Application of a breast milk-derived polypeptide in the preparation of bronchopulmonary dysplasia prevention and treatment drugs, health products and food additives

Abstract

The invention discloses an application of a human milk-derived polypeptide in preparing a bronchopulmonary dysplasia prevention and treatment drug, a health product and a food additive, belongs to the field of biomedicine, and provides a new use of the human milk-derived polypeptide in preparing a bronchopulmonary dysplasia prevention and treatment drug, a health product and a food additive; the invention treats newborn mice and A549 alveolar epithelial cells with high-concentration oxygen, and simultaneously intervenes with a polypeptide MDABP derivative, detects the repair of lung tissue pathological damage, and the expression levels of alveolar type II epithelial cell marker pulmonary surfactant-associated protein C (SP-C) and alveolar type I epithelial cell marker aquaporin 5 (AQP5), and finds that the polypeptide MDABP derivative can improve alveolar epithelial cell damage caused by hyperoxia exposure, reduce apoptosis, and improve BPD lung tissue pathological damage, indicating that the polypeptide MDABP derivative intervention has an improving effect on bronchopulmonary dysplasia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a breast milk-derived polypeptide in the preparation of bronchopulmonary dysplasia prevention and treatment drugs, health products and food additives. Background Art

[0002] Bronchopulmonary dysplasia (BPD) is a common complication of premature infants. With the rapid development of perinatal medicine and neonatal care technology, the birth rate and survival rate of premature infants, especially extremely premature infants and very premature infants, have increased significantly, but the incidence of BPD has also increased year by year. BPD has a long course and poor prognosis, which can lead to multiple complications such as respiratory diseases and neurodevelopmental disorders, seriously affecting the long-term quality of life of children and increasing the burden on families. At present, there is a lack of effective intervention measures for BPD. Therefore, it is urgent to find effective means of preventing and treating BPD.

[0003] Breastfeeding can significantly reduce the risk of BPD in premature infants, but its protective mechanism is still unclear. Studies have shown that breast milk contains a variety of active substances (such as exosomes, oligosaccharides and peptides, etc.), which have a protective effect on neonatal diseases. Previous studies have shown that peptides are important bioactive substances in breast milk, which have the effects of promoting growth and development, immunomodulation, and anti-infection. They are low in antigenicity and easy to absorb, and are a hot spot for new drug research and development. In recent years, the application prospects of peptides in lung diseases have also been supported by national policies and market demand. my country has included peptide drugs in the encouraged catalog and increased support for the biopharmaceutical industry. Studies have confirmed that peptides can be used as carriers for pulmonary drug delivery systems to improve the pulmonary absorption and bioavailability of protein peptide drugs and reduce side effects and immunogenicity. Recent studies have found that peptides also have significant therapeutic effects in the prevention and treatment of BPD. For example, recombinant human IGF-1 (rhIGF-1) / binding peptide 3 can promote pulmonary angiogenesis, reduce pulmonary epithelial-mesenchymal transition, and improve BPD lung structure and function. The above studies all indicate that peptides have great potential in the prevention and treatment of neonatal bronchopulmonary dysplasia. Summary of the invention

[0004] In order to solve the above problems, the present invention discloses the use of a breast milk-derived polypeptide in the preparation of bronchopulmonary dysplasia prevention and treatment drugs, health products and food additives.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention provides a human milk-derived polypeptide, wherein the sequence of the human milk-derived polypeptide is an amino acid sequence as shown in SEQ ID NO.1, and the structure of the human milk-derived polypeptide is as shown in Formula 1:

[0007]

[0008] The present invention also provides a human milk-derived polypeptide derivative, which is a chimeric peptide comprising an active amino acid sequence as shown in SEQ ID NO.1, and the chimeric peptide is formed by connecting the amino acid sequence as shown in SEQ ID NO.1 with a cell-penetrating peptide.

[0009] Furthermore, the breast milk-derived polypeptide derivative is formed by connecting the N-segment of the amino acid sequence shown in SEQ ID NO.1 to a cell-penetrating peptide, and the cell-penetrating peptide sequence is shown in SEQ ID NO.2.

[0010] SEQ ID NO.1:

[0011] QSVLTQPPSVSAAPGQKV;

[0012] Transmembrane sequence SEQ ID NO.2:

[0013] GRKKRRQRRRPPQQ.

[0014] The present invention also provides a use of the above-mentioned breast milk-derived polypeptide in the preparation of bronchopulmonary dysplasia prevention and treatment drugs, health products or food additives.

[0015] Furthermore, the concentration of the breast milk-derived polypeptide to exert biological activity is 10-20 mg / Kg.

[0016] The present invention also provides a use of the above-mentioned breast milk-derived polypeptide derivative in the preparation of bronchopulmonary dysplasia prevention and treatment drugs, health products or food additives.

[0017] Furthermore, the concentration of the breast milk-derived polypeptide derivative that exerts biological activity is 10-20 mg / Kg.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides the use of human milk-derived polypeptides in the preparation of drugs, health products or food additives for preventing and treating bronchopulmonary dysplasia, and provides a new use of human milk-derived polypeptides in the preparation of drugs for preventing and treating bronchopulmonary dysplasia; the role and mechanism of polypeptide MDABP derivatives in BPD are studied, and by using animal and cell models, by detecting the pathological changes of rat lung tissue, the expression of proliferation and apoptosis-related factors and the expression levels of alveolar type II epithelial cell markers - pulmonary surfactant-related protein C (SP-C) and alveolar type I epithelial cell markers - aquaporin 5 (AQP5), it can be seen that the intervention of polypeptide MDABP derivatives has a certain improvement effect on bronchopulmonary dysplasia, improves lung tissue pathological damage, promotes the proliferation of lung tissue alveolar epithelial cells, and reduces the apoptosis of lung tissue alveolar epithelial cells, indicating that polypeptide MDABP derivatives have an improvement effect on BPD, and provide a new idea for the treatment of BPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of peptide intervention in BPD rat and cell models;

[0021] Figure 2 The peptide alleviated the body weight and lung tissue growth in the BPD rat model;

[0022] Figure 3 The peptide alleviated the pathological damage of lung tissue in the BPD rat model;

[0023] Figure 4 The peptide alleviates the proliferation and apoptosis of alveolar epithelial cells in BPD rats;

[0024] Figure 5 The peptide promoted the protein expression level of alveolar epithelial cells in BPD rats;

[0025] Figure 6 The peptide promoted the proliferation level of the BPD-A549 alveolar epithelial cell model;

[0026] Figure 7 The peptide inhibited the apoptosis level of BPD-A549 alveolar epithelial cells;

[0027] Figure 8 The peptide promoted the protein expression level in BPD-A549 alveolar epithelial cells. DETAILED DESCRIPTION

[0028] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0029] Breast milk derived polypeptide SEQ ID NO.1 (synthesized by Shanghai Kepeptide Biotechnology Co., Ltd.):

[0030] MDABP(QSVLTQPPSVSAAPGQKV)

[0031] Breast milk derived polypeptide derivative SEQ ID NO.3 (Synthesized by Shanghai Kepeptide Biotechnology Co., Ltd.):

[0032] MDABP derivative (GRKKRRQRRRPPQQQSVLTQPPSVSAAPGQKV).

[0033] Figure 1 Figure A is a flow chart of peptide intervention in BPD rat model; Figure 1 Figure B is a flow chart of peptide intervention in the BPD cell model.

[0034] Figure 2-Figure 8 HYP+MDABP in the table indicates the HYP+MDABP derivative group.

[0035] Example 1

[0036] Effects of MDABP (Milk-Derived Anti-BPD Peptide, human breast milk-derived anti-BPD peptide) derivatives on body weight and lung tissue changes in BPD neonatal rat model

[0037] 1. Experimental Methods

[0038] Newborn SD rats weighing 7±1g within 24 hours after birth were randomly divided into three groups: control group (CTL group), BPD model group (HYP group) and BPD intervention group (HYP+MDABP derivative group), with 12 rats in each group, and the experiment was repeated 3 times.

[0039] CTL group: Newborn rats were placed in air after birth. During the experiment, the ambient temperature was maintained at 22-25°C and the humidity was maintained at 40-60%.

[0040] HYP group: Newborn rats were immediately placed in a sealed plexiglass transparent box with continuous high oxygen input. The oxygen concentration in the box was detected by an oxygen meter and maintained at about 85%. Ca(OH)2 was used to absorb CO2 in the box at the bottom to make its concentration less than 5%. During the experiment, the temperature in the box was maintained at 22-25°C and the humidity was maintained at 40-60%.

[0041] HYP+MDABP derivative group: In addition to the HYP group, the mice were intraperitoneally injected with 10 mg / kg of the peptide MDABP derivative once a day. The CTL group and the HYP group were intraperitoneally injected with an appropriate amount of PBS every day.

[0042] The entire experiment lasted for 7 days, during which the three groups of newborn rats were weighed every 2 days, and the survival status of each group of animals was recorded. On the 7th day, the rats were killed and lung specimens were obtained for measurement with a ruler and images were collected.

[0043] 2. Experimental results

[0044] Depend on Figure 2 It can be seen that the BPD rat model was established by using 85% oxygen concentration with newborn SD rats as the research object. Compared with the CTL group, the weight gain trend of newborn rats in the HYP group was significantly weakened, and the intervention of adding peptide MDABP derivatives (10 mg / kg) can partially alleviate the weight loss trend of newborn rats. At the same time, compared with the HYP group, the peptide MDABP derivatives (10 mg / kg) can also significantly improve the macroscopic lung damage caused by hyperoxia. * / #P<0.05, ** / ##P<0.01*vsCTL, #vs HYP.

[0045] Example 2

[0046] Effects of MDABP derivatives on lung tissue pathology in neonatal SD rat model of BPD

[0047] 1. Experimental Methods

[0048] SD rats weighing 7±1g within 24 hours after birth were randomly divided into three groups: CTL group, HYP group and HYP+MDABP derivative group, with 12 rats in each group. The experiment was repeated 3 times.

[0049] CTL group: Newborn rats were placed in air after birth. During the experiment, the ambient temperature was maintained at 22-25°C and the humidity was maintained at 40-60%.

[0050] HYP group: Newborn rats were immediately placed in a sealed plexiglass transparent box with continuous high oxygen input. The oxygen concentration in the box was detected by an oxygen meter and maintained at about 85%. Ca(OH)2 was used to absorb CO2 in the box at the bottom to make its concentration less than 5%. During the experiment, the temperature in the box was maintained at 22-25°C and the humidity was maintained at 40-60%.

[0051] HYP+MDABP derivative group: In addition to the HYP group, 10 mg / kg MDAB was intraperitoneally injected once a day. The CTL group and HYP group were intraperitoneally injected with appropriate amount of PBS every day.

[0052] The whole experiment lasted for 7 days. Rats were killed on the 7th day, lung tissues were dissected, and appropriate amounts of the same lung lobe tissue samples were cut for H&E sectioning: a. Fixing tissues: The samples collected after modeling were fixed in 4% paraformaldehyde (room temperature overnight); b. Making wax blocks: After dehydration using ethanol gradient, they were treated in 50% and 100% xylene, then wax-impregnated and embedded, and the wax blocks were allowed to air-dry completely; c. Slicing: After the wax blocks were completely air-dried, they were cut into 5 μm thick slices along the direction parallel to the lung tissue and placed in a 42°C water bath. After the slides were developed, they were dried in a 45°C thermostat; dH&E staining: the above sections were stained with hematoxylin for 5 minutes, and then separated with acid water and ammonia water for a few seconds; the sections were rinsed with running water for 1 hour and then placed in distilled water for a while, and dehydrated again with 70% and 90% ethanol for 10 minutes; then stained with ethanol eosin staining solution for 2-3 minutes; the stained sections were further dehydrated with 100% ethanol, and then made transparent with xylene, and then neutral resin was dripped in, and the coverslip was sealed. After the resin was slightly dry, a label was attached, and images were taken under a light microscope. The H&E-stained specimens were randomly photographed in 5 fields of view under a 100x microscope, and a "+" was drawn in the middle of each field of view. The average alveolar interval was calculated according to M=L / N, in μm. N represents the number of alveolar intervals intersecting on the cross line, and L represents the total length of the cross line. L was measured using ImageJ, and N was calculated using Image Proplus6.0. Five fields of view of the H&E-stained specimen were randomly photographed under a 100x microscope, and the number of alveoli cut along a vertical line from the center of the respiratory bronchioles to the nearest diaphragm or pleural edge was counted, which was the RAC value.

[0053] 2. Experimental results

[0054] like Figure 3 It can be seen that the BPD rat model was established using 85% oxygen concentration with newborn SD rats as the research object. The alveolar area of ​​the hyperoxia group (HYP) was significantly larger, the average linear intercept was widened, and the number of alveoli was reduced compared with the normal air group (CTL); and after intraperitoneal injection of MDABP derivatives (10 mg / kg), these changes were significantly improved compared with the HYP group; compared with the hyperoxia group (HYP), peptide intervention can partially alleviate the pathological damage of the lung tissue of newborn rats.

[0055] Example 3

[0056] Effects of MDABP derivatives on proliferation and apoptosis of alveolar epithelial cells in BPD model in vivo

[0057] 1. Experimental Methods

[0058] SD rats weighing 7±1g within 24 hours after birth were randomly divided into three groups: CTL group, HYP group and HYP+MDABP derivative group, with 12 rats in each group. The experiment was repeated 3 times.

[0059] HYP group: Newborn rats were immediately placed in a sealed plexiglass transparent box with continuous high oxygen input. The oxygen concentration in the box was detected by an oxygen meter and maintained at about 85%. Ca(OH)2 was used to absorb CO2 in the box at the bottom to make its concentration less than 5%. During the experiment, the temperature in the box was maintained at 22-25°C and the humidity was maintained at 40-60%.

[0060] HYP+MDABP derivative group: In addition to the HYP group, 10 mg / kg MDAB was intraperitoneally injected once a day.

[0061] The CTL group and the HYP group were intraperitoneally injected with appropriate amount of PBS every day.

[0062] The whole experiment lasted for 7 days. Rats were killed on the 7th day, lung tissues were dissected, and appropriate amounts of the same lung lobe tissue samples were cut for fluorescent Tunel experiment and Ki67 experiment: I. Cell proliferation: a. The specific steps of fixing tissue, wax block preparation, and slice preparation were the same as the H&E slice preparation process; b. Dewaxing: dewaxing with xylene for 4 minutes, and then dewaxing with xylene II for 6 minutes; c. Gradient alcohol dehydration: dehydration with 100% ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, distilled water for 2 minutes, and finally rinsed with PBS 2-3 times, each time for 5 minutes; d. Proteinase K repair: After the slices were slightly dried, a circle was drawn around the tissue with a tissue pen (to prevent the liquid from flowing away), and proteinase K working solution was dripped into the circle to cover the tissue, and incubated in a 37°C incubator for 22 minutes; the slides were placed in PBS and washed on a decolorizing shaker for 3 times, each time for 5 minutes; e. Membrane permeation: After the slices were slightly dried, a membrane permeation working solution (0.1% Trion) was dripped into the circle to cover the tissue, and the slices were often Incubate at room temperature for 20 minutes, place the slide in PBS and shake it on a decolorizing shaker to wash 3 times, 5 minutes each time; f. Slice and shake dry or absorb excess PBS with absorbent paper, and block with 5% BSA at room temperature for 10 minutes; g. Wipe off excess blocking solution with filter paper, add the prepared Ki67 protein primary antibody, put it in a wet box, and incubate it at 4°C overnight; h. Wash with PBS 3 times, 5 minutes each time, shake dry the slice to remove excess PBS, then drop an appropriate amount of fluorescent secondary antibody with appropriate concentration into a wet box and incubate it in the dark for 1 hour at room temperature, then wash with PBS 2-3 times, 5 minutes each time, and then shake dry slightly; i. DAPI counterstaining of cell nuclei: wash the slice with PBS 3 times, 5 minutes each time, remove PBS, add DAPI staining solution in the circle, and incubate at room temperature in the dark for 10 minutes; j. Seal the slide: wash the slide with PBS 3 times, 5 minutes each time, shake dry the slice, and seal it with anti-fluorescence quenching sealant; k. Microscopic examination and photography: observe the slice under a fluorescence microscope and collect images. 2. Cell apoptosis: a. Same as cell proliferation step ae; b. Room temperature equilibrium: After the slices are slightly dried, add Buffer to the circle to cover the tissue and incubate at room temperature for 10 minutes; c. Add reaction solution: according to the number of slices and tissue size, take an appropriate amount of TDT enzyme, dUTP, and Buffer in the Tunel kit and mix them in a ratio of 1:5:50, add them to the circle to cover the tissue, place the slices flat in a humidified box, incubate at 37℃ for 2 hours, and add a small amount of water to the humidified box to maintain humidity; d. Same as cell proliferation step ik.

[0063] 2. Experimental results

[0064] like Figure 4It can be seen that the BPD animal model was established using 85% oxygen concentration with newborn SD rats as the research object. Compared with the CTL group, the proliferation of alveolar epithelial cells in the HYP group was inhibited and apoptosis was significantly increased. After the MDABP derivatives intervened in the BPD animal model, the proliferation ability of alveolar epithelial cells was enhanced and apoptosis was improved. Peptide intervention can significantly enhance the proliferation ability of alveolar epithelial cells in newborn rats and inhibit their apoptosis.

[0065] Example 4

[0066] Effects of MDABP derivatives on protein expression of alveolar epithelial cells in BPD model in vivo

[0067] 1. Experimental Methods

[0068] SD rats weighing 7±1g within 24 hours after birth were randomly divided into three groups: CTL group, HYP group and HYP+MDABP derivative group, with 12 rats in each group. The experiment was repeated 3 times.

[0069] HYP group: Newborn rats were immediately placed in a sealed plexiglass transparent box with continuous high oxygen input. The oxygen concentration in the box was detected by an oxygen meter and maintained at about 85%. Ca(OH)2 was used to absorb CO2 in the box at the bottom to make its concentration less than 5%. During the experiment, the temperature in the box was maintained at 22-25°C and the humidity was maintained at 40-60%.

[0070] HYP+MDABP derivative group: In addition to the HYP group, 10 mg / kg MDAB was intraperitoneally injected once a day.

[0071] The CTL group and the HYP group were intraperitoneally injected with appropriate amount of PBS every day.

[0072] The whole experiment lasted for 7 days. Rats were killed on the 7th day, lung tissues were dissected, and appropriate amounts of the same lung lobe tissues were cut for Western blot experiments: a. Lysis: Take out appropriate amounts of lung tissue blocks, add RIPA, protease inhibitors and phosphorylase inhibitors, mix according to the (ratio in the instructions), take an appropriate volume and add it to the homogenizer tube containing lung tissue, tighten the lid, and place it in a homogenizer for homogenization and lysis; b. Measure protein concentration: After sufficient lysis, centrifuge at 4°C and 12000rpm for 30min, aspirate the supernatant into a clean 1.5mL EP tube, and measure the protein concentration by the BCA method (see Table 1 for the working solution configuration);

[0073] Table 1. BCA working solution (solution A: solution B = 200:4) and protein standard concentration gradient settings

[0074]

[0075] c. Sample cooking: Mix the samples in a ratio of protein: 5×SDS loading buffer = 1:4, then treat at 95℃ for 10min, cool to room temperature for later use; d. Prepare gel: According to the instructions of the kit, prepare separation gels with concentrations of 8%, 10%, 12% and 15% respectively according to the molecular weight of the protein, pour them into the glass plate in the clean and air-dried gel rack, seal the upper layer with anhydrous ethanol, leave it at room temperature for 15min, pour out the anhydrous ethanol after the lower separation gel is condensed, and then prepare the upper concentrated gel according to the instructions of the kit, insert a 10-hole tooth comb without leaving any bubbles, and leave it at room temperature for 30min. After the upper concentrated gel is completely condensed, place it in ddH2O at 4℃ for later use; e. Electrophoresis: Take out the gel prepared in advance, according to the corresponding protein According to the size of the molecule, choose a gel of appropriate concentration, calculate the loading volume according to the protein loading amount of 30μg / μL, add the sample to each channel with a pipette, and then select a constant voltage of 70V and 40min to concentrate the sample, and then adjust the voltage to 140V and 50min to separate the protein. After the electrophoresis is completed, carefully remove and cut off the excess gel; f. Transfer: according to the size of the gel, cut a PVDF membrane of appropriate size, activate it with methanol for 1min, prepare 1L transfer solution (1 package of transfer solution powder + 800mL double distilled water + 200mL methanol), soak the transfer clip in the transfer solution, and place it in the transfer clip in the order of gel at the positive electrode and membrane at the negative electrode. After removing the bubbles, clamp it and put it into the transfer tank. The transfer tank is placed in an ice box to avoid excessive temperature during the transfer process. Select 350mA, 1 hour or 350mA, 1.5 hours according to the molecular weight; g. After the transfer, prepare an appropriate amount of blocking solution (5% skim milk powder) and block it on a shaker at room temperature for 2 hours; h. Dilute the antibody stock solution to an appropriate concentration with the primary antibody diluent according to the antibody instructions, rinse the blocked PVDF membrane with TBST (TBS powder + 0.1% Tween-20, diluted with double distilled water), and then place the diluted primary antibody and the membrane in an incubation box and shake overnight at 4°C; the next day, wash the membrane 3 times with the configured TBST shaker at room temperature, each time for 10 minutes; after completion, discard the TBST, pour the configured secondary antibody into the incubation box, shake it for 1 hour at room temperature, and wash the membrane 3 times in the same way; i. Development: Mix the developer A solution and B solution in equal proportions (prepare and use now), and develop according to the operating instructions of the exposure instrument. Data analysis uses ImageJ software for quantitative analysis, and GraphPad Prism8.0 for statistical analysis, and the mean ± standard deviation is used to express it.

[0076] 2. Experimental results

[0077] like Figure 5It can be seen that the expression of surfactant-associated protein C (SP-C), a marker of alveolar type II cells, and aquaporin 5 (AQP5), a marker of alveolar type I epithelial cells, was downregulated in the hyperoxia group (HYP), and the expression of related proteins was upregulated after the intervention of MDABP derivatives under hyperoxia. * / #P<0.05, ** / ##P<0.01, *** / ###P<0.001*vs CTL, #vsHYP.

[0078] Example 5

[0079] Effects of MDABP derivatives on proliferation of A549 alveolar epithelial cells in an in vitro BPD model

[0080] 1. Experimental Methods

[0081] A549 lung epithelial cells were used as the research object, and FHC culture medium was prepared according to the instructions of ScienCell Research Laboratories, including: DMEM:F12 culture medium, 10% fetal bovine serum (exosomes removed), and double antibodies (penicillin 100U / ml, streptomycin 100μg / ml).

[0082] A549 cells were seeded into 6-well plates, with 1.5×105 cells per well. After incubation in a 37°C, 5% CO2 incubator for 24 hours, normoxic cells were used as the control group. The cells were placed in a glass oxygen chamber containing 85% oxygen concentration, which was set as the HYP group. On this basis, MDABP derivatives (1 μmol / L) or MDABP derivative scrambled peptides (Scr-MDABP derivatives) were intervened, which were set as the HYP+MDABP derivative and HYP+Scr groups, respectively. After incubation for 24 hours, half of the culture medium was removed, and an equal volume of 2xEdU working solution was added to make the final concentration of EdU 10 μM. The cells were placed in a 37°C incubator in the dark for 2 hours, all the culture medium was discarded, and 1 mL of 4% paraformaldehyde was added for fixation at room temperature for 15 minutes. The paraformaldehyde fixative was then discarded, and 1 mL of each well was used. The cells were washed with PBS 3 times, 5 min each time, PBS was discarded, and each well was incubated with 1 mL of permeabilization solution (PBS containing 0.3% TritonX-100) at room temperature for 15 min, the permeabilization solution was discarded, and the cells were washed with PBS 2 times, 5 min each time, and the washing solution in the previous step was discarded. 0.5 mL of Click reaction solution was added to each well, and incubated at room temperature in the dark for 30 min, the reaction solution was discarded, and the cells were washed with PBS 3 times, 5 min each time, and the washing solution was discarded. 1×Hoechst solution 1 mL / well was added, and incubated at room temperature in the dark for 30 min, and the Hoechst solution was discarded. The cells were washed with PBS 3 times, 5 min each time. The images were observed and collected under a fluorescence microscope, and GraphPad Prism8.0 was used for data analysis. The differences were compared and calculated using t-tests, and when P < 0.05, it was considered to be significantly different.

[0083] 2. Experimental results

[0084] like Figure 6 It can be seen that after the cells were placed in a glass oxygen chamber with 85% hyperoxia, the proliferation of A549 alveolar epithelial cells was significantly inhibited. The addition of MDABP derivative scrambled peptides for intervention failed to restore the proliferation ability of A549 cells, while the addition of MDABP derivatives for intervention could significantly restore the proliferation ability of A549 cells. * / #P<0.05, *vs CTL, #vs HYP.

[0085] Example 6

[0086] Effects of MDABP derivatives on apoptosis of A549 alveolar epithelial cells in an in vitro BPD model

[0087] 1. Experimental Methods

[0088] A549 lung epithelial cells were used as the research object, and FHC culture medium was prepared according to the instructions of ScienCell Research Laboratories, including: DMEM:F12 culture medium, 10% fetal bovine serum (exosomes removed), and double antibodies (penicillin 100U / ml, streptomycin 100μg / ml).

[0089] A549 cells were seeded into 6-well plates, with 1.5×105 cells in each well. After incubation in a 37°C, 5% CO2 incubator for 24 hours, the normoxic cells were used as the control group. The cells were placed in a glass oxygen chamber containing 85% oxygen concentration and designated as the HYP group. On this basis, the cells were intervened with MDABP derivatives (1 μmol / L) or MDABP derivative scrambled peptides (Scr-MDABP derivatives) and designated as the HYP+MDABP derivative and HYP+Scr groups, respectively, and incubated for 24 hours. Take an appropriate amount of 10×Binding Buffer and add deionized water to dilute it into 1×Binding Buffer; wash the cells twice with PBS buffer, absorb 0.25% trypsin (without EDTA) into the culture bottle to digest the cells, add 2% BSA to prevent over-digestion, and closely observe the digestion process under an inverted phase contrast microscope. When the cells are nearly round, discard the trypsin, absorb an appropriate amount of culture solution containing 10% FCS and add it to the culture bottle to terminate the digestion, and do not blow; absorb an appropriate amount of cell suspension, centrifuge at 2000rpm at room temperature for 5 to 10 minutes; resuspend the cells once with precooled PBS (4°C), precool the centrifuge to 4°C, centrifuge at 2000rpm for 5 to 10 minutes, and wash the precipitate several times; absorb 300μL of 1×Binding Buffer and add it to the centrifuge tube, blow it continuously to make the cells into a single cell suspension, and package the cells; absorb 5μL of Annexin in a light-proof environment V-FITC solution was mixed and incubated at room temperature for 15 minutes; PI labeling: 5 minutes before the machine, 5 μL of PI staining was added; Annexin V-FITC and PI single staining tubes were set at the same time, and 3 replicate tubes were set for the experimental tubes; before the machine, 200 μL of 1×Binding Buffer was added. Within 1 hour after treatment, the apoptosis rate of each sample was detected using the Accuri C6 flow cytometer, and the results were analyzed using the software provided by the Accuri C6 flow cytometer.

[0090] 2. Experimental results

[0091] Depend on Figure 7 It can be seen that after placing the cells in a glass oxygen chamber with 85% hyperoxia, it was found that the cell apoptosis in the hyperoxia group (HYP) was increased compared with the air control group (CTL). After adding the MDABP derivative scrambled peptide (HYP+Scr) under hyperoxia conditions, there was no obvious change in apoptosis, while after adding the MDABP derivative (HYP+MDABP derivative) under hyperoxia conditions, cell apoptosis was reduced compared with before.

[0092] Example 7

[0093] Effects of MDABP derivatives on protein expression in A549 alveolar epithelial cells in an in vitro BPD model

[0094] 1. Experimental Methods

[0095] A549 alveolar epithelial cells were used as the research object, and FHC culture medium was prepared according to the instructions of ScienCell Research Laboratories, including: DMEM:F12 culture medium, 10% fetal bovine serum (exosomes removed), and double antibodies (penicillin 100U / ml, streptomycin 100μg / ml).

[0096] A549 cells were seeded into 6-well plates, with 1.5×105 cells per well. After incubation in a 37°C, 5% CO2 incubator for 24 hours, normoxic cells were used as the control group (CTL). The cells were placed in a glass oxygen chamber with 85% high oxygen, which was set as the HYP group. On this basis, MDABP derivatives (1μmol / L) or MDABP derivative scrambled peptides (Scr-MDABP derivatives) were intervened, which were set as the HYP+MDABP derivative and HYP+Scr groups, respectively. After incubation for 24 hours, the adherent cells were placed on the operating table, the supernatant was discarded, and they were gently rinsed with PBS three times. a. Lysis: The lysis solution (prepared according to the instructions) was added to the six-well plate at a volume of 80μL / well, and lysed on ice for 20-30min; b. Protein concentration measurement: The cells were scraped off with a cell scraper and placed in a 1.5mL EP tube together with the lysis solution, centrifuged at 4°C and 12000rpm for 30min, and the supernatant of the sample after centrifugation was aspirated to a clean 1.5mL EP tube, BCA method to measure protein concentration; c. Cooking samples: Mix the samples according to the ratio of protein: 5×SDS loading buffer = 4:1, then treat at 95℃ for 10min, cool to room temperature for use; d. Prepare gel: According to the instructions of the kit, prepare separation gels with concentrations of 8%, 10%, 12% and 15% respectively according to the molecular weight of the protein, pour them into the glass plate in the clean and air-dried gel rack, seal the upper layer with anhydrous ethanol, leave it at room temperature for 15min, pour out the anhydrous ethanol after the lower separation gel is condensed, and then prepare the upper concentrated gel according to the instructions of the kit, insert a 10-hole comb without leaving any bubbles, leave it at room temperature for 30min, and after the upper concentrated gel is completely condensed, store it in ddH2O at 4℃ for use; e. Electrophoresis: Take out the pre-prepared 1. Glue: select the appropriate concentration of gel according to the size of the corresponding protein molecules, calculate the loading volume according to the protein loading amount of 30μg / μL, add the sample to each channel with a pipette, and then select a constant voltage of 70V and 40min to concentrate the sample, and then adjust the voltage to 140V and 50min to separate the protein. After the electrophoresis is completed, carefully remove and cut off the excess gel; f. Transfer: cut a PVDF membrane of appropriate size according to the size of the gel, activate it with methanol for 1min, prepare 1L transfer solution (1 package of transfer solution powder + 800mL double distilled water + 200mL methanol), place the transfer clip in the transfer solution and soak it, place it in the transfer clip in the order of gel at the positive electrode and membrane at the negative electrode, drive away the bubbles and put it into the transfer tank, and place the transfer tank in an ice box to avoid excessive temperature during the transfer process.Select 350mA, 1 hour or 350mA, 1.5 hours according to the molecular weight; g. After the transfer, prepare an appropriate amount of blocking solution (5% skim milk powder) and block it on a shaker at room temperature for 2 hours; h. Dilute the antibody stock solution to an appropriate concentration with the primary antibody diluent according to the antibody instructions, rinse the blocked PVDF membrane with TBST (TBS powder + 0.1% Tween-20, diluted with double distilled water), and then place the diluted primary antibody and the membrane in an incubation box and shake overnight at 4°C; the next day, wash the membrane 3 times with the configured TBST shaker at room temperature, each time for 10 minutes; after completion, discard the TBST, pour the configured secondary antibody into the incubation box, shake it for 1 hour at room temperature, and wash the membrane 3 times in the same way; i. Development: Mix the developer A solution and B solution in equal proportions (prepare and use now), and develop according to the operating instructions of the exposure instrument. Data analysis uses ImageJ software for quantitative analysis, and GraphPad Prism8.0 for statistical analysis, and the mean ± standard deviation is used to express it.

[0097] 2. Experimental results

[0098] like Figure 8 It can be seen that the expression of surfactant-associated protein C (SP-C), a marker of alveolar type II cells, and aquaporin 5 (AQP5), a marker of alveolar type I epithelial cells, was downregulated in the hyperoxia group (HYP). After the addition of MDABP derivative scrambled peptide (HYP+Scr) under hyperoxia, the expression of related proteins did not change significantly, while the expression of related proteins was upregulated after the addition of MDABP derivative intervention. * / #P<0.05, ** / ##P<0.01*vs CTL, #vs HYP.

[0099] It should be noted that the above content merely illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements can be made without departing from the principle of the present invention. These improvements all fall within the protection scope of the claims of the present invention.

Claims

1. A breast milk-derived polypeptide, characterized in that The sequence of the breast milk-derived polypeptide is the amino acid sequence shown in SEQ ID NO.1, and the structure of the breast milk-derived polypeptide is shown in Formula 1:

2. A breast milk-derived polypeptide derivative, characterized in that: The breast milk-derived polypeptide derivative is formed by connecting the N-segment of the amino acid sequence shown in SEQ ID NO.1 to a cell-penetrating peptide, and the cell-penetrating peptide sequence is shown in SEQ ID NO.

2.

3. Use of the breast milk-derived polypeptide according to claim 1 in the preparation of a drug for preventing and treating bronchopulmonary dysplasia.

4. Use of the breast milk-derived polypeptide according to claim 3 in the preparation of a drug for the prevention and treatment of bronchopulmonary dysplasia, characterized in that: The concentration of the breast milk-derived polypeptide at which biological activity is exerted is 10-20 mg / Kg.

5. Use of the breast milk-derived polypeptide derivative according to claim 2 in the preparation of a drug for preventing and treating bronchopulmonary dysplasia.

6. Use of the breast milk-derived polypeptide derivative according to claim 5 in the preparation of a drug for the prevention and treatment of bronchopulmonary dysplasia, characterized in that: The concentration of the breast milk-derived polypeptide derivative at which biological activity is exerted is 10-20 mg / Kg.

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