Application of polypeptide EN-9 in preparation of product for preventing and / or repairing intestinal barrier injury

Abstract

The invention relates to the technical field of biomedicine, and discloses application of polypeptide EN-9 in preparation of a product for preventing and / or repairing intestinal barrier injury. It is found that the natural polypeptide EN-9 (the amino acid sequence is ENDPRAVAF) from lactobacillus acidophilus can effectively improve the expression quantity of tight junction protein ZO-1 and Occludin protein, the intestinal barrier injury caused by LPS is relieved, the safety is high, a scientific basis and a practical foundation can be provided for development of drugs for treating diseases related to the intestinal barrier injury, and the natural polypeptide EN-9 has a wide application prospect. Good clinical application values are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of polypeptide EN-9 in the preparation of products for preventing and / or repairing intestinal barrier damage. Background Art

[0002] The intestinal barrier is a defense system composed of multiple layers in the intestine, responsible for selectively absorbing nutrients and preventing harmful substances (such as pathogens, toxins) from entering the body. It is jointly composed of a mechanical barrier (such as intestinal epithelial cells and tight junction proteins), a chemical barrier (such as gastric acid, antimicrobial peptides), an immune barrier (such as intestinal immune cells, secretory IgA), and a microbial barrier (such as symbiotic flora). Among them, intestinal epithelial cells regulate permeability through tight junctions (such as ZO-1, Occludin, Claudins), while the mucus layer and symbiotic bacteria (such as Bifidobacterium) can prevent pathogen invasion. As the first line of defense of the human body's defense system, they jointly maintain the integrity of the intestinal barrier, which is a key factor in protecting intestinal homeostasis. In addition, modern medicine has proposed that the destruction of intestinal barrier integrity is the pathological basis for the occurrence and development of nutritional and metabolic diseases, intestinal diseases, and various infectious diseases.

[0003] Lactic acid bacteria are a general term for Gram-positive bacteria that can ferment sugars to produce lactic acid, and are widely present in the human intestine, fermented foods, and natural environments. Lactic acid bacteria play an important role in maintaining the intestinal microecological balance and enhancing immunity by producing organic acids such as lactic acid and acetic acid to lower the environmental pH value and inhibit the growth of pathogenic bacteria. Lactobacillus acidophilus is an important representative strain among lactic acid bacteria and can stably colonize in the human digestive tract due to its excellent acid and bile salt tolerance. Research shows that Lactobacillus acidophilus mainly inhabits the small intestine and the female reproductive tract, and inhibits the growth of pathogenic bacteria by secreting antibacterial substances and competitive occupancy. It can not only improve the intestinal barrier function, relieve lactose intolerance symptoms, but also regulate the vaginal microenvironment and prevent urogenital tract infections. In related technologies, researchers found that the natural lactic acid bacteria antimicrobial peptide EN-9 (amino acid sequence: ENDPRAVAF) isolated from the fermentation supernatant of Lactobacillus acidophilus has a significant down-regulating effect on the gene expression of pro-inflammatory cytokines TNF-α and IL-1β produced in acne inflammatory cells, and can inhibit the growth of Propionibacterium acnes, and can be used to treat acne, but there is no report on its application in the repair of intestinal barrier damage.

[0004] Based on this, the present invention explores the intestinal barrier damage repair activity of EN-9 by establishing an effective LPS-induced Caco-2 intestinal barrier damage model, in order to provide a new treatment plan for the clinical application of related disease treatment. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides the use of polypeptide EN-9 in the preparation of a product for preventing and / or repairing intestinal barrier damage.

[0006] In a first aspect of the present invention, there is provided the use of polypeptide EN-9 in the preparation of a product for preventing and / or repairing intestinal barrier damage, wherein the amino acid sequence of the polypeptide EN-9 is as shown in SEQ ID NO.1.

[0007] The application according to the embodiments of the present invention has at least the following beneficial effects:

[0008] The present invention discovers that the natural polypeptide EN-9 derived from Lactobacillus acidophilus has good application potential in treating intestinal barrier damage caused by lipopolysaccharide (LPS). Specifically, the present invention experimentally proves that EN-9 can effectively increase the expression levels of tight junction proteins ZO-1 and Occludin, alleviate intestinal barrier damage caused by LPS, and has high safety. This discovery not only indicates the intestinal barrier repair effect of EN-9 in addition to existing studies such as antibacterial effects, but also provides a scientific basis and practical foundation for the development of therapeutic drugs for diseases related to intestinal barrier damage, and has good clinical application value.

[0009] Zonula occludens 1 (ZO-1) is one of the important components constituting tight junctions. Downregulation of its expression or reduction of its activity will affect the formation of tight junctions between cells, hinder the intestinal mucosa from exerting its important defense barrier function, and increase the risk of intestinal source infection caused by harmful bacteria and toxins penetrating the intestinal tract into the blood. Occludin is one of the important component proteins in tight junction proteins and has biological functions such as maintaining epithelial cell polarity, regulating cell adhesion, and receiving and transmitting cell signals. Therefore, increasing the expression levels of ZO-1 protein and Occludin protein helps to improve the effect of intestinal barrier repair.

[0010] In a second aspect of the present invention, there is provided the use of a substance specifically expressing polypeptide EN-9 in the preparation of a product for preventing and / or repairing intestinal barrier damage, wherein the amino acid sequence of the polypeptide EN-9 is as shown in SEQ ID NO.1.

[0011] In some embodiments of the present invention, the prevention and / or repair of intestinal barrier damage includes increasing the expression levels of ZO-1 protein and / or Occludin protein.

[0012] In some embodiments of the present invention, the substance specifically expressing polypeptide EN-9 includes at least one of the following:

[0013] A) A nucleic acid molecule encoding the polypeptide EN-9;

[0014] B) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule described in A).

[0015] In some embodiments of the present invention, the effective concentration of the polypeptide EN-9 is 250-550 μM. For example, it can be 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM or 550 μM.

[0016] In some embodiments of the present invention, the intestinal barrier injury includes intestinal barrier injury caused by lipopolysaccharide.

[0017] In some embodiments of the present invention, the product includes a drug.

[0018] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipient includes at least one of a diluent, excipient, filler, binder, disintegrant, absorption promoter, surfactant, adsorption carrier, lubricant, sweetener and flavoring agent.

[0020] In some embodiments of the present invention, the excipient includes water.

[0021] In some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0022] In some embodiments of the present invention, the binder includes at least one of cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone.

[0023] In some embodiments of the present invention, the wetting agent includes glycerol.

[0024] In some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate and sodium bicarbonate.

[0025] In some embodiments of the present invention, the absorption promoter includes quaternary ammonium compounds.

[0026] In some embodiments of the present invention, the surfactant includes cetyl alcohol.

[0027] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and saponite clay.

[0028] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0029] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is generally recognized for this purpose and serves as an inactive ingredient of the medicament. A compilation of pharmaceutically acceptable carriers can be found in reference books such as Handbook of Pharmaceutical Excipients (2nd Edition, edited by A. Wade and P. J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994).

[0030] In some embodiments of the present invention, the drug is administered via the gastrointestinal tract or / and parenteral administration routes.

[0031] In some embodiments of the present invention, the parenteral administration routes are selected from injection, respiratory administration, cutaneous administration, mucosal administration or cavity administration.

[0032] In some embodiments of the present invention, the dosage forms of the parenterally administered drug are selected from injections, sprays, aerosols, patches, etc.

[0033] In some embodiments of the present invention, the dosage forms of the drug administered via the gastrointestinal tract are selected from tablets, capsules, powders, granules, pills, solutions, emulsions or syrups, etc.

[0034] In some embodiments of the present invention, the drug exists in the form of oral preparations, injections or topical administration preparations.

[0035] In some embodiments of the present invention, the oral preparations include tablets, capsules, pills, powders, granules, syrups or solutions; the injections include injection dosage forms or freeze-dried powder for injection dosage forms; the topical administration preparations include creams, ointments, sprays, aerosols, gels, cataplasms or patches.

[0036] In some embodiments of the present invention, the carriers that can be selected in the preparation of oral preparations can be conventional pharmaceutical excipients such as starch, dextrin or cyclodextrin and various chemically modified cyclodextrins, sucrose, stearates, etc. When preparing freeze-dried powder for injection, it can be prepared by methods such as sterile spray drying, low-temperature vacuum drying, freeze drying, etc. The subsequent preparation processes and equipment for each preparation belong to the conventional technologies in the pharmaceutical field, and the present invention does not limit this.

[0037] Other features and advantages of the present invention will be described in the subsequent specification. Brief Description of the Drawings

[0038] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, where:

[0039] Figure 1 Statistical results of the experiment on the effect of the present invention's EN-9 on the proliferation activity of caco-2;

[0040] Figure 2 Statistical results of the experiment on the effect of LPS on the proliferation activity of caco-2;

[0041] Figure 3 Observation results of the present invention's ZO-1 protein under a laser confocal microscope;

[0042] Figure 4 Statistical results of the expression of the present invention's ZO-1 protein;

[0043] Figure 5 Observation results of the present invention's Occludin protein under a laser confocal microscope;

[0044] Figure 6 Statistical results of the expression of the present invention's Occludin protein. Specific embodiments

[0045] The concept of the present invention and the technical effects produced will be clearly and completely described below in conjunction with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0046] When a numerical range is disclosed herein, the above range is considered continuous and includes the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0047] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the related listed items.

[0048] In the description of the present invention, the description referring to terms such as "one embodiment", "some embodiments", "schematic embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0049] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0050] Example 1: Effect of EN-9 on the activity of colon adenocarcinoma cells

[0051] In this example, the human cloned colon adenocarcinoma cell line Caco-2 (hereinafter referred to as Caco-2 cells) was used as the experimental cell line. The structure and function of this cell line are similar to those of differentiated small intestinal epithelial cells and have structures such as microvilli, and are often used for experiments simulating intestinal transport in vivo. This example explored the effect of EN-9 on the activity of colon adenocarcinoma cells Caco-2. The specific experimental methods are as follows:

[0052] 1. Experimental materials

[0053] Experimental cells: The human cloned colon adenocarcinoma cell line Caco-2, purchased from Wuhan Punosai Life Science Co., Ltd.

[0054] Experimental instruments: Biological safety cabinet, centrifuge, 37 °C, 5% CO2 incubator, microscope, liquid nitrogen tank, constant temperature water bath, ultra-low temperature refrigerator, pipette, etc.

[0055] Experimental reagents: Natural polypeptide EN-9 (amino acid sequence: ENDPRAVAF, SEQ ID NO.1); Lipopolysaccharides (abbreviated as LPS); Sterile 1×PBS, DMEM complete medium (containing 10% FBS), digestive solution (containing 0.25% trypsin and 0.02% EDTA), cryopreservation solution (containing 90% FBS and 20% DMSO), serum-free DMEM complete medium, CCK-8 solution, etc.

[0056] Experimental consumables: Centrifuge tubes (15 mL), culture dishes (diameter 10 cm), disposable sterile pipettes, 1.8 mL cryopreservation tubes, programmable cooling boxes, 96-well cell culture plates.

[0057] 2. Experimental methods

[0058] (1) Caco-2 cell culture:

[0059] Cell resuscitation: First, preheat the water in the constant temperature water bath to 37°C, and put PBS and complete DMEM medium into the water bath for preheating. Then, take out the Caco-2 cells from the liquid nitrogen tank, put them into a thin film glove, and transfer them to a 37°C constant temperature water bath for rewarming. Shake the cryopreservation tube to increase the rewarming rate. Add 1 mL of complete DMEM medium (containing 20% FBS) to a 15 mL centrifuge tube, quickly aspirate the cells in the melted cryopreservation tube into the centrifuge tube, gently blow and mix evenly, and centrifuge at 1000 rpm for 3 min. Prepare a T25 culture flask, write the cell name, passage number, and date on it, and add 5 mL of complete DMEM medium containing 10% FBS. After centrifugation, discard the supernatant, resuspend the cells with 1 mL of complete medium, transfer the cell suspension to the culture flask, mix well, place it in a 37°C, 5% CO2 incubator for static culture, and observe the cell morphology and growth status at regular intervals. Change the medium when the medium turns yellow. When the growth density reaches about 85%, the cells can be passaged.

[0060] Cell passage: Observe the cell status under the microscope. Passage the cells when they adhere and grow to more than 85% of the bottom of the culture dish. In the biosafety cabinet, open the culture flask, discard the medium in the culture flask, wash it twice with 2 mL of 1×PBS, completely soak all areas of the culture dish, and discard the PBS. Then add 3 mL of complete DMEM medium and gently pipette the cells down. Then transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 3 min. At the same time, prepare a new T25 culture flask, make a mark, and add 5 mL of medium. After centrifugation, discard the supernatant, resuspend the pellet with 2 mL of medium, take 500 μL of the cell suspension and transfer it to the culture flask for passage culture. Place the culture flask horizontally, shake and mix well, and then place the culture flask in the incubator for static culture.

[0061] Cell culture: Take Caco-2 cells in the logarithmic growth phase of P1 generation, perform cell counting, dilute the cell suspension with complete DMEM medium (containing 10% FBS), and dilute the cell concentration to 1×10 5 cells / mL. Inoculate 100 μL of the cell suspension into a 96-well plate (do not seed cells in the outermost circle of the well plate, add 100 μL of 1×PBS), place it in a 37°C, 5% CO2 cell incubator for 12 - 24 h. When the cell density reaches more than 60%, drug administration can be carried out.

[0062] (2) Drug administration treatment:

[0063] Divide the experiment into a blank group, a control group, experimental group 1, and experimental group 2, with six replicate wells in each group. Among them:

[0064] ① Experimental group 1: Take the above Caco-2 cell suspension, and add 100 μL of EN-9 at different concentrations (the concentrations are set as 0, 20, 50, 100, 200, 300, 400, 600 μM. The EN-9 solution is prepared by dissolving it in serum-free DMEM medium), and culture it at 37 °C and 5% CO2 for 24 h; remove the supernatant, and then add 100 μL of a mixture of CCK-8 and serum-free DMEM medium with a volume ratio of 1:9.

[0065] ② Experimental group 2: Take the above Caco-2 cell suspension, and add 100 μL of LPS at different concentrations (0.1, 1.0, 10.0, 20.0, 50.0, 100.0, 150.0 μg / mL. The LPS solution is prepared by dissolving it in serum-free medium), and culture it at 37 °C and 5% CO2 for 24 h; remove the supernatant, and then add 100 μL of a mixture of CCK-8 and serum-free DMEM medium with a volume ratio of 1:9.

[0066] ③ Control group: Take the above Caco-2 cell suspension, just replace the EN-9 solution in the experimental group with an equal volume of serum-free DMEM medium, and the other conditions are the same as those in the experimental group.

[0067] ④ Blank group: Replace both the Caco-2 cell suspension and the EN-9 solution in the experimental group with an equal volume of serum-free DMEM medium, and the other conditions are the same as those in the experimental group.

[0068] Place the mixed culture solutions of the above groups in a cell culture incubator, incubate at 37 °C for 45 min, then perform on-machine detection, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at 450 nm, and calculate its cell survival rate.

[0069] Cell survival rate % = (OD of experimental well - OD of blank well) / (OD of control well - OD of blank well) × 100%.

[0070] 3. Experimental results

[0071] The experimental results of the effects of EN-9 and LPS on the proliferation activity of human cloned colon adenocarcinoma cell line caco-2 are as follows Figure 1 and Figure 2As shown in the figure, the results showed that there were no significant differences in the effects of adding different concentrations of EN-9 on the activity of Caco-2 cells compared with the blank group (P>0.05), indicating its high safety. Therefore, subsequent experiments were carried out with 200, 400, and 600 μM EN-9. Compared with the blank group, 1 μg / mL of LPS significantly reduced the cell activity of Caco-2 (P<0.05), while 150 μg / mL of LPS significantly increased the cell activity of Caco-2 (P<0.05), and other concentrations had no significant effect on the proliferative activity of Caco-2 cells (P>0.05). Therefore, subsequent experiments were carried out with 100 μg / mL LPS solution that had no effect on cell activity.

[0072] Example 2: Experiment on the effect of EN-9 on LPS-induced intestinal barrier injury

[0073] In this example, the effect of EN-9 on LPS-induced intestinal barrier injury was detected, which specifically included the following contents:

[0074] 1. Experimental method

[0075] (1) Cell culture:

[0076] Caco-2 cells were cultured according to the method of Example 1 above. Then, Caco-2 cells in the logarithmic growth phase were collected, counted, and DMEM complete medium was added to make the cell density 1×10 5 cells / mL. Then, 1 mL was inoculated into each well of a confocal dish and cultured overnight in a 5% CO2 incubator. When the cells adhered and covered 80% of the bottom of the dish, subsequent experiments were carried out.

[0077] (2) Experimental grouping:

[0078] The experiment was divided into a blank group (Control group), an LPS group (model group), and an experimental group (including a high-dose group, a medium-dose group, and a low-dose group), where:

[0079] ① High-dose group: Remove the old medium in the confocal dish, add an EN-9 solution with a final concentration of 600 μM (EN-9 was prepared with serum-free DMEM medium) to the dish, and add LPS with a final concentration of 100 μg / mL after 1 h. Then, it was cultured at 37°C and 5% CO2 for 24 h;

[0080] ② Medium-dose group: Remove the old medium in the confocal dish, add an EN-9 solution with a final concentration of 400 μM (EN-9 was prepared with serum-free DMEM medium) to the dish, and add LPS with a final concentration of 100 μg / mL after 1 h. Then, it was cultured at 37°C and 5% CO2 for 24 h;

[0081] ③ Low-dose group: Remove the old medium in the confocal dish, add EN-9 solution with a final concentration of 200 μM to the dish (EN-9 is prepared with serum-free DMEM medium), add LPS with a final concentration of 100 μg / mL after 1 h, and culture at 37°C and 5% CO2 for 24 h;

[0082] ④ LPS group: Remove the old medium in the confocal dish, add serum-free DMEM medium (equal volume to the EN-9 solution in the above experimental groups) and LPS with a final concentration of 100 μg / mL, and culture at 37°C and 5% CO2 for 24 h;

[0083] ⑤ Blank group: Replace both the EN-9 solution and LPS solution in the experimental group with an equal volume of serum-free medium, and the other conditions are the same.

[0084] (3) Immunofluorescence observation of the expression of ZO-1 protein:

[0085] Zonula occludens-1 (ZO-1) is one of the important components that make up tight junctions. Downregulation of its expression or reduction of its activity will affect the formation of tight junctions between cells, hinder the intestinal mucosa from exerting its important defense barrier function, and increase the risk of intestinal source infection caused by harmful bacteria and toxins penetrating the intestinal tract into the blood. After cell intervention treatment according to the above method in this experiment, aspirate the old medium and wash three times with pre-cooled PBS. Then, perform an immunofluorescence experiment according to the following steps to detect the expression of the tight junction protein ZO-1 of Caco-2.

[0086] Among them, the method of the immunofluorescence experiment is as follows:

[0087] ① Fixation: Fix with 10% formalin for 15 min, and wash three times with pre-cooled normal PBS;

[0088] ② Permeabilization: Permeate with 0.2% Triton X-100 for 15 min, and wash three times with pre-cooled normal PBS;

[0089] ③ Blocking: Block with 5% BSA (diluted with PBS) for 2 h, and wash three times with 1 mL PBS on a shaker for 1 min;

[0090] ④ Primary antibody incubation: Add 500 μL of ZO-1 antibody (antibody dilution ratio 1:200), incubate overnight on a shaker at 4°C, and wash three times with 1 mL PBS on a shaker for 1 min;

[0091] ⑤ Secondary antibody incubation: Goat anti-rabbit fluorescein isothiocyanate (FITC) (antibody dilution ratio 1:500), incubate for 1 hour at 37°C in the dark, and wash three times with 1 mL PBS on a shaker for 1 min;

[0092] ⑥ Nuclear staining: Add 500 μL of DAPI to stain the cell nuclei in the dark for 5 min. After aspiration, wash three times with 1 mL of PBS, and finally leave 1 mL of PBS in the dish for observation under a laser confocal microscope.

[0093] (4) Immunofluorescence observation of the expression of Occludin protein:

[0094] Occludin is one of the important component proteins in tight junction proteins and has biological functions such as maintaining the polarity of epithelial cells, regulating cell adhesion, and receiving and transmitting cell signals. After cell intervention treatment according to the model construction method in this experiment, aspirate the old culture medium and wash three times with pre-cooled PBS. Then, perform the immunofluorescence experiment according to the following steps to detect the expression of the tight junction protein Occludin in Caco-2 cells.

[0095] Among them, the immunofluorescence experiment method is as follows:

[0096] ① Fixation: Fix with 10% formalin for 15 min and wash three times with pre-cooled normal PBS.

[0097] ② Permeabilization: Permeabilize with 0.2% Triton X-100 for 15 min and wash three times with pre-cooled normal PBS.

[0098] ③ Blocking: Block with 5% BSA (diluted with PBS) for 2 h and wash three times with 1 mL of PBS on a shaker for 1 min.

[0099] ④ Primary antibody incubation: Add 500 μL of Occludin antibody (antibody dilution ratio 1:300), incubate overnight on a shaker at 4°C, and wash three times with 1 mL of PBS on a shaker for 1 min.

[0100] ⑤ Secondary antibody incubation: Alexa Fluor 488-labeled goat anti-rabbit IgG (antibody dilution ratio 1:1000), incubate for 1 hour in the dark at 37°C, and wash three times with 1 mL of PBS on a shaker for 1 min.

[0101] ⑥ Nuclear staining: Add 500 μL of DAPI to stain the cell nuclei in the dark for 5 min. After aspiration, wash three times with 1 mL of PBS, and finally leave 1 mL of PBS in the dish for observation with a laser confocal microscope.

[0102] 2. Experimental results

[0103] The laser confocal microscopic observation image of the tight junction protein ZO-1 is as Figure 3As shown in the figure, the results show that the tight junction protein ZO-1 in the normal control group is distributed along the cell membrane, with clear boundaries and a reticular structure, outlining the cell contours; after LPS treatment of Caco-2 cells, the fluorescence distribution between cells is disordered, the intensity is weakened, the cell gap is enlarged, and the cell contours are blurred, indicating that the Caco-2 cell tight junction injury model has been successfully constructed. When different concentrations of EN-9 solution are added, the fluorescence intensity is enhanced, and when EN-9 with a concentration of 400μM is added, the fluorescence intensity can be significantly increased, indicating that EN-9 can improve the disordered distribution of cell junction proteins and make the distribution of ZO-1 tend to be normal.

[0104] Furthermore, GraphPad Prim8.0 one-way ANOVA was performed based on the fluorescence intensity of ZO-1 protein. The results are as follows: Figure 4 As shown, LPS reduced the expression of ZO-1, and the addition of EN-9 at a concentration of 400 μM increased the expression of ZO-1 (P < 0.05), indicating that it helps to improve cell tight junctions and help repair intestinal barrier damage.

[0105] Laser confocal microscopy of tight junction protein Occludin Figure 5 As shown in the figure, the results show that the tight junction protein Occludin in the normal control group is distributed along the cell membrane, with clear boundaries and a reticular structure, outlining the cell contours. After LPS treatment of Caco-2 cells, the fluorescence distribution between cells is disordered, the intensity is weakened, the cell gap is enlarged, and the cell contours are blurred. The addition of EN-9 can increase the fluorescence intensity, improve the disordered distribution of cell junction proteins, and make the distribution of Occludin tend to be normal.

[0106] Furthermore, GraphPad Prim8.0 one-way ANOVA was performed based on the fluorescence intensity of Occludin protein. The results are as follows: Figure 6 As shown, when EN-9 was added at a concentration of 400 μM, the expression of Occludin was increased (P < 0.05).

[0107] The above results indicate that EN-9 can increase the expression of tight junction proteins ZO-1 and Occludin in the intestinal barrier injury model, and can be used to prevent or treat intestinal barrier injury.

[0108] In summary, the present invention provides the use of polypeptide EN-9 in the preparation of products for preventing and / or repairing intestinal barrier damage. The present invention found that polypeptide EN-9 can increase the expression levels of tight junction protein ZO-1 protein and Occludin protein in the intestinal barrier damage model, has high safety, and has no significant effect on the proliferation activity of small intestinal epithelial cells.

[0109] The above has made a detailed description of the embodiments of the present invention. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the gist of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant technical field. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of polypeptide EN-9 in the preparation of a product for preventing and / or repairing intestinal barrier injury, wherein the amino acid sequence of the polypeptide EN-9 is as shown in SEQ ID NO.

1.

2. Use of a substance specifically expressing polypeptide EN-9 in the preparation of a product for preventing and / or repairing intestinal barrier injury, wherein the amino acid sequence of the polypeptide EN-9 is as shown in SEQ ID NO.

1.

3. The application according to claim 2, characterized in that The substance specifically expressing polypeptide EN-9 includes at least one of the following: A) A nucleic acid molecule encoding the polypeptide EN-9; B) An expression cassette, vector or transgenic cell line containing the nucleic acid molecule described in A).

4. The application according to any one of claims 1 to 3, characterized in that, The prevention and / or repair of intestinal barrier injury includes increasing the expression level of ZO-1 protein and / or Occludin protein.

5. The application according to claim 4, characterized in that, The effective concentration of the polypeptide EN-9 is 250 - 550 μM.

6. The application according to claim 5, characterized in that, The intestinal barrier injury includes intestinal barrier injury caused by lipopolysaccharide.

7. The application according to claim 5, wherein The product includes a drug.

8. The application according to claim 6, characterized in that, The drug further contains a pharmaceutically acceptable excipient.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipient includes at least one of a diluent, excipient, filler, binder, disintegrant, absorption promoter, surfactant, adsorption carrier, lubricant, sweetener and flavoring agent.

10. The application according to claim 9, characterized in that, The drug is administered through the gastrointestinal tract or / and non-gastrointestinal administration routes.

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