Soothing anti-inflammatory polypeptide for inhibiting TNFa protein activity

Abstract

The invention provides an anti-inflammatory polypeptide for inhibiting the activity of TNFa (Tumor Necrosis Factor a) protein. Specifically, skeleton polypeptide with the sequence of PIYLGGVFQ is modified, polypeptide with high affinity with TNFa protein and high inhibition rate on NO, IL-6 and degranulation rate is screened out from 11 modified polypeptides, and the polypeptide has high stability. In addition, compared with hormone anti-inflammatory drugs, the polypeptide provided by the invention has higher safety and can be used for preparing soothing anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, this invention relates to a soothing and anti-inflammatory polypeptide that inhibits the activity of TNFα protein. Background Technology

[0002] Sensitive skin develops through a complex process involving the skin barrier, neurovascular response, and innate immune inflammation. The main factor causing sensitive skin is the inflammatory response triggered by various forms of external stimuli. Activated inflammatory pathways lead to paroxysmal or periodic burning, redness, stinging, itching, and tightness, with or without persistent erythema.

[0003] TNF-α (tumor necrosis factor-α), as a core inflammatory cytokine, plays a crucial role in skin inflammation. Its overexpression is directly related to various skin problems, such as redness, stinging, and barrier damage. TNF-α is a key cytokine in the upstream initiation stage of the inflammatory cascade, promoting the migration of activated neutrophils and T cells from the epidermis to the dermis. TNF-α activates the NF-κB and MAPK signaling pathways, inducing the release of pro-inflammatory factors such as IL-6 and IL-8 from keratinocytes and endothelial cells, and upregulating adhesion molecules (such as ICAM-1 and VCAM-1), leading to leukocyte infiltration, increased vascular permeability, and triggering redness, burning, and pain.

[0004] Inhibiting the TNF-α signaling pathway or regulating its expression can effectively alleviate symptoms of skin sensitivity, making it an important strategy in the field of skin soothing. Summary of the Invention

[0005] The purpose of this invention is to provide an effective method for relieving skin sensitivity symptoms by inhibiting the TNF-α signaling pathway or regulating its expression.

[0006] In a first aspect of the invention, an anti-inflammatory polypeptide that inhibits TNFα protein activity is provided, said polypeptide being selected from the group consisting of:

[0007] 1) A backbone polypeptide with the sequence PIYLGGVFQ;

[0008] 2) A polypeptide modified by acetylation based on the backbone polypeptide;

[0009] 3) A peptide modified by palmitoylation based on the aforementioned backbone peptide; or

[0010] 4) A polypeptide modified by cyclization based on the backbone polypeptide.

[0011] In another preferred embodiment, the cyclized polypeptide has the structure shown in Formula I:

[0012]

[0013] In another preferred embodiment, the acetylation modification is at the N-terminus of the skeletal polypeptide.

[0014] In another preferred embodiment, the acetylation modification is performed on the first amino acid residue at the N-terminus of the skeletal polypeptide.

[0015] In another preferred embodiment, the palmitoylation modification is performed at the N-terminus of the skeletal polypeptide.

[0016] In another preferred embodiment, the palmitoylation modification is performed on the first amino acid residue at the N-terminus of the skeletal polypeptide.

[0017] In another preferred embodiment, the C-terminus of the polypeptide has a -OH group.

[0018] In another preferred embodiment, the polypeptide has a high affinity for TNF-α protein.

[0019] In another preferred embodiment, the polypeptide is able to inhibit the level of NFκB(p65), thereby inhibiting the NFκB signaling pathway.

[0020] In another preferred embodiment, the polypeptide inhibits the expression of NFκB in U937 cells.

[0021] In another preferred embodiment, the peptide exhibits stability of up to 30 days.

[0022] In another preferred embodiment, the polypeptide significantly inhibits the NO content in cells.

[0023] In another preferred embodiment, the polypeptide significantly inhibits the NO content in Raw264.7 cells.

[0024] In another preferred embodiment, the polypeptide significantly inhibits the level of IL-6 in cells.

[0025] In another preferred embodiment, the polypeptide significantly inhibits the level of IL-6 in HaCaT cells.

[0026] In another preferred embodiment, the polypeptide significantly inhibits cell degranulation.

[0027] In another preferred embodiment, the polypeptide significantly inhibits mast cell degranulation.

[0028] In a second aspect of the invention, a composition is provided, the composition comprising:

[0029] 1) The polypeptide as described in the first aspect of the present invention; and

[0030] 2) Acceptable carriers in cosmetics or pharmaceuticals.

[0031] In a third aspect of the invention, the use of the polypeptide as described in the first aspect of the invention is provided for the preparation of a soothing and anti-inflammatory medicament.

[0032] In a fourth aspect of the invention, a method for relieving inflammation is provided, the method comprising: administering an effective amount of a polypeptide as described in the first aspect of the invention or a composition as described in the second aspect of the invention to a subject.

[0033] In a fifth aspect of the invention, a kit is provided, the kit comprising: a polypeptide according to the first aspect of the invention, a nucleic acid encoding the polypeptide according to the first aspect of the invention, a vector expressing the polypeptide according to the first aspect of the invention, or a composition according to the second aspect of the invention.

[0034] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0035] Figure 1 A schematic diagram illustrating the principle of biomembrane interference (BLI) technology is shown.

[0036] Figure 2 A schematic diagram illustrating the principle of protein interaction experiments is shown.

[0037] Figure 3 The NO content in Raw264.7 cells was measured.

[0038] Figure 4 The results show the detection of IL-6 levels in HACAT cells.

[0039] Figure 5 The degranulation rate of P815 cells was detected. Detailed Implementation

[0040] Through extensive and in-depth research, the inventors have developed an anti-inflammatory peptide that inhibits TNFα protein activity. This invention involves modifying a backbone peptide with the sequence PIYLGGVFQ, and screening from 12 modified peptides to identify a peptide with high affinity for TNFα protein, capable of inhibiting the NFκB signaling pathway, and exhibiting a high inhibition rate (16.94%) of NO and IL-6, with a stability of up to 30 days. This invention is based on this foundation.

[0041] the term

[0042] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0043] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0044] polypeptide

[0045] This invention uses a polypeptide with the sequence PIYLGGVFQ (SEQ ID NO.1) as the backbone polypeptide, which has a -OH group at its C-terminus. Eleven modifications were performed on the backbone polypeptide, including acetylation, palmitoylation, cyclization, phosphorylation, amidation, methylation, ethylation, formylation, PEG modification (PEG400), lauric acid modification, and oleic acid modification. The optimal modification methods were screened out. These modifications endow the backbone polypeptide with higher affinity for TNFα and improve its inhibition rate against NO and IL-6.

[0046] Peptide cyclization modification ([PIYLGGVFQ])

[0047] Cyclation: The amino group of the N-terminal Pro (proline) undergoes dehydration condensation with the carboxyl group of the C-terminal Gln (glutamine) to form a new amide bond, constituting a cyclic structure, as shown in Formula I.

[0048]

[0049] Composition

[0050] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned polypeptide and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0051] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0052] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described polypeptides of the present invention, along with a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the polypeptides of the present invention can also be used with other therapeutic agents.

[0053] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0054] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0055] Compared with the prior art, the main advantages of the present invention include:

[0056] 1. The anti-inflammatory peptides of the present invention target and bind to TNFα protein and inhibit its activity, thereby achieving a soothing and anti-inflammatory effect.

[0057] 2. The anti-inflammatory peptides of the present invention have higher safety compared with hormone-based anti-inflammatory drugs.

[0058] 3. The anti-inflammatory peptides of the present invention have a stability of up to 30 days.

[0059] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0060] Example 1. Peptide modification and preparation

[0061] Using G110101(PIYLGGVFQ-OH) peptide as the backbone, a series of modifications were performed to form 12 candidate peptides, as shown in Table 1.

[0062] Table 1. List of peptide modifications

[0063] serial number sequence Modification G110101 PIYLGGVFQ-OH Skeleton peptide (unmodified) G110102 Ac-PIYLGGVFQ-OH acetylation G110103 Palmitoyl-PIYLGGVFQ-OH Palmitylation G110104 [PIYLGGVFQ] cyclization G110105 PIYLGGVFQ(Tyr side chain phosphorylation)-OH Phosphorylation G110106 PIYLGGVFQ-NH2 amidation G110107 PIYLGGVFQ-OCH3 Methyl esterification G110108 PIYLGGVFQ-OC2H5 Ethyl esterification G110109 Hco-PIYLGGVFQ-OH Formylation G110110 PEG-PIYLGGVFQ-OH PEG modification (PEG400) G110111 Lauroyl-PIYLGGVFQ Lauric acid modification G110112 Oleoyl-PIYLGGVFQ Oleic acid modification

[0064] The preparation process of the above-mentioned polypeptides is shown in Table 2:

[0065] Table 2. Methods for preparing peptides

[0066]

[0067]

[0068] Example 2. Affinity screening of peptides

[0069] 1. Experimental Principle

[0070] The Octet platform is based on bio-layer interferometry (BLI) to detect and analyze biomolecular interactions.

[0071] Biomembrane interferometry (BLI) is a technique that detects surface reactions of a sensor by detecting shifts in the interference spectrum. When a beam of visible light is emitted from a spectrometer, two reflected spectra are formed at the two interfaces of the optical film at the sensor's end, creating an interference spectrum. Any changes in film thickness and density due to molecular bonding or dissociation can be reflected by the shift value of the interference spectrum, allowing for real-time monitoring of the response spectrum. Figure 1 ).

[0072] 2. Experimental Materials

[0073] Testing instruments: Octet RED96e, Startorius BioAnalytical Instruments Inc.

[0074] Consumables: 1.5ml EP tubes, 15mL centrifuge tubes, 96-well black opaque plates, and various sizes of pipette tips;

[0075] Chip: SA sensor (ForteBio, Cat: 18-5019);

[0076] Buffer DMSO, 1×PBST (1×PBS with 0.02% Tween-20 added); 10mM Glycine pH 1.8.

[0077] 3. Interactive Experiment

[0078] 1) Biotinylated proteins

[0079] (1) Add biotinylation reagent at a molar ratio of 1.5 to the antibody and react at room temperature for 1 h;

[0080] (2) Remove excess biotinylation reagent from the biotinylated protein using a gravity desalting column;

[0081] (3) Take biotinylated TNFα protein, dilute it, and then solidify it into an SA chip for affinity detection;

[0082] 2) Biotinylated TNF-α protein is specifically captured using the SA chip. Once the signal reaches saturation, it binds to the peptide. A schematic diagram of the principle is shown below. Figure 2 As shown.

[0083] 3) The 12 peptides were dissolved in the corresponding reconstitution buffer to a concentration of 5 mM, and then diluted with PBST buffer to a concentration of 1 mM.

[0084] 4) Add the sample to the 96-well plate in the following order, 200 μL per well.

[0085] 5) The affinity of the peptides is shown in Table 3. The three candidate peptides with the highest affinity were G110102 (Ac-PIYLGGVFQ-OH), G110103 (Palmitoyl-PIYLGGVFQ-OH) and G110104 ([PIYLGGVFQ]).

[0086] Table 3. Results of peptide affinity

[0087] serial number sequence Affinity G110101 PIYLGGVFQ-OH 5.30E-05 G110102 Ac-PIYLGGVFQ-OH 1.30E-05 G110103 Palmitoyl-PIYLGGVFQ-OH 2.20E-05 G110104 [PIYLGGVFQ] 3.40E-05 G110105 PIYLGGVFQ(Tyr side chain phosphorylation)-OH 1.30E-04 G110106 PIYLGGVFQ-NH2 6.30E-04 G110107 PIYLGGVFQ-OCH3 1.50E-04 G110108 PIYLGGVFQ-OC2H5 2.60E-04 G110109 Hco-PIYLGGVFQ-OH 4.30E-04 G110110 PEG-PIYLGGVFQ-OH 7.30E-04 G110111 Lauroyl-PIYLGGVFQ 4.20E-03 G110112 Oleoyl-PIYLGGVFQ 5.60E-03

[0088] Example 3: Peptide Inhibits NFκB Expression

[0089] Study on the level of NFκB (p65) expression in U937 cells stimulated by peptide inhibition of TNFα.

[0090] U937 cells were used at a rate of 2 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 100 cells / well (96-well plate) and stimulated for 1 hour at room temperature with a mixture of human TNFα (100 ng / ml) and peptide (100 μM). Then, the cells were treated with primary antibody and FITC-labeled secondary antibody as described above.

[0091] NFκB (p65) levels were detected in TNFα-stimulated cells, and the effects of peptides were assessed using Western blotting. β-actin protein levels served as a positive control for nuclear extracts. The inhibitory rates of peptides on NFκB nuclear translocation are shown in Table 4. The results indicated that the four candidate peptides with the highest inhibitory effects were G110102 (Ac-PIYLGGVFQ-OH), G110103 (Palmitoyl-PIYLGGVFQ-OH), G110104 ([PIYLGGVFQ]), and G110107 (PIYLGGVFQ-OCH3).

[0092] Table 4. Inhibition rate of peptides on NFκB expression levels

[0093] serial number sequence Inhibition rate (%) G110101 PIYLGGVFQ-OH 14％ G110102 Ac-PIYLGGVFQ-OH 25％ G110103 Palmitoyl-PIYLGGVFQ-OH 28％ G110104 [PIYLGGVFQ] 19％ G110105 PIYLGGVFQ(Ser / Thr / Tyr side chain phosphorylation)-OH 8％ G110106 PIYLGGVFQ-NH2 11％ G110107 PIYLGGVFQ-OCH3 15％ G110108 PIYLGGVFQ-OC2H5 12％ G110109 Hco-PIYLGGVFQ-OH 8％ G110110 PEG-PIYLGGVFQ-OH 14％ G110111 Lauroyl-PIYLGGVFQ -4％ G110112 Oleoyl-PIYLGGVFQ -12％

[0094] Example 4: Screening of peptide stability

[0095] The candidate peptides were prepared into 100 ng / mL solutions and incubated at 37℃ and 45℃ for 0 days, 14 days, and 30 days, respectively, to assess their stability. The testing conditions and detection items are shown in Table 5.

[0096] Table 5. List of items for peptide stability testing

[0097]

[0098] Appearance, observed visually

[0099] HPLC method description

[0100] Pump A: 0.065% trifluoroacetic acid solution in 100% water (v / v)

[0101] Pump B: 0.05% trifluoroacetic acid solution in 100% acetonitrile (v / v)

[0102] Total flow rate: 1 ml / min, wavelength: 220 nm

[0103] Table 6. HPLC gradient elution program

[0104] time Pump A (%) Pump B (%) 0.01 95 5 25.00 35 65 25.01 5 95 27.00 5 95 27.01 95 5 33.00 95 5

[0105] Purity was determined by HPLC, with chromatographic purity calculated using the area normalization method.

[0106] Peptide content was determined by HPLC, using standard curve solutions (20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL) to calculate sample concentration.

[0107] The results showed that the stability of the four candidate peptides G110101(PIYLGGVFQ-OH), G110102(Ac-PIYLGGVFQ-OH), G110103(Palmitoyl-PIYLGGVFQ-OH) and G110104([PIYLGGVFQ]) was satisfactory (Tables 7, 8, 9, and 10).

[0108] Table 7. Stability results of peptide G110101

[0109]

[0110] Table 8. Stability results of peptide G110102

[0111]

[0112] Table 9. Stability results of peptide G110103

[0113]

[0114] Table 10. Stability results of peptide G110104

[0115]

[0116] Example 5. Cellular efficacy of peptides

[0117] 1. NO emission detection

[0118] Cell seeding: Log-phase Raw264.7 cells (mouse mononuclear macrophage leukemia cells) were harvested and adjusted to a cell density of 6E5 / ml. 100 μL of each cell was seeded into a 96-well plate and cultured overnight at 37°C with 5% CO2. When the cell layer coverage reached approximately 50-60%, pre-treatment was performed. According to the experimental groups, the samples were diluted to the required concentration using Raw264.7 maintenance medium (2% FBS + DMEM + L-glutamine). After mixing, the culture plates were removed, the original medium was discarded, and 100 μL of the prepared sample solution was added to each well. The plates were then returned to the incubator for another 4 hours of incubation. After pre-treatment, 1 μg / ml LPS was added to the prepared pre-treated sample solution and vortexed. The culture plates were removed, the original medium was discarded, and 100 μL of the corresponding sample solution was added to each well according to the experimental groups. The plates were then returned to the incubator for another 18 hours of incubation. Cell culture supernatant was collected and tested according to the NO detection kit instructions. GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. T-tests were used for statistical analysis between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0119] Wherein, inhibition rate (%) = (model group - sample group) / model group × 100%.

[0120] Table 11. Test Groups

[0121]

[0122] Table 12. Summary of NO test results

[0123]

[0124]

[0125] The results are shown in Table 12 and Figure 3 As shown, compared with BC, the NO content of samples G110101, G110102, and G110104 decreased significantly, with inhibition rates of 13.64%, 16.94%, and 14.24%, respectively, demonstrating good skin-soothing effects.

[0126] 2. IL6 content detection

[0127] Cell seeding: Log-phase HaCaT cells were harvested, and the cell density was adjusted to 7E5 / ml. 1 mL of each cell was seeded into a 6-well plate and cultured overnight at 37°C with 5% CO2. When the seeding rate reached approximately 70-80%, synchronization was performed. The culture plate was removed, the original culture medium was discarded, and the plate was washed once with PBS. 1 mL of HaCaT cell maintenance medium (MEM + L-glutamine + sodium pyruvate) was added to each well, and the plate was returned to the incubator for 6 hours. After synchronization, drug pretreatment was performed. According to the experimental groups, the HaCaT cell maintenance medium was used to dilute the samples to the required concentration. After mixing, the culture plate was removed, the original culture medium was discarded, and 1 mL of the prepared sample solution was added to each well. The plate was returned to the incubator for 1 hour. After pretreatment, the culture plate was removed, and 50 ng / ml TNF-α was added to the corresponding experimental group. The mixture was gently shaken to mix, and the plate was returned to the incubator for 24 hours. Cell culture supernatant was collected and analyzed according to the IL-6 ELISA assay kit instructions. GraphPad Prism was used for plotting, and results are expressed as Mean ± SD. T-tests were used for statistical analysis between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0128] Table 13. Test Groups

[0129]

[0130] Table 14. Summary of IL-6 test results

[0131]

[0132] The results are as follows Figure 4 As shown in Table 14, compared with BC, the IL-6 content of samples G110101, G110102, and G110104 at a concentration of 20 ppm was significantly decreased, with inhibition rates of 31.98%, 46.96%, and 55.07%, respectively. Compared with the positive control dexamethasone, the IL-6 content of samples G110102 and G110104 at a concentration of 20 ppm was not significantly different, indicating that the soothing efficacy of these samples at a concentration of 20 ppm is comparable to that of dexamethasone, demonstrating good skin soothing effects.

[0133] 3. De-particle rate test

[0134] P815 cells with good growth and confluence of 80%-90% were digested, centrifuged, resuspended in high-glucose DMEM basal medium, and counted. The cells were then diluted to 1.2 × 10⁻⁶. 650 μL of cells / mL was seeded into each well of a 96-well plate. After the samples were prepared according to the groups, the 96-well plates were pre-treated according to the groups in Table 5, with 50 μL per well and 6 replicates per group. The plates were incubated in a CO2 incubator for 2 h at 37 °C and 5% CO2 concentration. After the pre-treatment, 40 μL of C48 / 80 working solution (25 μg / mL) or DMEM basal medium was added to the corresponding experimental groups. The plates were then incubated in a CO2 incubator for 40 min at 37 °C and 5% CO2 concentration. After C48 / 80 stimulation for 40 min, the plates were placed on ice for 10 min to stop the reaction. The degranulation was observed under a microscope (200× magnification) and photographed. Degranulated cells had rough and irregular cell membranes, low refractive index of granules in the cytoplasm, and exocytosis of granules visible in the cytoplasm or around the cell membrane, i.e., degranulation. Undegranulated cells had smooth, intact, and bright cell membranes, and homogeneous cytoplasm with almost no granules. The ImageJ image processing software was used to count the number of degranulated cells and the total number of cells in each image. The degranulation rate was calculated using the following formula:

[0135]

[0136] Table 15 Test Groups

[0137]

[0138] Table 16 Summary of De-particle Test Results

[0139]

[0140] The results are as follows Figure 5 As shown in Table 16, compared with NC, samples G110101, G110102, and G110104 showed significantly lower degranulation rates at a concentration of 20 ppm. Cells circled in red represent degranulated cells. The degranulation rates were 20.65%, 15.41%, and 12.11%, respectively, with G110102 and G110104 showing significantly lower degranulation rates than G110101. In conclusion, G110102 and G110104 exhibited the best inhibitory effect on P815 cell degranulation, indicating that these samples possess good anti-allergic skin efficacy.

[0141] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-inflammatory polypeptide that inhibits the activity of a TNFa protein, characterized in that, The polypeptide is a polypeptide selected from the group consisting of: 1) a backbone polypeptide having the sequence of PIYLGGVFQ; 2) a polypeptide having an acetylation modification on the basis of the backbone polypeptide; 3) a polypeptide having a palmitoylation modification on the basis of the backbone polypeptide; or 4) a polypeptide having a cyclization modification on the basis of the backbone polypeptide.

2. The polypeptide of claim 1, wherein, The polypeptide having the cyclization modification has a structure shown in Formula I:

3. The polypeptide of claim 1, wherein The acetylation modification and the palmitoylation modification are at the N-terminus of the backbone polypeptide.

4. The polypeptide of claim 1, wherein The polypeptide has a high affinity for TNF-α protein.

5. The polypeptide of claim 1, wherein The polypeptide is capable of inhibiting the level of NFκB, and thereby inhibiting the NFκB signaling pathway.

6. The polypeptide of claim 1, wherein The polypeptide is capable of inhibiting the level of NO, IL-6 and degranulation rate in cells.

7. A composition characterized in that, The composition comprises: 1) the polypeptide of claim 1; and 2) a cosmetically or pharmaceutically acceptable carrier.

8. Use of a polypeptide according to claim 1, characterized in that, A soothing anti-inflammatory medicament.

9. A method of soothing and anti-inflammatory, characterized by, The method comprises: administering an effective amount of the polypeptide of claim 1 or the composition of claim 7 to a subject.

10. A kit characterized in that, The kit comprises: the polypeptide of claim 1, a nucleic acid encoding the polypeptide of claim 1, a vector expressing the polypeptide of claim 1, or the composition of claim 7.

Images