Milk-derived polypeptide and application thereof in preparation of drugs, health-care products or food additives for treating necrotizing enterocolitis

Abstract

The invention discloses a milk-derived polypeptide and application thereof in preparation of drugs, health care products or food additives for treating necrotizing enterocolitis, and belongs to the technical field of biological medicines. The milk-derived polypeptide disclosed by the invention has an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence which has more than 80% of homology with the amino acid sequence as shown in SEQ ID NO.1. The milk-derived polypeptide disclosed by the invention can promote the recovery of intestinal villi in neonatal necrotizing enterocolitis, relieve intestinal barrier injury, reduce the expression level of intestinal inflammatory factors and regulate the immune function of a body. Therefore, the milk-derived polypeptide disclosed by the invention can be used for preparing medicines, health-care products or food additives for treating necrotizing enterocolitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a milk-derived polypeptide and its application in preparing a medicine, health product or food additive for treating necrotizing enterocolitis. Background Art

[0002] Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in newborns, especially premature infants, and is an important cause of intestinal perforation and systemic inflammatory response syndrome. NEC has a long course and poor prognosis. Surviving children are often accompanied by multiple sequelae such as short bowel syndrome, intestinal stenosis, and long-term neurodevelopmental disorders, which not only seriously affect the quality of life of children, but also bring a heavy burden to families and society. There is still a lack of effective prevention and treatment measures for NEC. Currently, clinical treatment mainly relies on broad-spectrum antibiotics, supportive care, and late surgery. There is no specific treatment for NEC. Therefore, seeking effective means of preventing and treating NEC is a hot topic and difficulty in current research.

[0003] Breast milk is a recognized natural peptide reservoir, rich in peptides, a unique class of bioactive substances with anti-infection, antioxidant, immune, neuromodulatory, hormonal, and metabolic regulatory properties. Furthermore, peptides have low molecular weight, high specific activity, low accumulation in the body, and ease of synthesis and modification. As promising molecular building blocks for pharmaceuticals, they have become a hot topic in recent years for new drug research and development.

[0004] Breast milk-derived peptides have significant effects in the prevention and treatment of NEC. For example, β-defensin-3 in breast milk can induce the migration of intestinal epithelial cells and has a certain effect on the repair of intestinal damage in newborn NEC rats when administered via a gastric tube. Intestinal trefoil factor 3 (TFF3) is a small molecule peptide present in breast milk that can downregulate intestinal inflammatory responses, reduce intestinal mucosal damage mediated by multiple inflammatory factors, and play a role in post-intestinal repair. Recombinant human TFF3 can alleviate intestinal inflammatory responses in rats with NEC and protect intestinal mucosal damage. These studies all indicate that peptides have promising therapeutic application prospects in neonatal intestinal injury and repair. Summary of the Invention

[0005] Purpose of the Invention: The present invention aims to address the shortcomings of existing technologies and provide a milk-derived polypeptide and its use in the preparation of a drug, health product, or food additive for treating necrotizing enterocolitis. The milk-derived polypeptide of the present invention can promote the recovery of intestinal villi in neonatal necrotizing enterocolitis, alleviate intestinal barrier damage, reduce the expression of intestinal inflammatory factors, and regulate the body's immune function. Therefore, the milk-derived polypeptide of the present invention can be used in the preparation of a drug, health product, or food additive for treating necrotizing enterocolitis.

[0006] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0007] The present invention provides a milk-derived polypeptide having an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having more than 80% homology with the amino acid sequence as shown in SEQ ID NO.1.

[0008] The milk-derived polypeptide of the present invention is a peptide chain consisting of 22 amino acid residues.

[0009] SEQ ID NO.1:

[0010] Lys-Asn-Pro-Thr-His-Gln-Ile-Tyr-Pro-Val-Thr-Gln-Pro-Leu-Ala-Pro-Val-His-Asn-Pro-Ile-Ser (LNPTHQIYPVTQPLAPVHNPIS). This polypeptide is derived from β-casein (lactoferrin precursor), amino acids 44 to 65, and can be prepared by chemical synthesis methods well known to those skilled in the art, such as liquid phase synthesis / solid phase synthesis of small molecule polypeptides.

[0011] Preferably, the milk-derived polypeptide has an amino acid sequence that is no less than 88% homologous to the amino acid sequence shown in SEQ ID NO.1.

[0012] In the present invention, sequence homology can refer to any definition of sequence homology known in the art. Sequence homology can be understood as homology sequences determined by BLAST provided by the National Center for Biotechnology Information (NCBI) before the filing date of this invention.

[0013] Those skilled in the art may make necessary modifications to the milk-derived polypeptide, including but not limited to protection / deprotection of specific groups, cyclization, N-methylation, phosphorylation, glycosylation, or PEGylation at one or more sites within the C-terminus, N-terminus, or intermediate residues. These modifications may be selected by those skilled in the art based on the desired hydrolysis rate, solubility, immune response, signal transduction, and other properties of the polypeptide, all of which are within the scope of the present invention.

[0014] Preferably, the milk-derived polypeptide is derived from β-casein.

[0015] The present invention also provides a milk-derived polypeptide derivative, which is a chimeric peptide formed by connecting a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 with a cell-penetrating peptide.

[0016] Preferably, the cell-penetrating peptide is one of TAT, Penetratin, Polyarginine, P22N, DPV3, and DPV6. Based on comprehensive considerations of cell-penetrating efficiency and biocompatibility, the present invention selects 8 or more arginine residues to ensure that oligoarginine exhibits good performance during cell-penetrating while minimizing potential cytotoxicity.

[0017] The present invention also provides the use of the milk-derived polypeptide in preparing a therapeutic drug for necrotizing enterocolitis.

[0018] The drug can effectively promote the recovery of intestinal villi in neonatal necrotizing enterocolitis, alleviate intestinal barrier damage, reduce the expression level of intestinal inflammatory factors and regulate the immune function of the intestine.

[0019] The present invention also provides the use of the milk-derived polypeptide in preparing health products or food additives.

[0020] The present invention also provides a polypeptide composition comprising the above-mentioned milk-derived polypeptide and a pharmaceutically acceptable carrier or excipient, or comprising the above-mentioned milk-derived polypeptide derivative and a pharmaceutically acceptable carrier or excipient.

[0021] The active ingredient of the polypeptide composition of the present invention contains the milk-derived polypeptide or milk-derived polypeptide derivative described above.

[0022] The carrier includes one or more of viruses, liposomes, and nanoparticles, and the carrier is selectively adjusted based on requirements such as intestinal absorption, mucus layer penetration, pH adjustment, enzyme activity adjustment, etc. The excipient includes one or more of mannitol, lactose, fatty acids, and polyethylene glycol.

[0023] The present invention also provides the use of the polypeptide composition in preparing a therapeutic drug for necrotizing enterocolitis.

[0024] The drug can promote the recovery of intestinal villi in neonatal necrotizing enterocolitis, alleviate intestinal barrier damage, reduce the expression level of intestinal inflammatory factors and regulate the body's immune function.

[0025] Beneficial effects:

[0026] The milk-derived polypeptides described in this invention can effectively promote the recovery of the intestinal villi, ensuring nutrient absorption; alleviate intestinal barrier damage and restore the intestinal environment; significantly reduce the expression of intestinal inflammatory factors, inhibiting inflammatory responses; and effectively regulate intestinal immune function and enhance the immune barrier. Therefore, the milk-derived polypeptides described in this invention can be used to prevent and treat NEC in neonates and to prepare therapeutic drugs, health products, or food additives for necrotizing enterocolitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The changes in body weight and survival rate in the mouse NEC in vivo model under the intervention of the polypeptide in Example 1;

[0028] Figure 2 This is an H&E staining image of intestinal tissue under the intervention of the polypeptide in the in vivo mouse NEC model in Example 2;

[0029] Figure 3 This is an image of immunohistochemical staining of tissue barrier proteins under the intervention of polypeptides in the in vivo mouse NEC model in Example 3;

[0030] Figure 4 This is a schematic diagram of the ELISA detection indicators of intestinal tissue inflammatory factors under the intervention of polypeptides in the mouse NEC in vivo model in Example 4;

[0031] Figure 5 This is a flow cytometric analysis of intestinal tissue immune cells under polypeptide intervention in the in vivo mouse NEC model of Example 5. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0033] The amino acid sequence of the milk-derived polypeptide in the following examples of the present invention is SEQ ID NO.1:

[0034] LNPTHQIYPVTQPLAPVHNPIS,

[0035] Synthesized by Shanghai Ketide Biotechnology Co., Ltd. with a purity of >95%.

[0036] Example 1 Effect of milk-derived polypeptides on body weight and survival rate of NEC model mice

[0037] When inducing the NEC model in mice, the mice were fed infant formula daily, ensuring daily hypoxia and hypothermia to mimic the clinical manifestations of apnea and bradycardia seen in neonatal NEC patients. Four days after modeling, patchy intestinal edema, inflammation, and necrosis developed, closely resembling the manifestations of human NEC.

[0038] Experimental animals: C57BL / 6J mice, 4-5 g, 5-6 days old, were obtained from the Experimental Animal Center of Nanjing Medical University.

[0039] Experimental methods:

[0040] C57BL / 6J mice weighing 4-5 g within 5-6 days after birth were randomly divided into three groups: Ctrl group, NEC group, and NEC+β-casein 65 group, with 18 mice in each group. The experiment was repeated 3 times.

[0041] NEC group: All mice were subjected to hypoxic asphyxiation (5% O2, 95% N2, 5 min) every 6 hours. Within 2 min after the end of hypoxia, they were gavaged with hypertonic milk (Wyeth stage 1 milk powder and Abbott newborn puppy milk powder in a ratio of 2:1, 50 μL / mg). At the same time, they were cold stimulated in a 4°C incubator for 10 min in the morning and evening.

[0042] Ctrl group: C57BL / 6J mice were placed in the same cage with their mothers and fed with breast milk.

[0043] NEC+β-casein 65 group: Based on the NEC model, milk-derived peptides (10 mg / kg) were added to the hypertonic milk administered orally daily.

[0044] The entire experiment lasted for 4 days, during which the three groups of mice were weighed daily and the survival status of the animals in each group was recorded.

[0045] Data were analyzed using GraphPad Pris9 and expressed as mean ± standard deviation (SD). Student's t-test was used to compare and calculate differences, and significant differences were considered when P < 0.05. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0046] Depend on Figure 1 As can be seen, using newborn mice as research subjects, the NEC animal model was established through hypoxia (5%), cold stimulation (4°C), and oral administration of hypertonic milk. Compared with the Ctrl group, the weight gain trend of newborn mice in the NEC group was significantly inhibited. Intervention with milk-derived peptides can partially alleviate the weight loss trend of newborn mice. Furthermore, compared with the NEC group, the milk-derived peptide LNPTHQIYPVTQPLAPVHNPIS also significantly improved the survival rate of newborn mice in the NEC group.

[0047] Example 2 Effects of milk-derived polypeptides on intestinal villi in NEC model mice

[0048] Experimental animals: C57BL / 6J mice, 3-4 g, 5-6 days old, were obtained from the Experimental Animal Center of Nanjing Medical University.

[0049] Experimental methods:

[0050] C57BL / 6J mice weighing 3-4 g within 5-6 days after birth were randomly divided into three groups: Ctrl group, NEC group, and NEC+β-casein 65 group, with 18 mice in each group. The experiment was repeated 3 times.

[0051] NEC group: All newborn mice were subjected to hypoxic asphyxiation (5% O2, 95% N2, 5 min) every 6-8 hours. Within 2 min after the end of hypoxia, they were gavaged with hypertonic milk (Wyeth stage 1 milk powder and Abbott newborn puppy milk powder in a ratio of 2:1, 50 μL / mg). At the same time, they were cold stimulated in a 4°C incubator for 10 min in the morning and evening.

[0052] Ctrl group: Newborn mice were placed in the same cage with their mothers and fed with breast milk.

[0053] NEC+β-casein 65 group: Based on the NEC model, the milk-derived polypeptide LNPTHQIYPVTQPLAPVHNPIS (10 mg / kg) was added to the hypertonic milk administered orally every day.

[0054] The entire experiment lasted for 4 days. Finally, the mice were uniformly killed by spinal dislocation. The gastrointestinal tissue was dissected gently and the proximal intestinal tissue 2 cm away from the terminal ileum was taken as a sample.

[0055] Prepare the sample for H&E sectioning:

[0056] a. Fix tissue: The samples collected after modeling were fixed in 4% paraformaldehyde (room temperature overnight);

[0057] b. Preparation of paraffin blocks: After dehydration using gradient ethanol, continue treatment in 50% and 100% xylene, then dip into wax, embed, and wait for the paraffin blocks to completely air dry;

[0058] c. Sectioning: After the paraffin blocks were completely air-dried, 5 μm thick slices were cut parallel to the intestinal cross section. The slices were spread in a 42°C water bath and dried in a fume hood for 24 hours. After treatment with xylene, the slices were treated with a gradient of ethanol (100% → 95% → 80% → 75%) and then washed with distilled water for 2 minutes.

[0059] H&E staining: The sections were stained with hematoxylin for 5 minutes and then rinsed with distilled water. The sections were then incubated with 1% hydrochloric acid ethanol for 1 minute, washed with distilled water for 2 minutes, and soaked in saturated lithium carbonate for 1 minute to return to blue. The sections were then treated with 0.5% eosin solution for 5 minutes and rinsed with distilled water. The sections were then treated with an ethanol gradient (95% → 95% → 100% ethanol) and finally mounted with neutral gum. [Image of H&E staining of intestinal tissue after peptide intervention in the mouse NEC model in vivo] Figure 2 .

[0060] Depend on Figure 2As can be seen, using newborn mice as research subjects, an NEC animal model was established through hypoxia (5%), cold stimulation (4°C), and hypertonic milk gavage. Compared with the Ctrl group, the intestinal tissue of the newborn mice in the NEC group showed obvious severe damage to the intestinal tissue structure, with hemorrhage, edema, and even rupture of the mucosal layer, submucosa, and lamina propria, and poor villus development and partial loss. Under the intervention of the milk-derived polypeptide LNPTHQIYPVTQPLAPVHNPIS, the intestinal tissue structure was significantly improved, with reduced hemorrhage and edema in the mucosal layer, submucosa, and lamina propria, but there was still moderate to severe separation of the mucosal layer and submucosa, and the villi were well developed, with a few villi shedding visible locally. Therefore, the above-mentioned milk-derived polypeptide can partially alleviate the pathological damage to the intestinal tissue of newborn mice and promote the recovery of intestinal villus tissue to a certain extent.

[0061] Example 3 Effects of milk-derived polypeptides on intestinal barrier proteins in NEC model mice

[0062] C57BL / 6J mice weighing 4-5 g within 5-6 days after birth were randomly divided into three groups: Ctrl group, NEC group, and NEC+β-casein 65 group, with 18 mice in each group. The experiment was repeated 3 times.

[0063] NEC group: All newborn mice were subjected to hypoxic asphyxiation (5% O2, 95% N2, 5 min) every 6-8 hours. Within 2 min after the end of hypoxia, they were gavaged with hypertonic milk (Wyeth stage 1 milk powder and Abbott newborn puppy milk powder in a ratio of 2:1, 50 μL / mg). At the same time, they were cold stimulated in a 4°C incubator for 10 min in the morning and evening.

[0064] Ctrl group: Newborn mice were placed in the same cage with their mothers and fed with breast milk.

[0065] NEC+β-casein 65 group: Based on the NEC model, the milk-derived polypeptide LNPTHQIYPVTQPLAPVHNPIS (10 mg / kg) was added to the hypertonic milk administered orally every day.

[0066] The entire experiment lasted for 4 days. Finally, the mice were killed by spinal dislocation. The gastrointestinal tissue was dissected gently and the proximal intestinal tissue 2 cm away from the terminal ileum was taken as a sample.

[0067] The intestinal tissue was further subjected to immunohistochemistry for the barrier protein Occludin:

[0068] a fixed tissue, wax block production, slice production specific steps with Example 2 H & E slice production process;

[0069] b. Hydrate the sections in a gradient of xylene and ethanol, then soak in sodium citrate antigen retrieval solution (sub-boiling temperature), then wash with PBS. Soak in 3% hydrogen peroxide (incubate in the dark for 15 minutes), then wash with PBS.

[0070] c. Incubate sections with primary antibody (Anti-Occludin, Abcam) overnight at 4°C. Wash with PBS. Continue incubation with HRP-conjugated secondary antibody (goat anti-rabbit IgG, HRP conjugate).

[0071] d. Add DAB colorimetric reagent to the labeled tissue and wash in running water. Continue staining with hematoxylin solution for 3 minutes, wash again in running water, and treat with separation solution to return the blue color.

[0072] e. Dehydrate in graded ethanol, clarify in xylene, and fix with neutral resin.

[0073] The immunohistochemical staining images of tissue barrier proteins under peptide intervention in the mouse NEC model in vivo are shown in Figure 3 .

[0074] Depend on Figure 3 As shown, the NEC animal model was established in neonatal mice by hypoxia (5%), cold stimulation (4°C), and oral administration of hypertonic milk. Compared with the control group, the expression of the barrier protein occludin in the intestinal tissue of the neonatal NEC mice was significantly reduced. However, the expression of the barrier protein occludin in the intestinal tissue of the newborn mice in the NEC group was significantly increased after intervention with the milk-derived peptide LNPTHQIYPVTQPLAPVHNPIS. Immunohistochemistry results for the barrier protein occludin showed that peptide intervention increased the expression of the barrier protein in the intestinal tissue of NEC mice compared with the NEC group.

[0075] Example 4 Effects of Milk-derived Peptides on Intestinal Inflammatory Factors in NEC Model Mice

[0076] C57BL / 6J mice weighing 4-5 g within 5-6 days after birth were randomly divided into three groups: Ctrl group, NEC group, and NEC+β-casein 65 group, with 5 mice in each group. The experiment was repeated 3 times.

[0077] NEC group: All newborn mice were subjected to hypoxic asphyxiation (5% O2, 95% N2, 5 min) every 6-8 hours. Within 2 min after the end of hypoxia, they were gavaged with hypertonic milk (Wyeth stage 1 milk powder and Abbott newborn puppy milk powder in a ratio of 2:1, 50 μL / mg). At the same time, they were cold stimulated in a 4°C incubator for 10 min in the morning and evening.

[0078] Ctrl group: Newborn mice were placed in the same cage with their mothers and fed with breast milk.

[0079] NEC+β-casein 65 group: Based on the NEC model, the milk-derived polypeptide LNPTHQIYPVTQPLAPVHNPIS (10 mg / kg) was added to the hypertonic milk administered orally every day.

[0080] The entire experiment lasted for 4 days. Finally, the mice were uniformly killed by spinal dislocation. The gastrointestinal tissue was dissected gently. The proximal intestinal tissue 2 cm from the terminal ileum was taken as a sample, added with 0.5 ml of pre-cooled PBS (containing 1% BSA, pH 7.2), and the homogenate was centrifuged. The supernatant was collected and diluted with PBS.

[0081] Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of TNF-α and IL-6 in the intestinal tissue supernatant:

[0082] a. Reagent Preparation

[0083] 1) Mouse TNF-α Standard: Reconstitute in distilled water to a concentration of 4000 pg / mL (stock solution). Initially dilute to 2000 pg / mL (highest concentration on the standard curve), then serially dilute with Standard Diluent in 2-fold increments. The standard concentrations after dilution are: 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL.

[0084] 2) Mouse IL-6 Standard: Reconstitute in distilled water to a concentration of 1000 pg / mL (stock solution). Initially dilute to 500 pg / mL (highest concentration on the standard curve), then serially dilute with Standard Diluent in 2-fold increments. The standard concentrations after dilution are: 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.63 pg / mL, and 7.81 pg / mL.

[0085] 3) 1x Assay Buffer: Dilute the 10x concentrated assay buffer to 1x assay buffer.

[0086] 4) Antibody working solution: Dilute the concentrated detection antibody 1:100 with 1x detection buffer.

[0087] 5) Horseradish peroxidase working solution (horseradish peroxidase-labeled streptavidin): Dilute concentrated horseradish peroxidase-labeled streptavidin 1:100 with 1x detection buffer.

[0088] 6) Washing solution: dilute the 20x concentrated washing solution to 1x washing solution.

[0089] b. Operation steps

[0090] Add 300 μL of 1x washing solution and let it soak for 30 seconds, discard the washing solution, and pat dry on absorbent paper; add 100 μL of 2-fold diluted standard to the duplicate wells, place 100 μL of sample in the sample well, and do not add sample to the blank control well; add 50 μL of diluted antibody working solution to each well; apply sealing film, shake at 300 rpm, and incubate at room temperature for 1.5 hours; remove the liquid in the well, wash each well 6 times with 300 μL of washing solution, shake and discard the washing solution; add 100 μL of diluted horseradish peroxidase working solution to each well; seal the plate with film Seal the plate, shake at 300 r / min, and incubate at room temperature for 45 minutes; wash the plate, wash each well 6 times with 300 μL of washing solution, and discard the washing solution; add 100 μL of colorimetric solution TMB to each well and incubate at room temperature in the dark for 20 minutes; quickly add stop solution to each well to terminate the reaction; read the plate using a microplate reader, measure the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm, and the calibrated OD value is the measured value at 450 nm minus the measured value at 570 nm. Analyze the cytokine content based on the standard curve.

[0091] c. Calculation

[0092] The concentration of each sample was calculated using the calibrated OD value as the horizontal axis and the standard concentration as the vertical axis. Data were analyzed using GraphPad Prism 9 and expressed as mean ± standard deviation (SD). Student's t-test was used to compare and calculate differences. P < 0.05 was considered significant. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0093] Depend on Figure 4 Compared with the Ctrl group, the expression of inflammatory factors TNF-α and IL-6 in the ileum of neonatal mice in the NEC group was significantly increased. However, intervention with the milk-derived peptide LNPTHQIYPVTQPLAPVHNPIS significantly reduced the expression of these inflammatory factors. ELISA results for intestinal tissue inflammatory factors showed that peptide intervention significantly reduced the secretion of inflammatory factors in the intestinal tissue of NEC mice compared with the NEC group.

[0094] Example 5 Effects of Milk-derived Peptides on Intestinal Immune Cells in NEC Model Mice

[0095] C57BL / 6J mice weighing 4-5 g within 5-6 days after birth were randomly divided into three groups: Ctrl group, NEC group, and NEC+β-casein 65 group, with 5 mice in each group. The experiment was repeated 3 times.

[0096] NEC group: All newborn mice were subjected to hypoxic asphyxiation (5% O2, 95% N2, 5 min) every 6-8 hours. Within 2 min after the end of hypoxia, they were gavaged with hypertonic milk (Wyeth stage 1 milk powder and Abbott newborn puppy milk powder in a ratio of 2:1, 50 μL / mg). At the same time, they were cold stimulated in a 4°C incubator for 10 min in the morning and evening.

[0097] Ctrl group: Newborn mice were placed in the same cage with their mothers and fed with breast milk.

[0098] NEC+β-casein 65 group: Based on the NEC model, the milk-derived polypeptide LNPTHQIYPVTQPLAPVHNPIS (10 mg / kg) was added to the hypertonic milk administered orally every day.

[0099] The entire experiment lasted for 4 days. Finally, the mice were killed by spinal dislocation. The gastrointestinal tissue was dissected gently and the whole intestinal tissue was taken as a sample.

[0100] Isolation of intestinal innate immune cells and detection of intestinal tissue immune cells using flow cytometry:

[0101] Place the small intestine in an ice bath of 1×PBS solution, remove excess fat tissue, use a syringe to draw up 1×PBS solution to flush and remove the intestinal contents, cut the intestine longitudinally and rinse, cut the small intestine into a centrifuge tube containing 1×HBSS solution with 3% FBS and shake, prepare the corresponding solution for the colon, remove the intestine with tweezers and centrifuge, treat the cells with 40% and 80% Percoll separation solution and centrifuge again, and finally aspirate the middle layer cells into a container containing PBS solution.

[0102] The volume required for loading is calculated based on the number of cells counted in each tube of sample, usually 1×10 6 Prepare the sample by transferring the loaded cells to a 1.5ml Eppendorf tube, filling to 1ml, and centrifuging at 5000 rpm for 2 minutes. Remove the supernatant, add premixed antibody labeling buffer (1× PBS, 30 μl / sample), and label at 4°C in the dark for 15 minutes. Add 500 μl of 1× PBS to each sample tube and centrifuge at 5000 rpm for 2 minutes to elute the unlabeled antibody. Remove the supernatant, resuspend each sample in 300 μl of 1× PBS, filter through a 200-mesh nylon mesh into a new 1.5ml Eppendorf tube, and load onto the flow cytometer for analysis or store at 4°C in the dark until analysis. All flow cytometric data were collected on a BD FACSVerse flow cytometer and analyzed using FlowJo v10.0.7 software.

[0103] Depend on Figure 5As can be seen, flow cytometry was used to analyze intestinal immune cells, with CD45 marking neutrophils, F4 / 80 marking total macrophages, CD11b marking M1 macrophages, and CD206 marking M2 macrophages. The experiment found that the number of macrophages in the NEC group and the milk-derived polypeptide LNPTHQIYPVTQPLAPVHNPIS group increased significantly. After the intervention of the polypeptide LNPTHQIYPVTQPLAPVHNPIS, the total number of macrophages increased compared with the NEC group ( Figure 5 A). After the intervention of peptide LNPTHQIYPVTQPLAPVHNPIS, the pro-inflammatory M1 macrophages were significantly reduced compared with the NEC group, while the anti-inflammatory M2 macrophages were significantly increased compared with the NEC group ( Figure 5 B. Figure 5 C). The results of intestinal tissue flow cytometry showed that peptide intervention could regulate intestinal tissue innate immune cells, reduce pro-inflammatory M1 macrophages, and increase anti-inflammatory M2 macrophages.

[0104] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A milk-derived polypeptide, characterized in that: The milk-derived polypeptide has an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having more than 80% homology with the amino acid sequence as shown in SEQ ID NO.

1.

2. The milk-derived polypeptide according to claim 1, characterized in that The milk-derived polypeptide is modified by cyclization, N-methylation, phosphorylation, glycosylation or PEG at one or more sites of its C-terminus, N-terminus and intermediate residues.

3. The milk-derived polypeptide according to claim 1, characterized in that The milk-derived polypeptide is derived from β-casein.

4. A milk-derived polypeptide derivative, characterized in that: The milk-derived polypeptide derivative is a chimeric peptide formed by connecting a polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 with a cell-penetrating peptide.

5. The milk-derived polypeptide derivative according to claim 4, characterized in that: The cell-penetrating peptide is one of TAT, Penetratin, Polyarginine, P22N, DPV3, and DPV6.

6. Use of the milk-derived polypeptide according to any one of claims 1 to 3 in the preparation of a therapeutic drug for necrotizing enterocolitis.

7. The use according to claim 6, characterized in that The drug promotes the recovery of intestinal villi in neonatal necrotizing enterocolitis, alleviates intestinal barrier damage, reduces the expression level of intestinal inflammatory factors and regulates the immune function of the intestine.

8. Use of the milk-derived polypeptide according to any one of claims 1 to 3 in the preparation of health products or food additives.

9. A polypeptide composition, characterized in that The invention comprises the milk-derived polypeptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient, or comprises the milk-derived polypeptide derivative according to claim 4 or 5 and a pharmaceutically acceptable carrier or excipient.

10. Use of the polypeptide composition according to claim 9 in the preparation of a therapeutic drug for necrotizing enterocolitis.

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