Polypeptide pep169L with anti-inflammatory effect and application thereof

Abstract

The invention discloses a polypeptide pep169L with an anti-inflammatory effect and an application of the polypeptide pep169L. The inhibition effect of the pep169L polypeptide on NF-kappa B reporter gene and p65 protein expression is identified, the inhibition effect of the pep169L polypeptide on inflammatory cytokine IL-1beta is detected, and the function of the pep169L polypeptide for reducing DSS-induced colitis in a mouse body is explored. The pep169L polypeptide can significantly inhibit the production of LPS-induced reporter genes NF-kB and p65 and inflammatory cytokines IL-1beta, can inhibit the secretion of DSS-induced inflammatory cytokines IL-6, IL-1beta and TNF-alpha in a mouse body, and improves DSS-induced clinical characterization, including colon length, disease activity index and pathological change. The polypeptide has the functions of inhibiting inflammatory response and reducing DSS-induced colitis, and a new thought is provided for design of novel anti-inflammatory small molecule drugs.

Description

Technical Field

[0001] The invention relates to a polypeptide pep169L with anti-inflammatory effect and application thereof, belonging to the field of polypeptides. Background Art

[0002] As the body's first line of defense, the innate immune system 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 diseases caused by abnormal immune system responses, which are mainly characterized by local tissue or systemic inflammatory responses. Such diseases include rheumatoid arthritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), psoriasis, etc. Although the inflammatory response is an important part of the body's self-protection to a certain extent, its uncontrolled or continuous activation may lead to tissue damage and the occurrence of various pathological conditions. The dextran sulfate sodium (DSS) model is an experimental model widely used to study enteritis and inflammatory bowel disease. DSS is a compound that can induce colonic inflammation, which causes intestinal inflammation by damaging intestinal epithelial cells. The advantages of this model are that it is easy to operate, the effect is controllable, and it can better simulate the pathological characteristics of inflammatory bowel disease. Therefore, the DSS model provides a good experimental platform for exploring the potential mechanism of action of peptides and other drugs to cure or alleviate inflammatory diseases.

[0004] Peptides exhibit a wide range of biological activities in anti-inflammatory and immunomodulatory aspects. Studies have shown that certain peptides can reduce DSS-induced intestinal inflammation by 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 also increasingly used in anti-inflammatory treatments due to their adjustability and specificity.

[0005] Studying the role of peptides in the DSS model can not only provide new ideas for the treatment of inflammatory diseases, but also provide data support for the evaluation of peptide characteristics. The mechanism of virus escape from the NF-κB signaling pathway also provides a theoretical basis for the study of future anti-inflammatory drugs. These studies help to deeply understand the role of peptides in inflammatory mechanisms and develop new therapeutic strategies for the treatment of 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 excessive activation of the NF-κB signaling pathway.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a polypeptide, whose amino acid sequence is RQIKIWFQNRRMKWKKVDFIAGINNLGEKIY.

[0008] The present invention also provides the use of the polypeptide in preparing a drug for treating and / or preventing inflammatory diseases.

[0009] Wherein, the inflammatory disease includes colitis.

[0010] These include significantly reducing the secretion of inflammatory cytokines and inhibiting the body's inflammatory response, making it a highly potential anti-inflammatory drug candidate.

[0011] Wherein, 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 drug for treating and / or preventing inflammatory diseases, which contains the polypeptide.

[0014] The present invention also provides the use of the polypeptide in preparing a medicine for improving blood in stool 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 new anti-inflammatory small molecule drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[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 blood in the stool of mice; Figure 2 B: Fecal occult blood test kit was used to measure the fecal occult blood of mice; Figure 2 C: DAI score of DSS-induced colitis in mice;

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

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

[0020] Figure 5 A: Observation of mouse colon pathological sections; Figure 5 B: Evaluation of colon pathological changes in mice. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0022] Example 1

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

[0024] (1) The peptide pep169L (RQIKIWFQNRRMKWKKVDFIAGINNLGEKIY) was synthesized, and the peptide CP (amino acid sequence: RQIKIWFQNRRMKWKKSLHGRGDPMEAFII) was set as a negative control.

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

[0026] HeLa cells (2×10 5cells / well), transfected with NF-κB-luc luciferase reporter gene plasmid after 16 hours, discarded the culture medium after 24 hours, washed the cells lightly with phosphate buffer, added DMEM culture medium or different polypeptides (pep169L or CP, 40μM) for 30 minutes, stimulated with 100ng / mL lipopolysaccharide (LPS), added 100μL cell lysis buffer (1×Cell Lysis Buffer) for lysis after 1 hour, blow the cells thoroughly, transfer them all into a finger-shaped tube, centrifuge at 12000rpm for 1min, and collect the supernatant. 100μL luciferase substrate (firefly luciferase) and 20μL cell lysate were thoroughly mixed and added to a non-transparent ELISA plate to detect the reporter gene activity. The results showed that pep169L could significantly inhibit the expression of LPS-induced NF-κB reporter gene ( Figure 1 A).

[0027] (3) Real-time fluorescence quantitative PCR identification of pep169L peptide inhibiting LPS-induced inflammatory cytokine production

[0028] HeLa cells were plated (24-well cell plates, 2×10 5 / well), discard the medium after overnight culture, add DMEM medium or different polypeptides (pep169L or CP, 40μM) and incubate for 30min, then stimulate with 100ng / mL LPS, lyse with Trizol after 1h of stimulation, extract RNA, remove DNA and reverse transcribe into cDNA using reverse transcription kit Hiscript III RT Super mix for qPCR, and detect the expression level of cytokine IL-1β by qRT-PCR. The results showed that LPS can induce the production of a large number of inflammatory cytokines, while polypeptide pep169L can significantly inhibit the production of inflammatory cytokines induced by LPS ( Figure 1 B).

[0029] (4) Western Blot analysis showed that pep169L peptide could significantly inhibit LPS-induced p65 protein expression

[0030] HeLa cells were plated (24-well cell plates, 2×10 5 / well), discard the culture medium after overnight culture, add DMEM culture medium or different polypeptides (pep169L or CP, 40μM) for incubation for 30min, stimulate with 100ng / mL LPS, use Western Blot and IP lysis buffer to lyse after 1h of stimulation, blow the cells thoroughly, transfer all of them into finger-shaped tubes, centrifuge at 12000rpm for 3min, collect the supernatant, add 5× loading buffer, cook the sample at 100℃ for 10min, separate the proteins by SDS-PAGE gel electrophoresis, transfer the gel strip to NC membrane, and put it into 5% skim milk blocking solution diluted with TBST for blocking for 2h. Wash with TBST 3 times, 5min each time. Add p65 protein and GAPDH monoclonal antibody (Biyuntian, AF1186) diluted with TBST containing 5% skim milk (p65 protein and GAPDH monoclonal antibody are diluted at 1:1000) at 4℃ overnight. Wash with TBST 5 times, 5min each time. Goat Anti-Rabbit IgG HRP goat anti-rabbit (Aibotek Biotechnology Co., Ltd., AS014-500ul) and Goat Anti-Mouse IgG HRP goat anti-mouse (Aibotek Biotechnology Co., Ltd., AS003-500ul) diluted 1:5000 in TBST containing 5% skim milk were added in turn and incubated at room temperature for 1 hour. Wash with TBST 6 times, 5 minutes each time. Use the chemiluminescent imaging system Amersham Image 600 for color development and exposure. The results showed that the pep169L peptide could significantly inhibit the expression of p65 protein induced by LPS ( Figure 1 C).

[0031] Example 2

[0032] The pep169L peptide inhibited dextran sodium sulfate (DSS)-induced colitis in mice in vivo, improved the clinical manifestations induced by DSS, including colon length, disease activity index and pathological changes, established a DSS-induced colitis model in mice, and evaluated the effect of the pep169L peptide on DSS-induced colitis in mice. 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 selected and 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 sterile water containing 4% DSS every day for 6 consecutive days; DSS+CP group: mice freely drank sterile water containing 4% DSS every day for 6 consecutive days, and the mice were intraperitoneally injected with polypeptide CP every day, with an injection dose of 100nmol / mouse; DSS+pep169L group: mice freely drank sterile water containing 4% DSS every day for 6 consecutive days, and the mice were intraperitoneally injected with 100nmol / mouse pep169L polypeptide every day; DSS+5-ASA: 5-ASA is a first-line drug for treating colitis, and the present invention uses it as a positive control for treating colitis. The mice drank sterile water containing 4% DSS every day for 6 consecutive days, and were orally gavaged with 5-ASA 10mg / ml every day, 300μL (dissolved in 0.5% CMC-Na) for each mouse, and the oral gavage dose was 100mg / kg; PBS group: mice freely drank sterile water every day.

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

[0036] The mice's diet, activity, and hair were observed every day. The mice's weight was weighed and recorded at the same time every day, and the weight change of the mice was expressed as a percentage of the original weight. The fecal characteristics of the mice were observed and recorded. In addition, the fecal occult blood test kit was used to measure the fecal occult blood of the mice to evaluate the severity of colitis. The results showed that the peptide pep169L could significantly improve the fecal blood and fecal occult blood of the mice ( Figure 2 A; Figure 2 B).

[0037] (3) Evaluation of the disease activity index (DAI) of colitis in mice

[0038] DAI was calculated based on the weight loss, stool characteristics and occult blood in the mice. The formula for calculating the percentage of weight loss in mice was: percentage of weight loss in mice (100%) = (measured weight of mice - weight at the beginning of the experiment) / weight of mice at the beginning of the experiment × 100%. Weight loss score: no weight loss, 0 points; weight loss of 0%-5%, 1 point; weight loss of 5%-10%, 2 points; weight loss of 10%-20%, 3 points; weight loss of more than 20%, 4 points. Stool characteristics score: normal stool, 0 points; loose stool, 2 points; watery stool, 4 points. Stool occult blood score: occult blood negative, 0 points; occult blood positive, 2 points; visible blood in the stool, 4 points. The disease activity index of each mouse was calculated according to the formula DAI score = (weight loss score + stool characteristics score + stool occult blood score) / 4. The DAI score of the mouse was recorded every day, and the relationship curve 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 mice with DSS-induced colitis

[0040] In mice with DSS colitis, the shortening of colon length can reflect the severity of inflammation to a certain extent. At the end of the sixth day of the experiment, the mice were killed by cervical dislocation and soaked in 75% alcohol for 3 minutes. The colon tissue was separated, the morphological changes of the colon were observed, the colon length was measured, and photos were taken for record. The results showed that compared with the normal control group, the colon of mice in different DSS treatment groups showed different degrees of significant shortening. The shortening length of the colon in the peptide pep169L treatment group was less than that in the peptide CP group, and there was a significant difference ( Figure 3 A; Figure 3 B), indicating that the peptide pep169L can inhibit DSS-induced shortening of mouse colon.

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

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

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

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

[0045] (7) ELISA determination of inflammatory cytokine levels in mouse colon tissue

[0046] R&D Mouse IL-6DuoSet ELISA, Mouse IL-1βDuoSet ELISA, and Mouse TNF-αDuoSet ELISA detection kits were used to detect changes in inflammatory factors in mouse intestinal tissues. The determination of inflammatory factors was performed according to the requirements of the detection kits. The results showed that the peptide pep169L could significantly reduce the content of IL-6, IL-1β, and TNF-α in the colon tissue of DSS-induced colitis mice, and reduce DSS-induced systemic inflammation ( Figure 4 B).

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

[0048] The pathological changes of colon tissue in mice with DSS-induced colitis were evaluated by pathological sections. Pathological sections of colon tissue were prepared according to conventional methods. Colon tissue samples fixed with 10% neutral formalin fixative were dehydrated with ethanol, paraffin-embedded after tissue dehydration, and then sliced ​​with a microtome and fixed on a glass slide. The thickness of the tissue sections was 5 μm. Colon tissue sections were stained with hematoxylin-eosin (H&E). Pathological changes in tissue sections were observed by optical microscopy and photographed and recorded. The colon structure of mice in the normal control group was intact, and no lesions were observed; the intestinal epithelium of the mice in the DSS-only treatment group was damaged and detached, the cell structure was incomplete, the glands were reduced and disappeared, and a large number of inflammatory cells infiltrated, accompanied by edema, showing severe lesions; intraperitoneal injection of the peptide pep169L can significantly reduce the infiltration of inflammatory cells, while the intestinal epithelial structure is relatively intact, the degree of gland damage is relatively mild, and the colon lesions are significantly reduced compared with the DSS-only treatment group; oral gavage of 5-ASA can also significantly reduce the occurrence of inflammatory cell infiltration. The above results further indicate that the peptide pep169L can inhibit the inflammatory response of DSS-induced colitis mice ( Figure 5 ).

Claims

1. A polypeptide, characterized in that Its amino acid sequence is RQIKIWFQNRRMKWKKVDFIAGINNLGEKIY.

2. Use of the polypeptide according to claim 1 in the preparation of a drug for treating and / or preventing inflammatory diseases.

3. The application according to claim 2, characterized in that: The inflammatory disease includes colitis.

4. The application according to claim 2, characterized in that: Including reducing the secretion of inflammatory cytokines.

5. The application according to claim 4, characterized in that: The inflammatory cytokines include IL-6, IL-1β, and TNF-α.

6. The use according to claim 2, characterized in that: Including inhibiting the expression of p65 protein.

7. A drug for treating and / or preventing inflammatory diseases, characterized in that: It contains the polypeptide according to claim 1.

8. Use of the polypeptide according to claim 1 in the preparation of a medicament for improving blood in stool and / or fecal occult blood.

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