CN113476461A - Medicine for improving chemotaxis of inflammation-inhibiting type macrophages and application thereof - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K38/19—Cytokines; Lymphokines; Interferons
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses a medicine for improving chemotaxis of inflammation-inhibiting macrophages and application thereof, in particular to application of glucosamine in preparing a medicine for improving chemotaxis of inflammation-inhibiting macrophages, and in vitro experiments show that glucosamine in vitro treatment does not influence proinflammatory or inflammation-inhibiting polarization of macrophages, but obviously improves chemotaxis of inflammation-inhibiting macrophages to CX3CL 1.
Description
Technical Field
The invention belongs to the medicine technology, and particularly relates to application of a compound in preparation of a medicine for improving chemotaxis of inflammation-inhibiting macrophages.
Background
Macrophages (macrophages) exhibit distinct functions upon stimulation by IFN-gamma or IL-4. IFN-gamma-stimulated macrophages differentiate into M1-type macrophages that promote inflammation, and IL-4-stimulated M2-type macrophages that appear to inhibit inflammation. This functional pluripotency is of great importance for various diseases, for example, macrophages in tumor tissues promote the elimination of cancer cells by immune cells of the body if they differentiate into M1 type macrophages, and conversely inhibit the immunity of the body if they differentiate into M2 type macrophages. The receptor of CX3C chemokine ligand 1[ CX3CL1] is CX3CR1, and plays an important role in migration, proliferation and angiogenesis of vascular endothelial cells. In vivo, CX3CL1 occurs in two distinct subtypes: membrane-bound and soluble (sFKN). Thus, it combines the properties of a chemotactic agent and an adhesion molecule. The presence of both forms of CX3CL1 in vivo suggests a particular role for it in a large family of chemokines, which is not comparable to any other chemokine to date, and in contrast to the abundant expression of other chemokines in peripheral blood cells, CX3CL1 is rarely or even not expressed in peripheral blood cells.
Disclosure of Invention
The invention discloses a new application of utilizing GlcN to improve chemotactic capacity of inflammatory-inhibiting macrophages. Macrophages regulate the proliferation and differentiation of vascular endothelial cells in the microenvironment through chemotaxis and secretion of cytokines to influence the generation of new blood vessels, and chemokines play a role in indirectly regulating angiogenesis by combining with corresponding receptors on the surfaces of the macrophages.
The invention adopts the following technical scheme:
a medicament for improving chemotaxis of inflammation-inhibiting macrophages comprises glucosamine as an active ingredient.
Application of glucosamine in preparation of medicines or health products for improving chemotaxis of inflammation-inhibiting macrophages.
Application of glucosamine in preparation of medicines or health products for improving chemotaxis of inflammation-inhibiting macrophages to CX3CL 1.
The invention discloses a method for improving chemotaxis of inflammation-inhibiting macrophages to CX3CL1, which utilizes glucosamine to stimulate macrophages and improves chemotaxis of inflammation-inhibiting macrophages to CX3CL 1. Furthermore, glucosamine and cytokines are used to stimulate macrophages, and the chemotaxis of inflammation-inhibiting macrophages to CX3CL1 is improved.
The invention discloses application of glucosamine and cytokines in preparing a medicament for improving chemotaxis of inflammation-inhibiting macrophages to CX3CL 1.
In the present invention, the cytokine is IL-4 or IL-13.
Glucosamine (glcne) is an amino-containing sugar in the human body, widely present in articular cartilage and connective tissue, and is an essential component of cartilage matrix and synovial fluid. Glucosamine has been used in europe for more than twenty years as an over-the-counter drug for the treatment of osteoarthritis, and no significant side effects have been observed with long-term use. Glucosamine has been approved by various countries as a dietary supplement for alleviating arthritis, but studies on the improvement of chemotaxis of inflammation-inhibiting macrophages by glucosamine have not been reported so far. The method comprises the steps of treating macrophages with IL-4 or IL-13 to induce polarization, stimulating the macrophages with PBS or GlcN for 24 hours respectively, after counting the cells, inoculating the same number of cells in each group into an upper chemotactic (transwell) chamber, wherein the lower chamber is a basic culture medium added with CX3CL1, continuing conventional culture for 4 hours, wiping the cells which are not chemotactic at the upper part of the chamber, fixing 4% PFA for 15 minutes, washing the PBS for 2 times, dripping an anti-fluorescence attenuating agent containing DAPI at the bottom of the chamber, taking a picture by an inverted fluorescence microscope, counting the number of cells in each field under a 10-fold mirror by imageJ, observing fluorescence of each group under the fluorescence microscope, and finding that the number of chemotactic macrophages in a GlcN control group is obviously more than that of the PBS group. The ability of glucosamine to improve chemotaxis of inflammatory-suppressor macrophages towards CX3CL1 was demonstrated. The results of the experiment comprehensively prove that: GlcN in vitro treatment does not affect either pro-inflammatory or anti-inflammatory polarization of macrophages, but significantly increases chemotaxis of anti-inflammatory macrophages.
Drawings
FIG. 1 shows that glucosamine treatment significantly increases chemotaxis of suppressor macrophages towards CX3CL 1;
FIG. 2 shows that glucosamine treatment does not affect macrophage pro-inflammatory polarization;
FIG. 3 shows that glucosamine treatment did not affect macrophage inflammatory-suppressive polarization.
Detailed Description
The following examples are presented only to assist those skilled in the art in a more complete understanding of the present invention, and are not intended to limit the invention in any way.
The main materials and sources used are as follows:
laser confocal microscopy (LSM 880, Zeiss); flow cytometry (Millipore Guava easyCyte); real-time fluorescent quantitative PCR instrument (ABI Stepone Plus); a micro spectrophotometer (Nanodrop 2000); inverted fluorescence microscope (Olympus, IX 51); glucosamine (Sigma, usa); LPS (Sigma, usa); IFN- γ (PeproTech, USA); IL-4 (PeproTech, USA); IL-13 (PeproTech, USA); CX3CL1 (pepotech, usa); iNOS-PE antibody (Biolegend, USA); CD80-PE antibody (Biolegend, USA); CD86-PE antibody (Biolegend, USA); CD117-PE antibody (Biolegend, USA); transwell chambers (8.0- μm pore size, Corning). The specific experimental method and the testing method are conventional technologies, and the experimental group and the control group are parallel experiments.
Example one glucosamine treatment significantly increased chemotaxis of suppressor macrophages to CX3CL1
Macrophages (iBMDM) were treated with IL-4 (40 ng/mL) while stimulated with PBS buffer or GlcN (3 mM) for 24h, specifically: experimental groups: adding IL-4 and GlcN into RPMI 1640 basic culture medium, inoculating macrophage, and culturing conventionally (37 deg.C, 5% CO)2) 24 hours; control group: IL-4 and PBS are added into RPMI 1640 basic culture medium, then macrophage is inoculated, and conventional culture is carried out for 24 hours.
Then, a conventional cell chemotaxis (transwell) experiment was performed, specifically: after cell counting, respectively inoculating the same number of cells in each group into an upper transwell chamber, wherein the lower chamber is an RPMI 1640 basic culture medium added with CX3CL1, wiping cells which are not chemotactic at the upper part of the chamber after continuously culturing for 4h in a conventional way, fixing by 4% PFA for 15min, washing by PBS for 2 times, dripping an anti-fluorescence attenuation agent containing DAPI at the bottom of the chamber, taking a picture by an inverted fluorescence microscope, and counting the number of cells in each visual field under a 10-fold lens by imageJ, wherein the result is shown in figure 1.
Example two
Macrophages (iBMDM) were treated with IL-13 (20 ng/mL) while stimulated with PBS buffer or GlcN (3 mM) for 24h, specifically: experimental groups: adding IL-13 and GlcN into an RPMI 1640 basic culture medium, inoculating macrophages, and culturing for 24 hours conventionally; control group: IL-13 and PBS were added to RPMI 1640 basal medium, and macrophages were inoculated and cultured routinely for 24 hours.
Routine cell chemotaxis (transwell) experiments were then performed as in the first example, and the results are shown in FIG. 1.
Referring to fig. 1A, fluorescence of each group is observed under a fluorescence microscope, and it is found that the number of chemotactic macrophages of the GlcN control group is significantly larger than that of the PBS group, a representative picture of each group of samples is shown in a picture, a scale shows 100 μm, and fig. 1B is data after statistics.
Example Triglucosamine treatment did not affect macrophage pro-inflammatory polarization
Macrophages (iBMDM) were treated with LPS/IFN-. gamma. (20 ng/mL) while stimulated with PBS or GlcN (3 mM) for 24h, respectively, F4/80 and iNOS immunofluorescent staining were performed according to the conventional procedure, fluorescent secondary antibodies labeled Alexa Fluor 594 and Alexa Fluor 488 were added, and iNOS signals (green), F4/80 signals (red) and DAPI signals (blue) were simultaneously observed and photographed with a confocal microscope using an anti-fluorescence attenuation blocking agent containing DAPI. Referring to FIG. 2A, the fluorescence of each group was observed under a confocal laser microscope, and it was found that the number of F4/80+ iNOS + macrophages was almost not different in the GlcN group from that in the PBS group. The pictures are displayed as representative pictures in each group of samples, and the scales are displayed as 20μm.
Macrophages (iBMDM) were treated with LPS/IFN-. gamma. (20 ng/mL) while stimulated with PBS or GlcN (3 mM) for 24h, respectively, collected in 1.5mL EP tubes, incubated with iNOS, CD80, and CD86 flow antibodies, respectively, for 30min at room temperature, examined by flow cytometry, and analyzed for experimental results by FlowJo software. Referring to fig. 2B, C, D, flow cytometry results showed that the average fluorescence intensity of iNOS +, CD80+, CD86+ of the GlcN group was almost not different from that of the PBS group. The photographs show representative pictures from each group of samples.
Macrophages (iBMDM) were treated with LPS/IFN-. gamma. (20 ng/mL) while stimulated with PBS or GlcN (3 mM), respectively, for 24h, and RNA was extracted, reverse transcribed and QPCR by the conventional procedure, with 18S as an internal control. Referring to FIG. 2E, the expression level of proinflammatory cytokines from macrophages was measured for QPCR, and it can be seen that the GlcN group was almost not different from the PBS group.
Example Tetraglucosamine treatment did not affect macrophage inflammatory-suppressive polarization
Macrophages (iBMDM) were treated with IL-4 (40 ng/mL) while stimulated with PBS or GlcN (3 mM) for 24h, respectively, F4/80 and CD206 immunofluorescent staining was performed according to the conventional procedure, Alexa Fluor 594-labeled and Alexa Fluor 488-labeled fluorescent secondary antibodies were added, and mounted with DAPI-containing anti-fluorescence attenuation mounting medium, and CD206 signal (green), F4/80 signal (red) and DAPI signal (blue) were simultaneously observed and photographed in a confocal microscope. Referring to FIG. 3A, the fluorescence of each group was observed under a confocal laser microscope, and it was found that the number of F4/80+ CD206+ macrophages was almost not different in the GlcN group from that in the PBS group. The pictures are displayed as representative pictures in each group of samples, and the scales are displayed as 20μm.
Macrophages were treated with IL-4 (40 ng/mL) while stimulated with PBS or GlcN (3 mM) for 24h, respectively, collected in 1.5mL EP tubes, incubated with CD206 flow antibody, respectively, for 30min at room temperature, detected by flow cytometry, and analyzed by FlowJo software. Referring to fig. 3B, flow cytometry results showed that the mean fluorescence intensity of CD206+ for the GlcN group was almost not different from that of the PBS group. The photographs show representative pictures from each group of samples.
Macrophages were treated with IL-4 (40 ng/mL) while stimulated with PBS or GlcN (3 mM), respectively, for 24h, RNA was extracted, reverse transcription and QPCR were performed according to the conventional procedure, with 18S as an internal control. Referring to FIG. 3C, a graph of macrophage proinflammatory inhibitory cytokine expression levels for QPCR was shown, and it can be seen that the GlcN group had little difference from the PBS group.
The results of the above experiments comprehensively prove that: GlcN in vitro treatment does not affect either pro-inflammatory or anti-inflammatory polarization of macrophages, but significantly increases chemotaxis of anti-inflammatory macrophages.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (10)
1. A medicament for improving chemotaxis of inflammatory-inhibiting macrophages, which comprises glucosamine as an active ingredient.
2. Use of glucosamine in the manufacture of a medicament for improving chemotaxis of inflammatory-inhibiting macrophages to CX3CL 1.
3. Application of glucosamine in preparing medicine for improving chemotaxis of macrophage with inflammation.
4. Application of glucosamine in preparation of health products for improving chemotaxis of inflammation-inhibiting macrophages to CX3CL 1.
5. Application of glucosamine in preparing health products for improving chemotaxis of inflammation-inhibiting macrophages.
6. The use according to claim 2, 3, 4 or 5, wherein the pharmaceutical or nutraceutical is a liquid formulation.
7. A method for improving chemotaxis of an anti-inflammatory macrophage toward CX3CL1, which comprises stimulating the macrophage with glucosamine to improve chemotaxis of the anti-inflammatory macrophage toward CX3CL 1.
8. The method of claim 7, wherein the chemotaxis of the suppressor macrophages towards CX3CL1 is improved by stimulating macrophages with glucosamine and cytokines.
9. The method of claim 8, wherein the cytokine is IL-4 or IL-13.
10. The application of glucosamine and cytokines in preparing a medicament for improving chemotaxis of inflammation-inhibiting macrophages to CX3CL 1.
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2021
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