CN114344451B

CN114344451B - Application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia

Info

Publication number: CN114344451B
Application number: CN202210085094.1A
Authority: CN
Inventors: 万晓玉; 肖永陶; 王伟鹏
Original assignee: Shanghai Institute Of Pediatric Research; Shanghai Pulmonary Hospital
Current assignee: Shanghai Institute Of Pediatric Research; Shanghai Pulmonary Hospital
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2023-09-26
Anticipated expiration: 2042-01-25
Also published as: CN114344451A

Abstract

The invention provides application of Reg4 antibacterial peptide in treating pseudomonas aeruginosa infectious pneumonia.

Description

Application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia

Technical Field

The invention relates to the field of new medicine application, in particular to application of a Reg4 antibacterial peptide in treating pseudomonas aeruginosa infectious pneumonia.

Background

Pseudomonas aeruginosa (Pseudomonas aeruginosa, PAO 1), also known as Pseudomonas aeruginosa, is a unidirectional movement type gram negative opportunistic pathogen, distributed in various environments and organisms such as soil, water, plants and animals.

Clinically, PAO1 is one of the major pathogenic bacteria in hospital acquired and respiratory tract infections, and can cause acute and chronic infections in the respiratory tract, urinary tract and burn sites of patients, leading to serious morbidity and mortality. Particularly in patients with underlying diseases such as cancer, cystic fibrosis, ventilator-associated pneumonia, or other immunodeficiency, can lead to a significant incidence of infection and even mortality.

Pseudomonas aeruginosa is one of the leading causes of morbidity and mortality in cystic fibrosis patients. Studies have shown that PAO1 is the second leading cause of hospital-acquired pneumonia (Hospital-acquired pneumonia, HAP), accounting for 14% -16%.

The main treatment regimens for PAO1 infection are antibiotics and supportive therapy. But in recent years, due to excessive abuse of antibiotics, resistance has been demonstrated to a variety of antibiotics, which severely limits the therapeutic regimens available for treating PAO1 infected patients. Once a patient is infected with a pan-resistant PA01 strain, the therapeutic effect is very limited even if different antibiotics are changed. Carbapenem antibiotics, including imipenem and meropenem, are the last means of treating gram-negative bacterial infections. However, the situation is optimistic, and statistics are monitored according to the Chinese bacterial drug resistance monitoring network: in 2019, 27.5% of the strains of Pseudomonas aeruginosa clinically isolated from third-class hospitals developed resistance to imipenem antibiotics. The novel antibiotics have longer development period, and the novel antibiotics can not effectively solve the problem of PAO1 drug resistance. Therefore, there is an urgent need to develop new effective non-antibiotic treatment strategies for PAO1 infection to control and treat refractory infections caused by multi-drug resistant bacteria.

Disclosure of Invention

The invention aims to overcome the defects and provide an ideal substitute for antibiotics, namely, the Reg4 antibacterial peptide.

The invention provides an application of an active ingredient, which is characterized in that:

the active ingredients are Reg4 protein;

the Reg4 protein is shown as SEQ ID NO. 1:

>sp|Q9D8G5|REG4_MOUSE Regenerating islet-derived protein 4 OS＝Mus musculus OX＝10090 GN＝Reg4 PE＝2 SV＝1

MetAlaSerLysGlyValArgLeuLeuLeuLeuLeuSerTrpValAlaGlyProGluVal

LeuSerAspIleLeuArgProSerCysAlaProGlyTrpPheTyrTyrArgSerHisCys

TyrGlyTyrPheArgLysLeuArgAsnTrpSerHisAlaGluLeuGluCysGlnSerTyr

GlyAsnGlySerHisLeuAlaSerValLeuAsnGlnLysGluAlaSerValIleSerLys

TyrIleThrGlyTyrGlnArgAsnLeuProValTrpIleGlyLeuHisAspProGlnLys

LysGlnLeuTrpGlnTrpThrAspGlySerThrAsnLeuTyrArgArgTrpAsnProArgThrLysSerGluAla

ArgHisCysAlaGluMetAsnProLysAspLysPheLeuThrTrp

AsnLysAsnGlyCysAlaAsnArgGlnHisPheLeuCysLysTyrLysThr

such uses include the preparation of a medicament for treating or alleviating the symptoms of PAO1 infection.

Further, the invention provides an application of an active ingredient, which is further characterized in that:

the above uses also include at least one of the following uses:

use a. for the preparation of a medicament for the treatment and alleviation of a pulmonary inflammatory response induced by PAO 1;

use B. for the preparation of a medicament for the treatment and alleviation of lung injury caused by PAO1.

the use also includes the preparation of a medicament for the treatment and alleviation of weight loss conditions caused by PAO1 infection.

the use also includes use in the preparation of a medicament capable of binding PAO1.

the application comprises the preparation of the medicine for inhibiting the growth of PAO1 bacteria.

the above uses also include the preparation of a medicament for reducing PAO1 adhesion and invasion of alveolar epithelial cells.

the application also comprises the preparation of the medicine for reducing PAO1 colonization in the lung.

the application also comprises the application in preparing a medicine capable of inhibiting PAO1 migration.

the application also comprises the preparation of the medicine for relieving the invasion of PAO1 to the spleen.

In addition, the PAO1 chronic infection model experimental model provided by the invention is characterized by being modeled by the following steps:

s1, preparing an agar bead suspension containing PAO1 bacteria;

s2, injecting the agar beads into specific parts of the target model.

The invention has the following functions and effects:

the Reg4 (regenerating family, reg 4) is secreted protein expressed by the organism, and the research of the invention shows that the Reg4 has antibacterial activity on PAO1.

In the study of the invention, the Reg4 protein is incubated with PAO1, and the Reg4 protein can obviously inhibit the PAO1 from growing.

In the research of the invention, through successfully establishing a mouse PAO1 chronic infection model, the Reg4 is found to reduce the colonization of the PA01 in the lung and improve the lung pathological injury and inflammatory response of the PAO1 infected mouse.

Therefore, the invention considers that the Reg4 protein has stronger antibacterial activity, has the characteristics of small molecular mass, protease degradation resistance, broad antibacterial spectrum, different antibacterial mechanism from the traditional antibiotics, better antibacterial activity on super drug-resistant strains generated by the antibiotics, and the like, and is superior to the Reg4 protein which belongs to one of human antibacterial peptides, does not cause rejection reaction of organisms, and has no risks such as drug residues.

Therefore, the Reg4 protein can be used as a novel antibacterial peptide to replace the traditional antibiotics, improve the PAO1 chronic pulmonary infection, become the best medicament for treating the pseudomonas aeruginosa infection in the future, and provide a novel treatment strategy for clinical treatment of the PAO1 infection.

Drawings

FIG. 1Reg4 protein inhibits PAO1 growth

Wherein, fig. 1A is a graph of PAO1 bacterial growth kinetics (×pa01+pbs vs. pao1+10 μg/ml, p < 0.0001).

FIG. 1B shows the co-incubation of Reg4 protein with PAO1 bacteria for 4h OD ₆₀₀ And measuring the result.

FIG. 1C shows the results of plating a 24h bacterial plate by co-incubation of the Reg4 protein with PAO1 bacteria.

FIG. 1D shows the measurement of bacterial numbers at 24h of co-incubation of the Reg4 protein with PAO1 bacteria.

FIG. 2 flow chart of PAO1 infected mice and weight change of mice

Wherein, FIG. 2A is a flow chart of PA01 lung chronically infected mice.

FIG. 2B shows the change in Reg expression after PA01 infection.

FIG. 2C shows the body weight change of PA01 lung infected mice.

FIG. 3 pathological changes in the lung of PAO1 infected mice

Wherein, FIG. 3A is a mouse lung HE staining mirror (magnification:. Times.200 for each group).

FIG. 3B is a Masson's staining lens (magnification:. Times.200 for each group) of mice lungs.

FIG. 4 detection of the level of expression of related inflammatory factors in mouse Lung and alveolar lavage fluid

Among them, FIG. 4A shows the detection of the content of Reg4 in the alveolar lavage fluid of mice by ELISA.

FIG. 4A is an ELISA for detecting IL-22 content in mouse alveolar lavage fluid.

FIG. 4C shows the expression level of IL-1β in colon mucosal tissue of mice.

FIG. 4D shows the expression level of IL-10 in colon mucosal tissue of mice.

FIG. 4E shows the expression levels of CCL-28 in mouse colonic mucosal tissue.

FIG. 4F shows CXCL-2 expression levels in colon mucosal tissue of mice.

FIG. 5 bacterial content of mice lung, spleen and alveolar lavage fluid

Among them, FIG. 5A is the effect of recombinant murine Reg4 protein on PAO1 bacterial content in lung, spleen and alveoli.

FIG. 5B is a comparison of PAO1 bacteria content in the lungs;

FIG. 5C is a comparison of PAO1 bacteria content in spleen;

FIG. 5D is a comparison of PAO1 bacteria content in alveolar lavage fluid;

FIG. 6 influence of Reg4 protein on PAO1 adhesion and invasion of MLE 12 cells

Among them, FIG. 6A is the effect of recombinant murine Reg4 protein on PAO1 adhesion to MLE 12 cells.

FIG. 6B is a comparison of PAO1 bacteria content of adherent MLE 12 cells.

FIG. 6C is a graph showing the effect of recombinant murine Reg4 protein on PAO1 invasion of MLE 12 cells.

FIG. 6D is a comparison of PAO1 bacteria content of invading MLE 12 cells.

FIG. 7 influence of Reg4 protein on PAO1 adhesion and invasion of MH-S cells

Among them, FIG. 7A is the effect of recombinant murine Reg4 protein on PAO1 adhesion to MH-S cells.

FIG. 7B is a comparison of PAO1 bacteria content adhering to MH-S cells.

FIG. 7C is the effect of recombinant murine Reg4 protein on PAO1 invasion of MH-S cells.

FIG. 8 binding of Reg4 to PAO1

FIG. 8A shows the detection of PAO1 binding to Reg4 protein by ELISA.

FIG. 8B shows the PAO1 migration results in semi-solid agarose plates.

FIG. 8C shows the results of halo diameter measurements of PAO1 migration in semi-solid agarose plates.

Detailed Description

EXAMPLE 1 experiment of Reg4 in vitro inhibition of PAO1 growth

1.1 main materials

Pseudomonas aeruginosa PAO1 (ATCC-15692): bio-82076, available from beijing Bai-o-bordetella biotechnology limited.

1.2 method

1.2.1 Pseudomonas aeruginosa culture

0.5ml of TSA liquid culture was pipetted with a sterile pipette based on complete solubilization of the lyophilized powder in the lyophilization tube. The dissolved bacterial suspension was transferred to a tube containing 5ml of liquid medium and mixed well, and 100. Mu.l was transferred to solid medium. And (5) standing and culturing the liquid test tube and the inclined test tube. 100. Mu.l of PA01 bacteria solution was added to 1000ml of TSA medium containing 50. Mu.g/ml kanamycin, and the culture was carried out overnight at 37℃until the logarithmic phase, 4000rpm,20min, after which time the bacteria were collected by centrifugation, resuspended in PBS buffer, and the total number of bacterial colonies was measured by plate count method and the bacteria were stored at-80℃for use.

1.2.2 Reg4 protein concentration modulation

The drug concentration was adjusted to 1mg/ml with sterilized PBS buffer. All protein solutions were sterilized by filtration using a 0.22 μm filter before use and stored in sterile 1.5ml EP tubes.

1.2.3 Co-incubation of Reg4 protein with PAO1

10 μl of the resuscitated PA01 bacterial suspension was inoculated into a total of 200 μl containing different concentrations (0, 2 or 10 μg/ml) of Reg4 protein. By OD every 2 hours ₆₀₀ The measurement monitors the growth of bacteria.

PAO1 was diluted to a final concentration of 2X 10 with sterile PBS buffer ⁵ CFU/ml. 4 tubes of 15ml centrifuge tubes were taken and arranged in a row, with 1980. Mu.l of TSA medium and 20. Mu.l of diluted PAO1 broth (as control group), with 1978. Mu.l of TSA medium, 20. Mu.l of broth and 2. Mu.l of recombinant murine Reg4 protein (as 1. Mu.g/ml group) added to tube 1, with 1960. Mu.l of TSA medium, 20. Mu.l of broth and 20. Mu.l of recombinant murine Reg4 protein (as 10. Mu.g/ml group) added to tube 3, and mixed well. The lid was covered and incubated in a 37℃incubator for 16 hours.

1.2.4 determination of bacterial colony count

Continuously diluting the incubated bacterial liquid with sterilized water to a final concentration of 1×10 ⁷ CFU/ml. 100 μl of the incubated bacterial liquid was uniformly smeared on a kanamycin agar plate. The flat plate is placed on an ultra-clean bench for 30 minutes, so that the bacterial liquid permeates into the surface layer of the culture medium. The plate is placed in an incubator at 37 ℃ for cultivation for 24 hours, the number of colonies on the plate is calculated after 24 hours, and the number of colonies in each group is obtained after multiplication by dilution.

1.3 results

1.3.1 determination of bacterial colony count

Bacterial plate counts were performed according to GB 4789.2-2016. PAO1 colony count was 8.3X10 ⁹ CFU/ml。

1.2.2 recombinant Reg4 protein inhibits PAO1 bacterial growth

PAO1 was incubated with recombinant murine Reg4 protein at different concentrations, respectively, for OD ₆₀₀ Measurement (FIGS. 1A-B). The results of the bacterial growth kinetics curve show that the PAO1 growth can be effectively inhibited when the concentration of the Reg4 protein reaches 10 mug/ml. PAO1 was incubated with 0. Mu.g/ml, 1. Mu.g/ml and 10. Mu.g/ml recombinant murine Reg4 protein, respectively, for 16h and the total bacterial colony was determined (FIGS. 1A-B). The results showed that the total number of bacteria in the PAO1+0. Mu.g/ml Reg4 group was 8.37X10 ⁸ CFU/ml, after administration of 1. Mu.g/ml recombinant murine Reg4 proteinThe total bacterial count is reduced to 6.73X10 ⁸ CFU/ml reduced the total number of recombinant murine Reg4 protein bacteria by about 5-fold given 10. Mu.g/ml, with a total of 1.63×10 ⁹ CFU/ml (p=0.0023) (fig. 1C-D). The result shows that the Reg4 recombinant protein can effectively inhibit the growth of PAO1.

Example 2 in vivo bacteriostasis experiments

2.1 Main Material

(1) C57BL/6 mice: shanghai Ji Hui laboratory animal feeding Co.Ltd

(2) Lavage needle 8: GWZ-8-45, shanghai Ming Dynasty Biotech Co., ltd

2.2 method

2.2.1 Regulation of protein concentration

The drug concentration was adjusted to 1mg/ml with sterilized PBS buffer. All proteins were sterilized by filtration using a 0.22 μm filter before use and stored in sterile 1.5ml EP tubes.

2.2.2 preparation of PAO1 agar beads suspension

(1) 100ul of frozen stock solution was pipetted onto TSA plates and incubated overnight (37 ℃,200rpm, about 16 h)

(2) Single colonies were inoculated into 5ml TSB broth (15 ml centrifuge tube) and cultured overnight (37 ℃ C., 200rpm, about 16 h)

(3) Bacterial overnight cultures were taken and the OD was measured by diluting the bacteria with PBS (1:50) ₆₀₀ Values.

(4) The bacteria overnight culture was diluted: the 2OD bacteria were taken and placed in a tube containing 20ml of fresh TSB and incubated at 37℃and 200rpm for about 3-4 hours until 10-15OD (bacteria reached log phase).

(5) TSA plates (TSB+1.5% agar) were prepared, autoclaved and equilibrated in a water bath at 50 ℃. 150ml of pre-autoadhesion heavy mineral oil was added in a water bath at 50 ℃.

(6) When the Pseudomonas aeruginosa reached the log phase, bacterial cells were collected at 2700 Xg at 4℃for 15min by centrifugation and the supernatant was discarded.

(7) The bacterial pellet was resuspended in 1ml sterile PBS and resuspended.

(8) 1ml of bacterial suspension was mixed with 9ml of TSA liquid pre-heated at 50 ℃. 10ml of TSA-Pseudomonas aeruginosa mixture was added to heavy mineral oil (preheated at 50 ℃) and stirred immediately at room temperature for 6 minutes. Agitation must create a visible vortex in the oil.

(9) The mixture was cooled to 4 ℃ and stirred at minimum speed for 35 minutes.

(10) The agar-bead-oil mixture was left in ice for an additional 20 minutes.

(11) The agar beads were transferred to 50ml Falcon tubes and centrifuged at 2 Xg for 15min at 4 ℃. The mineral oil was removed by six washes with sterile PBS. After three washes, the beads may be pelleted by gravity without the use of a centrifuge. After the last wash, the agar beads were resuspended in 20-30ml PBS.

(12) A small aliquot of beads (about 0.5 ml) was taken and aseptically homogenized.

(13) 100ul of homogenized beads were taken and diluted in 900ul of sterile PBS. Dilution to 10 is continued at 1:10 ^-6 。

(14) Serial dilution of plates on TSA plates, including dilution to 10 ^-6 The samples were double undiluted and incubated at 37 ℃.

(15) The bead diameter was measured in several areas using an inverted light microscope. The beads must be between 100 and 200 meters in diameter.

(16) The beads were stored overnight at 4 ℃.

2.2.3 mice chronic infection experiments

Wild Type (WT) C57BL/6 mice (female: male=1:1) of 8 weeks old were taken as the infection subjects, randomly divided into 6 groups, and grouped as follows: the experimental procedure is shown in fig. 2A, including Con group (n=10), pao1+pbs group and pao1+reg4 group (n=10).

Plate coating method to determine the number of bacteria in agar bead suspension, dilution with PBS to 4X 10 ⁷ CFU/ml agar bead suspension. Mice were anesthetized with 1% sodium pentobarbital solution, placed in the supine position, the fur disinfected, the skin exposed trachea vertically incised, and cannulated with a sterile, flexible 22G intravenous catheter, with care taken to remove the probe while moving down into the trachea. The catheter is inserted into the trachea (not too deep) and stopped before reaching the carina (bifurcation). Immediately, 50ul of the agar bead suspension was taken with a 1ml syringe and connected to the catheter. Lightly push the injectionThe plunger of the injector allows the beads to be implanted into the lungs. The mice were stood up for 1 minute after dosing to ensure that bacterial fluid entered the lungs on both sides. The incision is sutured. The animals were placed on the heating pad until fully awake. From day 1 post-infection, mice given daily injections of 100 μl of Reg4 protein (10 μg/ml) into the intraperitoneal cavity of PAO1+Reg4 groups, con groups and PAO1+PBS groups were given metered doses of sterile PBS solution for intraperitoneal injection.

Mice body weight was recorded daily at regular intervals and material was drawn on day 28 of infection. The eyeballs were bled after anesthesia of the mice. Intact lungs were dissected out, left lung fixed in 4% paraformaldehyde, right lung used to perform lung tissue homogenization to count bacterial load (CFU). Part of the mice were spleen fixed in 4% paraformaldehyde and part of the mice were spleen used for tissue homogenization to count bacterial load.

A portion of mice was collected from alveolar lavage: the mice were anesthetized, the airways and lung tissues were fully exposed by surgery, a "v" incision was made along the airways, catheters were inserted into the incisions, and were fixed, 700 μl of sterile PBS was aspirated by a syringe, and alveolar lavage fluid was repeatedly perfused, aspirated, and collected.

2.2.4 measurement of organ bacterial load

The lungs and spleens of the mice were taken with sterile scissors and forceps, and a sterile PBS solution was added according to the weight, and homogenization treatment was performed in a sterile environment. The homogenates were then serially diluted in gradient, 100. Mu.l of each homogenate was pipetted onto TSA plates containing kanamycin, incubated overnight, and colonies were observed and counted. 100. Mu.L of alveolar lavage was plated on TSA plates containing kanamycin, cultured overnight, and colonies were observed and counted.

2.2.6 Lung histopathological scoring

The lung of the mouse is taken and fixed in 4% paraformaldehyde, and the pathological changes such as the integrity of the lung result, inflammatory cell infiltration and the like are observed under a microscope through conventional dehydration, embedding, slicing, HE staining and Masson staining.

2.2.7 detection of inflammatory Lung reactions

The amounts of Reg4 and IL-22 in the mouse alveolar lavage fluid were measured according to the protocol provided by the reagent manufacturer. RNA extraction kit is used for extracting RNA of mouse lung tissue, and the expression level of mouse lung related inflammatory genes including IL-1 beta, IL-10, CXCL-2, CCL-8 and CCL-2 is detected by quantitative PER.

2.3 results

2.3.1 Reg4 protein can improve weight loss of mice infected with PAO1

The results of the ELISA kit for detecting the content of Reg4 in the alveolar lavage fluid of each group of mice show that the content of Reg4 in the alveolar lavage fluid of PAO1 infected mice is obviously increased compared with that of PBS group of mice as shown in FIG. 2B. Weight loss for mice in each group as shown in fig. 2C, mice in the Con group not given PAO1 infection had a slow increase in weight, while mice in each group with PAO1 infection had a different degree of weight loss. On day 2 post-infection, the weights of mice in pao1+pbs group and pa01+reg4 group were 92.6% and 93% of the initial weight, respectively, significantly lower than in Con group (p both < 0.01). On day 3 post-infection, mice in pao1+reg4 and PA01 group began to gain in body weight. From day 22 to day 28 of infection, the weight of the pao1+reg4 group mice was significantly better than the pao1 group mice (p < 0.05). The results show that the Reg4 protein treatment can improve the weight condition of mice infected with PA 01.

2.3.2 Reg4 protein can relieve lung injury caused by PAO1

The pathology results are shown in fig. 3A-C, and compared with the ileum and colon tissues of the Con mice, the pao1+pbs mice were stained for pulmonary edema, the alveoli were stained for a large number of neutrophil infiltrates, the Masson stained for pulmonary fibrosis, and the pulmonary fibrosis was stained for a large number of fiber deposits. And the lung injury of mice in PAO1+Reg4 group is improved, inflammatory cell infiltration in the lung is reduced, and pulmonary fibrosis is reduced. Quantitative analysis found that neutrophil scores and Ashcroft score were lower in lung tissue of mice in pao1+reg4 group than in pao1 group. The results show that the Reg4 protein can effectively relieve chronic injury of lung caused by PAO1.

2.3.3 Reg4 protein can improve PAO 1-induced pulmonary inflammatory response

The IL-22 content of the alveolar lavage fluid of each group of mice was measured using ELISA kit. The results are shown in FIG. 4A, wherein the IL-22 content of PAO1 infected small alveolus lavage fluid is obviously increased compared with that of mice in the uninfected group, and the IL-22 content in serum can be obviously reduced by the intervention of Reg4 protein. The expression level of the mouse lung-related inflammatory gene was detected using quantitative PCR, and the results are shown in FIGS. 4B-F. The expression levels of IL-1 beta, IL-10, CXCL-2 and CCL-28 in lung tissues of PA-infected mice are obviously increased compared with those of uninfected mice, and the expression levels of IL-1 beta, IL-10, CXCL-2, CCL-8 and CCL-2 in lung tissues of mice can be obviously reduced by the intervention of the Reg4 protein. The above results indicate that Reg4 protein can improve PAO 1-induced pulmonary inflammatory response.

2.3.4 Reg4 protein can reduce PAO1 colonization in lung

The number of PAO1 bacteria in the lung and alveolar lavage fluid of each group of mice was examined and the results are shown in FIGS. 5A-C. No PAO1 was detected in both the lungs and alveolar lavage of the Con group mice, while a large amount of PAO1 was detected in both the small lungs and alveolar lavage of the PAO1 group. The PAO1 numbers in lung and alveolar lavage fluid were significantly reduced in the pao1+reg4 group following reg4 protein treatment (p < 0.05).

2.3.5 The Reg4 protein can reduce the invasion condition of PAO1 on the spleens of mice, and the number of PAO1 bacteria in the spleens of each group of mice can be detected, and the results are shown in figures 5A and D. PAO1 was not detected in the lung external organs of Con group, and PAO1 was found to colonize in large amounts in the spleen of PAO 1-infected mice. The number of PAO1 in the spleen was significantly reduced in the PAO1+Reg4 group compared to the PA01 group. The results show that the Reg4 protein can effectively inhibit PAO1 from invading other organs and tissues outside the lung.

EXAMPLE 3 Reg4 protein reduces PAO1 adhesion and invasion of alveolar epithelial cells

3.1 Main Material

(1) Mouse alveolar epithelial cells (mouse alveolar epithelial cells, MLE-12) C432, shanghai Yubo Biotechnology Co., ltd

(2) Mouse alveolar macrophage (mouse alveolar macrophages, MH-S) C4704, shanghai Yu Bobo Biotechnology Co., ltd

(2) 24-well cell standard culture plate: 3524 Shanghai Baisai Biotechnology Co., ltd

(3) Gentamicin sulfate: PXTG80002, shanghai round-the-sea Biotechnology Co., ltd

3.2 method

(1) MLE-12 was routinely subcultured in DMEM containing 10% fetal bovine serum,In a cell culture medium of 100U/mL streptomycin and 100U/mL penicillin, the cells were cultured in a CO2 incubator (5% CO2, 95% air) at 37℃and the culture medium was changed every 48 hours. After the cells are grown and fused to 70% -80%, the cells are digested and passaged by 0.25% pancreatin-EDTA, and the cells are passaged for about 5 times, and the cells are tested, and the concentration is adjusted to be about 5 multiplied by 10 ⁵ cell/ml was ready for use.

(2) Effect of Reg4 on PAO1 adhesion to MLE-12 cells

Cell suspension (1 mL/well) was added to a 24-well cell culture plate, incubated at 37℃in a 5% CO2 incubator, and when cells were confluent into a monolayer (cell number: about 5X 10) ⁵ cells/well), PBS was washed 3 times, and PAO1 was co-cultured with MLE-12 cells at an infection ratio of 100:1 for 2h (centrifugation at 500 Xg for 5min to promote binding of bacteria to epithelial cells). 10. Mu.g/ml of Reg4 protein was added to the Reg4 group 10min after the bacteria addition, and an equal amount of PBS solution was added to the PBS group. After the incubation, the cells were washed 3 times with PBS to wash out bacteria that were not attached to the cells. Then, 100. Mu.L of 0.5% Triton X-100 was added thereto and the mixture was allowed to act for 5 minutes, followed by repeated pipetting to lyse the cells. Lysates were serially diluted 10-fold with PBS buffer, and appropriate dilutions were plated on Luria-Bertani (LB) plates and incubated at 37℃for 24h for colony counting, with 3 replicate wells.

(3) Effect of Reg4 on PAO1 invasion of MLE-12 cells

Culture methods were as above, after PAO1 was incubated with cells for 2 hours, the cells were rinsed 3 times with PBS to remove non-adherent bacteria, and DMEM medium containing gentamicin (100. Mu.g/mL) was added thereto, and incubated in a 5% CO2 cell incubator at 37℃for 2 hours to kill non-invasive PAO1 adherent to the cell surface. Cells were washed 3 times with PBS, 100. Mu.L of 0.5% Triton X-100 was added to each well and allowed to act for 5min, and the cells were lysed and detached by repeated blowing. Serial dilution of the lysate in 10 times ratio is carried out, a proper amount of diluent is inoculated on an LB plate for viable bacteria plate counting, and 3 repeated holes are arranged.

(4) MH-S was routinely subcultured in cell culture medium containing 10% fetal bovine serum RPMI-1640medium, 100U/mL streptomycin and 100U/mL penicillin, and incubated in a CO2 incubator (5% CO2, 95% air) at 37℃with medium changes every 48 h. The concentration is adjusted to be about 5 multiplied by 10 ⁵ cell/mL ready for use. (5) Reg4 adhesion of MH-S cells to PAO1Influence of (2)

Cell suspension (1 mL/well) was added to 24-well cell culture plates, incubated at 37℃in a 5% CO2 incubator, and PAO1 was co-cultured with MLE-12 cells at an infection ratio of 100:1 for 2h (centrifugation at 500 Xg for 5min to promote binding of bacteria to epithelial cells). 10. Mu.g/ml of Reg4 protein was added to the Reg4 group 10min after the bacteria addition, and an equal amount of PBS solution was added to the PBS group. After the incubation, the cells were washed 3 times with PBS to wash out bacteria that were not attached to the cells. Then, 100. Mu.L of 0.5% Triton X-100 was added thereto and the mixture was allowed to act for 5 minutes, followed by repeated pipetting to lyse the cells. Lysates were serially diluted 10-fold with PBS buffer, appropriate dilutions were plated on LB plates, incubated at 37℃for 24h for colony counting, and 3 replicate wells were made.

(6) Effect of Reg4 on PAO1 invasion of MH-S cells

Culture method as above, after PAO1 and cells were incubated for 2 hours, PBS was used for washing 3 times to remove non-adherent bacteria, and then RPMI-1640medium containing gentamicin (100. Mu.g/mL) was added thereto, and incubated in a 5% CO2 cell incubator at 37℃for 2 hours to kill non-invasive PAO1 adhering to the cell surface. Cells were washed 3 times with PBS, 100. Mu.L of 0.5% Triton X-100 was added to each well and allowed to act for 5min, and the cells were lysed and detached by repeated blowing. Serial dilution of the lysate in 10 times ratio is carried out, a proper amount of diluent is inoculated on an LB plate for viable bacteria plate counting, and 3 repeated holes are arranged.

3.3 results

3.3.1 Reg4 protein reduces PAO1 adhesion and invasion of MLE-12 cells

Reg4 was incubated with MLE-12 cells and the bacterial content of adherent and invasive cells was measured by plate counting, and the results are shown in FIGS. 6A-D. In the Reg4 protein treated group, the number of PAO1 that adhered to and invaded MLE-12 cells was smaller than in the PBS treated group, indicating that Reg4 protein reduced PAO1 adhesion and invasion of MLE-12 cells.

3.3.2 Reg4 protein can reduce adhesion of PAO1 and invasion of MH-S cells

Reg4 was incubated with MH-S cells and the bacterial content of adherent and invasive cells was measured by plate counting and the results are shown in figures 7A-D. In the Reg4 protein-treated group, the number of PA01 adhering to and invading MH-S cells was smaller than in the PBS-treated group, indicating that Reg4 protein reduced PAO1 adhesion and invading MH-S cells.

EXAMPLE 4 binding of Reg4 protein to PAO1 inhibiting its migration

4.1 Main Material

(1) 96-well EIA/RIA plate: 9018, shanghai Yu Ind Biotech Co., ltd

(2) Reg4 antibody: bs-10036R, shanghai Kogya Biotechnology Co., ltd

(3) TMB color development liquid: p0209-100ml, shanghai Biyun biotechnology Co., ltd

4.2 method

4.2.1 ELISA assay detection of binding to Reg4 protein

ELISA plates were incubated with 100ul of a PA01 bacterial suspension (bacterial count 5X 10) ⁷ CFU/ml) ug/ml Reg4 protein or BSA were coated overnight (4 ℃ C., 16 h). The plates were washed 3 times with PBS, and different concentrations of Reg4 protein and mutant Reg4 protein (0, 2, 4, 6, 8 and 10 ug/ml) were formulated with PBS containing 1% BSA, and the protein-containing solutions were incubated with ELISA plates for 2h (37 ℃). Plates were washed 3 times with PBS, reg4 antibody (1:1000) was added and incubated for 2h. The plates were washed 3 times with PBS, secondary antibodies (1:5000) were added and incubated for 2h at 37 ℃. PBS was used to wash the plate 3 times, TMB was added for color development, and OD was measured ₄₅₀ Values.

4.2.2 Effect of Reg4 protein on PAO1 migration

PA01 in the logarithmic growth phase was incubated with Reg4 protein for 1h. 10ul of the culture was inoculated on a semi-solid agar plate containing 0.3% agar, and after incubation at 37℃for 10 hours, the bacteria moved to form a halo, and the size of the halo diameter was measured.

4.3 results

4.3.1 Reg4 protein and PAO1 binding to each other

The binding of PAO1 flagella to Reg4 was detected using ELISA assay and the results are shown in FIG. 8A. PAO1+BSA group OD ₄₅₀ No significant change in the values was seen, but PAO1+Reg4 group OD ₄₅₀ The value increased with increasing dose of Reg4 protein, suggesting that Reg4 protein binds to PAO1.

4.3.2 Reg4 protein inhibits PAO1 migration

The migration of PAO1 in semi-solid agar plates was compared and the results are shown in FIGS. 8B-C. PBS group PAO1 migrated on semi-solid agar plates to form halos with a diameter of (7.75.+ -. 0.27) cm, whereas after administration of Reg4 protein, reg4+PAO1 group bacteria had a halo diameter reduced to (6.21.+ -. 0.46) cm, suggesting that Reg4 protein could inhibit PAO1 migration.

Sequence listing

<110> Shanghai Lung department Hospital

Shanghai Institute of Pediatrics

<120> application of Reg4 antibacterial peptide in treatment of pseudomonas aeruginosa infectious pneumonia

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 157

<212> PRT

<213> Artificial

<400> 1

Met Ala Ser Lys Gly Val Arg Leu Leu Leu Leu Leu Ser Trp Val Ala

1 5 10 15

Gly Pro Glu Val Leu Ser Asp Ile Leu Arg Pro Ser Cys Ala Pro Gly

20 25 30

Trp Phe Tyr Tyr Arg Ser His Cys Tyr Gly Tyr Phe Arg Lys Leu Arg

35 40 45

Asn Trp Ser His Ala Glu Leu Glu Cys Gln Ser Tyr Gly Asn Gly Ser

50 55 60

His Leu Ala Ser Val Leu Asn Gln Lys Glu Ala Ser Val Ile Ser Lys

65 70 75 80

Tyr Ile Thr Gly Tyr Gln Arg Asn Leu Pro Val Trp Ile Gly Leu His

85 90 95

Asp Pro Gln Lys Lys Gln Leu Trp Gln Trp Thr Asp Gly Ser Thr Asn

100 105 110

Leu Tyr Arg Arg Trp Asn Pro Arg Thr Lys Ser Glu Ala Arg His Cys

115 120 125

Ala Glu Met Asn Pro Lys Asp Lys Phe Leu Thr Trp Asn Lys Asn Gly

130 135 140

Cys Ala Asn Arg Gln His Phe Leu Cys Lys Tyr Lys Thr

145 150 155

Claims

1. Use of an active ingredient characterized in that:

the active ingredient is Reg4 protein;

the application comprises the steps of preparing a medicament for treating or relieving the infection symptom of pseudomonas aeruginosa;

wherein the sequence of the Reg4 protein is shown as SEQ ID NO. 1.

2. Use of an active ingredient according to claim 1, characterized in that:

the use further includes at least one of the following uses:

use a. for the preparation of a medicament for the treatment and alleviation of pulmonary inflammatory reactions induced by pseudomonas aeruginosa;

the application B is used for preparing medicines for treating and relieving lung injury caused by pseudomonas aeruginosa.

3. Use of an active ingredient according to claim 1, characterized in that:

the use also includes the preparation of a medicament for the treatment and alleviation of weight loss conditions caused by pseudomonas aeruginosa infection.

4. Use of an active ingredient according to claim 1, characterized in that:

the application comprises the preparation of a medicament for inhibiting the bacterial growth of pseudomonas aeruginosa.

5. Use of an active ingredient according to claim 1, characterized in that:

the use also includes a method for the preparation of a medicament for reducing pseudomonas aeruginosa adhesion and invasion of alveolar epithelial cells.

6. Use of an active ingredient according to claim 1, characterized in that:

the application also comprises the preparation of the medicine for reducing the colonization of the lung by pseudomonas aeruginosa.

7. Use of an active ingredient according to claim 1, characterized in that:

the application also comprises the application in preparing a medicine capable of inhibiting the migration of pseudomonas aeruginosa.

8. Use of an active ingredient according to claim 1, characterized in that:

the application also comprises the preparation of the medicine for relieving the spleen invasion of the pseudomonas aeruginosa.