Agrocybe cylindracea immunoactive peptide with function of relieving colitis as well as preparation method and application thereof

Abstract

The invention discloses agrocybe cylindracea immunoactive peptide with a function of relieving colitis as well as a preparation method and application thereof, and relates to the technical field of active peptide. The invention provides an agrocybe cylindracea immunocompetent peptide. The amino acid sequence of the agrocybe cylindracea immunocompetent peptide is Leu-Ala-Leu-Trp-Pro. The agrocybe cylindracea immune active peptide can be prepared through artificial chemical synthesis and can also be obtained through separation and purification of agrocybe cylindracea protein enzymatic hydrolysate. The agrocybe cylindracea immunocompetent peptide has an immunoregulation effect of regulating polarization of macrophages, has biological activity of relieving colitis, and can repair an intestinal mucosa barrier function and regulate intestinal immunity so as to relieve colitis.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to an immunomodulatory peptide from *Agrocybe aegerita* that has the function of relieving colitis, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing, nonspecific inflammatory disease of the intestine. Its exact pathogenesis remains unclear, but it is generally accepted that it involves an abnormal immune response and chronic intestinal inflammation resulting from complex interactions between genetic and environmental factors (such as diet, smoking, and physiological stress), the gut microbiota, and the host immune system. Clinically used drugs for UC treatment include aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. However, these drugs have limitations due to drug resistance and side effects, often leading to recurrent flare-ups and imposing a heavy economic burden and societal pressure on patients. Intestinal mononuclear-macrophages, as the core hub of UC pathology, drive disease progression through multidimensional mechanisms when their function is abnormal; therefore, therapeutic strategies targeting macrophages have become a hot topic in current UC research.

[0003] Edible fungi possess high nutritional value and significant biological functional potential. Currently, polysaccharides are the most widely studied and reported macromolecules in edible fungi. As core bioactive components equally important as polysaccharides, edible fungi proteins and polypeptides, with their precise targeting and multi-level mechanisms of action, are triggering systemic breakthroughs from basic research to industrial transformation in the fields of functional foods and biomedicine.

[0004] Among numerous edible fungi, *Agrocybe aegerita* (tea tree mushroom) has attracted much attention due to its unique metabolomics characteristics and its dual nature as both food and medicine. It is not only rich in basic nutrients such as dietary fiber, various vitamins, and minerals, but its special value also lies in its content of polypeptide bioactive components with antioxidant activity, anti-inflammatory properties, and immunomodulatory functions. Chinese Patent CN118440217A discloses a novel *Agrocybe aegerita* polysaccharide, its preparation method, and its application in the preparation of products for repairing intestinal mucosal barrier damage. The novel *Agrocybe aegerita* polysaccharide exhibits infrared spectroscopy at 3432.51 cm⁻¹. -1 The invention exhibits an absorption peak due to the stretching vibration of the OH group. The *Agrocybe aegerita* polysaccharide of this invention possesses novel structural features. Experiments have demonstrated that this novel *Agrocybe aegerita* polysaccharide can repair the intestinal mucosal barrier function and improve colitis symptoms by regulating the intestinal mechanical, chemical, immune, and biological barriers. Furthermore, research indicates that *Agrocybe aegerita* polysaccharide can induce lysosome-mediated apoptosis in colorectal cancer cells through histone modification, demonstrating its anti-cancer potential in the treatment of colorectal cancer. *Agrocybe aegerita* polysaccharide can prevent diet-induced obesity by regulating the intestinal microbiota and its related metabolites, suggesting that it can be used as a prebiotic for the prevention of diet-induced obesity.

[0005] Current research on tea tree mushrooms mainly focuses on its polysaccharide active components, while there is no relevant research on whether its polypeptide components have the function of relieving colitis. Summary of the Invention

[0006] The purpose of this invention is to provide tea tree mushroom immunoactive peptides with colitis-relieving function, their preparation methods, and applications.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides an immunomodulatory peptide from *Agrocybe aegerita*, wherein the amino acid sequence of the immunomodulatory peptide is Leu-Ala-Leu-Trp-Pro.

[0008] In another aspect, the present invention provides a method for preparing the above-mentioned immunomodulatory peptides of Agrocybe aegerita, the method comprising artificial chemical synthesis and separation and purification of Agrocybe aegerita protease hydrolysate.

[0009] Specifically, the method for artificially synthesizing immunoactive peptides from *Agrocybe aegerita* includes the following steps: S1. The Fmoc-Pro-Wang-Resin resin is subjected to swelling and deprotection treatment; S2. Add the next amino acid Fmoc-Trp-OH to the reactor to carry out the condensation reaction, and wash after the reaction is completed. S3. Repeat the deprotection in step S1 and step S2 until all amino acids are sequentially linked to obtain Leu-Ala-Leu-Trp-Pro-Wang-Resin resin. S4. Use cutting fluid to treat and obtain crude polypeptide; S5. High-purity peptides were obtained by reversed-phase high-performance preparative liquid chromatography.

[0010] Furthermore, the swelling conditions in step S1 are as follows: resin is added to the reaction column, N,N-dimethylformamide (DMF) is injected to completely immerse the resin, and the mixture is gently stirred or shaken at room temperature for 0.5-1 hour to allow the resin particles to fully expand.

[0011] Furthermore, the deprotection treatment conditions in step S1 are as follows: use a 20% piperidine DMF solution, treat for 10-30 minutes, and repeat twice.

[0012] Furthermore, the conditions for the condensation reaction in step S2 are as follows: an excess of the protected amino acid and the condensing agent are dissolved in DMF to activate the carboxyl group of the amino acid, which then reacts with the free amino group on the resin to form an amide bond.

[0013] Furthermore, the condensing agent is TBTU.

[0014] Furthermore, in step S4, the cutting fluid is a reduced E solution (TFA + anisole sulfide + phenol + EDT + water).

[0015] Further, in step S4, pre-cooled cutting fluid is added at a ratio of 10-15 ml per gram of resin, and the mixture is placed on a shaker at room temperature for 2.5-4 hours. After the reaction is completed, the cutting fluid containing crude peptides is separated from the resin by filtration.

[0016] Furthermore, the reversed-phase high-performance liquid chromatography purification in step S5 includes the following steps: (1) Sample preparation: Dissolve the crude peptides in a strong solvent and then filter them through a filter membrane; (2) Chromatographic conditions: An Accucore™ C18 HPLC column was used. 1 L of mobile phase A (elution buffer A) was prepared as an aqueous solution containing 0.1% TFA, and mobile phase B was prepared as an acetonitrile solution containing 0.09% TFA. Linear gradient elution was used (gradually increasing from 100% eluent A to 100% eluent B; gradient rate 0.66% eluent B / min, gradient range and time: 0→100% eluent B, 90 min; flow rate: 7.5 ml / min) to separate peptide components of different polarities.

[0017] (3) Collection and identification: The eluent is monitored by an ultraviolet detector (214 nm wavelength), and the target components are collected manually or automatically according to the chromatographic peaks; (4) Post-processing: The eluents containing the target peptides are combined, most of the acetonitrile is removed by rotary evaporation under reduced pressure, and then freeze-dried to finally obtain high-purity peptide products.

[0018] Furthermore, the strong solvent is a ddH2O solution containing 0.1% TFA.

[0019] Specifically, the method for separating and purifying the enzyme hydrolysate of *Agrocybe aegerita* to obtain immunoactive peptides includes the following steps: (1) Preparation of dried protein powder from tea tree mushrooms; (2) Add pepsin to the dried protein powder of tea tree mushroom and adjust the pH to 1.5-2.5 for enzymatic hydrolysis; (3) Adjust the pH to 7.0-7.5, add trypsin, perform enzymatic hydrolysis, filter after enzyme inactivation, and then perform ultrafiltration to obtain the product.

[0020] According to some embodiments of the present invention, 4% by mass of pepsin is added in step (2).

[0021] Furthermore, in step (2), after adding pepsin, the pH is adjusted to 2 before enzymatic hydrolysis.

[0022] Furthermore, the enzymatic hydrolysis conditions in step (2) are 1-3 hours at 37°C; Furthermore, the enzymatic hydrolysis conditions in step (2) are 2 hours at 37°C.

[0023] Furthermore, in step (3), after adjusting the pH to 7, trypsin is added.

[0024] According to some embodiments of the present invention, in step (3), 4% by mass of pancreatic enzyme is added.

[0025] Furthermore, the enzymatic hydrolysis conditions in step (3) are 1-3 hours at 37°C; Furthermore, the enzymatic hydrolysis conditions in step (3) are 2 hours at 37°C.

[0026] According to some embodiments of the present invention, the enzyme inactivation condition is to inactivate the enzyme in a boiling water bath for 10 minutes.

[0027] Furthermore, in step (3), the filtration is performed using a 0.22 μm filter.

[0028] Furthermore, in step (3), ultrafiltration is performed using a 3kDa molecular weight ultrafiltration membrane.

[0029] Further, step (1) of preparing the dried protein powder of *Agrocybe aegerita* includes the following steps: 1) Dissolve the tea tree mushroom powder in water, stir magnetically at 20-25℃ for 3-4 hours, centrifuge, and collect the supernatant; 2) Add ammonium sulfate to the supernatant, let stand at 4°C for 15-20 hours, centrifuge, take the precipitate, and redissolve it in water; 3) Dialyze the product in a dialysis bag and dry it to obtain dried tea tree mushroom protein powder.

[0030] Furthermore, in step 1), the ratio of tea tree mushroom powder to water is 1:14-16, in g:mL.

[0031] Furthermore, in step 1), the ratio of tea tree mushroom powder to water is 1:15, in g:mL.

[0032] According to some embodiments of the present invention, after adding water, the mixture is stirred at room temperature for 3 hours and then centrifuged.

[0033] Specifically, the centrifugation conditions in step 1) are 4℃, 3000-5000g, and centrifugation for 10-20 minutes.

[0034] Furthermore, the centrifugation conditions in step 1) are 4°C, 4000g, and centrifugation for 15 minutes.

[0035] Specifically, in step 2), the amount of ammonium sulfate added is to achieve 70-80% saturation; Furthermore, in step 2), the amount of ammonium sulfate added is up to 75% saturation.

[0036] According to some embodiments of the present invention, after adding ammonium sulfate in step 2), the mixture is stirred for 0.5 hours and then left to stand at 4°C for 18 hours.

[0037] Specifically, the centrifugation conditions in step 2) are 4℃, 5000-7000g, and centrifugation for 10-20 minutes; Furthermore, the centrifugation conditions in step 2) are 4°C, 6000g, and centrifugation for 15 minutes.

[0038] Specifically, in step 3), dialysis is performed in a 7000D dialysis bag at 4°C for 24 hours.

[0039] Specifically, the drying in step 3) is freeze drying.

[0040] In another aspect, the present invention provides the application of the above-mentioned tea tree mushroom immunoactive peptides in the preparation of a drug for treating colitis.

[0041] Specifically, the colitis mentioned includes ulcerative colitis, infectious colitis, ischemic colitis, radiation colitis, and Crohn's disease.

[0042] Specifically, the tea tree mushroom immunomodulatory peptide is the active ingredient in the drug.

[0043] Specifically, the drug has the following effects: (1) Alleviate macrophage inflammatory damage; (2) Improves symptoms of colitis; (3) Repairing the damaged intestinal mucosal barrier; (4) Regulates intestinal immunity and relieves intestinal inflammation.

[0044] Furthermore, in (1), alleviating macrophage inflammatory damage specifically involves reducing IL-1. β IL-6, TNF-α a Levels, increase IL-10 levels.

[0045] Furthermore, (2) specifically improves colitis symptoms by: shortening colon length, reducing weight loss, and lowering DAI score.

[0046] Furthermore, (3) specifically repairs the damaged intestinal mucosal barrier by increasing the expression levels of Claudin1, Occludin, ZO-1 and Muc2 in the colon, promoting mucin secretion, and repairing the mechanical barrier and mucus barrier.

[0047] Furthermore, in (4), regulating intestinal immunity and alleviating intestinal inflammation specifically means reducing the infiltration of M1 type inflammatory macrophages and promoting the polarization of colonic macrophages towards the M2 phenotype.

[0048] Furthermore, the drug may also include a pharmaceutically acceptable carrier.

[0049] Furthermore, the pharmaceutically acceptable carriers include, but are not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, and fillers.

[0050] Specifically, the dosage form of the drug, depending on the route of administration, includes, but is not limited to, gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0051] Furthermore, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0052] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0053] The beneficial effects of this invention are as follows: The LALWP immunomodulatory peptide from *Agrocybe aegerita* identified in this invention possesses a novel structural sequence and is a previously unreported natural polypeptide. It can be obtained through artificial chemical synthesis or by separation and purification from *Agrocybe aegerita* protease hydrolysate. This invention demonstrates, through in vivo and in vitro experiments, that LALWP has immunomodulatory effects in regulating macrophage polarization and exhibits bioactivity in alleviating colitis, repairing intestinal mucosal barrier function, and regulating intestinal immunity, thereby alleviating colitis. Attached Figure Description

[0054] Figure 1 This is a flowchart of the extraction process for polypeptides from enzymatic hydrolysis of *Agrocybe aegerita*.

[0055] Figure 2 Flowchart for screening the immunomodulatory peptide Leu-Ala-Leu-Trp-Pro from *Agrocybe aegerita*.

[0056] Figure 3 This is a secondary mass spectrum of the immunomodulatory peptide Leu-Ala-Leu-Trp-Pro from *Agrocybe aegerita*.

[0057] Figure 4 To investigate the protective effect of Leu-Ala-Leu-Trp-Pro, an immunomodulatory peptide from Agrocybe aegerita, on DSS-induced colitis in mice.

[0058] Figure 5To investigate the effects of Leu-Ala-Leu-Trp-Pro, an immunomodulatory peptide from Agrocybe aegerita (tea tree mushroom), on the mechanical and mucous barrier functions of the intestinal mucosa in mice with DSS-induced colitis.

[0059] Figure 6 The effect of Leu-Ala-Leu-Trp-Pro, an immunomodulatory peptide from Agrocybe aegerita (tea tree mushroom), on intestinal mucosal immunity in mice with DSS-induced colitis. Detailed Implementation

[0060] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0061] Source of experimental materials: The tea tree mushrooms were purchased from Guangdong Yuewei Edible Fungus Technology Co., Ltd.

[0062] Example 1: Screening process for Leu-Ala-Leu-Trp-Pro, an immunomodulatory peptide from *Agrocybe aegerita* (tea tree mushroom). Preparation of dried protein powder from tea tree mushrooms: Protein was extracted from tea tree mushrooms using the 75% ammonium sulfate precipitation method.

[0063] The specific process is as follows: After drying, the tea tree mushrooms are pulverized into powder using a high-speed grinder. The powder is then dissolved in distilled water at a material-to-liquid ratio of 1:15 (g / mL). The mixture is stirred at room temperature for 3 hours, followed by centrifugation at 4°C and 4000g for 15 minutes. The supernatant is collected, and ammonium sulfate is added to 75% saturation. After stirring for 0.5 hours, the mixture is placed at 4°C and allowed to stand for 18 hours. Subsequently, it is centrifuged at 4°C and 6000g for 15 minutes, and the precipitate is collected and reconstituted with distilled water. The precipitate is then dialyzed in a 7000D dialysis bag at 4°C for 24 hours. After dialysis, it is freeze-dried to obtain the tea tree mushroom protein powder.

[0064] Preparation of *Agrocybe aegerita* enzymatic hydrolysate: A 10 mg / mL protein solution was prepared using *Agrocybe aegerita* protein powder. 4% pepsin was added, and the pH was adjusted to 2.0. Enzymatic hydrolysis was carried out at 37°C for 2 hours. The pH was then adjusted to 7.0, and 4% trypsin was added. Enzymatic hydrolysis was carried out at 37°C for 2 hours. Afterward, the enzyme was inactivated by boiling in a water bath for 10 minutes. After cooling to room temperature, the solution was filtered through a 0.22 μm filter, dispensed, and stored at -80°C. The extraction flow chart of enzymatically hydrolyzed peptides from *Agrocybe aegerita* is shown below. Figure 1 As shown.

[0065] Isolation and purification of bioactive peptides from *Agrocybe aegerita* and screening of immunomodulatory peptides: Ultrafiltration was performed using a 3 kDa ultrafiltration membrane. The peptide sequences in the <3 kDa fraction were identified by LC-MS / MS. Peptide activity was scored using the peptide ranker assay and cell penetration ability was scored using the Cpp pred assay. Peptides containing tyrosine, tryptophan, alanine, leucine, and arginine were given priority. Finally, the top three monomeric peptides were selected: Leu-Ala-Leu-Trp-Pro (LALWP, SEQ ID NO:1), Leu-Gln-Leu-Pro-Leu (LQLPL, SEQ ID NO:2), and Leu-Gln-Pro-Phe (LQPF, SEQ ID NO:3).

[0066] Example 2: Artificial solid-phase synthesis of Leu-Ala-Leu-Trp-Pro, Leu-Gln-Leu-Pro-Leu, and Leu-Gln-Pro-Phe Taking Leu-Ala-Leu-Trp-Pro as an example, the Fmoc-Pro-Wang-Resin resin is first subjected to swelling and deprotection treatment.

[0067] The specific conditions for swelling are as follows: Add resin to the reaction column, then inject sufficient N,N-dimethylformamide (DMF) at 15 ml / g to completely submerge the resin. Gently stir or shake at room temperature for 0.5 to 1 hour to allow the resin particles to fully expand, providing a larger contact area for subsequent reactions.

[0068] The deprotection treatment conditions are as follows: add 15 ml of 20% piperidine / DMF solution (15 ml / g) and treat for 10-30 minutes. This process is usually repeated twice to ensure complete removal.

[0069] The next amino acid, Fmoc-Trp-OH, was then added to the reactor for a condensation reaction. The condensation reaction conditions were as follows: using 3 molar amounts of Fmoc-Trp-OH and 3 molar amounts of condensing agent TBTU dissolved in DMF to activate the carboxyl group of the amino acid, which then reacted with the free amino group on the resin to form an amide bond.

[0070] After the condensation reaction is completed, the product is washed, and the steps of deprotection, feeding, reaction and washing are repeated until all amino acids are sequentially linked to obtain the Leu-Ala-Leu-Trp-Pro-wang-Resin peptide.

[0071] The product was then treated with a cutting fluid, specifically reduced solution E (trifluoroacetic acid: anisole: 1,2-ethylenedithiol: phenol: water = 87.5:5:2.5:2.5:2.5), added at a ratio of 10-15 mL per gram of resin to the pre-cooled cutting fluid (operation in a fume hood is recommended). The reaction was carried out at room temperature on a shaker for 2.5 to 4 hours. After the reaction, the cutting fluid containing the crude peptide was separated from the resin by vacuum filtration. The peptide was cleaved from the resin while simultaneously removing the side-chain protecting groups to obtain the crude peptide.

[0072] Finally, the target peptide was purified by reversed-phase high-performance liquid chromatography to obtain a purity higher than 95%.

[0073] Reversed-phase high-performance preparative liquid chromatography purification includes the following steps: (1) Sample preparation: Dissolve the crude polypeptide product in 10 ml of strong solvent (ddH2O containing 0.1% TFA). If necessary, sonication can be used to aid dissolution. Then filter the solution through a filter membrane. (2) Chromatographic conditions: An Accucore™ C18 HPLC column was used. 1 L of mobile phase A (elution buffer A) was prepared as an aqueous solution containing 0.1% TFA, and mobile phase B was prepared as an acetonitrile solution containing 0.09% TFA. Linear gradient elution was used (gradually increasing from 100% eluent A to 100% eluent B; gradient rate: 0.66% eluent B / min; gradient range and time: 0→100% eluent B, 90 min; flow rate: 7.5 ml / min) to separate peptide components of different polarities.

[0074] (3) Collection and identification: The eluent is monitored by a UV detector (214 nm wavelength), and the target components are collected manually or automatically according to the chromatographic peaks. Each component needs to be identified by mass spectrometry (MS) to confirm whether it is a peptide of the required molecular weight.

[0075] (4) Post-processing: The eluents containing the target peptides are combined, most of the acetonitrile is removed by rotary evaporation under reduced pressure, and then freeze-dried to finally obtain high-purity peptide products.

[0076] The artificial solid-phase synthesis methods for Leu-Gln-Leu-Pro-Leu and Leu-Gln-Pro-Phe are the same as those described above.

[0077] Example 3: Protective effect of artificially synthesized Leu-Ala-Leu-Trp-Pro, Leu-Gln-Leu-Pro-Leu, and Leu-Gln-Pro-Phe on inflammatory macrophages. Detection of the protective effect of peptide samples on LPS-stimulated BMDMs cell inflammatory injury model: BMDMs cells were processed at a ratio of 2×10 6Cells were seeded in 6-well plates and, after induction of maturation, three peptides (Leu-Ala-Leu-Trp-Pro, Leu-Gln-Leu-Pro-Leu, and Leu-Gln-Pro-Phe, 100 μg / mL) and LPS (100 ng / mL) were added. After culturing for 24 hours, cellular RNA was extracted for the detection of inflammatory factor mRNA levels. Each group was configured with three replicates. The qPCR results showed that, compared with Leu-Gln-Leu-Pro-Leu and Leu-Gln-Pro-Phe, Leu-Ala-Leu-Trp-Pro reduced IL-1 levels. β IL-6, TNF-α a The trend of increasing IL-10 levels was more pronounced, suggesting that Leu-Ala-Leu-Trp-Pro is the most immunomodulatory active peptide from *Agrocybe aegerita* (tea tree mushroom) and can alleviate macrophage inflammatory damage. Figure 2 The secondary mass spectrum of the immunomodulatory peptide Leu-Ala-Leu-Trp-Pro from *Agrocybe aegerita* is shown below. Figure 3 As shown.

[0078] Example 4: Leu-Ala-Leu-Trp-Pro (LALWP), an immunomodulatory peptide from *Agrocybe aegerita*, improves symptoms of DSS-induced colitis in mice. Six- to eight-week-old male C57BL / 6J mice (weighing 18-20g) were randomly divided into six groups of six mice each after seven days of acclimatization: control group, model group (DSS group), low-dose LALWP group (10 mg / kg / d), medium-dose LALWP group (20 mg / kg / d), high-dose LALWP group (30 mg / kg), and 5-aminosalicylic acid (5-ASA) group (200 mg / kg / d).

[0079] The blank control group was given free access to sterile water starting from the first day of modeling, and 200 μL of PBS was administered by gavage daily.

[0080] The model group mice were given free access to an aqueous solution containing 2.5% DSS for 6 days. On the 7th day, DSS was discontinued and replaced with normal sterile drinking water. At the same time, 200 μL of PBS was administered by gavage daily.

[0081] The LALWP and 5-ASA treatment groups were administered different doses of LALWP solution and 5-ASA solution (200 μL / day) by gavage on the first day after modeling, and were given drinking water containing 2.5% DSS for 6 days. DSS was discontinued on the 7th day.

[0082] Mice were monitored and their body weight changes, fecal characteristics, and fecal blood loss were recorded daily to calculate the Disease Activity Index (DAI). Specifically, mouse body weight was monitored daily: a decrease of 1-5% was scored as 1 point, 6-10% as 2 points, 11-15% as 3 points, and more than 15% as 4 points. Fecal characteristics were assessed: normal was 0 points, loose stools as 2 points, and watery diarrhea as 4 points. Fecal blood loss was examined: no bleeding was 0 points, occult blood was 1-2 points, and visible blood loss was 3-4 points. The DAI score was the average of the percentage of body weight loss, fecal characteristics score, and fecal blood loss score.

[0083] After modeling, mice were anesthetized with pentobarbital and euthanized. The abdominal cavity was dissected, and the colonic tissue from the cecum to 1 cm from the anus was separated. The attached mesentery was carefully dissected, photographed, and its length measured. One cm of distal colonic tissue was taken and fixed in paraformaldehyde solution for subsequent hematoxylin-eosin (H&E) staining and histopathological observation. After fixation, the tissue was dehydrated, cleared, impregnated with paraffin, and embedded to prepare paraffin sections. The sections were dewaxed with xylene and hydrated with a gradient of ethanol, followed by H&E staining: hematoxylin staining of the nuclei for 1 minute, followed by rinsing with running water; eosin staining of the cytoplasm for 20 seconds; then dehydrated with a gradient of ethanol, cleared with xylene, and finally mounted with neutral resin. The tissue morphology and structure were observed and analyzed under a microscope.

[0084] The results are as follows Figure 4 As shown, compared with the DSS group, LALWP gavage treatment significantly alleviated colitis symptoms in mice, specifically by improving colon length shortening, reducing weight loss, and lowering DAI scores. HE staining results showed that, compared with the DSS group, the LALWP-treated mice exhibited repaired colonic mucosal epithelial integrity, more regular crypt arrangement, and no obvious inflammatory cell infiltration. From the above mouse symptom assessment and HE results, it can be seen that high-dose LALWP can achieve or even surpass the therapeutic effect of 5-ASA on colitis, and the alleviating effect of high-dose LALWP on colitis is superior to that of medium and low doses, indicating that the alleviating effect of LALWP on colitis is dose-dependent.

[0085] Example 5: LALWP repairs intestinal mechanical and mucus barrier function in DSS-induced colitis mice. Intestinal barrier dysfunction is a core pathological feature of colitis (UC), primarily manifested as downregulated expression of intestinal epithelial tight junction proteins (such as ZO-1, Occludin, and the Claudin family) and reduced mucus layer thickness (Muc2 secretion deficiency). To further evaluate the mucosal barrier repair effect of LALWP in colitis-affected mice, we assessed the impact of LALWP on the mechanical barrier of the intestinal tract in colitis-affected mice by immunofluorescence detection of Claudin1, Occludin, and ZO-1 expression. We assessed chemical barrier function by immunofluorescence detection of Muc2 expression and alcine blue staining to detect mucus protein secretion.

[0086] Immunofluorescence staining: After dewaxing and hydration of paraffin sections, antigen retrieval was performed to expose the antigenic epitopes. The sections were placed in a retrieval chamber containing retrieval solution and treated using autoclaving for 8 minutes, followed by natural cooling. After drying the sections, a histochemical pen was used to draw a circle around the tissue, and 5% BSA blocking solution was added to completely cover the tissue. The sections were then blocked in a humidified chamber at 37°C for 1 hour. After blocking, the blocking solution was removed, and primary antibody was prepared at the concentration recommended in the antibody manufacturer's instructions. 10 μL of primary antibody was added to the histochemical circle to cover the tissue, and the sections were incubated overnight in a humidified chamber at 4°C. The next day, the sections were removed, warmed to room temperature, and washed three times with PBST on a shaker for 10 minutes each time. After washing, the sections were dried, and 10 μL of secondary antibody prepared according to the manufacturer's instructions was added. The sections were incubated in a humidified chamber at room temperature for 1 hour, and then washed three more times with PBST using the same method. Finally, the sections were dried, and 10 μL of mounting medium containing DAPI was added. The sections were incubated at room temperature in the dark for 20 minutes before mounting, and the sections were observed and images were acquired under an upright fluorescence microscope.

[0087] Alcian blue staining: Dissect mice and free the colon tissue, cut off a 1 cm segment of intestine (preserving intestinal contents to maintain the integrity of the mucus layer), and immediately fix it overnight in Carnoy's fixative (methanol:chloroform:glacial acetic acid = 6:3:1). Subsequently, the tissue was dehydrated sequentially by 100% methanol I, 100% methanol II, 100% ethanol I, and 100% ethanol II, 30 minutes each; then cleared in xylene I and xylene II for 30 minutes each. Afterward, the tissue was immersed in paraffin I and paraffin II for 1 hour each, followed by paraffin embedding, sectioning, dewaxing, and hydration according to standard methods. For staining, delineate the tissue area with a histochemical pen, add Alcian acidification solution for staining for 3 minutes, gently shake off the stain, add Alcian staining solution for staining for 30 minutes, rinse with running water; shake dry, add nuclear solid red staining solution to counterstain cell nuclei for 5 minutes, and rinse again with running water. The sections were then dehydrated sequentially with 80%, 90%, 100% I, and 100% II alcohols, 3 minutes each, followed by xylene clearing for 30 minutes. Finally, the sections were mounted and observed under a microscope.

[0088] The results are as follows Figure 5The results showed that, compared with the DSS group, LALWP significantly increased the expression levels of Claudin1, Occludin, ZO-1, and Muc2 in the colon of colitis mice. Alixin blue staining also indicated that LALWP could promote mucin secretion and repair the mucus barrier in colitis mice. These results demonstrate that LALWP can repair the intestinal mechanical and mucus barriers in colitis mice.

[0089] Example 6: LALWP modulates intestinal immune function in DSS-induced colitis mice Intestinal mucosal immune dysfunction is a crucial aspect of the pathogenesis of ulcerative colitis (UC). In UC patients, the intestinal epithelial barrier is impaired due to various factors. Continuous stimulation by intestinal antigens prompts macrophages and dendritic cells to release large amounts of cytokines, further damaging the intestinal mucosal immune barrier and exacerbating the inflammatory response. As the most numerous immune cells in the lamina propria, macrophages are considered key regulators of intestinal immune homeostasis. Macrophages are highly plastic and can be activated in response to signals from the local microenvironment, cytokines, and metabolites, differentiating into different functional phenotypes, mainly including the pro-inflammatory M1 type and the anti-inflammatory M2 type. M1 macrophages induce intestinal barrier dysfunction and promote inflammation progression; while M2 macrophages have been shown to alleviate intestinal inflammation and promote tissue repair. With the deepening research into the disease mechanism of UC, regulating macrophage polarization towards the M2 type will undoubtedly become an important target for clinical and experimental UC treatment strategies.

[0090] As an immunomodulatory peptide that regulates macrophage activity, we further investigated the regulatory effect of LALWP on macrophage polarization phenotype in colitis mice in vivo.

[0091] from Figure 6 Immunofluorescence staining results of colon tissue A in the DSS group showed that, compared with the Control group, the number of F4 / 80+CD86+ macrophages in the colon tissue of the DSS group was significantly increased, while the number of F4 / 80+CD86+ macrophages was significantly reduced after LALWP treatment. This indicates that in colitis mice, the infiltration of M1 type inflammatory macrophages is increased, while LALWP can significantly reduce the infiltration of M1 type inflammatory macrophages. Figure 6 As shown in Figure B, the number of F4 / 80+CD206+ macrophages significantly increased after LALWP treatment, indicating that LALWP can promote the polarization of colonic macrophages in colitis mice towards the M2 phenotype. This suggests that LALWP can induce the polarization of colonic M1 inflammatory macrophages in DSS colitis mice towards the M2 pro-repair phenotype, thereby regulating intestinal immunity, repairing the immune barrier, and alleviating intestinal inflammation.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kind of immunomodulatory peptide from *Agrocybe aegerita*, characterized in that, The amino acid sequence of the *Agrocybe aegerita* immunomodulatory peptide is Leu-Ala-Leu-Trp-Pro.

2. The method for preparing the immunomodulatory peptides from *Agrocybe aegerita* according to claim 1, characterized in that, The preparation method includes artificial chemical synthesis and separation and purification of the tea tree mushroom protease hydrolysate.

3. The preparation method according to claim 2, characterized in that, The method for artificially synthesizing immunoactive peptides from *Agrocybe aegerita* includes the following steps: S1. The Fmoc-Pro-Wang-Resin resin is subjected to swelling and deprotection treatment; S2. Add the next amino acid Fmoc-Trp-OH to the reactor to carry out the condensation reaction, and wash after the reaction is completed. S3. Repeat the deprotection in step S1 and step S2 until all amino acids are sequentially linked to obtain Leu-Ala-Leu-Trp-Pro-Wang-Resin resin. S4. Use cutting fluid to treat and obtain crude polypeptide; S5. High-purity peptides were obtained by reversed-phase high-performance preparative liquid chromatography.

4. The preparation method according to claim 2, characterized in that, The method for separating and purifying the enzyme hydrolysate of *Agrocybe aegerita* to obtain immunoactive peptides includes the following steps: (1) Preparation of dried protein powder from tea tree mushrooms; (2) Add pepsin to the dried protein powder of tea tree mushroom, adjust the pH to 1.5-2.5, and carry out enzymatic hydrolysis; (3) Adjust the pH to 7.0-7.5, add trypsin, perform enzymatic hydrolysis, filter after enzyme inactivation, and then perform ultrafiltration to obtain the product.

5. The preparation method according to claim 4, characterized in that, Step (1) of preparing tea tree mushroom protein powder includes the following steps: 1) Dissolve the tea tree mushroom powder in water, stir magnetically at 20-25℃ for 3-4 hours, centrifuge, and collect the supernatant; 2) Add ammonium sulfate to the supernatant, let stand at 4°C for 15-20 hours, centrifuge, take the precipitate, and redissolve it in water; 3) Dialyze the product in a dialysis bag and dry it to obtain dried tea tree mushroom protein powder.

6. The use of the tea tree mushroom immunoactive peptide according to claim 1 in the preparation of a medicament for treating colitis.

7. The application according to claim 6, characterized in that, The colitis mentioned includes ulcerative colitis, infectious colitis, ischemic colitis, radiation colitis, and Crohn's disease.

8. The application according to claim 6, characterized in that, The aforementioned drug contains tea tree mushroom immunomodulatory peptides as its active ingredient.

9. The application according to claim 6, characterized in that, The drug has the following effects: (1) Alleviate macrophage inflammatory damage; (2) Improves symptoms of colitis; (3) Repairing the damaged intestinal mucosal barrier; (4) Regulates intestinal immunity and relieves intestinal inflammation.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the tea tree mushroom immunomodulatory peptide as described in claim 1.

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