Bioactive heptadecapeptide and use thereof

Abstract

The present application provides a kind of bioactive seventeen peptides and its application, belong to bioactive peptide technical field, the amino acid sequence of the seventeen peptides is as shown in SEQ ID NO.1;Seventeen peptides provided by the present application can significantly reduce immune damage, improve immune organ index, reduce spleen tissue damage, can significantly promote the secretion level of immunoglobulin and cytokine in blood;The polypeptide has immunoregulation, can improve immunity, can be used for preparing the product of enhancing immunity.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a bioactive heptadecapeptide and its applications. Background Technology

[0002] The core functions of the immune system include defense, surveillance, homeostasis, and tolerance. Immune dysfunction can occur in many situations, such as HIV infection, radiotherapy and chemotherapy, chronic diseases, and malnutrition. Prolonged or extreme mental stress, overwork and overtraining, smoking, and excessive alcohol consumption can also adversely affect the immune system. Preventing and restoring weakened immune function is crucial for maintaining health. Using immunomodulators to enhance the host's defense response is an effective way to improve disease resistance. Existing technologies already have drugs that can be used as immunostimulants. However, due to cost and side effects, most of these drugs are difficult to use long-term or preventively. Finding safe, low-toxicity immunomodulators is of great significance for enhancing immunity and preventing disease.

[0003] Cyclophosphamide (CTX) is one of the most widely used alkylating agents in chemotherapy. Since the late 1950s, it has become an important drug in clinical research and treatment of various cancers due to its high therapeutic index and broad-spectrum antitumor activity. However, CTX exhibits non-selective cytotoxicity against both tumor and normal cells, and has a significant inhibitory effect on rapidly proliferating immune cells. Common side effects include leukopenia, bone marrow suppression, and immune dysfunction. CTX has inhibitory effects on both humoral and cellular immunity. Short-term administration of specific doses of CTX can significantly reduce the immune function of mice, and therefore it has been widely used to construct immunosuppressive animal models.

[0004] The spleen, thymus, and liver are important immune organs, and their functions are closely related to cellular and humoral immunity. These organs are not only key sites for lymphocyte differentiation, development, and maturation, but also coordinate various aspects of the body's immune response. Cyclophosphamide (CTX) can inhibit lymphocyte differentiation, reduce the number of lymphocytes in immune organs, and consequently cause a decrease in the size of the spleen, thymus, liver, and even overall body weight. Therefore, the thymus index, spleen index, and liver index are often used as important indicators for assessing the body's immune regulatory status.

[0005] Bioactive peptides are highly favored in the development of functional foods and pharmaceuticals due to their excellent processing properties and broad range of biological activities. Researching and developing bioactive peptides with immune-enhancing effects is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a bioactive heptapeptide and its applications.

[0007] A bioactive heptadecapeptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] TDTNNNDNQLDQFPRRFSEQ ID NO.1.

[0009] The application of the above-mentioned bioactive heptapeptide in the preparation of products that enhance immunity.

[0010] Preferably, the product is a health supplement or a medicine.

[0011] Preferably, the product also includes auxiliary materials.

[0012] Preferably, the above-mentioned bioactive heptapeptide is used in the preparation of immune enhancers.

[0013] A product containing the aforementioned bioactive heptadecapeptide.

[0014] Preferably, the product is a health supplement or a medicine.

[0015] Preferably, the product also includes auxiliary materials.

[0016] The beneficial effects of the present invention include at least the following: This invention provides a bioactive heptadecapeptide that can significantly reduce immune damage, increase immune organ indices, alleviate spleen tissue damage, and significantly promote the secretion levels of immunoglobulins (IgM, IgA, IgG) and cytokines (TNF-α, IFN-γ, IL-2) in the blood, thereby enhancing humoral immunity. This peptide has immunomodulatory effects, can improve the body's immunity, and can be used to prepare products that enhance immunity. Attached Figure Description

[0017] Figure 1 This is the mass spectrum of the synthesized heptadecapeptide.

[0018] Figure 2 Figure showing the effects of synthetic peptides on mouse body weight and immune organs; In the figure: A is a curve showing the change in body weight; B is a graph showing the spleen index, thymus index, and liver index of mice; C is a HE staining image of spleen tissue; data are expressed as mean ± standard value (n=3 or 6); different superscript characters indicate statistical significance determined by ANOVA; compared with the CK group. ## P<0.01, compared with the CTX group: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0019] Figure 3Figure showing the effects of synthetic peptides on humoral and cellular immunity in mice; In the figure: A represents the serum secretion levels of immunoglobulins A, G, and M; B represents the secretion levels of cytokines TNF-α, IFN-γ, and IL-2; data are expressed as mean ± standard value (n=3), and different superscript characters indicate statistical significance determined by ANOVA; compared with the CK group. ## P<0.01, compared with the CTX group: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0020] Figure 4 This is a diagram of transcriptome sequencing and bioinformatics analysis. In the figure: A is the differential gene GO analysis diagram of the cyclophosphamide (CTX) group vs. the polypeptide (H) group, and B is the differential gene KEGG analysis diagram of CTX vs. HC.

[0021] Figure 5 This is a diagram showing the effect of synthetic peptides on the expression of related genes; In the figure: A represents the expression of inflammation-related genes, and B represents the expression of genes related to the JAK-STAT signaling pathway; data are expressed as mean ± standard value (n=3); different superscript characters indicate statistical significance determined by ANOVA; compared with the CK group. ## P<0.01, compared with the CTX group: * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0023] All details not described in the embodiments are based on existing technology in the field.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments are commercially available.

[0025] Experimental materials Cyclophosphamide and levamisole hydrochloride were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals and reagents used were of analytical grade.

[0026] The heptadecapeptide (TDTNNNDNQLDQFPRRF) provided by the present invention can be synthesized by itself or synthesized commercially by a company.

[0027] Example 1 During the research on polypeptides, the inventors synthesized a heptadecapeptide. The crude product of the sequence was obtained by solid-phase synthesis in a peptide synthesizer (Agilent 1200, Agilent Technologies, Inc., Santa Clara, California, USA), and the polypeptide was purified by a high-performance liquid chromatograph (LC6000N, Beijing Innovation Tongheng Technology Co., Ltd., Beijing). The amino acid sequence of the synthesized heptadecapeptide is TDTNNNDNQLDQFPRRF (SEQ ID NO.1), and the purity is 97.03%. The mass spectrum of the synthesized heptadecapeptide is as Figure 1 shown.

[0028] Example 2 1 Method 1.1 Animal experiments and treatments Six- to eight-week-old male mice (BALB / c) without specific pathogens were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), license number: SCXK(Beijing) 2021-0011. The average body weight (bw) of the mice was 20 ± 2 g, and the mice were maintained at a controlled temperature of 20-25°C and a relative humidity of 50 ± 10%. Animal experiments were conducted in accordance with the principles of laboratory animal care and were approved by the Ethics Committee of the Laboratory Animal Center of Shandong University.

[0029] Ten mice were in each group. The mice were divided into a CK (blank control) group, a CTX (cyclophosphamide model) group, an LMS (levamisole hydrochloride positive control) group, and an HC (synthetic peptide) group (Table 1). The experimental groups were intraperitoneally injected with 200 μL of cyclophosphamide at 80 mg / kg once a day for 3 days; the CK group was given 200 μL of normal saline. Three days after administration, the HC group was orally administered the synthetic peptide powder (the heptadecapeptide synthesized in Example 1) at 100 mg / kg, the positive control group was given levamisole hydrochloride at 40 mg / kg, and the CK control group and the model group were gavaged with normal saline. The gavage dose was 200 μL once a day for 7 days, and the body weight of the mice was recorded every day.

[0030] Table 1 Grouping and drug administration of animal experiments

[0031] After the experiment, blood was collected from the eyeballs of mice. The blood samples were left at room temperature for 2 hours, then centrifuged at 2000 rpm for 10 minutes to obtain serum samples. Simultaneously, the mice were euthanized by cervical dislocation, and their immune organs, including the thymus, spleen, and liver, were immediately weighed. The thymus, spleen, and liver indices were calculated using the following formula: Index (mg / g) = Thymus, spleen, or liver weight (mg) / Body weight (g). A portion of the spleen was fixed in 4% paraformaldehyde general-purpose tissue fixative, while other portions of the spleen and liver were frozen in liquid nitrogen and then stored at -80°C for later use.

[0032] 1.2 Histopathological Analysis Spleen tissue fixed in 4% paraformaldehyde was embedded in paraffin, cut into 5 mm continuous sections, and stained with hematoxylin and eosin (HE) according to standard procedures. The sections were then examined histopathologically under an optical microscope (Nikon, Tokyo, Japan).

[0033] 1.3 Determination of blood cells in peripheral blood The levels of hemoglobin, red blood cells, white blood cells, platelets, and lymphocytes in the peripheral blood of mice were measured using a fully automated animal blood cell analyzer (BC-2800 Vet, Shenzhen Mindray Animal Medical Technology Co., Ltd., Shenzhen).

[0034] 1.4 Determination of Immunoglobulins After the obtained blood was left at room temperature for two hours, it was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected. The levels of IgM, IgA, and IgG in the serum of mice in each group were detected using a double-antibody sandwich ELISA method. The ELISA kit (Beinlai, Wuhan, China) was followed according to the instructions. The absorbance was measured at 450 nm, and the Logistic curves were plotted using ELISA calc software based on the concentration and absorbance of the standard wells to calculate the levels of IgM, IgA, and IgG in the serum.

[0035] 1.5 Measurement of Cytokines After the obtained blood was left at room temperature for two hours, it was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected. The levels of TNF-α, IFN-γ, and IL-2 in the serum of mice in each group were detected using a double-antibody sandwich ELISA method. The ELISA kit (Beinlai, Wuhan, China) was followed according to the instructions. The absorbance was measured at 450 nm, and the Logistic curves were plotted using ELISA calc software based on the concentration and absorbance of the standard wells to calculate the levels of IL-2, IFN-γ, and TNF-α in the serum.

[0036] 1.6 RNA extraction and RT-qPCR detection Four samples were randomly selected from each group to detect the mRNA expression of relevant genes in spleen tissue. Total RNA was extracted from spleen tissue using an RNA extraction kit (Takara, Japan), and cDNA was synthesized using the PrimeScript™ RT reagent Kit withgDNA Eraser kit (Takara, Japan). Detection was performed using a SYBR® Premix Ex Taq™ II (Takara, Japan) real-time PCR quantification system (QuantStudio 3 Real-Time RCR Systems, Thermo Fisher, USA). TNF-α, IL-4, IL-5, IFN-γ, IL-1β, IL-2, IL-2ra, IL-2rg, JAK3, STAT5, SOCS3, CCND1 and CCND2 mRNA expression levels of the gene. Primers are shown in Table 2. Use... GAPDH The gene was used as a reference, and the final expression level of the RT-qPCR product was calculated using the 2-ΔΔCt method.

[0037] Table 2 qPCR primer sequences

[0038] 1.7 Transcriptome sequencing (RNA-Seq) analysis of differentially expressed genes Transcriptome sequencing was performed on mice from the CTX and HC groups, with four biological replicates in each group. Raw sequencing data (Raw Reads) were generated using Illumina HiSeq™ 2000 (Illumina, USA). After quality assessment and filtering, high-quality sequencing data (Clean Reads) were obtained for downstream analysis. The HISAT2 alignment software was used to align the sequencing data with reference genes. Transcripts were reconstructed using StringTie, and the expression levels of all genes in each sample were calculated using the FPKM method. Differential gene expression analysis was performed using DESeq2. The screening criteria for significantly differentially expressed genes were: |log2FoldChange|>1 and Adjusted P value<0.05. GO enrichment and KEGG enrichment analyses of differentially expressed genes were performed using ClusterProfiler.

[0039] 1.8 Western Blot Detection Mouse liver tissue was homogenized in liquid nitrogen and placed in 10 times the tissue volume of RIPA lysis buffer (RIPA:cocktail:PMSF:phosphoprotease inhibitor A:phosphoprotease inhibitor B = 100:2:1:1:1). Lysis was performed on ice for 30 min. After lysis, the tissue was centrifuged at 12000 r / min for 10 min at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA kit (Solepro, Beijing, China). The protein solution was added to 5× reducing protein loading buffer at a 4:1 ratio, denatured in a metal bath at 95°C for 10 min, cooled, and then separated on a 4%–12% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked for 1 h at room temperature with TBST containing 5% skim milk (containing 0.1% Tween-20), and then incubated overnight at 4°C with the following primary antibodies: STAT5 (1:1000) and β-actin (1:2000). After incubation with the primary antibody, the membrane was incubated with horseradish peroxidase-conjugated IgG secondary antibody (1:2000) at room temperature for 1 h. The PVDF membrane was stained with ECL chemiluminescent solution, and the bands were visualized using a gel imaging analysis system (JY04S-3C, Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd., Beijing, China). The gray values ​​of the bands were quantified using ImageJ software, with β-actin as an internal control.

[0040] 1.9 Statistical Analysis GraphPad Prism version 9.0 (La Jolla, CA, USA) was used for graphical analysis. All data are expressed as mean ± standard deviation (SD). One-way ANOVA was used for statistical analysis, with P < 0.05 indicating statistical significance and P < 0.01 indicating highly statistical significance.

[0041] 2 Results and Analysis 2.1 Effects of synthetic peptides on mouse body weight and immune organs The health status of mice can be directly assessed through changes in body weight. For example... Figure 2 As shown in Figure A, after three consecutive days of intraperitoneal injection of cyclophosphamide, the body weight of mice in all three groups decreased significantly, reaching 16-18g on the fifth day, a decrease of 3-4g compared to the initial weight. The normal control group (CK group) showed no significant change in body weight, indicating the successful establishment of the immunosuppression model. After treatment began, the body weight of the LMS group (positive control group) and the HC group increased over time, while the CTX group showed slow weight gain. After one week of treatment, the body weight of the LMS and HC groups returned to normal levels, indicating that the synthetic peptide had a certain restorative effect on CTX-induced immunosuppression in mice.

[0042] The spleen, thymus, and liver are major immune organs, and organ indices can partially reflect the body's immune function, showing a positive correlation within a certain range. For example... Figure 2 As shown in Figure B, the spleen and thymus indices in the CTX group were significantly lower than those in the CK group (P<0.05), indicating that CTX induced atrophy of immune organs and confirming the effectiveness of the established immunosuppression model. Compared with the model group, the spleen, thymus, and liver indices in the HC group were significantly increased (P<0.05). These experimental results demonstrate that synthetic peptides can promote the growth of immune organs in immunosuppressed mice.

[0043] A breakdown of the immune system is often accompanied by damage to immune organs. The spleen is the largest lymphatic organ in the human body. After HE staining, the red pulp in spleen tissue appears purplish-red, while the white pulp appears blue. Figure 2 The CK group mice showed normal red and white pulp structure in their spleen tissue, with tightly packed lymphocytes and prominent lymph nodes. In contrast, the CTX group mice exhibited disordered spleen tissue structure, blurred boundaries between the red and white pulp, reduced lymphocyte count, and increased reticular cell count. Compared to the model group, the HC group mice showed clearer red and white pulp in their spleen tissue, increased lymphocytes, and denser, more orderly arranged spleen cells. This indicates that intervention with synthetic peptides can effectively inhibit pathological changes in spleen tissue.

[0044] 2.2 Effects of synthetic peptides on hematological parameters in mice White blood cells and lymphocytes are important components of the body's innate immunity, and the total white blood cell and lymphocyte counts can reflect the body's immune function status. When the body's overall immune function is low, the proliferation of white blood cells and lymphocytes is also inhibited, resulting in a decrease in the total white blood cell count. As shown in Table 3, compared with the CK group, the CTX group showed a significant decrease in platelets, red blood cells, white blood cells, hemoglobin, and lymphocytes (P<0.05), indicating that cyclophosphamide has an adverse effect on blood function and inhibits bone marrow hematopoietic function in mice. After treatment, the platelet counts, red blood cells, and hemoglobin in the LMS and HC groups were significantly different from those in the model group (P<0.05). White blood cells and lymphocytes in all treatment groups showed significant recovery, but the recovery level in the HC group was closer to that of the normal group. These results indicate that synthetic peptides can effectively restore bone marrow hematopoietic function in mice and significantly improve peripheral blood cell counts.

[0045] Table 3. Complete blood cell count (CBC) parameters in different treatment groups

[0046] Data are presented as mean ± standard deviation (n=3). Different superscript characters indicate statistical significance determined by ANOVA. Compared with the CK group ## P<0.01, compared with the CTX group: * P<0.05, ** P<0.01,*** P<0.001, **** P<0.0001.

[0047] 2.3 Effects of synthetic peptides on humoral and cellular immunity in mice The levels of IgA, IgG, and IgM in the serum of mice in each group were measured to determine the effect of the synthetic peptide on humoral immunity. Figure 3 The results showed that, compared with the control group, the serum IgG, IgA, and IgM levels in the model group mice were significantly decreased (P<0.05). After treatment, the serum IgA, IgM, and IgG levels in the HC group mice were significantly increased (P<0.05), approaching the levels of the control group. These results indicate that synthetic peptides can restore the serum IgG, IgA, and IgM levels in immunosuppressed mice.

[0048] Cytokines play a driving role in the immune response, and their secretion levels are closely related to immune regulation. They enhance the overall efficiency and power of the immune response by coordinating, activating, and strengthening the function of immune cells. Figure 3 As shown in Figure B, compared with the normal group, the levels of TNF-α, IFN-γ, and IL-2 in the CTX group were significantly decreased (P<0.05), decreasing from 273.405 pg / ml to 79.57 pg / ml, 270.155 pg / ml to 57.11 pg / ml, and 360.019 pg / ml to 97.45 pg / ml, respectively. This indicates that cyclophosphamide can inhibit the secretion of cytokines in mouse blood, thereby reducing the immunity of mice. Compared with the CTX group, the production of TNF-α in the LMS and HC groups was significantly increased (P<0.05), increasing to 122.909 pg / ml and 159.709 pg / ml, respectively. The secretion levels of IFN-γ and IL-2 in the LMS group were close to those in the control group. The results indicate that synthetic peptides have the effect of regulating the secretion of multiple cytokines in mouse serum.

[0049] 2.4 Transcriptome sequencing (RNA-Seq) and GO / KEGG analysis Functional analysis of differentially expressed genes (DEGs) obtained from transcriptome sequencing was performed using the Clusterprofile database. GO enrichment analysis showed that DEGs obtained from the CTX and HC treatments were significantly enriched in biological processes such as immune response, cell migration, cardiac development, adaptive immune response, cell surface receptor signaling pathways, and T cell receptor signaling pathways. Figure 4KEGG pathway analysis revealed that the DEGs obtained from the CTX and HC groups were mainly enriched in pathways such as Cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, and Hematopoietic cell lineage signaling pathway. Figure 4 (B)

[0050] 2.5 Study on the immunomodulatory function of synthetic peptides and the JAK-STAT pathway To further validate the transcriptome sequencing results, RT-qPCR and Western blot were performed to detect relevant inflammatory genes and JAK-STAT pathway-related genes and proteins. Figure 5 As shown in Figure A, compared with the CK group, the CTX group TNF-α, IL-1β, IFN-γ γ and IL-5 The mRNA expression of the gene was significantly downregulated (P<0.05), while that of the HC group was significantly upregulated (P<0.01), consistent with the ELISA results. To demonstrate that synthetic peptides enhance mouse immunity by activating the JAK-STAT signaling pathway, nine key genes in this pathway were selected. IL-2, IL-2ra, IL-2rg, IL-4, JAK3, STAT5, SOCS3, CCND1 and CCND2 ) Detect relative mRNA expression, such as Figure 5 As shown in Figure B, the synthetic peptide can effectively enhance the expression of these key genes. To further demonstrate whether the JAK-STAT signaling pathway is activated, the expression level of STAT5 protein was analyzed by Western blot and ImageJ grayscale value analysis. The band in the model group was lighter, while the band of the synthetic peptide was darker and had a higher grayscale value, showing a significant difference compared to the model group (P<0.01). These results indicate that the synthetic peptide may enhance mouse immunity by activating the JAK-STAT signaling pathway.

[0051] The heptadecapeptide provided by this invention can alleviate weight loss in immunosuppressed mice, improve cyclophosphamide-induced atrophy of immune organs, and has a significant immune-enhancing effect on immune organs. The heptadecapeptide can maintain the cellular immune system and regulate immune function by promoting the secretion of various cytokines. The heptadecapeptide can also activate the JAK-STAT signaling pathway. The heptadecapeptide provided by this invention can improve the body's immunity and can be used to prepare products that enhance immunity.

Claims

1. A biologically active heptadecapeptide, characterized in that, The amino acid sequence of the heptadecapeptide is shown in SEQ ID NO.

1.

2. The use of the bioactive heptadecapeptide of claim 1 in the preparation of products that enhance immunity.

3. Use according to claim 2, wherein the compound is ###0002### The product in question is a health supplement or a medicine.

4. The use according to claim 2, wherein the compound is ###0002### The product also includes auxiliary materials.

5. The use according to claim 2, wherein the compound is ###0002### The application of the bioactive heptadecapeptide of claim 1 in the preparation of an immune enhancer.

6. A product characterized in that, It contains the bioactive heptadecapeptide as described in claim 1.

7. The product as described in claim 6, characterized in that, The product in question is a health supplement or a medicine.

8. The product as described in claim 6, characterized in that, The product also includes auxiliary materials.

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