CN114712404A - Use of nocardia rubra cell wall skeleton as neutrophil regulator - Google Patents

Abstract

The present application relates to the use of nocardia rubra cell wall skeleton as a neutrophil modulator. The present application provides a neutrophil regulator comprising a component derived from the cell wall of nocardia rubra, which can regulate chemotactic, phagocytic, bactericidal functions (reactive oxygen species ROS production, degranulation) of neutrophils. The neutrophil modulators of the present application may also improve the local immune microenvironment of chronically infected wounds.

Description

Use of nocardia rubra cell wall skeleton as neutrophil regulator

The present application claims priority from chinese patent application (application No. 202110724324X) filed on 29.06/29/2021.

Technical Field

The present disclosure relates to the fields of medicine, microbiology, and biopharmaceutical industry. In particular, the present application relates to the use of nocardia rubra cell wall skeleton as a neutrophil modulator.

Background

Nocardia rubra (Nocardia rubra) is one of Nocardia. The red nocardia cell wall skeleton can be prepared by fermenting, cell breaking and protease degrading the red nocardia thallus.

In the prior art, the Nocardia rubra cell wall skeleton is commercially available, specifically from Liaoning Gersted biopharmaceutical GmbH (Nr-CWS). Nocardia rubra cell wall skeleton has been used for the treatment of cervical erosion, precancerous lesions of cervical cancer (CN101073583A), anti-human papilloma virus (CN1935262A), skin lesions or skin ulcers (CN101209267A), fungal infections, herpes simplex, herpes zoster (CN 1879661A).

However, the role of nocardia rubra cell wall scaffolds in the regulation of neutrophils and in the local immune microenvironment that improves chronically infected wounds has not been reported in the prior art.

Disclosure of Invention

According to some embodiments of the present application, there is provided the use of nocardia rubra cell wall skeleton as a neutrophil modulator or bacteriostatic agent.

According to further embodiments of the present application, there is provided a use of nocardia rubra cell wall scaffold in the manufacture of a medicament for treating a chronically infected wound.

In some embodiments, the neutrophil modulator or drug is prepared in any one of the dosage forms selected from the group consisting of: suppository, unguent, cream, lotion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, and powder; wherein the paste is selected from: ointment, plaster, cream.

According to some embodiments, there is provided a method of modulating neutrophils under in vitro culture conditions, comprising the steps of: providing an effective amount of nocardia rubra cell wall skeleton to neutrophils in an inflammatory state.

According to some embodiments, there is provided a method of modulating neutrophils under in vitro culture conditions, comprising the steps of: neutrophils in an inflammatory state are contacted (e.g., 30 minutes) with an effective amount of nocardia rubra cell wall skeleton.

Methods for modulating neutrophils in vitro culture conditions are not useful for the prevention, diagnosis, treatment of disease in animals, particularly humans.

In some embodiments, the neutrophil modulator or drug comprises:

-nocardia rubra cell wall skeleton, and

-a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier is selected from, but not limited to: fillers, stabilizers (e.g., trehalose, glycine), flavoring agents (e.g., xylitol), disintegrating agents (e.g., sodium carboxymethylcellulose), binders (e.g., gelatin), lubricants (e.g., magnesium stearate).

In some embodiments, the stabilizing agent is selected from one or a combination of: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, and dextran.

In some embodiments, the flavoring agent is selected from one or a combination of the following: sucrose, monosaccharide, saccharin sodium, aspartame, sorbitol, xylitol and mannitol.

In some embodiments, the binder is selected from one or a combination of: sodium carboxymethylcellulose, hypromellose, and gelatin.

In some embodiments, the lubricant is selected from one or a combination of: comprises talcum powder, magnesium stearate and superfine silica powder.

In some specific embodiments, pharmaceutically acceptable carriers suitable for use herein may also be mentioned, such as, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethylcellulose, erythritol, gelatin, magnesium stearate, a propellant, a humectant, a solvent, a solubilizer, an emulsifier, an antioxidant, a pH regulator and a preservative. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropyl alcohol, tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose acetate isobutyrate, sucrose anhydride tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-tert-butyl hydroxytoluene, potassium sorbate, phosphate buffer solution, Triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

In some embodiments, the pharmaceutically acceptable carrier is dextran.

In some embodiments, the neutrophil modulator or medicament of the present application is administered 1-3 times a day, or once every two days. Different dosages are used depending on the area and extent of the lesion in the patient, and usually 1. mu.g/unit dose/dose to 1000. mu.g/unit dose/dose. Specifically, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μ g per unit dose per time, and ranges between any two of the foregoing values.

In some embodiments, the administration cycle lasts from 2 days to 6 months, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, or more, and ranges between any two of the foregoing.

In some embodiments, the nocardia rubra cell wall skeleton is a commercially available nocardia rubra cell wall skeleton (e.g., trade name "nocardia rubra cell wall skeleton (Nr-CWS)").

In other embodiments, the nocardia rubra cell wall skeleton is obtained by a process comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) optionally, subjecting the disruption product to a lipid removal operation;

3.2) optionally, subjecting the disruption product to a nucleic acid removal operation;

3.3) optionally, subjecting the disruption product to a protein removal operation;

3.4) optionally, subjecting the disruption product to a cell membrane removal operation;

3.5) obtaining a product derived from the cell wall of the red nocardia;

4) optionally, freeze-drying the product derived from the nocardia rubra cell wall;

5) optionally, subpackaging;

wherein the content of the first and second substances,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged in sequence or in parallel,

step 4) and step 5) can be interchanged in sequence;

the average particle size of the disruption is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500 nm;

preferably, the dispensing means into containers;

the container is selected from: bottles, tubes, bags, pouches, plates, ampoules, injection devices, aluminum film packaging, dressings, pouches, films.

For disruption of nocardia rubra, the aim is to remove intracellular material. Therefore, the techniques of ultrasonic crushing, high-pressure homogenizer crushing, lysozyme and the like can be adopted. The skilled artisan will appreciate that any known or future method suitable for disrupting gram-positive bacteria is suitable for use in the presently disclosed embodiments.

The skilled person has the ability to adapt the specific parameters and equipment of culturing, disruption, separation, collection, removal of impurities, packaging in response to the subsequent application (e.g. topical application) of the active ingredient (cell wall and its constituent components) in order to avoid introduction of factors in the preparation step that affect the subsequent application.

In some embodiments, the lipids in the disrupted product are removed using an organic solvent. In some embodiments, the DNA and RNA in the disruption products are removed using a nuclease. In some embodiments, the protein in the disruption product is degraded using a hydrolase. In some embodiments, the cell membranes in the disruption products are removed using a surfactant.

In some embodiments, the average particle size of the disruption is from 10nm to 1000 nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190nm ± 10nm, and ranges between any two of the foregoing values. The methods of particle size testing are well known in the art.

In some specific embodiments, the average particle size of the disruption is from 10nm to 800 nm.

In other specific embodiments, the average particle size of the disruption is from 10nm to 500 nm.

In specific embodiments, the dispensing means into a bottle or ampoule. Just prior to use, a solvent (e.g., sterile water) is added to the vial or ampoule. As an example, the bottle is a vial (visual, made of borosilicate glass or soda lime glass).

In some specific embodiments, the subject is an animal other than a human, e.g., a farm animal, a pet, a work animal, an ornamental animal, a production animal, a laboratory animal (e.g., rat, mouse, guinea pig, rabbit, dog, primate).

Drawings

Fig. 1A to 1D: the effect of the cell wall skeleton on neutrophil chemotactic function.

Fig. 2A to 2C: the cell wall skeleton partially restores the chemotactic function of damaged neutrophils in an inflammatory state. P <0.05, P <0.01, P <0.001, P <0.0001, ns means no statistical significance (note: RC is a group of cell wall scaffolds).

Fig. 3A to 3B: the cell wall skeleton can reverse the decrease of the phagocytic function of the neutrophil caused by inflammation and increase the phagocytic efficiency of the neutrophil to bacteria.

FIG. 4: mean fluorescence intensity of reactive oxygen species ROS of neutrophils.

Fig. 5A to 5C: mean fluorescence intensity of each grade of particle of neutrophils.

Fig. 6A to 6D: cytokine expression in control and cell wall scaffold treated groups.

Fig. 7A to 7D: expression of chemokines in control and cell wall scaffold treated groups.

Detailed Description

Cell wall skeleton

Constituents that constitute the major structure of the cell wall; but not to be understood as merely representing cross-linked network entities within the cell wall, the skilled person will appreciate that the term does not exclude other cell wall components adsorbed, bound, carried on the cross-linked network entities.

Nocardia rubra

Nocardia rubra refers to the species Nocardia rubra (Nocardia rubra) belonging to the genus Nocardia.

Nocardia rubra cell wall

In the present disclosure, "nocardia rubra cell wall" can be understood as both an intact cell wall and an incomplete cell wall (e.g., disrupted, or partially degraded). The skilled artisan, in light of the present disclosure, will appreciate that the component exhibiting the desired activity is derived from the cell wall of nocardia rubra (e.g., is the cell wall itself or a component thereof). Therefore, various forms of intact cell walls, disrupted cell walls, incomplete degradation products of cell walls, constituents of cell walls, extracts of cell walls, etc., which are allowed to be used in clinical applications, are included in the scope of the present disclosure.

In a specific example, the nocardia rubra cell wall skeleton is the product of the bacteria after disruption and removal of impurities (proteins, nucleic acids, cell membranes, lipids).

In another specific example, the nocardia rubra cell wall skeleton is a commercially available product, and an example is a product represented by national drug standard S20030009.

The skilled artisan will appreciate that S20030009 is an administrative license number issued by the drug administration, which number will vary with the renewal of the certificate, the law, and the adjustment of numbering rules. However, the product standard, product parameters, production process and quality requirements represented by the changed number are not changed. Thus, S20030009 is to be understood in this disclosure as S20030009 and equivalents thereof.

Neutrophil modulators

A neutrophil modulator refers to a substance that is capable of modulating the activity, function, behavior of neutrophils in vivo, in vitro, or ex vivo.

"modulating" according to some embodiments refers to any one or combination of:

-restoring the chemotactic function of damaged neutrophils in an inflammatory state;

-increasing the number of chemotactic cells;

-reversing the inflammatory-induced decrease in neutrophil phagocytic function;

-increasing the phagocytic efficiency of neutrophils;

-inhibiting the production of reactive oxygen species ROS;

-inhibiting degranulation of neutrophils;

-inhibiting over-activation of neutrophils.

In particular examples, the neutrophil modulator comprises an effective amount of a cell wall scaffold of the present disclosure.

Unit dose

The neutrophil modulators of the present disclosure may be prepared in the form of a unit dose (or unit formulation).

"optional" means that the subsequently described events thereof can occur, but need not occur; as the case may be. For example, "optionally, to dispense" means to allow product to be dispensed, but not necessarily; whether the split charging or not does not influence the realization of the technical effect.

The terms "a", "an", "the", and "the" include plural references unless expressly stated otherwise.

When referring to a range of values (e.g., 60 μ g to 120 μ g), this is a shorthand way of referring specifically to each value in the range, including fractional and integer values.

The present disclosure is further described below in conjunction with the examples. These examples are not intended to limit the scope of the present disclosure. When the specific conditions are not specified, the operation is carried out under the conventional conditions, as recommended by the raw material supplier. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1 commercially available Nocardia rubra cell wall skeleton

Red Nocardia cell wall skeleton (Nr-CWS) was purchased from Liaoning Gersted biopharmaceuticals GmbH, and is available in Chinese Standard S20030009 (solid content of cell wall skeleton in each bottle should be no less than 60 μ g, wherein muramic acid content is no less than 1.0 μ g, sugar content is no less than 4.0 μ g, and reconstitution volume is 2.0 ml).

Example 2 preparation of cell wall skeleton of Nocardia rubra

The preparation method of the cell wall skeleton of the Chinese medicine standard character S20030009 is basically not obviously different from the following steps, but is adjusted due to different production scales.

1. The cells were cultured according to a known method and collected. The cells are disrupted (e.g., by sonication or high pressure homogenizer). It is also permissible to crush the cells by any suitable method known in the art. The condition of the fragmentation is checked under a microscope, the number of the visible bacteria in each visual field is not more than 5, and the condition that a plurality of visual fields (10 to 30) meet the standard is checked to be qualified.

2. Removing nucleic acid: the disrupted supernatant was centrifuged, and DNase and RNase were added to the obtained precipitate to remove nucleic acids according to the procedures recommended by the enzyme supplier.

3. Removing protein: the precipitate is added with a common protease (e.g. trypsin) and the protein is removed according to the recommended procedures of the enzyme supplier.

4. Removing lipid: adding organic reagent (such as one or combination of acetone, diethyl ether, and ethanol) into the precipitate, and removing lipid according to conventional operation in the art.

5. Removing cell membranes: TritonX-100 was added to the precipitate, and the precipitate was collected by centrifugation and rinsed with PBS.

It should be understood that, between the above steps of removing impurities, the skilled person can adjust the order so as to make the steps compatible. After removing the non-cell wall components, the precipitate was redissolved in water for injection for use. Optionally, it can be sterilized at 115 ℃ for 20-30 minutes as a stock solution of the cell wall skeleton (comprising mainly the cell wall skeleton and its constituents).

Example 3 exemplary preparation of neutrophilic granulocyte modulators

1. The product obtained in example 2 (active ingredient 60. mu.g to 120. mu.g, for example, 60. mu.g, 70. mu.g, 80. mu.g, 90. mu.g, 100. mu.g, 110. mu.g, 120. mu.g) or the commercial product of example 1 is coated on a dressing (for example, sterile gauze) to prepare a drug for external use.

2. The product obtained in example 2 (60 μ g of active ingredient) was made into lyophilized powder.

3. Lotion preparation methods well known in the art may also be employed, for example: the lotion mostly takes water and alcohol as dispersion media; is prepared from active component, electrolyte, isoosmotic regulator, etc. in dispersing medium.

4. The product obtained in example 2 (active ingredient 60 μ g to 120 μ g, e.g. 60 μ g, 70 μ g, 80 μ g, 90 μ g, 100 μ g, 110 μ g, 120 μ g) or the commercial product of example 1 was prepared as enteric capsules.

Test example

Drugs and main agents

Test drugs: the red nocardia rubra cell wall skeleton (Chinese medicine standard S20030009, specification 60 mug/branch) is externally used.

Test example 1 Effect on neutrophil function

I. The research content is as follows:

the effect of the cell wall skeleton on neutrophil (PMN) function was studied and explored.

Analyte:

a single lyophilized powder (example 1) was dissolved using 1ml of physiological saline as a cell wall matrix stock solution.

Test methods

1. Chemotaxis experiment:

1) the cell wall skeleton was examined for chemotaxis towards PMNs.

Preparing the stock solution into different concentrations (dilution times of 0, 1:2, 1:10, 1:100, 1:1000 and 1:10000), performing a PMN agarose chemotaxis experiment by using cell wall skeletons with different concentrations as chemotactic agents, and observing the chemotaxis function of PMN after 2 h.

The grouping is as follows: control group (chemotactic tripeptide fMLP as chemotactic agent, which is a known PMN chemokine), cell wall backbone as chemotactic agent (undiluted group, 1:2 diluted group, 1:10 diluted group, 1:100 diluted group, 1:1000 diluted group, 1:10000 diluted group).

2) PMNs were intervened with the cell wall scaffold and their chemotactic function for fMLP was observed.

The experimental method comprises the following steps: the PMN was pretreated with LPS (1. mu.g/ml) for 30min, excess LPS was washed off, and then cell wall skeleton intervention was performed, LPS stimulation was performed for 30min, and chemotactic function of PMN was observed after 2 h. When each group of cells intervenes, the cell culture concentration is 1X 10⁶One per ml.

The grouping is as follows: control group, cell wall skeleton single intervention group (3 groups: 1:100, 1:1000, 1:10000), LPS pretreatment group (LPS), cell wall skeleton intervention group after LPS pretreatment (3 groups: LPS + cell wall skeleton 1:100, LPS + cell wall skeleton 1:1000, LPS + cell wall skeleton 1: 10000).

2. And (3) detecting Reactive Oxygen Species (ROS):

and (3) washing each group of treated cells, washing out the culture medium, and preparing an active oxygen excitation and detection solvent (the concentration of phorbol ester PMA is 1nmol/ml, and the concentration of ROS probe DCFH-DA is 1 mu mol/ml) by using a 1640 culture medium (serum-free). Add 200. mu.l of the prepared reagents to each group of cells, incubate for 30min in a cell incubator (37 ℃/5% CO2), wash excess probe with PBS (twice), and resuspend the cells in a 300. mu.l system for flow detection.

3. And (3) particle removal detection:

each group of treated cells was centrifuged, cell supernatant was taken for subsequent detection by ELISA (antimicrobial proteins MPO, HBP, NE, MMP9), cells were washed twice with PBS, PMN was labeled with secretory vesicle marker (CD35-BB515) and azurophilic granule marker (CD63-PE/Cy7), incubated at room temperature for 30min, excess antibody was washed off with PBS, and cells were resuspended in a 300. mu.l system for flow detection.

4. And (3) detecting the phagocytic function:

each treated group of cells was centrifuged at 5X 10⁵ Add 10. mu.l RFP-pHrodo to each PMN^TMRed, in a cell incubator (37 ℃/5% CO)₂) After incubation for 30min, excess RFP was washed out with PBS and the cells were resuspended in a 300. mu.l system for flow detection.

Results IV

1. The stock solution is diluted into different concentrations and used as a chemotactic agent to carry out a neutrophilic granulocyte chemotaxis experiment, and the dilution times are 0, 1:2, 1:10, 1:100, 1:1000 and 1: 10000. In the non-inflammatory state, the cell wall skeleton did not induce chemotaxis of PMNs (fig. 1A to 1D).

Stimulating PMN with LPS for 0.5h, washing off redundant LPS, interfering with cell wall skeleton for 0.5h, washing cells, and then suspending the cells with 1640RIPM culture medium for chemotaxis (LPS stimulation concentration: 1 μ g/ml, cell wall skeleton interference concentrations are respectively diluted at 1:100, 1:1000 and 1:10000, and interference time: 30 min). Statistical analysis was performed on chemotaxis indicators in each set of chemotaxis experiments. The cell wall skeleton can partially restore the damaged chemotactic function of neutrophils in an inflammatory state, so that the chemotactic distance of the neutrophils is increased, the number of the chemotactic cells is obviously increased, and the cell wall skeleton is diluted to the optimal intervention concentration by 1:1000 (figures 2A to 2C).

3. Interfering the cell wall skeleton with PMN pretreated by LPS, and observing the change of the phagocytic function of the PMN. When the cell wall skeleton is diluted by 1:1000, the reduction of PMN phagocytosis caused by inflammation can be reversed, and the bacterial phagocytosis efficiency of PMN is increased (fig. 3A-3B).

4. And (3) interfering the cell wall skeleton with PMN pretreated by LPS, and observing the change of the sterilization function of PMN active oxygen. Under the inflammatory state, the cell wall skeleton can inhibit the excessive generation of Reactive Oxygen Species (ROS), reduce oxidative stress and reduce the peroxidation damage of tissues and organs caused by excessive reactive oxygen species (figure 4).

5. The cell wall skeleton was interfered with LPS-pretreated PMNs, changes in the degranulation bactericidal function of PMNs were observed, as well as the expression levels of markers (secretory vesicle marker: CD35, stimulatory granule marker: CD66b, azurophil/primary granule marker: CD 63). Under inflammatory conditions, neutrophil over-activation; the cell wall skeleton can inhibit over-activation of neutrophils, inhibit over-degranulation of neutrophils, and relieve damage of the neutrophils degranulation to cells, organs and the like in an inflammatory state (fig. 5A to 5C).

Test example 2 Regulation of the cell wall skeleton on the immune microenvironment of chronic wounds

I. Establishment of chronic wound model

Selecting healthy adult male Wistar rats with SPF grade, and weighing 200 g-250 g. After 7 days of acclimatization, the results were randomized: blank group, wound surface group, cell wall skeleton treatment group. The rats in the wound group and the cell wall skeleton treatment group establish a chronic wound model on the back. The blank group was only treated for back skin preparation.

Pentobarbital sodium (30mg/kg) was anesthetized in the abdominal cavity, and fine hair on the back was removed by shaving the skin on the back using depilatory cream. Then, a full-layer skin defect wound surface with the diameter of 20mm and the depth reaching the deep fascia is punched on the back of the mouse by using a puncher, 80mg/kg of hydrocortisone acetate is injected immediately, and an immune defect type chronic wound surface model difficult to heal is established.

Grouping II

1. Blank group: no drug was administered.

2. Wound surface group: control (0.9% sodium chloride injection, 20. mu.l/mouse) was added dropwise to the rat wound every 1 day.

3. Cell wall skeleton treatment group: the cell wall skeleton of example 1 (200. mu.l/mouse, 30. mu.g of cell wall skeleton in 200. mu.l physiological saline) was added dropwise to the rat wound every 1 day.

Test methods

1. 3, 7, 10 and 14 days after molding, respectively taking 3 rats of pentobarbital sodium (100mg/kg) from each group, carrying out anesthesia sacrifice, cutting the skin of the rats along the wound, photographing and recording the wound healing condition of the back of each mouse (photographing the wound edge with a scale), and calculating the wound healing rate. Calculating the wound healing rate by adopting a formula: wound healing rate = (initial wound area-non-healed wound area)/initial wound area. The sample is divided into 4 portions, two portions are stored at the temperature of 80 ℃, two portions are fixed by 10 percent formaldehyde, and the sample is wrapped by wax blocks.

2. Treating the wound surface after modeling:

control group: the wound surface was sterilized with 75% alcohol, and then wrapped with 2 layers of wet gauze containing 0.9% sodium chloride injection and 2 layers of sterile dry gauze, and fixed, and dressing change was performed 2 times per day.

Cell wall skeleton treatment group: after the wound surface is disinfected by 75% alcohol, 2 layers of wet gauze containing cell wall skeletons and 2 layers of sterile dry gauze are sequentially covered, bound and fixed, and the dressing change is carried out for 2 times every day.

Appropriate maintenance measures are taken for experimental animals and wounds, and rats are prevented from biting skin defect areas.

3. Wound evaluation:

1) the healing rate is as follows:

3 mice in the 3 rd, 7 th and 14 th rhizoma gastrodiae intoxication experimental groups and the control group respectively after the injury are photographed, the healing condition of the back wound surface of each mouse is recorded, and computer graphic processing is carried out by adopting NIHImageJ Java1.8.0 software to calculate the healing rate of the injury.

2) Wound surface observation:

the wound surface has changed size, whether exudate is accumulated, whether bleeding occurs, whether scabbing occurs, etc. Wound healing can be divided into: inflammatory reaction, granulation tissue formation, re-epithelialization, and post-wound closure molding. Evaluation can be made by regenerated epithelium length and granulation tissue thickness. The length of the regenerated epithelium refers to the distance between the first hair follicle appearing on the skin at the edge of the wound surface as the starting point and the foremost end of the regenerated epithelium as the end point.

3) Evaluation of tissue level: whether epithelialization occurs or not; the presence or absence of granulation tissue formation; how thick granulation tissue is formed; whether the development of subcutaneous hair follicles in the wound healing area of the experimental group is finished or not is judged; collagen fiber and fibroblast arrangement occurs; mature fibrous connective tissue grows.

4) Evaluation at cell level: the number and the form of neutrophils, endothelial cells, fibroblasts, lymphocytes and macrophages change; the positional relationship between neutrophils and the above cells is changed; expression of chemotaxis-related molecules (CXCR-1, CXCR2) and immune-related molecules (PD-L1) on cells was observed.

4. Collecting samples:

on the 3 rd, 7 th and 14 th days of treatment, 6 rats in each group were randomly selected and sacrificed, and the wound surface and periwound tissues (the boundary between the wound surface and normal tissues) reaching deep under the fascia within 0.5cm of the wound margin were excised. According to the requirements of each experiment, the samples are divided into:

grinding the tissue, extracting neutrophils (macrophages, lymphocytes and the like) of the tissue, and preserving grinding fluid (if the wound surface has seepage, preserving the seepage for subsequent detection);

② the paraffin wax is used for slicing and dyeing;

and thirdly, rapidly placing the mixture in a freezing tube, placing the mixture in a liquid nitrogen tank, and placing the mixture in a refrigerator at the temperature of minus 80 ℃ for freezing storage for later experimental detection.

5. Detection indexes are as follows:

1) the concentrations of IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma inflammatory factors were determined by ELISA/CBA.

2) Hematoxylin-eosin staining:

on days 3, 7 and 14 after the wound, the wound healing skin tissue and the adjacent whole skin tissue are cut off and washed by sterile normal saline. Then soaking in 4% by volume neutral formaldehyde solution (pH 7.2-7.4) for 24 hr at 4 deg.C, and storing the tissue in 75% ethanol at 4 deg.C. And carrying out gradient dehydration on the wound tissue fixed by the formaldehyde solution. And (4) permeabilizing the dehydrated wound tissues, and then embedding paraffin. The sections were cut successively with a microtome to a thickness of 5 μm. The slices were thoroughly developed in water at 46 ℃ and then taken out with a glass slide and baked at 51 ℃ on a baking machine. The slides were immersed in a xylene solution. The slides were immersed in the ethanol solution and then rinsed. The slides were stained in hematoxylin stain and rinsed with water. The slide was developed by placing it in a 1% ethanol hydrochloric acid solution, followed by rinsing with water. The slide was then immersed in 80% ethanol solution. The slides were stained in eosin. The slides were then dehydrated by immersion in an ethanol solution. The tissue sections were observed under a microscope.

3) Evaluating the spatial relationship between the neutrophils and endothelial cells, fibroblasts, T cells, lymphocytes and macrophages through multicolor immunofluorescence two-dimensional reconstruction: the dewaxed and hydrated slide was placed in a repair cup and immersed in 1 × antigen repair solution. The repair cup is placed in a microwave oven to boil. Naturally cooling to room temperature. The diluted primary antibody solution was added dropwise and incubated at room temperature for 1 hr. The slides were washed with 1 × TBST buffer. The HRP secondary antibody working solution was added dropwise, incubated for 10min, and the slides were washed with 1 XTSST buffer. 100. mu.l of 1 XTY dye working solution was dropped onto the slide, incubated at room temperature for 10min, and the slide was washed with 1 XTSST buffer. Repairing with microwave, and cooling to room temperature. The slides were washed 1 time with sterile water and 1 × TBST buffer. And after dyeing is finished, mounting for observation or carrying out subsequent dyeing. The stained tissue pieces were observed under a fluorescent microscope and interpreted and statistically analyzed using software.

IV. results

1. The expression of cytokines (IL-1 beta, IL-6, IL-10, TNF-alpha) in the control and cell wall skeleton treated groups on days 3, 7, 10, 14.

At day 3, each factor had a downward trend in the cell wall scaffold group. At day 7, each factor had a tendency to increase in the cell wall scaffold group. P <0.05, P < 0.0001. RC represents the cell wall skeleton treatment group (fig. 6A to 6D).

2. Chemokine expression in control and cell wall skeleton treated groups at 3, 7, 10, 14 days.

At day 7, each factor had a tendency to rise significantly in the cell wall scaffold group. P <0.05, P < 0.00001. In the cell wall skeleton treatment group, the chemokine increased at day 7, and the cytokine was also increased at day 7 (wherein IL-8 is neutrophil chemokine, CCL3 is monocyte chemokine, XCL1 is lymphocyte chemokine, CX3CL1 can chemotact various immune cells). RC represents the cell wall skeleton treatment group (fig. 7A to 7D).

3. Compared with a control group, the Ly6G histochemistry and the fluorescent staining of the cell wall skeleton treatment group have hair follicle staining, and a positive control staining is normal. Indicating that the wound tissue has a certain number of neutrophils.

4. The cell wall skeleton treated group had a higher number of CD8 cells compared to the control group. The cell wall skeleton has the functions of recruiting lymphocytes, increasing the number of local immune cells and enhancing the immune function.

Claims (10)

1. Use of a nocardia rubra cell wall scaffold in the preparation of a neutrophil modulator; preferably, the modulator is for any one or combination selected from the group consisting of:

-restoring the chemotactic function of damaged neutrophils in an inflammatory state;

-increasing the number of chemotactic cells;

-reversing the inflammatory-induced decrease in neutrophil phagocytic function;

-increasing the phagocytic efficiency of neutrophils;

-inhibiting the production of reactive oxygen species ROS;

-inhibiting degranulation of neutrophils;

-inhibiting over-activation of neutrophils.

2. Use of a nocardia rubra cell wall scaffold in the preparation of a medicament for the treatment of a chronically infected wound; preferably, the medicament is for improving the local immune microenvironment of a chronically infected wound.

3. The use according to claim 1 or 2, wherein the neutrophil modulator or medicament is prepared in a dosage form selected from any one of: ointment, cream, emulsion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder, dressing, bandage, membrane, patch, and suppository.

4. The use according to claim 1 or 2, wherein the unit dose of the neutrophil modulator or medicament comprises from 1 μ g to 1000 μ g nocardia rubra cell wall skeleton; preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μ g.

5. The use according to claim 1 or 2, wherein the nocardia rubra cell wall skeleton is the national drug standard S20030009 or its update-numbered nocardia rubra cell wall skeleton.

6. Use according to claim 1 or 2, wherein the nocardia rubra cell wall skeleton is obtainable by a method comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) carrying out an operation of removing lipid on the crushed product;

3.2) carrying out nucleic acid removal operation on the crushed product;

3.3) carrying out protein removal operation on the crushed product;

3.4) carrying out cell membrane removal operation on the crushed product;

3.5) obtaining a product derived from the cell wall of the red nocardia;

4) optionally, subpackaging;

5) optionally, freeze-drying the product derived from the cell wall of nocardia rubra;

wherein the content of the first and second substances,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged in sequence or in parallel,

step 4) and step 5) can be interchanged in sequence;

the average particle size of the disruption is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500 nm;

preferably, the dispensing means dispensing into containers;

preferably, the container is selected from: bottles, tubes, bags, pouches, plates, ampoules, injection devices, aluminum film packaging, dressings, pouches, films.

7. A method of modulating neutrophils under in vitro culture conditions comprising the steps of:

contacting neutrophils with an effective amount of nocardia rubra cell wall skeleton, said contacting lasting for at least 30 minutes;

the neutrophils are neutrophils in an inflammatory state;

the method is not used for disease prevention use in animals or humans;

the method is not used for disease diagnostic use in animals or humans;

the method is not for disease treatment use in animals or humans;

preferably, the concentration of the nocardia rubra cell wall skeleton is 6ng/ml to 6 μ g/ml; more preferably 6ng/ml to 600ng/ml, most preferably 60 ng/ml.

8. The method of claim 7, wherein the nocardia rubra cell wall skeleton is the national drug standard S20030009 or its updated numbering nocardia rubra cell wall skeleton.

9. The method according to claim 7, wherein the nocardia rubra cell wall skeleton is obtained by a process comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) carrying out an operation of removing lipid on the crushed product;

3.2) carrying out nucleic acid removal operation on the crushed product;

3.3) carrying out protein removal operation on the crushed product;

3.4) carrying out cell membrane removal operation on the crushed product;

3.5) obtaining a product derived from the cell wall of the red nocardia;

4) optionally, subpackaging;

5) optionally, freeze-drying the product derived from the nocardia rubra cell wall;

wherein the content of the first and second substances,

steps 3.1), 3.2), 3.3), 3.4) can be interchanged in sequence or in parallel,

step 4) and step 5) can be interchanged in sequence;

the average particle size of the disruption is from 10nm to 1000nm, preferably from 10nm to 800nm, more preferably from 10nm to 500 nm;

preferably, the dispensing means into containers;

preferably, the container is selected from: bottles, tubes, bags, pouches, plates, ampoules, injection devices, aluminum film packaging, dressings, pouches, films.

10. A neutrophil culture medium comprising a nocardia rubra cell wall skeleton;

the red nocardia cell wall skeleton is a national standard S20030009 or a red nocardia cell wall skeleton with a renewed serial number; or

The nocardia rubra cell wall skeleton obtained by the steps defined in claim 6 or 9.

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