the Alzheimer's disease model Caenorhabditis elegans and significantly reduce the paralysis rate of transgenic CL4176 nematodes. It has significant anti-Alzheimer's activity and is expected to be a new drug for the treatment of Alzheimer's disease.

[0014] 3. The polypeptide provided by this invention has a significant inhibitory effect on the secretion of IgE by U266 cells. This polypeptide can achieve an anti-allergic effect by inhibiting the production of IgE, and is expected to become a new drug for the treatment of allergies. Attached Figure Description

[0015] Figure 1 The results show the purity analysis of the polypeptide with the amino acid sequence TFSHAEGT (SEQ ID NO.1).

[0016] Figure 2 The mass spectrometry results are for the polypeptide with the amino acid sequence TFSHAEGT (SEQ ID NO.1); RT: 5.74 min; Area: 1327159; Height: 271652. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments 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 some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1

[0019] Peptide Synthesis and Identification

[0020] This embodiment describes the solid-phase synthesis of a polypeptide with the amino acid sequence TFSHAEGT (SEQ ID NO.1) (assisted by Zhengzhou Huazan Pharmaceutical Technology Co., Ltd.), and the purity of the synthesized polypeptide was analyzed and identified by mass spectrometry. Figure 1 As shown, in the polypeptide of this invention, the main peak accounts for the majority, with almost no impurity peaks, and the purity is above 95%. Figure 2 As shown, the polypeptide of this invention has [M+3H]. 3+ The value was 851.58, which is basically consistent with its theoretical molecular weight (848.86). These results indicate that the polypeptide with the amino acid sequence shown in SEQ ID NO.1 synthesized in this embodiment has a purity that meets the requirements for subsequent experiments.

[0021] Example 2

[0022] The effect of the polypeptide of the present invention on the paralysis model of CL4176 nematode was obtained by the polypeptide prepared in Example 1.

[0023] I. Experimental Materials

[0024] MgSO4 was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; agar powder, yeast powder, cholesterol, tryptone, NaCl, and CaCl2 were all purchased from Beijing Solarbio Science & Technology Co., Ltd.; Na2HPO4·12H2O and KH2PO4 were all purchased from Sinopharm Chemical Reagent Co., Ltd.; the transgenic AD model of Caenorhabditis elegans (C. elegans) CL4176[dvIs27[myo-3p::A-Beta(1-42)::let-851 3'UTR)+rol-6(su1006)]X] and the uracil-deficient Escherichia coli OP50 were both purchased from the Caenorhabditis elegans Genetic Center in the United States.

[0025] II. Solution Preparation

[0026] M9 buffer: Weigh 0.6g disodium hydrogen phosphate, 0.3g potassium dihydrogen phosphate, 0.5g sodium chloride, and 0.025g magnesium sulfate (dehydrated), dissolve in 100mL deionized water, sterilize at high temperature, and set aside for use.

[0027] NGM culture plate: Weigh 3.0g sodium chloride, 2.5g tryptone, and 17g agar into an Erlenmeyer flask, add 975mL deionized water, and autoclave. When the temperature of the sterilized solution drops to approximately 55℃, add 0.5mL of 1M calcium chloride solution, 1mL of 5mg / mL cholesterol solution, 1mL of 1M magnesium sulfate solution, and 25mL of potassium phosphate buffer solution in sequence. Use a pipette to add the prepared culture medium liquid to the corresponding petri dishes or well plates, and allow to cool and solidify overnight.

[0028] Stock solution of the polypeptide to be tested: Weigh an appropriate amount of polypeptide and dissolve it in DMSO to prepare a 10mM solution.

[0029] III. Experimental Methods

[0030] 1. Effects of peptides on the survival rate of wild-type Caenorhabditis elegans

[0031] The polypeptide stock solution was diluted with M9 buffer to 0.25 mM, 0.5 mM, and 1 mM, respectively. 500 μL of each solution was evenly spread onto freshly prepared NGM plates and allowed to air dry at room temperature. 50 μL of OP50 bacterial suspension was added to the center of each plate, and the plates were dried overnight. Wild-type *C. elegans* were inoculated onto the drug-containing plates. A control group without the polypeptide was also included. After 48 hours of culture, the effect of different polypeptide concentrations on the survival rate of wild-type *C. elegans* was observed. Each group contained at least 30 nematodes, and each experiment was repeated three times. The 48-hour survival rate was calculated.

[0032] 48-hour nematode survival rate % = (Total number of surviving nematodes after 48 hours / Total number of surviving nematodes at 0 hours) × 100%

[0033] 2. Effects of peptides on the transgenic AD model Caenorhabditis elegans strain CL4176

[0034] The polypeptide stock solution was diluted with M9 buffer to 0.25 mM, 0.5 mM, and 1 mM. 500 μL of each solution was evenly spread onto freshly prepared NGM plates and allowed to air dry at room temperature. 50 μL of OP50 bacterial suspension was added to the center of each plate, and the plates were dried overnight. Synchronized CL4176 *C. elegans* nematodes were inoculated onto the drug-containing plates. A group without the polypeptide was also set up as a model group.

[0035] CL4176 *C. elegans*, after being synchronized, were cultured at 16°C for 48 hours, reaching the L3 stage. The culture plate was then transferred to 25°C to induce Aβ gene transcription. Under these conditions, the number of paralyzed *C. elegans* within 36 hours was observed and recorded. Paralyzed individuals exhibited stiffness in the trunk, leading to motor impairment or even loss of motor function; the head could be twisted. Each group contained at least 30 nematodes, and each experiment was repeated three times. The paralysis rate at 36 hours was calculated as follows: 36-hour paralysis rate % = (Number of paralyzed nematodes / Total number of nematodes) × 100%.

[0036] 3. Statistical Analysis

[0037] Data are expressed as mean ± SD. The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0038] IV. Experimental Results

[0039] 1. Effects of peptides on the survival rate of wild-type Caenorhabditis elegans

[0040] The survival rate of nematodes after 48 hours under different peptide concentrations is shown in Table 1. Compared with the control group, there was no significant difference in the survival rate of wild-type nematodes under different peptide concentrations, indicating that the peptides provided by this invention are safe and have no side effects.

[0041] Table 1. Effects of different concentrations of peptides on nematode survival rate.

[0042]

[0043]

[0044] Note: ns indicates no significant difference compared to the control group, P>0.05

[0045] 2. Effects of peptides on the transgenic AD model Caenorhabditis elegans strain CL4176

[0046] Transgenic CL476 nematodes can induce human Aβ at 25°C. 1-42 In muscle expression, with Aβ 1-42 As the nematodes accumulate, they gradually become paralyzed and lose their ability to move. Therefore, the CL4176 transgenic nematode is widely used as a biological model to explore the anti-Alzheimer's disease effects of drugs.

[0047] The results of the nematode paralysis rate detection within 36 hours are shown in Table 2. Compared with the model group, the paralysis rate of nematodes in different concentrations of peptide groups was significantly reduced (P < 0.05, P < 0.01, P < 0.001), and a good dose-response relationship was observed. These results indicate that the peptide containing the amino acid shown in SEQ ID NO. 1 provided by this invention can effectively inhibit the occurrence of paralysis in the AD model *C. elegans*, exhibiting significant anti-AD activity, and is expected to be further developed as a drug for treating AD.

[0048] Table 2. Nematode paralysis rate in each group

[0049] Group Paralysis rate (%) Model group 89.33±7.68 0.25mM peptide 71.24±6.22* 0.5mM peptide 68.24±8.09** 1mM peptide 53.24±8.76***

[0050] Note: * This indicates a comparison with the model group. * P < 0.05 ** P < 0.01, *** P < 0.001.

[0051] Example 3

[0052] Study on the IgE inhibitory activity of the peptides of this invention

[0053] I. Experimental Materials

[0054] Peptide stock solution: Weigh the peptide shown in SEQ ID NO.1 and prepare a 10 mM solution with DMSO. Store at 4°C. Dilute with DMSO according to the required concentration during the experiment.

[0055] Phosphate-buffered saline (PBS), fetal bovine serum (FBS), RPMI 1640 medium, trypan blue, and dimethyl sulfoxide (DMSO) were all purchased from Beijing Solarbio Science & Technology Co., Ltd. U266 cell lines were obtained from the laboratory of Henan University of Traditional Chinese Medicine, and IgE detection kits were purchased from Mabtech Corporation.

[0056] II. Experimental Methods

[0057] U266 cryopreserved cell lines were revived in a 37°C water bath. After adding 10 mL of PBS and centrifuging to remove DMSO, the cells were resuspended in RPMI 1640 medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. Once the cells regained their growth state, the U266 cell density was counted using a cell counting chamber, and the cells were diluted to 1×10⁶ cells / mL using pre-prepared RPMI 1640 complete medium (without antibiotics) containing 10% fetal bovine serum. 5 The 96-well plates were divided into groups: a blank group, a model group, and experimental groups with different concentrations. Each group had three replicates. The blank group was added with 100 μL of complete culture medium, while the model and experimental groups were added with 100 μL of 1×10⁻⁶ medium. 5Cell suspensions of U266 cells / mL were prepared. Different treatments were then applied: the blank and model groups were supplemented with 100 μL of complete culture medium, while the experimental groups were supplemented with 100 μL of complete culture medium containing the tested peptide stock solution, bringing the final drug concentrations in the 96-well plates to 10 μM, 20 μM, and 40 μM, respectively. The culture plates were incubated at 37°C for 72 h. After incubation, U266 cells were in suspension. 50 μL of the mixture from each well was removed, and trypan blue staining was used to determine cell viability after treatment with different drug concentrations. The culture plates were then centrifuged at 1000 rpm for 5 min, and the supernatant was collected. Total IgE levels were measured using an ELISA kit, and the IgE inhibition rate was calculated.

[0058] Cell viability (%) = (Total number of live cells / (Total number of live cells + Total number of dead cells)) × 100%

[0059] IgE experimental data are expressed as inhibition rate (I), where E 实验 E represents the detected value of IgE in each experimental group. 模型 E represents the average IgE level in the U266 cells of the model group. 空白 The formula representing the blank group detection value is as follows:

[0060] Inhibition rate Ic for each group = 100% - (E 实验 -E 空白 ) / (E 模型 -E 空白 )×100%.

[0061] III. Experimental Results

[0062] The results of U266 cell survival rate detection under different peptide concentrations are shown in Table 3. Compared with normally cultured U266 cells, there was no significant difference in U266 cell survival rate under different peptide concentrations, indicating that the peptides provided by the present invention have no side effects.

[0063] Table 3. Effects of different concentrations of peptides on cell viability.

[0064] Concentration (μM) Cell viability (%) 0 95.83±5.01 10 <![CDATA[92.13±7.81 ns ]]> 20 <![CDATA[90.83±8.43 ns ]]> 40 <![CDATA[89.69±7.93 ns ]]>

[0065] Note: ns indicates no significant difference compared to the control group, P>0.05.

[0066] As shown in Table 4, within the concentration range of 10-40 μM, the peptides significantly inhibited IgE secretion from U266 cells, exhibiting a favorable dose-response relationship. These results indicate that the peptides provided by this invention can achieve anti-allergic effects by inhibiting IgE production, and hold promise as a novel drug for treating allergies.

[0067] Table 4. Inhibition rate of IgE by different concentrations of peptides

[0068] Concentration (μM) IgE inhibition rate (%) 10 23.33±7.65 20 35.23±8.22 40 49.88±6.23

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is TFSHAEGT.

2. The use of the polypeptide as described in claim 1 in the preparation of a drug for treating Alzheimer's disease.

3. The application according to claim 2, characterized in that, The drug uses the polypeptide of claim 1 as the active ingredient and is formulated into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier excipient; the dosage form includes injections and powders.

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