A peptide hydrogel, its preparation method and its application in treatment

Abstract

The present application aims to provide a peptide hydrogel and an amphiphilic peptide capable of forming the peptide hydrogel, which are suitable for autoimmune diseases caused by cancer chemotherapy, radiotherapy and immunotherapy, including various organ immune inflammation and related manifestations, and mucosal damage and ulcer of skin, oral cavity and digestive system. The amphiphilic peptide is a general formula (1) Cm-IDR-Z or a physiologically acceptable salt thereof, wherein C m , IDR, Z are as defined in the specification. The peptide hydrogel has a positive characteristic peak in the range of 190-210 nm, a negative single peak in the range of 210-230 nm in CD spectrum, and after the peptide hydrogel is dispersed into 1% SDS (sodium dodecyl sulfate) solution, the CD spectrum has a positive characteristic peak in the range of 190-200 nm, and a negative double peak in the range of 200-210 nm and 215-230 nm.

Description

Technical Field

[0001] This invention relates to a biomimetic host defense peptide hydrogel, its preparation method, and its applications. Specifically, the biomimetic host defense peptide hydrogel possesses a β-sheet fibrous structure and, under lipid membrane conditions, can selectively and specifically modulate the body's immune response through conformational changes. This invention relates to the application of the biomimetic host defense peptide hydrogel in autoimmune diseases, indications, or syndromes induced by cancer chemotherapy, radiotherapy, and immunotherapy, including immune-related inflammation in various organs, as well as mucosal damage and ulcers of the skin, oral cavity, and digestive tract, belonging to the field of biomaterials. Background Technology

[0002] Inflammation is an adaptive immune response, a defense mechanism of the host against damaged tissues or infectious pathogens. Inflammation is closely associated with a variety of diseases, especially chronic inflammation, including asthma, inflammatory bowel disease, cancer, type II diabetes, lipid metabolism disorders, cardiovascular diseases (such as hypertension and hyperlipidemia), and central nervous system-related conditions (such as Parkinson's disease and cognitive impairment). The incidence of these chronic inflammatory diseases is increasing year by year, becoming a global health concern. Inflammation is a normal defensive response of the host to damaging factors; it is a complex regulated process involving multiple cells (such as macrophages, lymphocytes, and monocytes) and multiple factors (signal transduction and transcription activators, vasoactive amines, cytokines, chemokines, and inflammation-related enzymes).

[0003] Bioactive peptides (such as β-defensins and cathelicidins) expressed in mammalian epithelial cells and neutrophils serve as the first line of defense against inflammation, acting as "natural antibiotics." These are called host defense peptides (HDPs) and can combat inflammation through their direct antimicrobial properties and / or by influencing the host's immune response, including inducing cytokine and chemokine expression, promoting wound healing from infection, leukocyte activation, and macrophage / dendritic cell differentiation. Inspired by this, the development of in-depth clinical applications for these peptides has attracted researchers' attention. Over the past three decades, there has been considerable interest in the therapeutic use of HDPs, with over 5,000 articles published in this research field since 2017. These include articles introducing the potential clinical uses of HDPs, ranging from infections caused by multidrug-resistant bacteria to chronic inflammatory diseases such as arthritis, asthma, and colitis, as well as cancer treatment. Currently, HDP-based therapies in clinical trials are primarily used to treat respiratory, oral, and catheter-related infections, as well as wound healing.

[0004] Patent CN 107580502 B provides an anti-inflammatory peptide (amino acid sequence: KFRKAFKRFF) for the treatment of pancreatic cancer, as pancreatic cancer has a strong inflammatory component, resulting in an increase in B cells and a decrease in macrophages in pancreatic tumors; Patent CN 111701011B provides a composition of host defense peptides Caerin1.1 and Caerin1.9 peptides isolated from the skin secretions of Australian frogs and toads for the treatment of bacterial infections of the skin and mucous membranes; Patent CN 105722852B provides an innate defense regulator peptide (RIVPA) that can effectively treat chemotherapy-induced mucositis, radiation-induced mucositis, neutropenia-related infections, and colitis. The RIVPA peptide mimics the function of natural mucosal defense peptides; it does not have direct antibiotic activity but modulates the host response, increases survival rates after infection with large numbers of bacterial Gram-negative and Gram-positive pathogens, and accelerates recovery from tissue damage following continued exposure to various agents, including bacterial pathogens, trauma, and chemotherapy. Patent CN106749595B provides a defensive peptide (Cathelicidin-PP) derived from tree frogs, which has a significant inhibitory effect on the growth of bacteria and fungi caused by Escherichia coli, Salmonella paratyphi A, Pseudomonas aeruginosa, and Candida glabrata.

[0005] However, existing defensive peptides are mostly derived from biological extraction, resulting in low yields and the risk of non-specific immunogenicity, which limits their widespread application. Secondly, there is significant sequence and structural diversity among defensive peptides from different species, leaving considerable room for further exploration. Studies (Cytokine Growth Factor Rev. 35, 37–45 (2017); Nat. Rev. Immunol. 16, 599–611 (2016); Cancer Biol. Ther. 18, 94–100 (2017); Cell 167, 829–842 (2016)) have shown that arginine metabolism also plays a crucial role in T cell activation and the regulation of immune responses. For example, during the resolution of inflammation, immunomodulatory cells promote arginine degradation through the expression of the catabolic enzyme arginase 1 (ARG1). Therefore, providing arginine-containing materials and / or preventing arginine degradation in tumor-associated macrophages (TAMs) can re-stimulate T-cell-mediated and NK-cell-mediated immune responses.

[0006] Furthermore, the practical efficacy of naturally occurring HDPs is often strongly influenced by environmental factors. For example, many naturally occurring HDPs lose their direct anti-inflammatory effects in the presence of physiological salt concentrations (e.g., high salt content, especially divalent cations) or certain host factors (such as anionic polysaccharides, apolipoproteins, DNA, F-actin, and glycosaminoglycans). Coupled with issues such as poor absorption, biodistribution, metabolism, and excretion characteristics, HDPs have failed to demonstrate sufficient efficacy in in vitro clinical trials. Secondly, naturally occurring HDPs may also exhibit other functions undesirable for drug development, such as the ability to induce mast cell degranulation, release histamine and prostaglandins, and activate complement factors.

[0007] Therefore, it is crucial to develop novel biomimetic HDPs by effectively reducing their cytotoxic effects through biomimetic design, while optimizing their beneficial immunomodulatory functions and anti-inflammatory activities. Summary of the Invention

[0008] To address the aforementioned background and the shortcomings and limitations of existing technologies, this invention aims to provide peptide hydrogels and amphiphilic peptides capable of forming such hydrogels for autoimmune diseases, indications, or syndromes induced by cancer chemotherapy, radiotherapy, and immunotherapy, including related manifestations of immune inflammation in various organs, as well as mucosal damage and ulcers of the skin, oral cavity, and digestive tract. Host defense peptides can participate in the regulation of innate and adaptive immunity by modulating immune effector cells. Studies have found that host defense peptides can influence the entire signaling network of the immune response; they are expressed throughout the body and mediate a wide range of physiological responses, associating them with various inflammatory and autoimmune diseases. The biomimetic host defense peptide hydrogel proposed in this invention is composed of microfibers forming β-sheets. This allows it to shield its own positive charge before reaching the lesion site, thereby reducing side effects such as hemolysis; upon encountering immune effector cells, its conformation changes to an α-helical structure, exposing its positive charge and thus exhibiting specific targeting effects. Furthermore, the micro / nano structure of the hydrogel can also exert a clustering effect, enhancing its immunomodulatory function.

[0009] The present invention adopts the following technical solution:

[0010] In a first aspect, the present invention relates to 1. a peptide hydrogel, wherein the peptide has a structure of general formula (1), characterized in that the peptide hydrogel has a positive characteristic peak in the range of 190-210 nm and a negative characteristic single peak in the range of 210-230 nm in the CD spectrum, and after the peptide hydrogel is dispersed in a 1% SDS (sodium dodecyl sulfate) solution, the CD spectrum has a positive characteristic peak in the range of 190-200 nm and a negative double peak in the ranges of 200-210 nm and 215-230 nm;

[0011] The general formula (1) is Cm-IDR-Z or a physiologically acceptable salt thereof.

[0012] This includes all its isomers or diastereomers, including L-type isomers, D-type isomers, or a mixture of L-type and D-type isomers, stereoisomers, and isomers.

[0013] Or any of the aforementioned compounds, wherein the “physiologically acceptable salt” refers to a salt prepared from a non-toxic alkali or acid, including non-toxic alkali metal, alkaline earth metal, and ammonium salts such as sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and zinc protamine salts; non-toxic acid salts include, but are not limited to, hydrochloride, ethanolamine salts, trifluoroacetate, maleate, malate, benzenesulfonate, acetate, tartrate, sulfate, lactate, oxalate, and phosphate; particularly useful are the forms of acetate, hydrochloride, and phosphate.

[0014] C m It is a C8 to C 18 Acyl groups, wherein C8 to C9 18 It contains a straight-chain or branched alkyl group;

[0015] n is the number of times the peptide sequence is repeated, and in each case, it is an arbitrary integer from 2 to 5 independently.

[0016] Z is a C-terminated group, which in each case is independently selected from -COOH or -CONH2;

[0017] On the other hand, it provides a biomimetic host defense peptide of general formula (2).

[0018] The general formula (2) is C m -(X1X2RR) n X3-Z;

[0019] Wherein, R represents arginine and K represents lysine; X1, X2, and X3 are amino acids; X1 is selected from any one of the following amino acids: alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), and phenylalanine (Phe or F); X2 is selected from any one of the following amino acids: alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), proline (Pro or P), or phenylalanine (Phe or F); X3 is selected from any one of the following amino acids: alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), phenylalanine (Phe or F), tyrosine (Tyr or Y), or tryptophan (Trp or W).

[0020] Other variables are specified as in general formula (1).

[0021] On the other hand, it provides a biomimetic host defense peptide of general formula (3).

[0022] The general formula (3) is C m -X1X2RKX1X2KRX3-Z;

[0023] All variables are specified as in general formulas (1) and (2).

[0024] On the other hand, it provides a biomimetic host defense peptide of general formula (4).

[0025] The general formula (4) is C m -X1X2KRX1X2RKX3-Z;

[0026] All variables are specified as in general formulas (1) and (2).

[0027] The biomimetic host defense peptide possesses anti-infective immunomodulatory (inducing chemokine expression), induction activity in immune cells (neutrophils, macrophages, dendritic cells, and natural killer T cells), anti-inflammatory activity, wound healing, pro-angiogenic activity, pro-apoptotic activity in epithelial cells and regulatory T cells, and anti-apoptotic activity in neutrophils.

[0028] The biomimetic host defense peptide is the assembly building block for forming the biomimetic host defense peptide hydrogel. Its unique secondary structure allows the biomimetic host defense peptide molecule to further form a three-dimensional structure that can serve as a molecular recognition, enhancing its spatial topological response.

[0029] Preferably, the biomimetic host defense peptide is selected from the following sequences:

[0030]

[0031]

[0032] In a second aspect, the present invention provides a biomimetic host defense peptide hydrogel, characterized in that it is a hydrogel assembled in a physiologically acceptable medium from structures of general formula (1), general formula (2), general formula (3) or general formula (4) as described in the first aspect.

[0033] Compared to non-hydrogel biomimetic host defense peptides, biomimetic host defense peptide hydrogels reduce the hemolytic side effects of nonspecific membrane disruption and enhance immunomodulatory capabilities, including net inhibition of potentially harmful pro-inflammatory responses and multi-pathway immunomodulatory capabilities (regulating both pro-inflammatory and anti-inflammatory responses). Therefore, they can act as immunomodulators for both innate and adaptive immune responses. Upon contact with hydrophobic structures such as the cell membrane, they spontaneously transform from a β-sheet structure to an α-helix structure.

[0034] After forming a biomimetic host defense peptide hydrogel, the biomimetic host defense peptide exhibits better chemical stability and improved safety (increased specificity and reduced hemolysis rate) compared to previous biomimetic host defense peptides, and enhanced pharmacological properties (such as half-life, absorption, and efficacy).

[0035] During the differentiation of macrophages and monocytes, the presence of the aforementioned biomimetic host defense peptide hydrogel leads to the polarization of macrophages from the pro-inflammatory M1 phenotype to the M2 phenotype, altering the differentiation of dendritic cells and thereby promoting enhanced adaptive immunity.

[0036] Thirdly, this invention provides a method for preparing a biomimetic host defense peptide hydrogel as described in the second aspect. It mainly includes the following steps:

[0037] (1) A biomimetic host defense peptide is dispersed in an aqueous medium solution at a certain concentration and heated or sonicated until dissolved.

[0038] (2) Adjust the pH of the solution system or increase the ionic strength of the solution, and after static aging, obtain a biomimetic host defense peptide hydrogel.

[0039] Specifically, the certain concentration in step (1) refers to the concentration of host defense peptides being 0.1-100 mg / mL, preferably 0.5-20 mg / mL, and more preferably 1-10 mg / mL;

[0040] The aqueous medium solution can be pure water, physiological saline, glucose, PBS solution, or Tris buffer solution, etc.

[0041] If heating is required, the heating temperature should be controlled between 50-95℃;

[0042] If ultrasound is required, the ultrasound time is 0.5-30 minutes;

[0043] In step (2), the pH of the solution is adjusted to 4-10, preferably 5.5-8;

[0044] Increasing the ionic strength of the solution in step (2) refers to adding one or more commonly used physiological metal ion salts, such as sodium salts, potassium salts, iron salts and calcium salts.

[0045] The static aging time is 0.1-72 hours, preferably 1-24 hours;

[0046] Fourthly, the present invention provides an application of the above-mentioned hydrogel, the application including the prevention and treatment of diseases such as oral diseases, autoimmune diseases, inflammatory diseases, and wound infections.

[0047] The biomimetic host defense peptide hydrogel of the present invention is used to treat infectious diseases caused by microorganisms, including but not limited to periodontitis, oral mucosal healing disorders, etc.

[0048] The interaction between the host and the microbiome contributes to human health and disease. The human body surface is primarily covered by the epithelium, which acts as a physical barrier, serving as the first line of defense against pathogen invasion and a response to the symbiotic microbiome. However, the oral mucosa is an exception because teeth are essentially transmucosal organs, and the lack of a tight, intact interface between each tooth and the mucosa makes them vulnerable to infection by oral bacteria. Periodontitis is considered one of the most common infectious diseases. Therefore, unlike the microbiota in other mucosal sites (such as the gut and skin), the oral microbiota may have a direct and unique impact on the immune system, as well as the host's health and well-being.

[0049] The biomimetic host defense peptide hydrogel of the present invention is used to treat inflammatory diseases and inflammatory conditions.

[0050] The inflammatory conditions include some form of arthritis, including but not limited to osteoarthritis, rheumatoid arthritis, septic arthritis, TNF receptor-associated periodic syndrome, and inflammatory bowel disease, including Crohn's disease and ulcerative colitis.

[0051] The diseases mentioned include certain forms of inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, irritable bowel syndrome, collagenous colitis, lymphocytic colitis, ischemic colitis, diverted colitis, Behçet's syndrome, infectious colitis, and undifferentiated colitis.

[0052] The autoimmune diseases mentioned are caused by a disorder or abnormality of the autoimmune system, including but not limited to systemic syndromes such as systemic lupus erythematosus, Sjögren's syndrome, psoriasis, ankylosing spondylitis, scleroderma, rheumatoid arthritis, and polymyositis, or a syndrome that affects only a local body system, such as the endocrine system (type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, etc.), the skin system (pemphigus vulgaris), the hematologic system (autoimmune hemolytic anemia), or the nervous system (multiple sclerosis).

[0053] On the other hand, the present invention provides a treatment for immune activation under immunosuppression, which has high stability and safety due to its β-sheet structure under physiological conditions, and can therefore be administered by conventional intravenous, subcutaneous or intramuscular injection delivery systems, and may also include, but is not limited to, oral delivery systems, nasal delivery systems and mucosal delivery systems.

[0054] The biomimetic host defense peptide and its hydrogel provided by this invention show great promise as a low-cost and effective anti-inflammatory agent. It possesses at least one, several, or all of the following beneficial effects:

[0055] 1. The hydrogel of the present invention has good biocompatibility and low toxicity;

[0056] 2. The hydrogel of the present invention has multi-pathway immunomodulatory capabilities (regulating pro-inflammatory and anti-inflammatory responses), and can regulate both innate and adaptive immunity, thereby accelerating the body's healing;

[0057] 3. The hydrogel of the present invention has good in vivo stability and anti-enzymatic hydrolysis ability, and can exert a longer-lasting effect;

[0058] 4. The hydrogel of the present invention can be prepared on a large scale through solid-phase synthesis, with controllable cost and quality.

[0059] Detailed description of the invention:

[0060] 1. Definition

[0061] Before proceeding with the description of this invention, certain terms are defined as set forth herein.

[0062] In the sequence given for the peptide according to the invention, as given in Chapter 2400 of the 8th edition of the Manual of Patent Examining Procedure, the amino acid residues have their conventional meanings. Thus, alanine is “Ala” or A, valine is “Val” or V, leucine is “Leu” or L, isoleucine is “Ile” or I, phenylalanine is “Phe” or F, proline is “Pro” or P, tyrosine is “Tyr” or Y, tryptophan is “Trp” or W, and so on.

[0063] 2. Indications

[0064] "Inflammatory disease" or "inflammatory condition" refers to a condition characterized in part by inflammatory mechanisms, such as specific T lymphocyte responses or antibody-antigen interactions, which lead to the recruitment of inflammatory cells and endogenous mediator chemicals, including but not limited to cytokines, such as one or more of increased NF-κB activity, increased TNF-α production, increased IL-1β production, and increased IL-6 production.

[0065] 3. Production methods

[0066] Typically, the biomimetic host defense peptides described in this invention can be synthesized using solid-phase synthesis methods and purified according to methods known in the art. Any well-known procedure utilizing various resins and reagents can be used to prepare these biomimetic host defense peptides of this invention.

[0067] Solid-phase peptide synthesis methods are well-known and practiced in the art. The biomimetic host defense peptide synthesis of the present invention can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain, based on the general principles of solid-phase methods.

[0068] 4. A method for predicting the formation of β-sheet fiber microstructures in a biomimetic host defense tower.

[0069] Taking into account the hydrophilicity / hydrophobicity of peptide sequences and their tendency to form β-sheet secondary structures, conformational relationship analysis was performed to screen peptide sequences and obtain the amino acid sequences of potential defensive peptide hydrogels.

[0070] The beta-sheet tendency parameters and hydrophobic parameters of each amino acid in the peptide sequence are derived from: An algorithm for protein secondary structure prediction based on class prediction, Protein Engineering 1:289-294 (1987); and Analysis of membrane and surface protein sequences with the hydrophobic moment plot, J Mol Biol. 1984 Oct 15; 179(1):125-142. The reference values ​​for each amino acid are shown in Table 1.

[0071]

[0072]

[0073] Calculation method reference: The hydrophobicity parameter value of the peptide sequence is the average value of the hydrophobicity parameter values ​​corresponding to each amino acid in the peptide sequence;

[0074] The β-sheet tendency parameter of a peptide sequence is the average of the summation values ​​of the β-sheet tendency parameter for each amino acid in the peptide sequence.

[0075] Table 2: Hydrophobicity parameter values ​​of the host defense peptides involved in this invention

[0076]

[0077]

[0078] Table 3: β-sheet tendency parameters of host defense peptides involved in this invention

[0079]

[0080] The simulation data shows that the hydrophobicity parameter and the β-sheet tendency parameter can form a fibrous structure with a β-sheet structure within a certain range. The results show that the biomimetic host defense peptide of the present invention has a high tendency to form a β-sheet structure, which is conducive to the further formation of a hydrogel structure.

[0081] 5. Efficacy evaluation and determination methods

[0082] The biomimetic host defense peptide hydrogel of the present invention can be tested using various testing systems and animal models to determine its structure, functional status and efficacy.

[0083] Inflammatory bowel disease (IBD) is essentially an immune-related disease resulting from an imbalance between symbiotic bacteria in the gut and the mucosal immune system. It serves as an important disease model for validating host defense peptides. Active IBD is characterized by significant infiltration into the lamina propria of innate immune cells (neutrophils, macrophages, dendritic cells, and natural killer T cells) and adaptive immune cells (T cells and B cells). Increased numbers and activation of these cells in the intestinal mucosa elevate local levels of cytokines involved in the TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (IFN-γ), and the interleukin-23-Th17 pathway.

[0084] In IBD, the pro-inflammatory cytokine TNF-α has been observed to play a crucial role in the inflammatory cascade that triggers chronic inflammation. Elevated levels of circulating IL-6 are observed in various inflammatory diseases, including Crohn's disease. IL-6 is a key regulator of the inflammatory response. Influencing the production of this cytokine can alter the balance of effector CD4+ T cell subsets and induce B cell antibody production. Since IL-6 is largely produced by innate cells such as neutrophils, macrophages, and mast cells, it serves as a bridge between the innate and adaptive immune systems.

[0085] 5.1 Cell Experiments

[0086] 5.1.1 MTT assay

[0087] Cell viability was assessed using the MTT assay to evaluate the safety of the host defense peptide hydrogel and to exclude its influence on subsequent experiments. Human intestinal epithelial cell line Caco-2 cells were used. The host defense peptide hydrogel was treated with 0.5 mg / mL or 1 μg / mL LPS, and the procedure was performed according to the standard MTT protocol.

[0088] 5.1.2 LPS-induced construction of an in vitro inflammation model

[0089] An in vitro inflammation model of intestinal bronchiectasis (IBD) was established using Caco-2 cells transformed with lipopolysaccharide (LPS), and the effect of host defense peptide hydrogels was evaluated. LPS has been recognized as a potent, rapid, and continuous stimulant of pathological inflammation and apoptosis in intestinal epithelial cells, which can exacerbate intestinal epithelial barrier dysfunction and weaken the mucosal defense function against pathogens. Conversely, low-level expression of TLR4 in epithelial cells can limit the recognition of LPS signals and participate in the low responsiveness of normal mucosa to intestinal bacteria.

[0090] LPS intervention experimental method: Grouped cells were inoculated into 96-well plates. Cells were pre-cultured with the test sample (0.5 mg / mL) for 2 h, followed by stimulation with LPS at a concentration of 1 μg / mL for 48 h. The normal control group was given only an equal volume of PBS solution.

[0091] After the intervention was completed, the culture supernatant was aspirated, and the levels of cytokines TNF-α, IL-6, and PGE2 secreted by the cells were measured by ELISA, following the standard procedure of the kit.

[0092] The levels of nitric oxide (NO) in the supernatant of each group were measured. When LPS or T cells activate macrophages and polymorphonuclear leukocytes in vivo, they can produce large amounts of inducible nitric oxide synthase (NOS) and superoxide anion free radicals, thereby synthesizing large amounts of NO and H2O2. This plays a very important role in killing invading bacteria, fungi, and other microorganisms, as well as tumor cells, organic foreign bodies, and in inflammatory damage. The experimental procedures were performed according to the instructions for use of the kit.

[0093] Intestinal oxidative stress damage has a promoting effect on the pathogenesis and progression of IBD. The production of ROS in the intestinal mucosa of Crohn's disease (CD) patients is significantly increased, and the upregulated oxidative stress response in intestinal tissue damages the barrier function of intestinal epithelial cells, increasing their permeability. Detection of cellular ROS content: Caco-2 cells treated with host defense peptide hydrogel / LPS were collected by digestion, washed twice with serum-free DMEM medium, and the cellular ROS content was determined according to the Beyotime reagent manufacturer's instructions.

[0094] Western blot was used to determine the expression levels of inflammation-related proteins and apoptosis-related proteins, including COX-2 (an inducible synthase responsible for the production of inflammatory mediators), TLR4, and NF-κB.

[0095] 5.1.3 Evaluation of host defense peptide hydrogel-induced M1 / M isotype in RAW264.7 cells

[0096] In inflammatory diseases, macrophages induced into the M1 subtype via the classical activation pathway release pro-inflammatory factors, promoting inflammation and leading to damage to nervous system tissues. However, when induced into the M2 subtype via the alternative activation pathway, they exhibit anti-inflammatory effects and promote tissue repair. Examining the differentiation of mouse macrophages RAW264.7 induced by host defense peptide hydrogels can help analyze their mechanism in the treatment of inflammatory diseases. The classic method uses iNOS, CD86, and CD40 to identify M1 macrophages, and MR, CD206, and Arg I to identify M2 macrophages. The specific procedure is as follows: RAW264.7 cells are seeded in 24-well plates, and each well is stimulated with LPS (10 μg / mL). Then, the test sample (0.5 mg / mL) is added for co-incubation for 48 h, followed by fixation with 4% paraformaldehyde. Cells were incubated overnight at 4°C with primary antibodies such as anti-CD68 (1:200, pan-macrophage marker), CD86 (1:100, M1 marker), or CD206 (1:100, M2 marker), and then incubated with the corresponding fluorescently labeled secondary antibodies for 30 min. The mRNA expression of M1 and M2 macrophage markers in RAW264.7 cells under each condition was assessed by qRT-PCR quantitative analysis.

[0097] 5.2 In vivo studies:

[0098] 5.2.1 Establishment of an animal model of intestinal inflammation by induction with sodium dextran sulfate (DSS)

[0099] Female BALB / c mice, 6-8 weeks old and weighing approximately 25g, were housed for 3 days in an animal room at 25°C with alternating light / dark cycles every 12 hours. They were fasted for 24 hours prior to the experiment, with free access to water, and a DSS-induced ulcerative colitis model was established. DSS-induced inflammation occurs by disrupting the epithelial barrier, exposing the lamina propria to luminal contents and bacterial antigens, leading to vascular and mucosal damage. This exposure triggers activation of inflammatory pathways, resulting in increased production of inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-10, IL-12, and IFN-γ. DSS administration increased the expression of integrin-αM (ITGAM), integrin-αX (ITGAX), and IL-17. Long-term DSS administration increased the expression of IL-4 and IL-5, indicating that DSS-induced colitis is mediated by Th1 and Th2 immune mechanisms. Extensive data have demonstrated that the etiology, clinical symptoms, pathological changes, and treatment response of the DSS colitis model are similar to those of human ulcerative colitis (UC). Therefore, the DSS colitis model plays a key role in studying the etiology and pathogenesis of UC, and has become an important treatment method. It is also one of the most widely used ulcerative colitis models.

[0100] The effects of biomimetic host defense peptide hydrogels were evaluated using a DSS-induced colitis mouse model.

[0101] Examine the colon for ulcerative colitis and score according to Table 4 below:

[0102] 0 No damage 1 Local ulceration 2 Linear ulcers without severe inflammation 3 A linear ulcer with inflammation at a single point 4 Pain or inflammation at two or more points 5 Large ulcers or inflammations larger than 1 cm

[0103] 5.2.2 Establishment of an animal model of intestinal inflammation using trinitrobenzenesulfonic acid (TNBS)

[0104] Male Wistar rats were housed for 3 days in an animal room at 25°C with alternating light and dark conditions every 12 hours. They were fasted for 24 hours before the experiment, but had free access to water. A TNBS-induced ulcerative colitis model was used. According to literature reports, TNBS dissolved in 50% ethanol can induce intestinal lesions, manifested as colonic shortening, intestinal hemorrhage, epithelial necrosis leading to crypt destruction, and increased Th1 / Th17 immune response in the colon leading to transmural inflammation, similar to Crohn's disease (CD), and can be used to study the treatment of CD. Specific procedures: After anesthetizing the rats with ether, a 2mm inner diameter polyurethane tube was slowly inserted through the anus, with the tube extending approximately 8cm into the anus. Approximately 0.8mL of TNBS solution (10mg / kg) was slowly injected into the rat colon through the tube to induce the ulcerative colitis model. After 3 days, the rats exhibited symptoms such as diarrhea and weight loss. Rats with an 80% weight loss compared to pre-induction levels were considered to have successfully developed the model. Normal rats were used as a control group, and the same method was used to inject 50% ethanol solution into their colons.

[0105] 5.2.3 Construction and Evaluation of Animal Models of Periodontitis

[0106] To verify the effect of biomimetic host defense peptide hydrogel in treating periodontitis in vivo, an acute periodontitis model in rats was first established by ligation induction with ligature wire. Six-week-old Wistar rats, weighing approximately 200g, were selected. After anesthetizing the rats with 10% chloral hydrate (3.5mL / kg) via intraperitoneal injection, the rats were fixed in a supine position on the operating table. The rats' upper and lower teeth were expanded with a retractor, and the rats' oral cavity was disinfected with 75% ethanol. Then, a 0.25mm orthodontic ligature wire was wrapped and fixed around the cervical region of the left maxillary second molar. After one week of circumferential ligation, the rats were examined according to the clinical diagnostic criteria for periodontitis: (1) the color, shape, and texture of the gingiva; (2) bleeding on periodontal probing; (3) periodontal probing to see if there is the formation of deep periodontal pockets; (4) whether there is attachment loss, the formation of attachment loss is an indicator to distinguish periodontitis from gingivitis. If the above four criteria were met, the modeling was successful. After that, the wire was removed, and the rats were grouped. Attached image description:

[0107] Figure 1 Macroscopic and TEM images of the hydrogel prepared in Example 1.

[0108] Figure 2 Macroscopic and TEM images of the hydrogel prepared in Example 2.

[0109] Figure 3 Macroscopic and TEM images of the hydrogel prepared in Example 7.

[0110] Figure 4 The CD spectra of the hydrogel prepared in Example 1 and Comparative Example 1 are shown.

[0111] Figure 5 The CD spectra of the hydrogels prepared in Examples 2, 3, 4, 5, 6 and 7.

[0112] Figure 6 The CD spectra of the hydrogels prepared in Comparative Examples 2, 3, 4, 5, 6 and 7 in SDS solution are shown.

[0113] Figure 7 The rheological curves of the hydrogels prepared in Examples 7, 8, and 9 are shown.

[0114] Figure 8 The self-healing and shear thinning characteristic curves of the hydrogels prepared in Examples 8 and 9 are shown.

[0115] Figure 9 The study investigated the effect of host defense peptide hydrogels on LPS-induced cell survival.

[0116] Figure 10 The effects of host defense peptide hydrogel on LPS-induced secretion levels of inflammatory factors A) TNF-α, B) IL-8, C) PGE2 and D) NO in Caco-2 cells.

[0117] Figure 11 The effect of host defense peptide hydrogel on LPS-induced ROS secretion levels in Caco-2 cells.

[0118] Figure 12 The effect of host defense peptide hydrogel on LPS-induced differentiation of mouse macrophages RAW264.7 M1 / M2.

[0119] Figure 13 This is a photograph of the colon obtained after dissection following DSS-induced enteritis treatment.

[0120] Figure 14 This shows the changes in colon length after DSS-induced enteritis treatment.

[0121] Figure 15 The effect of host defense peptides on body weight in DSS-induced experimental colitis model animals.

[0122] Figure 16 The DAI score of an experimental colitis model animal on day 7 of DSS intervention for host defense peptides.

[0123] Figure 17 This describes the changes in colonic index after TNBS-induced enteritis treatment.

[0124] Figure 18 Three-dimensional reconstruction images of the left maxillary molars of rats in each group are shown. a represents the blank group, b represents the periodontitis control group, c represents the comparative treatment group, and d represents the example treatment group.

[0125] Figure 19 The microstructural parameters of the trabeculae in each group are BV / TV, Tb.N, Tb.Sp., and Tb.Th. Where ### indicates p < 0.001 compared to the control group; *p < 0.05, **p < 0.001 compared to the periodontitis group.

[0126] Figure 20 For analysis of alveolar bone resorption. A. The marked line represents the distance from the cementoenamel junction to the alveolar ridge crest (CEJ-ABC). B. The distance of CEJ-ABC for each group of rats.

[0127] Figure 21 The table shows the serum levels of inflammatory factors TNF-α and IL-1β in rats from each group in the examples. Wherein, ### indicates p < 0.001 compared to the control group; **p < 0.01 indicates p < 0.01 compared to the periodontitis group. Detailed Implementation

[0128] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the implementation of the invention is not limited thereto. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0129] In the following descriptions, "peptide," "peptide compound," "peptide sequence," and "bionic host defense peptide" all refer to the peptide compound used in this invention. It should be noted that the amino acid sequences of the peptides used in the embodiments of this invention are as described in the sequence listing, but the peptides used also include hydrophobic chains; therefore, their specific structures are as described above in this specification.

[0130] Example 1

[0131] Preparation method of SEQ ID No. 1 peptide hydrogel:

[0132] (1) The biomimetic host defense peptide with sequence SEQ ID No.1 was dispersed in 0.01M Hepes buffer at a concentration of 5 mg / mL and heated in an 85°C water bath for 2 hours until completely dissolved.

[0133] (2) Add calcium chloride to the above system. The concentration of calcium chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0134] The resulting hydrogel is transparent. Figure 1 a), TEM Figure 1 b) Characterization results show that it is composed of a fiber network. Further analysis using CD spectroscopy reveals the secondary structure of the hydrogel fibers, such as... Figure 4 As shown, it exhibits a typical characteristic peak of β-sheet at 220 nm.

[0135] When the hydrogel fiber was dispersed in a 1% SDS solution, the CD spectrum ( Figure 4 The secondary structure of the hydrogel undergoes a transformation, exhibiting a typical α-helical structure. This conformational change helps the hydrogel maintain stability in physiological solutions. When it encounters a lipid membrane, it can quickly transform into an α-helical structure with a stronger membrane-breaking ability.

[0136] Example 2

[0137] Preparation method of SEQ ID No. 2 peptide hydrogel:

[0138] (1) The biomimetic host defense peptide with sequence SEQ ID No.2 was dispersed in an aqueous solution at a concentration of 20 mg / mL and heated in an 80°C water bath for 2 hours until completely dissolved.

[0139] (2) Add disodium hydrogen phosphate and sodium dihydrogen phosphate (mass ratio 5:1) to the above system, wherein the concentration of sodium salt solution is 10mM, cool naturally to room temperature, and allow to stand for aging for 12h to obtain a biomimetic host defense peptide hydrogel that can stand upside down and support itself.

[0140] The resulting hydrogel is transparent. Figure 2 a), TEM Figure 2 b) Characterization results show that it is composed of a fiber network. Its CD spectrum is as follows: Figure 5 As shown in figure a, it exhibits typical characteristic peaks of the β-sheet.

[0141] Example 3

[0142] Preparation method of SEQ ID No. 3 peptide hydrogel:

[0143] (1) The biomimetic host defense peptide with sequence SEQ ID No.3 was dispersed in 0.01M Hepes buffer at a concentration of 10 mg / mL and heated in an 85°C water bath for 2 hours until completely dissolved.

[0144] (2) Sodium chloride was added to the above system. The concentration of sodium chloride in the solution was 2mM. After naturally cooling to room temperature and aging for 18h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself was obtained.

[0145] Its CD spectrum is as follows Figure 5 As shown in b, it exhibits typical characteristic peaks of the β-sheet.

[0146] Example 4

[0147] Preparation method of SEQ ID No. 4 peptide hydrogel:

[0148] (1) The biomimetic host defense peptide with sequence SEQ ID No.4 was dispersed in 0.01M Hepes buffer at a concentration of 5 mg / mL and heated in an 85°C water bath for 2 hours until completely dissolved.

[0149] (2) Add calcium chloride to the above system. The concentration of calcium chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 6 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0150] Its CD spectrum is as follows Figure 5 As shown in c, it exhibits typical characteristic peaks of the β-sheet.

[0151] Example 5

[0152] Preparation method of SEQ ID No. 5 peptide hydrogel:

[0153] (1) The biomimetic host defense peptide with sequence SEQ ID No.5 was dispersed in 0.01M Hepes buffer at a concentration of 1 mg / mL and sonicated until completely dissolved.

[0154] (2) Sodium chloride was added to the above system. The concentration of sodium chloride in the solution was 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself was obtained.

[0155] Its CD spectrum is as follows Figure 5 As shown in d, it has typical characteristic peaks of the β-sheet.

[0156] Example 6

[0157] Preparation method of SEQ ID No. 6 peptide hydrogel:

[0158] (1) The biomimetic host defense peptide with sequence SEQ ID No.6 was dispersed in 0.01M Hepes buffer at a concentration of 5 mg / mL and heated in an 80℃ water bath for 2 hours until completely dissolved.

[0159] (2) Sodium dihydrogen phosphate was added to the above system. The concentration of sodium dihydrogen phosphate in the solution was 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself was obtained.

[0160] Its CD spectrum is as follows Figure 5 As shown in e, it exhibits typical characteristic peaks of the β-sheet.

[0161] Example 7

[0162] Preparation method of SEQ ID No. 7 peptide hydrogel:

[0163] (1) The biomimetic host defense peptide with sequence SEQ ID No.7 was dispersed in water at a concentration of 5 mg / mL and heated in an 85°C water bath for 0.5 hours until completely dissolved.

[0164] (2) Add 10mM Na2HPO4 and 20mM Tris to the above system, pH 7, cool naturally to room temperature, and allow to stand for aging for 24 hours to obtain a biomimetic host defense peptide hydrogel that can stand upside down and support itself.

[0165] The resulting hydrogel is transparent. Figure 3 a), TEM Figure 3 b) Characterization results show that it is composed of a fiber network. Its CD spectrum is as follows: Figure 5 As shown in f, it exhibits typical characteristic peaks of the β-sheet. When dissolved in 1% SDS solution and allowed to stand for 24 hours, its CD spectrum is as follows... Figure 6 As shown in f, it exhibits typical characteristic peaks of the α-helix.

[0166] Example 8

[0167] Preparation method of SEQ ID No. 8 peptide hydrogel:

[0168] (1) The biomimetic host defense peptide with sequence SEQ ID No.8 was dispersed in 0.01M Hepes buffer at a concentration of 3 mg / mL and heated in a 75°C water bath for 2 hours until completely dissolved.

[0169] (2) Add potassium chloride to the above system. The concentration of potassium chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 2 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0170] Example 9

[0171] Preparation method of SEQ ID No. 9 peptide hydrogel:

[0172] (1) The biomimetic host defense peptide with sequence SEQ ID No.9 was dispersed in 0.01M sodium chloride solution at a concentration of 2.5 mg / mL and sonicated until completely dissolved.

[0173] (2) Add 100mM Tris to the above system, adjust the pH of the system to 7.5, cool naturally to room temperature, and let it stand for 24 hours to age, and obtain a biomimetic host defense peptide hydrogel that can stand upside down and support itself.

[0174] Example 10

[0175] Preparation method of SEQ ID No. 10 peptide hydrogel:

[0176] (1) The biomimetic host defense peptide with sequence SEQ ID No10 was dispersed in 0.01M Tris-HCl buffer at a concentration of 5 mg / mL and heated in a 65℃ water bath for 2 hours until completely dissolved.

[0177] (2) Add 10mM Na2HPO4 and 20mM NaH2PO4 to the above system, cool naturally to room temperature, and allow to stand for aging for 4 hours to obtain a biomimetic host defense peptide hydrogel that can stand upside down and support itself.

[0178] Example 11

[0179] Preparation method of SEQ ID No. 11 peptide hydrogel:

[0180] (1) The biomimetic host defense peptide with sequence SEQ ID No.11 was dispersed in 0.01M Hepes buffer at a concentration of 5 mg / mL and heated in a 70℃ water bath for 2 hours until completely dissolved.

[0181] (2) Add calcium chloride to the above system. The concentration of calcium chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0182] Example 12

[0183] Preparation method of SEQ ID No. 12 peptide hydrogel:

[0184] (1) The biomimetic host defense peptide with sequence SEQ ID No.12 was dispersed in 0.01M Hepes buffer at a concentration of 15 mg / mL and heated in a 65°C water bath for 2 hours until completely dissolved.

[0185] (2) Add ferric chloride to the above system. The concentration of ferric chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0186] The prepared hydrogel was used to evaluate a mouse skin abscess model, and the results are as follows: Figure 16 As shown.

[0187] Example 13

[0188] Preparation method of SEQ ID No. 33 peptide hydrogel:

[0189] (1) The biomimetic host defense peptide with sequence SEQ ID No.33 was dispersed in 0.01M physiological saline at a concentration of 5 mg / mL and heated in a 75℃ water bath for 2 hours until completely dissolved.

[0190] (2) Add 80mM Tris to the above system, adjust the pH of the system to 7.8, cool naturally to room temperature, and allow to stand for aging for 12 hours to obtain a biomimetic host defense peptide hydrogel that can stand upside down and support itself.

[0191] Example 14

[0192] Preparation method of SEQ ID No. 24 peptide hydrogel:

[0193] (1) The biomimetic host defense peptide with sequence SEQ ID No.24 was dispersed in 0.01M glucose solution at a concentration of 5 mg / mL and heated in a 55℃ water bath for 2 hours until completely dissolved.

[0194] (2) Add calcium chloride to the above system. The concentration of calcium chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0195] Example 15

[0196] Preparation method of peptide gel SEQ ID No. 35:

[0197] (1) The biomimetic host defense peptide with sequence SEQ ID No.35 was dispersed in 0.01M Tris buffer at a concentration of 2.5 mg / mL and heated in a 65°C water bath for 1 hour until completely dissolved.

[0198] (2) Add calcium chloride to the above system. The concentration of calcium chloride in the solution is 1 mM. After naturally cooling to room temperature and aging for 24 h, a biomimetic host defense peptide hydrogel that can stand upside down and support itself is obtained.

[0199] Comparative Example 1

[0200] The host defense peptide from Example 1 was dispersed in a 1% SDS solution and allowed to stand for 24 hours to obtain Comparative Example 1. Its CD spectrum is shown below. Figure 5 As shown in b, it exhibits typical characteristic peaks of the α-helix.

[0201] Comparative Example 2

[0202] The host defense peptide from Example 2 was dispersed in a 1% SDS solution and allowed to stand for 24 hours to obtain Comparative Example 2. Its CD spectrum is shown below. Figure 6 As shown in figure a, it exhibits typical characteristic peaks of the α-helix.

[0203] Comparative Example 3

[0204] The host defense peptide from Example 3 was dispersed in a 1% SDS solution and allowed to stand for 24 hours to obtain Comparative Example 3. Its CD spectrum is shown below. Figure 6 As shown in b, it exhibits typical characteristic peaks of the α-helix.

[0205] Comparative Example 4

[0206] The host defense peptide from Example 4 was dispersed in a 1% SDS solution and allowed to stand for 24 hours to obtain Comparative Example 4. Its CD spectrum is shown below. Figure 6 As shown in c, it exhibits typical characteristic peaks of the α-helix.

[0207] Comparative Example 5

[0208] The host defense peptide from Example 5 was dispersed in a 1% SDS solution and allowed to stand for 24 hours to obtain Comparative Example 5. Its CD spectrum is shown below. Figure 6 As shown in d, it exhibits typical characteristic peaks of the α-helix.

[0209] Comparative Example 6

[0210] The host defense peptide from Example 6 was dispersed in a 1% SDS solution and allowed to stand for 24 hours to obtain Comparative Example 6. Its CD spectrum is shown below. Figure 6 As shown in Figure e, it exhibits typical characteristic peaks of the α-helix.

[0211] Example 16

[0212] The safety of the hydrogels in Examples 1-8 was evaluated according to the method described in 5.1.1, wherein the dosage was 0.5 mg / mL. The results are as follows: Figure 9 As shown in the figure. The results indicate that, at the applied dose, the host defense peptide hydrogels all exhibited good safety and had no significant impact on cell viability.

[0213] Example 17

[0214] Following the method described in 5.1.2, the inflammatory regulatory mechanisms of the hydrogels of Examples 3, 4, and 5 and their corresponding comparative examples 3, 4, and 5 were evaluated using an LPS-induced in vitro inflammation model. The results are as follows: Figure 10-11 As shown. From Figure 10 Results analysis showed that in each experimental group, the levels of cytokines THF-α, IL-8, PGE2, and NO were significantly lower than in the inflammation model group (LPS group), demonstrating an anti-inflammatory effect. The hydrogel effects in each example were stronger than their control groups, indicating that host defense peptide hydrogels can enhance anti-inflammatory efficacy. Figure 11 The evaluation of the ROS scavenging ability of different treatment groups showed that the hydrogel group had better ROS scavenging ability and could more effectively protect cells from oxidative damage.

[0215] Example 17

[0216] Following the method described in 5.1.3, the induction of macrophage M1 / M2 differentiation in Examples 7, 12, and 16 was evaluated, and the results are as follows: Figure 12 As shown. The host defense peptide hydrogels prepared in each embodiment can significantly inhibit M1 subtype macrophages while promoting the differentiation of M2 type macrophages, demonstrating their potential to suppress inflammatory diseases.

[0217] Example 18

[0218] Dextran sulfate sodium (DSS) was used to induce an animal model of intestinal inflammation for the anti-inflammatory evaluation of host defense peptide hydrogels.

[0219] Specific operating steps: Prepare 3% DSS (Mw: 36000~50000) drinking water with sterile water, and then filter it with a 0.22μm filter membrane; give it to mice for 7 consecutive days. The mice will show symptoms such as diarrhea, mucus-like stool, fecal occult blood, gross bloody stool, weight loss, reduced activity, and poor coat color. Determine whether the model is successful according to the evaluation criteria in the table below.

[0220] The Disease Activity Index (DAI) score is used to assess and score three aspects: weight, stool viscosity, and fecal occult blood. The DAI score is the sum of these three indicators.

[0221] Table 5. DAI Scoring Details

[0222] 0 No change in weight normal Negative 1 1-5％ soft stool Light blue 2 5-10％ mucus-like stool blue 3 10-20％ Thin liquid stool Deep Blue 4 ＞20％ watery stool Naked bloody stool

[0223] Grouping and Drug Administration: Mice with consistent DAI scores were selected for subsequent experiments. Mice that successfully developed the model were divided into 6 groups: control group, model group, and Example 1 group, with 5 mice in each group. The control and model groups were administered physiological saline (10 mg / kg) by gavage; the other groups were administered 10 mg / kg of the biomimetic host defense peptide gel sample.

[0224] The improvement of DSS-induced inflammatory bowel disease by Examples 1-4 and their comparative examples 1-4 was evaluated according to the above method. One week after administration, anesthetized mice were sacrificed, dissected, and their colons were photographed and measured for length. The results are as follows. Figure 13 and 14 As shown in the figure, the colon length of mice in the normal group was 8.0 ± 0.2 cm, while that in the colitis model group was 5.6 ± 0.1 cm, significantly higher than that in the normal group (p < 0.01). After administration of the biomimetic host defense peptide and its hydrogel, the colon length was significantly reduced compared to the model group, and the colon length was even shorter in the hydrogel group than in the group without hydrogel formation, indicating that the hydrogel better alleviated the degree of inflammation.

[0225] The improvement of DSS-induced inflammatory bowel disease in Examples 8-11 was evaluated according to the above method. Mouse body weight was measured daily after administration, and the DAI score of each group was reassessed after 7 days of treatment. The evaluated DAI score for each group was calculated. Figure 15 As shown, the body weight of mice in the normal group gradually increased, while the body weight of mice in the colitis model group continuously decreased, reaching a loss of nearly 80% after 7 days. After administration of the biomimetic host defense peptide and its hydrogel, the trend of body weight loss was significantly alleviated compared to the model group. The corresponding DAI score (e.g.) Figure 16 The results were similar, indicating that hydrogels can effectively reduce the symptoms of colitis.

[0226] Pathological scoring of the colon: The slides were examined under a microscope in a blind manner and scored according to the Sykes criteria.

[0227] Table 6: Sykes Standard Scoring

[0228]

[0229] Example 19: Establishing an animal model of intestinal inflammation using trinitrobenzenesulfonic acid (TNBS) for anti-inflammatory evaluation of host defense peptide hydrogels.

[0230] Grouping and Administration: Rats with consistent DAI scores were selected for subsequent experiments. Rats that successfully established the model were divided into 6 groups: control group, model group, and Example 1 group, with 5 rats in each group. The control and model groups were administered physiological saline (10 mg / kg) by gavage; the other groups were administered 10 mg / kg of the biomimetic host defense peptide gel sample.

[0231] The rats' body weight was measured daily, and the DAI scores of each group of mice were evaluated again 7 days after administration, and the evaluation DAI score of each group was calculated.

[0232] Ten days after administration, rats in each group were weighed, anesthetized and euthanized, and the colon was removed. The colon was washed with physiological saline to remove intestinal contents, the length of the colon was measured and weighed, the colonic index (C / B ratio) was calculated, and the degree of colonic loss was observed by the naked eye.

[0233] like Figure 16 The colonic index of rats in each group was measured after administration of different biomimetic host defense peptide hydrogels. The colonic index of the normal group was 6.45±0.47 mg / g, while that of the colitis model group was 15.9±0.32 mg / g, which was significantly higher than that of the normal group (p<0.01). After administration of the biomimetic host defense peptide and its hydrogel, the colonic index was significantly lower in both groups compared with the model group. Furthermore, the colonic index in the hydrogel group was even lower than that in the group without hydrogel formation, indicating that the hydrogel effectively reduced the degree of inflammation. Specific data are shown in Table 7 below.

[0234] control group 6.45±0.47 TNBS Group 15.9±1.32 TNBS + Comparative Example 1 9.1±0.3 TNBS+ Example 1 7.2±0.67 TNBS + Comparative Example 2 11.9±0.25 TNBS+ Example 2 8.6±0.89 TNBS + Comparative Example 3 10.2±0.56 TNBS+ Example 3 7.8±0.31 TNBS+ Comparative Example 4 11.1±0.92 TNBS+ Example 4 8.2±0.29

[0235] Example 20: Construction and Evaluation of an Animal Model of Periodontitis

[0236] An animal model of periodontitis was constructed according to the method listed in the instructions. Grouping and administration: Five rats that had not undergone any treatment served as the control group; among the rats that had successfully developed the model, they were randomly divided into three groups of five each.

[0237] Control group: 50uL of physiological saline was injected into the gingiva of the model teeth;

[0238] Control group: 50 μL of physiological saline was injected into the gingiva of the model teeth of mice with periodontitis;

[0239] Example 1: 50 μL of product was injected into the gingiva of the model teeth of a mouse model of periodontitis.

[0240] Comparative Example 1: 50 μL of Comparative Example 1 was injected into the gingiva of the model teeth of a mouse model of periodontitis.

[0241] Administer once a week for 4 consecutive weeks.

[0242] (1) Microcomputed tomography (Micro-CT) was used to examine the alveolar bone of rats. Specifically, alveolar bone samples from each group of rats were scanned using Micro-CT. To ensure consistency in measurement standards, the viewing angle of all images was adjusted so that all cusps were on the same plane, and the occlusal plane was not visible from either the buccal or palatal side. Three-dimensional images were reconstructed to quantify the degree of bone destruction. In each sample, the alveolar bone surrounding the maxillary first molar was selected as the Region of Interest (ROI) for study. The anterior boundary of the ROI was the mesial root of the left maxillary first molar; the posterior boundary was the distal root of the left maxillary first molar; the upper boundary was the line connecting the bases of the root bifurcations of the first molar; and the lower boundary was the line connecting the mesial and distal apical roots of the first molar. The following bone microstructural parameters were analyzed within the ROI: Bone volume per trabecular volume (BV / TV) is the most commonly used parameter for measuring bone mass in clinical and basic research.

[0243] Compared to bone mass, trabecular microstructural parameters are direct indicators of bone quality, such as trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Alveolar bone loss (ABL) was measured along the long axis of the root of the left maxillary first molar. The vertical distance from the cementum enamel junction (CEJ) to the alveolar bone crest (ABC) represents the degree of alveolar bone loss. Distances were measured at six anatomical sites, including three palatal sites (mesial, middle, and distal) and three buccal sites (mesial, middle, and distal). Results showed that after 4 weeks, as... Figure 18 As shown, the control group rats showed significant alveolar process resorption, lower alveolar ridge crest, and exposed root bifurcation with communication between the two sides. In contrast, the host defense peptide hydrogel group showed less exposed root bifurcation with no communication between the two sides, and alveolar bone resorption was significantly lower than that of the control group. Quantified parameters also support this conclusion. Figure 19 and Figure 20 ).

[0244] (2) The expression levels of inflammatory mediators TNF-α and IL-1β in rat serum were measured. After rats were injected with DhHP-6, the changes in the content of TNF-α and IL-1β in the serum of each group of rats were detected. The specific method was as follows: serum samples of each group of rats were taken and the operation was carried out in accordance with the instructions of Solarbio TNF-α and IL-1β ElASE kit to detect the content of TNF-α and IL-1β in the serum of each group of rats.

[0245] The results are as follows Figure 21 As shown, the serum levels of TNF-α and IL-1β in the periodontitis group were significantly higher than those in the blank control group (p < 0.001, p < 0.001). In contrast, the levels of TNF-α and IL-1β in both treatment groups were significantly lower than those in the periodontitis group (p < 0.01, p < 0.01, p < 0.05). Furthermore, the hydrogel and solution groups showed even lower levels of TNF-α and IL-1β, indicating that the hydrogel can significantly improve the inflammatory response of periodontitis in rats and slow down alveolar bone resorption.

[0246] Statistical analysis

[0247] SPSS statistical software was used to perform statistical analysis on the experimental data. All results are expressed as mean ± standard deviation (Mean ± SD). Analysis of variance was used for comparisons among multiple groups. p < 0.05 was considered statistically significant, and p < 0.01 was considered statistically significant.

[0248] Summary of biological data

[0249] This indicates that the biomimetic host defense peptide hydrogel possesses anti-inflammatory activity, significantly reducing the levels of TNF-α and IL-8, and demonstrating good efficacy in chronic inflammatory diseases such as gastrointestinal inflammation. Furthermore, the biomimetic host defense peptide hydrogel exhibits a dual effect of anti-inflammatory and healing-promoting properties, thus demonstrating significant efficacy in the presence of both infection and inflammation. It can be used to treat inflammation in the absence of infection, and to prevent inflammation in the presence of infection. Simultaneously, the hydrogel can prevent the potential vicious cycle between chronic bacterial colonization, inflammation, and epithelial damage.

[0250] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A peptide hydrogel, characterized in that, The peptide hydrogel has a positive characteristic peak in the range of 190-210 nm and a negative single peak in the range of 210-230 nm in the CD spectrum. After the peptide hydrogel is dispersed in a 1% sodium dodecyl sulfate solution, the CD spectrum has a positive characteristic peak in the range of 190-200 nm and negative double peaks in the ranges of 200-210 nm and 215-230 nm. The peptide is selected from one of the following sequences: SEQ ID No.1: C8-LLRRLLRRI-CONH2 SEQ ID No. 2: C 12 -LLRRLLRRLLRRI-CONH2 SEQ ID No. 3: C 18 -LLRRLLRRLLRRLLRR I-CONH2 SEQ ID No. 4: C8-IIRRIIRRI-CONH2.

2. A class of amphiphilic peptide molecules, characterized in that, The peptide is selected from one of the following sequences: SEQ ID No.1: C8-LLRRLLRRI-CONH2 SEQ ID No. 2: C 12 -LLRRLLRRLLRRI-CONH2 SEQ ID No. 3: C 18 -LLRRLLRRLLRRLLRR I-CONH2 SEQ ID No. 4: C8-IIRRIIRRI-CONH2.

3. The peptide hydrogel according to claim 1, characterized in that, It is a hydrogel formed by the assembly of the peptides in a physiologically acceptable medium, excluding cross-linking agents.

4. The peptide hydrogel according to claim 1, characterized in that, The peptide forms a nanofiber structure, and the nanofiber has a β-sheet structure.

5. A method for preparing the peptide hydrogel according to any one of claims 1, 3-4, comprising the following steps: (1) Disperse the peptide in an aqueous medium solution and heat or sonicate until dissolved; (2) Adjust the pH of the solution system or increase the ionic strength, and after aging, obtain peptide hydrogel; in, The concentration of the peptide in step (1) is 0.1-100 mg / mL; The aqueous medium solution is selected from at least one or a combination of multiple of pure water, physiological saline, PBS solution, glucose or tris(hydroxymethyl)glycine buffer solution; The heating temperature should be controlled between 50-95℃; or the ultrasonic time should be 0.5-30 minutes. In step (2), the pH of the solution is adjusted to 4-10; the aging time is 0.1-72h.

6. The use of the peptide hydrogel according to any one of claims 1, 3-4 or the amphiphilic peptide molecule according to claim 2 in the preparation of a medicament for the prevention and / or treatment of colitis in humans and animals.

7. The use of the peptide hydrogel of any one of claims 1, 3-4 or the amphiphilic peptide molecule of claim 2 in the preparation of a medicament for the prevention and / or treatment of periodontitis in humans and animals, wherein the peptide sequence is SEQ ID No. 1.

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