Application of antibacterial peptide CRAMP in preparation of product for inhibiting enterobacter and / or indirectly promoting growth of lactobacillus

Abstract

The invention provides application of an antibacterial peptide CRAMP in preparation of a product for inhibiting enterobacter and / or indirectly promoting growth of lactobacillus, and belongs to the technical field of intestinal microecological regulation and control. Results of an in-vitro bacterial experiment and a mouse in-vivo experiment show that the antibacterial peptide CRAMP optimizes the intestinal micro-ecological environment by inhibiting the proliferation of enterobacter and indirectly promoting the growth of lactobacillus. Therefore, the invention provides application of the antibacterial peptide CRAMP in preparation of a product for inhibiting enterobacter and / or indirectly promoting growth of lactobacillus.

Description

Technical Field

[0001] The invention belongs to the technical field of intestinal microecology regulation, and specifically relates to the application of antimicrobial peptide CRAMP in preparing products for inhibiting the growth of enterobacteria and / or indirectly promoting the growth of lactobacilli. Background Art

[0002] The intestinal micro-ecosystem is a complex system composed of a variety of microorganisms. Different flora interact with each other in a dense ecosystem such as the intestine, either symbiotically or antagonistically, to form a relatively stable flora structure. Among them, Enterobacter and Lactobacillus play an important role in maintaining host health. Overgrowth of Enterobacter can disrupt intestinal balance, causing inflammation and other diseases, while Lactobacillus helps inhibit harmful bacteria and promote intestinal health by producing lactic acid.

[0003] The antimicrobial peptide CRAMP (Cathelicidin-Related Antimicrobial Peptide) is a broad-spectrum antimicrobial peptide found in mice and belongs to the cathelicidin family. It is an active peptide expressed by a precursor protein and generated by proteolysis, and is widely involved in the defense mechanism of innate immunity. At present, CRAMP has a broad-spectrum antimicrobial activity against a variety of pathogens (including bacteria, fungi and viruses), and is also involved in inflammatory-related diseases, cardiac ischemic re-attention injury and asthma. There are currently no reports that the antimicrobial peptide CRAMP inhibits the growth of Enterobacteriaceae and / or indirectly promotes the growth of Lactobacilli. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide the use of the antimicrobial peptide CRAMP in the preparation of products that inhibit the growth of Enterobacteriaceae and / or indirectly promote the growth of Lactobacillus, which can effectively optimize the intestinal microecological environment.

[0005] The invention provides application of antimicrobial peptide CRAMP in preparing products for inhibiting the growth of enterobacteriaceae and / or indirectly promoting the growth of lactobacilli.

[0006] Preferably, the Enterobacterium includes Escherichia coli (E. coli).

[0007] Preferably, the lactobacillus comprises Lactobacillus reuteri.

[0008] Preferably, the product includes at least one of the following: an intestinal microecological regulator, a medicine, and a health product.

[0009] Preferably, the formulation types of the product include injection powder and / or injection solution.

[0010] Preferably, the concentration of the antimicrobial peptide CRAMP in the product is not less than 1 mg / mL or 1 mg / g.

[0011] The present invention provides the use of antimicrobial peptide CRAMP in preparing medicines for preventing and / or treating diseases related to enterobacterial infection.

[0012] Preferably, the Enterobacter infection-related diseases include diseases caused by Escherichia coli infection.

[0013] Preferably, the diseases caused by the E. coli infection include at least one of the following: digestive system diseases, urinary system diseases and gynecological inflammation.

[0014] Preferably, the digestive system diseases include acute gastroenteritis and / or chronic enteritis;

[0015] The urinary system disease includes at least one of the following: urethritis, cystitis, pyelonephritis, cholecystitis, appendicitis and peritonitis;

[0016] The gynecological inflammation includes at least one of the following: pelvic inflammatory disease, adnexitis and endometritis.

[0017] The present invention provides the use of the antimicrobial peptide CRAMP in the preparation of a product that inhibits the growth of Enterobacteriaceae and / or indirectly promotes the growth of Lactobacillus. The present invention experiments have shown that the antimicrobial peptide CRAMP can specifically and effectively inhibit the proliferation of Enterobacteriaceae under in vitro conditions, and the inhibition strength is correlated with the concentration. At the same time, in vivo mouse experiments show that the abundance of Lactobacillus increases significantly on the 10th day after intraperitoneal injection of CRAMP, but the abundance of Enterobacteriaceae decreases significantly on the 2nd day. This shows that the CRAMP can significantly inhibit the excessive growth of Enterobacteriaceae in vivo, and indirectly lead to the proliferation of Lactobacillus, regulating the development of intestinal flora in a beneficial direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the Blast alignment result of Escherichia coli (E.coli);

[0019] Figure 2 This is the Blast comparison result of Lactobacillus reuteri;

[0020] Figure 3 The results of the co-culture of Escherichia coli and Lactobacillus reuteri by mouse CRAMP or control solution (Vehicle) in vitro; A is the plate colony growth result, and B is the colony growth statistics chart;

[0021] Figure 4 These are the results of detection of Lactobacillus and Enterobacteriaceae in mouse feces after injection of CRAMP or control solution (Vehicle), where A is the result of change in Lactobacillus abundance, and B is the result of change in Enterobacteriaceae abundance. DETAILED DESCRIPTION

[0022] The invention provides application of antimicrobial peptide CRAMP in preparing products for inhibiting the growth of enterobacteriaceae and / or indirectly promoting the growth of lactobacilli.

[0023] In the embodiment of the present invention, the antimicrobial peptide CRAMP is purchased from AlphaDiagnostic International, with the product number of SP-88328-1, the amino acid is Gly-Leu-Leu-Arg-Lys-Gly-Gly-Glu-Lys-Ile-Gly-Glu-Lys-Leu-Lys-Lys-Ile-Gly-Gln-Lys-Ile-Lys-Asn-Phe-Phe-Gln-Lys-Leu-Val-Pro-Gln-Pro-Glu-Gln (SEQ ID NO: 1), and the specification is 1 mg high-purity lyophilized powder. The antimicrobial peptide CRAMP includes the following forms: antimicrobial peptide CRAMP dry powder or antimicrobial peptide CRAMP PBS solution.

[0024] In the present invention, the enterobacteriaceae preferably include Escherichia coli (E.coli). The lactobacillus preferably includes Lactobacillus reuteri (L.reuteri). In one embodiment of the present invention, the in vitro antibacterial experiment shows that the aqueous solution of the antimicrobial peptide CRAMP at 40μg / mL and 100μg / mL can effectively inhibit the proliferation and growth of Escherichia coli. At a concentration of 40μg / mL, the antibacterial rate of CRAMP on Escherichia coli is more than 70%, and the antibacterial rate of CRAMP on Escherichia coli is more than 90% at 100μg / mL. At the same time, the PBS solution of the antimicrobial peptide CRAMP has no inhibitory effect on Lactobacillus reuteri. This shows that the PBS solution of the antimicrobial peptide CRAMP has specific antibacterial activity against bacteria. In another embodiment of the present invention, in an in vivo animal experiment, CRAMP was intraperitoneally injected into mice at 5mg / kg, and qPCR amplification was performed using specific primers for Lactobacillus and Enterobacteriaceae, and the relative abundance changes of Enterobacteriaceae and Lactobacillus in the experimental group and the control group were compared. The abundance of Lactobacillus increased significantly on the 10th day after CRAMP, but the abundance of Enterobacteriaceae decreased significantly on the 2nd day. The experimental results show that CRAMP can significantly inhibit the overgrowth of Enterobacteriaceae in vivo, leading to the proliferation of Lactobacillus and regulating the intestinal flora to develop in a beneficial direction. Therefore, the antimicrobial peptide CRAMP is also preferably used to prepare in vitro products for inhibiting or killing Enterobacteriaceae, including disinfectants, disinfected daily necessities, such as disinfectant hand sanitizers, antibacterial soaps, laundry detergents or other common toiletries.

[0025] In the present invention, the product preferably includes at least one of the following: an intestinal microecological regulator, a drug, and a health product. The intestinal microecological regulator can reduce the abundance of harmful bacteria such as Enterobacteriaceae, increase the abundance of beneficial bacteria such as Lactobacillus, and regulate the balance of intestinal microbial flora. The preparation type of the product preferably includes injection powder and / or injection solution. The concentration of the antimicrobial peptide CRAMP in the product is preferably not less than 1 mg / mL or 1 mg / g, and can be 5-100 mg / mL or 5-100 mg / g, and can also be 10-80 mg / mL or 10-80 mg / g, and can also be 20-50 mg / mL or 20-50 mg / g.

[0026] The present invention provides the use of antimicrobial peptide CRAMP in preparing medicines for preventing and / or treating diseases related to enterobacterial infection.

[0027] In the present invention, the diseases related to enterobacteriaceae infection preferably include diseases caused by Escherichia coli infection. The diseases caused by Escherichia coli infection preferably include at least one of the following: digestive system diseases, urinary system diseases and gynecological inflammation. The enterobacteriaceae have the function of secreting enterotoxins, which act on the intestines to cause abdominal pain, diarrhea and other problems in the body. At the same time, after Escherichia coli infects the intestines, intestinal dysfunction such as fever, nausea, vomiting, stomach discomfort and indigestion problems may occur.

[0028] In the present invention, the digestive system diseases preferably include acute gastroenteritis and / or chronic enteritis. The urinary system diseases preferably include at least one of the following: urethritis, cystitis, pyelonephritis, cholecystitis, appendicitis and peritonitis. Enterobacter bacteria use antigens and pili to stimulate the body to produce specific antibodies, which can invade the surface of the intestinal mucosa and cause inflammation.

[0029] In the present invention, the gynecological inflammation preferably includes at least one of the following: pelvic inflammatory disease, adnexitis and endometritis. When immunity is reduced or physical fitness is weak, the self-cleaning ability of the vagina is reduced, and Escherichia coli near the vagina can cause bacterial vaginitis.

[0030] In the present invention, the dosage form of the drug preferably includes at least one of the following dosage forms: injection powder, injection solution, suppository, lotion, etc.

[0031] In the present invention, the concentration of the antimicrobial peptide CRAMP in the drug is not less than 10 mg / mL or 10 mg / g, and can be 15-100 mg / mL or 15-100 mg / g, or 20-80 mg / mL or 20-80 mg / g, or 30-50 mg / mL or 30-50 mg / g. The present invention has no particular limitation on the preparation method of the drug, and the preparation method of the drug known in the art can be used.

[0032] The application of the antimicrobial peptide CRAMP provided by the present invention in the preparation of products that inhibit the growth of Enterobacteriaceae and / or indirectly promote the growth of Lactobacilli is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

[0034] All in vitro bacterial experiments were performed under sterile conditions.

[0035] 1 Antimicrobial peptide CRAMP: purchased from a commercial supplier (AlphaDiagnostic International), the specification is 1 mg high-purity lyophilized powder. It was dissolved in sterile low-endotoxin PBS to prepare a 1 mg / mL stock solution, which was aliquoted and stored at -20°C for subsequent use.

[0036] 2 Experimental strain isolation and purification

[0037] Collect feces from C57BL / 6 mice, weigh the feces samples aseptically, and fully homogenize (using a grinding rod) according to 1 mL of sterile PBS for every 100 mg. After centrifugation at 700g for 5 minutes, aspirate the supernatant and dilute 100 times, apply it to LB (Luria-Bertani) solid medium and MRS (de Man, Rogosa and Sharpe medium) solid medium, and invert it in a 37°C incubator for culture. After 24 hours, pick a single colony and amplify it in 2 mL of culture medium, place it in a 37°C incubator, and after 24 hours, there is a white precipitate at the bottom of the test tube, which is the amplified bacteria. Aspirate 200 μL of bacterial suspension for genomic DNA extraction. The specific steps are as follows: centrifuge the aspirated bacterial solution at 12000 rpm for 1 minute, discard the supernatant, add 500 μL PBS for washing, centrifuge at 12000 rpm for 1 minute, and wash twice. Finally, discard the supernatant, add 40 μL of sterile water, resuspend the bacterial pellet, boil in boiling water for 10 minutes, freeze for 2 minutes, centrifuge at 12000 rpm for 10 minutes, and aspirate the supernatant as the genome.

[0038] The PCR specific primers are as follows: L. reuteri-F1: 5'-CAGACAATCTTTGATTGTTTAG-3' (SEQ ID NO: 2) and E. coli-R1: 5'-GCTTGTTGGTTTGGGCTCTTC-3' (SEQ ID NO: 3); E. coli-F1: 5'-CATGCCGCGTGTATGAAGAA-3' (SEQ ID NO: 4) and E. coli-R1: 5'-CGGGTAACGTCAATGAGCAAA-3' (SEQ ID NO: 5). PCR amplification was performed using the above specific primers, and the target size band was obtained by agarose gel electrophoresis. 200 μL of the positive strain that produced the target size band was taken and sent to Qingke Biotechnology Co., Ltd. (Xi'an) for 16s sequencing, and then the sequencing results were compared using the Blast tool. It was identified as Escherichia coli (E. coli) ( Figure 1 ) and Lactobacillus reuteri (L. reuteri) Figure 2 ).

[0039] 3 Experimental methods

[0040] Escherichia coli was cultured in LB medium and Lactobacillus reuteri was cultured in MRS medium in a 37°C incubator overnight. After overnight culture, the bacterial solution was diluted to 5×10 3 CFU / mL (determined by optical density OD at 600nm and referenced to the standard curve). Prepare different concentrations of CRAMP working solution (final concentration 0, 40, 100 μg / mL) and dilute with sterile PBS to the required concentration for the experiment. Mix 100 μL of the diluted bacterial suspension with different concentrations of CRAMP solution and add it to a 96-well plate. Add bacterial suspension and CRAMP solvent control (100 μL) to the control group. Cultivate in a 37°C incubator for 2 hours. Dilute the culture solution 5 times and spread it on the corresponding culture medium plate (Escherichia coli-LB solid culture medium; Lactobacillus reuteri-MRS solid culture medium), culture at 37°C for 24 hours, and count the colony forming units (CFU). The antibacterial or proliferation effect of CRAMP on Escherichia coli and Lactobacillus reuteri was evaluated by CFU counting.

[0041] 4 Experimental results

[0042] As the concentration of CRAMP increased (40 to 100 μg / mL), the number of viable E. coli colonies decreased significantly. At a concentration of 40 μg / mL, the inhibition rate of E. coli reached more than 70%, and at 100 μg / mL it reached more than 90% ( Figure 3A and B), the data were statistically analyzed (such as one-way analysis of variance, ANOVA), proving that CRAMP has an inhibitory effect on Escherichia coli. However, CRAMP had no inhibitory effect on Lactobacillus reuteri at concentrations of 40 μg and 100 μg / mL ( Figure 3 (B) This experiment confirmed the specific inhibitory effect of CRAMP on Escherichia coli, but had no significant inhibitory effect on Lactobacillus reuteri. This laid the foundation for the potential application of CRAMP as a regulator of intestinal microecology.

[0043] Example 2

[0044] In vivo mouse experiments

[0045] 1Healthy SPF-grade C57BL / 6 female mice, 8 weeks old, weighing about 20g, were selected and divided into an experimental group (CRAMP) and a control group (Vehicle), with 5 mice in each group. Mouse-derived CRAMP was provided by a commercial supplier and dissolved in sterile low-endotoxin PBS at a concentration of 1mg / mL, ready for use. Each mouse in the experimental group was intraperitoneally injected with 5mg / kg CRAMP solution, and each mouse in the control group was intraperitoneally injected with an equal volume of sterile PBS (Vehicle). The mice were injected once every 4 days, and feces were collected once every 2 days. DNA was extracted from the mouse feces using a DNA extraction kit. After extraction, the DNA concentration was determined using Qubit4. After DNA quantification, qPCR amplification was performed using specific primers of Lactobacillus (LabF362: 5'-AGCAGTAGGGAATCTTCCA-3', SEQ ID NO: 6 and LabR677: 5'-CACCGCTACACATGGAG-3', SEQ ID NO: 7) and Enterobacter (Uni515F: 5'-GTGCCAGCAGCCGCGGTAA-3', SEQ ID NO: 8 and Ent826R: 5'-GCCTCAAGGGCACAACCTCCAAG-3', ', SEQ ID NO: 9). The reaction procedure used cDNA as a template, PCR amplification was performed using specific primers, and qPCR detection was performed using a two-step method. The reaction system is shown in Table 1 below.

[0046] Table 1 Reaction system

[0047] GoTaqqPCRMix(2x) 10μL Upstream primer (10 μM) 0.4μL Downstream primer (10 μM) 0.4μL DNA template 2μL(100ng) Nuclease-Free Water 7.2μL Total volume 20μL

[0048] The amplification procedure included: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and 40 cycles. The melting curve was set at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. In the final experimental data analysis, a standard curve was drawn based on the DNA concentration after different bacterial liquid dilutions and its corresponding Ct value (threshold cycle number), and calculations were performed based on this to compare the relative abundance changes of Enterobacteriaceae and Lactobacillus in the experimental group and the control group.

[0049] 2 In vivo results:

[0050] Statistical analysis was performed using Prism 9 software (GraphPad Software, La Jolla, CA). One-way analysis of variance (ANOVA) was used to compare the abundance differences between the two groups, with a significance level set at p < 0.05. Data are presented as mean ± standard deviation (Mean ± SEM). The results showed that the abundance of Lactobacillus increased significantly on day 10 after intraperitoneal injection of CRAMP ( Figure 4 A), but the abundance of Enterobacteriaceae decreased significantly on the second day ( Figure 4 B). The experimental results show that CRAMP can significantly inhibit the overgrowth of Enterobacteriaceae in vivo, leading to the proliferation of Lactobacillus, and regulating the intestinal flora to develop in a beneficial direction. The present invention provides a scientific basis for the development of CRAMP as a new intestinal microecological regulator, especially its potential application in the treatment of Enterobacteriaceae-related diseases (such as inflammatory bowel disease).

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the antimicrobial peptide CRAMP in the preparation of a product for inhibiting the growth of Enterobacteriaceae and / or indirectly promoting the growth of Lactobacilli.

2. The application according to claim 1, characterized in that: The Enterobacteriaceae include Escherichia coli (E. coli).

3. The application according to claim 1, characterized in that: The lactobacillus includes Lactobacillus reuteri.

4. The use according to claim 1, characterized in that: The product includes at least one of the following: an intestinal microecological regulator, a medicine, and a health product.

5. The use according to claim 1, characterized in that: The formulation types of the product include injection powder and / or injection solution.

6. The use according to any one of claims 1 to 5, characterized in that: The concentration of the antimicrobial peptide CRAMP in the product is not less than 1 mg / mL or 1 mg / g.

7. Use of the antimicrobial peptide CRAMP in the preparation of drugs for preventing and / or treating diseases related to Enterobacter infection.

8. The use according to claim 7, characterized in that: The enterobacterial infection-related diseases include diseases caused by Escherichia coli infection.

9. The use according to claim 8, characterized in that: The diseases caused by the E. coli infection include at least one of the following: digestive system diseases, urinary system diseases and gynecological inflammation.

10. The use according to claim 9, characterized in that: The digestive system diseases include acute gastroenteritis and / or chronic enteritis; The urinary system disease includes at least one of the following: urethritis, cystitis, pyelonephritis, cholecystitis, appendicitis and peritonitis; The gynecological inflammation includes at least one of the following: pelvic inflammatory disease, adnexitis and endometritis.

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