A polypeptide pep169l with anti-inflammatory effect and application thereof

Abstract

The application discloses a polypeptide pep169L with anti-inflammatory effect and application thereof. The application identifies the inhibitory effect of the pep169L polypeptide on NF-kappa B reporter gene and p65 protein expression, detects the inhibitory effect of the polypeptide on inflammatory cytokine IL-1beta, and explores the function of the pep169L polypeptide in reducing DSS-induced colitis in mice. The pep169L polypeptide can significantly inhibit the production of LPS-induced reporter gene NF-kappa B, p65 and inflammatory cytokine IL-1beta, can inhibit the secretion of DSS-induced inflammatory cytokines IL-6, IL-1beta and TNF-alpha in mice, and can improve the DSS-induced clinical characteristics, including colon length, disease activity index and pathological changes. The polypeptide has the functions of inhibiting inflammatory reaction and reducing DSS-induced colitis, and the application of the polypeptide can provide a new idea for the design of a new anti-inflammatory small molecule drug.

Description

Technical Field

[0001] This invention relates to a polypeptide pep169L with anti-inflammatory effects and its applications, belonging to the field of polypeptides. Background Technology

[0002] The innate immune system, as the body's first line of immune defense, can recognize pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), activate the nuclear factor κB (NFκB) signaling pathway, and induce the production of pro-inflammatory cytokines and antimicrobial peptides to resist the invasion of pathogenic microorganisms.

[0003] Inflammatory diseases are a class of conditions caused by abnormal immune system responses, primarily characterized by localized or systemic inflammatory reactions. These diseases include rheumatoid arthritis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), and psoriasis. While inflammatory responses are an important part of the body's self-protection mechanism to some extent, uncontrolled or persistent activation can lead to tissue damage and various pathological states. The dextran sulfate sodium (DSS) model is a widely used experimental model for studying enteritis and inflammatory bowel disease. DSS is a compound that can induce colonic inflammation by damaging intestinal epithelial cells, leading to intestinal inflammation. The advantages of this model are its ease of operation, controllable effects, and ability to effectively simulate the pathological characteristics of inflammatory bowel disease. Therefore, the DSS model provides an excellent experimental platform for exploring the potential mechanisms by which peptides can cure or alleviate inflammatory diseases.

[0004] Peptides exhibit a wide range of biological activities, including anti-inflammatory and immunomodulatory effects. Studies have shown that certain peptides can alleviate DSS-induced intestinal inflammation through mechanisms such as inhibiting the expression of pro-inflammatory factors, regulating cytokine levels, or enhancing intestinal barrier function. For example, some naturally derived peptides, such as collagen peptides and lactoferrin peptides, have been shown to improve the pathological state of DSS model mice. In addition, synthetic peptides are increasingly used in anti-inflammatory therapy due to their tunability and specificity.

[0005] Studying the role of peptides in DSS models not only provides new insights into the treatment of inflammatory diseases but also offers data support for peptide characterization. The mechanism by which viruses escape the NF-κB signaling pathway also provides a theoretical basis for future research on anti-inflammatory drugs. These studies contribute to a deeper understanding of the role of peptides in inflammatory mechanisms and the development of new therapeutic strategies for inflammatory diseases. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a polypeptide pep169L with anti-inflammatory effect and its application, thereby inhibiting the overactivation of the NF-κB signaling pathway.

[0007] Technical solution: To solve the above technical problems, the present invention provides a polypeptide with the amino acid sequence RQIKIWFQNRRMKWKKVDFIAGINNLGEKIY.

[0008] The present invention also provides the use of the said polypeptide in the preparation of medicaments for treating and / or preventing inflammatory diseases.

[0009] The inflammatory diseases mentioned include colitis.

[0010] Among its many benefits is the ability to significantly reduce the secretion of inflammatory cytokines and inhibit the body's inflammatory response, making it a highly promising candidate for anti-inflammatory drugs.

[0011] The inflammatory cytokines include IL-6, IL-1β, and TNF-α.

[0012] This includes inhibiting the expression of p65 protein.

[0013] The present invention also provides a medicament for treating and / or preventing inflammatory diseases, which contains the said polypeptide.

[0014] The present invention also provides the use of the polypeptide in the preparation of a medicament for improving hematochezia and / or fecal occult blood.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the polypeptide has the function of inhibiting inflammatory response and reducing DSS-induced colitis, which will provide new ideas for the design of novel anti-inflammatory small molecule drugs. Attached Figure Description

[0016] Figure 1 A: Dual-luciferase reporter assay to detect the expression of NF-κB reporter gene; Figure 1 B: qPCR assay to detect the production of inflammatory cytokines; Figure 1 C: Western blot analysis of p65 protein expression;

[0017] Figure 2 A: Observe and record the bloody stool in the mice; Figure 2 B: Use a fecal occult blood test kit to determine fecal occult blood in mice; Figure 2 C: DAI score of DSS-induced colitis in mice;

[0018] Figure 3 A: Measurement of colon length in mice; Figure 3 B: Statistical analysis of mouse colon length; Figure 3C: Determination of MPO in mouse colon tissue;

[0019] Figure 4 A: qPCR experiment to detect the expression of inflammatory cytokines in the mouse intestine; Figure 4 B: ELISA was used to measure the levels of IL-6, IL-1β, and TNF-α in colon tissue;

[0020] Figure 5 A: Observation of pathological sections of mouse colon; Figure 5 B: Assessment of pathological changes in the mouse colon. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example 1

[0023] The pep169L peptide was used to inhibit inflammatory signaling pathways in vitro. This method employed dual-luciferase reporter assays, real-time quantitative PCR, and Western blotting to identify that the pep169L peptide significantly inhibited LPS-induced expression of the NF-κB reporter gene, production of the inflammatory cytokine IL-1β, and expression of p65 protein. The specific steps are as follows:

[0024] (1) Synthesize peptide pep169L (RQIKIWFQNRRMKWKKVDFIAGINNLGEKIY) and set up peptide CP (amino acid sequence: RQIKIWFQNRRMKWKKSLHGRGDPMEAFII) as a negative control.

[0025] (2) Dual-luciferase reporter assay to detect the inhibition of LPS-induced NF-κB reporter gene expression by pep169L peptide.

[0026] HeLa cells (2 × 10⁻⁶) were seeded in 24-well plates. 5Cells / well were transfected with the NF-κB-luc luciferase reporter gene plasmid after 16 h. After 24 h, the culture medium was discarded, and the cells were gently washed once with phosphate buffer. DMEM medium or different peptides (pep169L or CP, 40 μM) were added and incubated for 30 min. The cells were then stimulated with 100 ng / mL lipopolysaccharide (LPS). After 1 h, 100 μL of cell lysis buffer (1×Cell Lysis Buffer) was added for lysis. The cells were thoroughly pipetted and transferred to finger tubes. The cells were centrifuged at 12000 rpm for 1 min, and the supernatant was collected. 100 μL of luciferase substrate (firefly luciferase) was thoroughly mixed with 20 μL of cell lysis product and added to a non-transparent microplate for reporter gene activity detection. The results showed that pep169L significantly inhibited LPS-induced NF-κB reporter gene expression. Figure 1 A).

[0027] (3) Real-time quantitative PCR was used to identify the inhibition of LPS-induced inflammatory cytokine production by the pep169L peptide.

[0028] HeLa cells were plated (24-well cell plates, 2×10⁶ cells / wells). 5 After overnight culture (number per well), the culture medium was discarded, and DMEM or different peptides (pep169L or CP, 40 μM) were added and incubated for 30 min. Then, the cells were stimulated with 100 ng / mL LPS for 1 h. Trizol was used to lyse the cells, RNA was extracted, DNA was removed, and cDNA was reverse transcribed using the Hiscript III RT Super mix for qPCR. The expression level of the cytokine IL-1β was detected by qRT-PCR. The results showed that LPS induced the production of a large number of inflammatory cytokines, while the peptide pep169L significantly inhibited the production of LPS-induced inflammatory cytokines. Figure 1 B).

[0029] (4) Western blot analysis showed that the pep169L peptide significantly inhibited LPS-induced p65 protein expression.

[0030] HeLa cells were plated (24-well cell plates, 2×10⁶ cells / wells). 5Cells were cultured overnight (cells / well), then the culture medium was discarded. DMEM medium or different peptides (pep169L or CP, 40 μM) were added and incubated for 30 min. Cells were then stimulated with 100 ng / mL LPS for 1 h. After stimulation, cells were lysed using Western blotting and IP lysis buffer. Cells were thoroughly pipetted and transferred to finger tubes. The cells were centrifuged at 12000 rpm for 3 min, and the supernatant was collected. 5× loading buffer was added, and the cells were boiled at 100℃ for 10 min. Proteins were separated by SDS-PAGE gel electrophoresis. The gel strips were then transferred to NC membranes and blocked in 5% skim milk blocking buffer diluted with TBST for 2 h. Cells were washed three times with TBST for 5 min each. p65 protein and GAPDH monoclonal antibody (Beyotime, AF1186) diluted with TBST containing 5% skim milk were added (p65 protein and GAPDH monoclonal antibody were both diluted 1:1000) and incubated overnight at 4℃. Cells were washed five times with TBST for 5 min each. Goat Anti-Rabbit IgG HRP (Aibot Biotechnology Co., Ltd., AS014-500ul) and Goat Anti-Mouse IgG HRP (Aibot Biotechnology Co., Ltd., AS003-500ul), diluted 1:5000 with TBST containing 5% skim milk, were added sequentially and incubated at room temperature for 1 h. The mixture was washed 6 times with TBST for 5 min each time. The mixture was then developed and exposed using an Amersham Image 600 chemiluminescence imaging system. The results showed that the pep169L peptide significantly inhibited LPS-induced p65 protein expression. Figure 1 C).

[0031] Example 2

[0032] The pep169L peptide inhibited DSS-induced colitis in mice in vivo, improved DSS-induced clinical manifestations, including colon length, disease activity index, and pathological changes, established a DSS-induced mouse colitis model, and evaluated the effect of the pep169L peptide on DSS-induced mouse colitis. The specific steps are as follows:

[0033] (1) Establishment of DSS-induced colitis model and experimental grouping

[0034] The concentration of dextran sulfate sodium DSS used to induce colitis was 4%. Male C57BL / 6 mice aged 6-8 weeks were randomly divided into 5 groups, including PBS group, DSS group, DSS+CP group, DSS+pep169L group, and DSS+5-ASA group. DSS group: Mice freely drank sterilized water containing 4% DSS for 6 consecutive days; DSS+CP group: Mice freely drank sterilized water containing 4% DSS for 6 consecutive days, and were simultaneously injected intraperitoneally with polypeptide CP at a dose of 100 nmol / mouse daily; DSS+pep169L group: Mice freely drank sterilized water containing 4% DSS for 6 consecutive days, and were simultaneously injected intraperitoneally with 100 nmol / mouse of pep169L polypeptide daily; DSS+5-ASA: 5-ASA is a first-line drug for the treatment of colitis. In this invention, it is used as a positive control for the treatment of colitis. Mice freely drank sterilized water containing 4% DSS for 6 consecutive days, and were simultaneously orally administered 5-ASA 10 mg / ml orally, 300 μL per mouse (dissolved in 0.5% CMC-Na), with an oral gavage dose of 100 mg / kg; PBS group: Mice freely drank sterilized water daily.

[0035] (2) Observation of symptoms and signs of DSS-induced colitis in mice

[0036] Mice were observed daily for their diet, activity, and coat condition. Their weight was measured and recorded at the same time each day, with weight changes expressed as a percentage of their original body weight. Fecal characteristics were observed and recorded. Furthermore, a fecal occult blood test kit was used to determine the presence of fecal occult blood and assess the severity of colitis. Results showed that the peptide pep169L significantly improved fecal hemorrhage and fecal occult blood in mice. Figure 2 A; Figure 2 B).

[0037] (3) Assessment of disease activity index (DAI) in mice with colitis

[0038] Disease Activity Index (DAI) was calculated based on mouse weight loss, stool characteristics, and fecal occult blood test. The percentage of weight loss was calculated using the formula: Percentage of weight loss (100%) = (Measured mouse weight - Initial weight) / Initial mouse weight × 100%. Weight loss scores were as follows: no weight loss, 0 points; weight loss 0%-5%, 1 point; weight loss 5%-10%, 2 points; weight loss 10%-20%, 3 points; weight loss exceeding 20%, 4 points. Stool characteristics scores were as follows: normal stool, 0 points; loose stool, 2 points; watery stool, 4 points. Fecal occult blood test scores were as follows: negative occult blood, 0 points; positive occult blood, 2 points; visible blood in stool, 4 points. The DAI score for each mouse was calculated using the formula: DAI score = (weight loss score + stool characteristics score + fecal occult blood score) / 4. The DAI score was recorded daily, and a curve showing the relationship between the DAI score and time was plotted. The results showed that the peptide pep169L could reduce the DAI score of DSS-induced colitis and delay the clinical manifestations of colitis. Figure 2 C).

[0039] (4) Changes in colon length in DSS-induced colitis mice

[0040] In DSS-treated colitis mice, the shortening of colon length can reflect the severity of inflammation to some extent. At the end of the sixth day of the experiment, mice were euthanized by cervical dislocation and immersed in 75% alcohol for 3 minutes. Colonic tissue was dissected, and morphological changes were observed. Colon length was measured and photographed. Results showed that compared with the normal control group, the colons of mice in different DSS treatment groups were significantly shortened to varying degrees. The colon shortening length in the pep169L peptide treatment group was less than that in the pep169L peptide treatment group, and the difference was statistically significant. Figure 3 A; Figure 3 B) indicates that the peptide pep169L can inhibit DSS-induced shortening of the mouse colon.

[0041] (5) Determination of myeloperoxidase (MPO) in mouse colon tissue

[0042] After colon length measurement, 100 mg of colon tissue was taken and placed in pre-cooled PBS (w / v, 1:5), and homogenized using a homogenizer. The homogenized tissue homogenate was centrifuged at 12000 rpm at 4℃ for 5 min, and the supernatant was collected for cytokine assay. Myeloperoxidase (MPO) is an indicator for quantitatively evaluating the intensity of inflammatory responses and is widely used to detect the degree of intestinal inflammation. The MPO content in colon tissue was determined using the Mouse MPO ELISA Kit. The results of MPO assay in colon tissue showed that peptide pep169L could significantly inhibit the intestinal inflammatory response in DSS-induced colitis mice. Figure 3 C).

[0043] (6) qPCR determination of inflammatory cytokine levels in mouse colon tissue

[0044] Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 3 minutes to separate colon tissue. 100 mg of colon tissue was taken and homogenized using a homogenizer in a buffer RL. RNA was then extracted, DNA was removed, and cDNA was reverse transcribed. The expression levels of cytokines IL-6, IL-1β, and TNF-α were detected by qRT-PCR. The results showed that the peptide pep169L significantly inhibited the production of DSS-induced inflammatory cytokines. Figure 4 A).

[0045] (7) ELISA was used to measure the levels of inflammatory cytokines in mouse colon tissue.

[0046] The changes in inflammatory factors in mouse intestinal tissue were detected using R&D Mouse IL-6 DuoSet ELISA, Mouse IL-1β DuoSet ELISA, and Mouse TNF-α DuoSet ELISA kits. The inflammatory factor assays were performed according to the kit instructions. Results showed that the peptide pep169L significantly reduced the levels of IL-6, IL-1β, and TNF-α in the colonic tissue of mice with DSS-induced colitis, and decreased DSS-induced systemic inflammation. Figure 4 B).

[0047] (8) Pathological changes in colon tissue of DSS-induced colitis mice

[0048] The pathological changes in the colonic tissue of mice with DSS-induced colitis were evaluated using pathological sections. Pathological sections of colonic tissue were prepared according to standard methods. Colonic tissue samples fixed in 10% neutral formalin were dehydrated with ethanol, then embedded in paraffin, sectioned using a microtome, and fixed onto glass slides. The tissue sections were 5 μm thick. Hematoxylin-eosin (H&E) staining was performed on the colonic tissue sections. Pathological changes in the tissue sections were observed and photographed using an optical microscope. In the normal control group, the colon of mice showed intact structure and no lesions. In the DSS-treated group alone, the intestinal epithelium of the colon was extensively damaged and sloughed off, with incomplete cell structure, reduced or absent glands, and extensive infiltration of inflammatory cells accompanied by edema, exhibiting severe lesions. Intraperitoneal injection of peptide pep169L significantly reduced inflammatory cell infiltration, while maintaining relatively intact intestinal epithelial structure and less glandular damage, resulting in significantly reduced colonic lesions compared to the DSS-treated group alone. Oral administration of 5-ASA also significantly reduced inflammatory cell infiltration. These results further indicate that peptide pep169L can inhibit the inflammatory response in DSS-induced colitis mice. Figure 5 ).

Claims

1. A polypeptide, characterized in that, RQIKIWFQNRRMKWKKVDFIAGINNLGEKIY.

2. Use of the polypeptide of claim 1 in the preparation of a medicament for treating and / or preventing colitis.

3. Use according to claim 2, characterized in that, including reducing secretion of inflammatory cytokines.

4. Use according to claim 3, characterized in that, The inflammatory cytokines include IL-6, IL-1β, TNF-α.

5. The use according to claim 2, characterized in that, including inhibiting expression of p65 protein.

6. A medicament for treating and / or preventing an inflammatory disease, characterized by, which contains the polypeptide of claim 1.

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