CN117137939A - Application of macrophage-promoting polarization material in chronic wound surface difficult to heal - Google Patents

Abstract

The macrophage-promoting polarization material provided by the application is applied to chronic wound surfaces which are difficult to heal, and the macrophage-inducing material M1 polarization and the macrophage-inducing material M2 polarization are sequentially segmented and used for the tissue proliferation period of the early stage of chronic wound surface generation and the beginning of reconstruction of skin tissue; the material for inducing macrophage M1 polarization induces acute immune response to generate in early wound healing, engulfs pathogenic microorganisms, shortens inflammatory phase and promotes smooth transition to proliferation phase; the material for inducing macrophage M2 polarization eliminates necrotic tissue in proliferation period, dissolves inflammation and promotes tissue repair and regeneration, thus providing optimum wound immune microenvironment for different wound healing periods, recovering M1/M2 type macrophage phenotype balance in chronic difficult-to-heal wound, and accelerating the healing of chronic difficult-to-heal wound on body surface.

Description

Application of macrophage-promoting polarization material in chronic wound surface difficult to heal

Technical Field

The application relates to the technical field of wound healing, in particular to application of macrophage-promoting polarization material in chronic wound difficult to heal.

Background

The chronic difficult-to-heal wound surface of the body surface refers to a wound surface which can not heal and has no healing tendency after more than 1 month of treatment, wherein diabetic feet and pressure injuries (such as pressure sores and bedsores) are typical chronic difficult-to-heal wound surfaces, and how to promote the healing of the chronic difficult-to-heal wound surface of the body surface is a serious health problem of people who are urgently needed to be solved in implementing health China strategy.

According to statistics of International wound repair and regeneration, the incidence rate of diabetic foot is 19-34% in diabetic patients, and the amputation rate caused by the diabetic foot is about 20%; the number of deaths within 5 years after diabetic foot was 2.5 times the number of people who had only diabetes. The incidence rate of the pressure injury in the crowd is 0.75%, wherein the incidence rate of the old in China is up to 5-27%, and the mortality rate of the patient due to bedsores and complications thereof is up to more than 40%. The clinical treatment of the chronic difficult-to-heal wound surface is accompanied by high recurrence rate, high amputation rate and high death rate due to the characteristics of long-term chronic inflammation, continuous bacterial infection, hypoxia, abnormal collagen metabolism, weak cell migration capability and the like of the patient with the chronic difficult-to-heal wound surface, and the core of the clinical treatment is to understand the mechanism of wound surface healing and to select proper materials to promote the chronic difficult-to-heal wound surface.

Classical wound repair involves a complex and orderly series of vital activities, including four phases that overlap and differ from one another: hemostasis, inflammation, proliferation, remodeling. Macrophages are key participants in the wound healing process and can be classified into an M1 pro-inflammatory phenotype (classical activated type) and an M2 anti-inflammatory phenotype (alternative activated type) according to the difference between phenotype and secreted cytokines, and the content of the two types can be changed along with the change of the healing stage. According to the publication of science conversion medicine, the phenotype of macrophages in wounds is gradually changed from M1 to M2, and the macrophage has important effects of dissolving inflammation, angiogenesis and re-epithelialization.

However, in chronic difficult-to-heal wounds such as diabetic feet, the high blood sugar level seriously damages the functions of macrophages, so that the balance among macrophages with different phenotypes in each healing stage of the wound surface is destroyed. The imbalance in macrophage phenotype severely affects the cells and signal molecules involved in the wound healing process, disrupting the normal physiological healing process and thus preventing diabetic wound healing. How to make up and recover the functions of macrophages, regulate and control the balance between M1 and M2 anti-inflammatory phenotype macrophages, and promote the smooth transition from an inflammatory phase to a proliferation phase is one of the keys for promoting the healing of diabetic wounds.

Patent CN202111425932.7 discloses a medical dressing with antibacterial and wound healing promoting effects, and the publication date is 2023, 4 and 11; the technical proposal is disclosed that PEGylated chitosan is used for inducing macrophage polarization into an M2 anti-inflammatory phenotype with anti-inflammatory repair function so as to accelerate wound healing; it does not disclose a means of polarizing macrophages to the M1 pro-inflammatory phenotype; based on the basis, the inventor provides an application of macrophage-promoting polarization material in chronic wound surface difficult to heal, which is different from the prior art, through research and improvement innovation on the basis of original patent.

Disclosure of Invention

The application provides an application of macrophage-promoting polarization material in a chronic difficult-to-heal wound surface of a body surface, which aims at solving the technical problems of shortening the wound healing time, accelerating the wound healing speed, providing an optimal immune microenvironment for the healing period of the diabetic wound surface and recovering the M1/M2 type macrophage balance in the chronic difficult-to-heal wound surface of the diabetes.

The technical scheme adopted by the application is as follows:

the application of the macrophage-promoting polarization material in the chronic wound surface difficult to heal comprises the macrophage-promoting M1 polarization material and the macrophage-promoting M2 polarization material;

the application sequentially segments the material capable of inducing the polarization of the macrophage M1 and the material capable of inducing the polarization of the macrophage M2 for the tissue proliferation period of early stage of chronic difficult-to-heal wound surface generation and the beginning of reconstruction of skin tissue: inducing acute immune response to generate in the early stage of wound healing, phagocytizing pathogenic microorganisms, and pushing the inflammatory phase to smoothly transition to the proliferation phase; and the necrotic tissue is removed in the middle and late stages of wound healing, the inflammation is dissolved, the repair and regeneration of the tissue are promoted, the M1/M2 type macrophage balance in the chronic difficult-to-heal wound is recovered, and the optimal wound immune microenvironment is provided for different wound healing periods, so that the healing of the chronic difficult-to-heal wound on the body surface is quickened.

Further, the macrophage-promoting M1 polarization material is biguanide chitosan (i.e., DICY-CS);

the biguanide chitosan is prepared by the following method:

s1, dissolving chitosan with a molecular weight of 10,000-1,000,000Da raw material in hydrochloric acid solution to prepare chitosan hydrochloride solution with a concentration of 0.1 mol/L;

s2, adding dicyandiamide with the amount of 2-4 times of the amino groups of the raw chitosan in S1 into the chitosan hydrochloride solution in S1 under the stirring condition to obtain a mixture B;

s3, reacting the mixture B at the reflux of 90 ℃ for 6-48 hours to obtain an intermediate reaction product A

S4, transferring the intermediate reaction product A into a dialysis bag with the molecular weight cutoff of 3,500-14,000Da, dialyzing in deionized water, changing water once every 6-12 hours, changing water for 6-10 times, and freeze-drying to obtain the biguanide chitosan.

Wherein the solubility and antibacterial property of the biguanide chitosan for promoting macrophage M1 polarization are better than those of natural chitosan; in chronic wound surfaces difficult to heal, early inflammatory reaction is not obvious, M1 type macrophages are insufficient, the biguanide chitosan has good biocompatibility, can promote the polarization of the macrophages M1, induce acute inflammation to appear, accelerate the arrival of inflammatory phase, and smoothly start classical wound healing procedures; experiments show that moderate inflammation caused by biguanide chitosan does not further accumulate and develop into excessive inflammation, and the healing process of chronic difficult-to-heal wound surfaces on the body surface can be reasonably promoted.

Preferably, the reaction time in the step S3 is 12-24 hours, and the inventor experiments show that the reaction is insufficient due to short reaction time, and the chitosan main chain skeleton is broken and degraded due to long reaction time.

Further, the macrophages include macrophage line RAW264.7, macrophage line THP-1, macrophage line U937, bone marrow-induced macrophage BMDM, peripheral blood-derived mononuclear macrophage PBMC, mammalian tissue-derived primary macrophages.

Further, the biguanide chitosan is biguanide chitosan solution, biguanide chitosan spray and biguanide chitosan gel.

Further, the macrophage-promoting M2 polarization material is polyethylene glycol chitosan (i.e., PEG-CS).

Further, the biguanide chitosan has a biguanide substitution degree of 5-20%;

preferably, the biguanide chitosan has a biguanide substitution degree of 10-15%;

the substitution degree of the biguanide on the chitosan and the dissolubility thereof are in a direct proportion relation, and the biguanide chitosan has better dissolubility and antibacterial property than natural chitosan, is favorable for being prepared into various solutions, gels, or sprays and the like to be dissolved in solvents, and is convenient for application in promoting wound healing; in addition, the chitosan in the macrophage-promoting M1 polarization material needs 5-20% of biguanide groups to stimulate macrophages, so that the effect of promoting the M1 polarization of the biguanide chitosan is ensured, and the effect of the application is realized.

Further, the macrophage-promoting M1 polarization material is applied to the surface chronic difficult-to-heal wound surface to initially occur until a granulation tissue formation stage; the macrophage-promoting M2 polarization material is applied to the granulation tissue proliferation phase of the surface chronic wound skin tissue difficult to heal and the regeneration of the surface chronic wound skin tissue is started.

Further, after the macrophage-promoting M1 polarization material is applied to the chronic difficult-to-heal wound surface of the body surface, the macrophages appear as A and/or B and/or C:

A. enhancing transcription of M1-type macrophage genes;

B. enhancing secretion of inflammatory factors;

C. the expression level of the co-stimulatory factor CD86 is increased.

Further, the inflammatory factor in B includes one or more of tumor necrosis factor alpha, interleukin-6 and interleukin-12.

Further, the chronic wound surface difficult to heal comprises diabetic foot, pressure sore, venous ulcer of lower limb, burns and scalds accompanied with serious infection or wound surface with serious unbalance of macrophage functions and macrophage phenotype and obvious insufficient inflammatory reaction in early stage of wound.

Compared with the prior art, the application has the following advantages:

the macrophage-promoting polarization material provided by the application is applied to chronic wound surfaces difficult to heal, and the macrophage-inducing material M1 polarization and the macrophage-inducing material M2 polarization are sequentially segmented and used for tissue proliferation phases of early healing occurrence of the chronic wound surfaces and starting reconstruction of skin tissues; the material for inducing macrophage M1 polarization induces acute immune response to generate in early wound healing, engulfs pathogenic microorganisms, shortens inflammatory phase and promotes smooth transition to proliferation phase; the material for inducing macrophage M2 polarization eliminates necrotic tissue in proliferation period, dissolves inflammation and promotes tissue repair and regeneration, thus providing optimal wound immune microenvironment for different wound healing periods of diabetes, recovering M1/M2 macrophage phenotype balance in chronic difficult-to-heal wound of diabetes, and accelerating the healing of chronic difficult-to-heal wound of diabetes body surface.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the description of the embodiments will be briefly described below.

FIG. 1 is a graph showing the percentage distribution of CD86 positive cells (M1 type macrophages) in Raw264.7 after induction by sample stimulation in example 1;

FIG. 2 is a graph showing the percentage of CD86 positive cells (M1 type macrophages) in macrophages after induction by sample stimulation in example 2;

FIG. 3a is a bar graph of the percentage of CD86 protein positive cells of the negative control group, the M1 type polarization positive control group, the M1 type polarization experimental group, and the M2 type polarization experimental group in example 3;

FIG. 3b is a bar graph of the percentage of CD206 protein positive cells of the negative control group, the M2 type polarization positive control group, the M1 type polarization experimental group, and the M2 type polarization experimental group in example 3;

FIG. 4a is a bar graph showing the levels of TNF-. Alpha.in the culture supernatants of the negative control, M1-type polarization positive control, M1-type polarization experimental group, and M2-type polarization experimental group of example 4;

FIG. 4b is a bar graph showing the content of interleukin-6 in the culture supernatants of the negative control group, the M1-type polarization positive control group, the M1-type polarization experimental group, and the M2-type polarization experimental group in example 4;

FIG. 4c is a bar graph showing the amounts of vascular endothelial growth factor in the culture supernatants of the negative control group, the M2-type polarization positive control group, the M1-type polarization experimental group, and the M2-type polarization experimental group in example 4;

FIG. 4d is a bar graph showing the content of interleukin-10 in the culture supernatants of the negative control group, the M2-type polarization positive control group, the M1-type polarization experimental group, and the M2-type polarization experimental group in example 4;

FIG. 5a is a bar graph showing the amounts of TNF-. Alpha.IL-1β, IL-6, iNOS in inflammatory genes of the negative control group, M1 type polarization positive control group, M1 type polarization experimental group, and M2 type polarization experimental group in example 5;

FIG. 5b is a bar graph showing the content of anti-inflammatory genes Stat6, mcr1, IL-10, arg1 in the negative control group, M2-type polarization positive control group, M1-type polarization experimental group, and M2-type polarization experimental group in example 5;

FIG. 6 is a graph showing comparison of wound surfaces of rats at days 0, 7, 10 and 14 in example 6;

FIG. 7 is a graph showing changes in wound area of rats at days 0, 7, 10 and 14 in example 6;

FIG. 8a is a graph of CD86 fluorescent markers in the placebo, commercial, DICY-CS, PEG-CS and combination groups of example 7 on day 7 after injury to rats;

FIG. 8b is a graph of CD86 fluorescent markers in the blank, commercial, DICY-CS, PEG-CS, and combination groups at day 14 post-injury for rats in example 7;

FIG. 8c is a bar graph of TNF- α concentration in the placebo, commercial, DICY-CS, PEG-CS, and combination groups of example 8 at day 7 post-injury rats;

FIG. 8d is a bar graph of TNF- α concentration in the day 14 post-injury blank, commercial control, DICY-CS, PEG-CS, and combination groups of the rats of example 8;

FIG. 9a is a graph of fluorescence labeling of CD68 and CD206 in the blank, commercial, DICY-CS, PEG-CS, and combination groups of example 9 on day 7 after injury to rats;

FIG. 9b is a graph of CD68CD206 fluorescent markers in the blank, commercial, DICY-CS, PEG-CS, and combination groups of example 9 at day 14 post-injury rats;

FIG. 9c is a bar graph of IL-10 concentration in the placebo, commercial, DICY-CS, PEG-CS, and combination groups of example 10 at day 7 post-injury rats;

FIG. 9d is a bar graph of IL-10 concentration in the day 14 post-injury blank, commercial control, DICY-CS, PEG-CS, and combination groups for rats of example 8;

FIG. 10a is a graph of H & E staining in example 11;

FIG. 10b is a map of Masson staining in example 12;

FIG. 10c is a graph of CD31 fluorescent markers in example 13;

FIG. 10d is a bar graph of a blank, commercial, DICY-CS, PEG-CS, and combination group made according to the wound area of FIG. 10 a;

FIG. 10e is a bar graph of a blank, commercial control, DICY-CS, PEG-CS, and combination group made according to the area of the collagen deposition zone in FIG. 10 b;

FIG. 10f is a bar graph of blank, commercial control, DICY-CS, PEG-CS, and combination groups made according to the percentage of fluorescence labeled positive areas in FIG. 10 c;

FIG. 11 is a chart showing the comparison of wound surfaces of diabetic rats 1, 5, 7, 14 and 21 in example 14.

Detailed Description

The following describes the specific technical scheme of the present application in connection with specific examples 1 to 14:

example 1

Preparation of biguanide chitosan:

s1, dissolving 2.5g of chitosan with a molecular weight of 200,000Da into 250mL of hydrochloric acid solution with a mass concentration of 0.1mol/L to prepare chitosan hydrochloride solution;

s2, adding 4g of dicyandiamide into the chitosan hydrochloride solution in the step S1 under the stirring condition to obtain a mixture B1;

s3, reacting the mixture B1 at the reflux of 90 ℃ for 24 hours to obtain an intermediate reaction product A1;

s4, transferring the intermediate reaction product A1 into a dialysis bag with the molecular weight cutoff of 7,000Da, dialyzing in deionized water, changing water once every 6-8 hours, changing water for 6-8 times, and freeze-drying to obtain the biguanide chitosan.

S5, inoculating 1 mL/hole of mouse mononuclear macrophage Raw264.7 cell suspension in a 12-hole plate, and keeping the number of cells in each hole at 10,000-20,000;

s6.1, treatment of experimental group: after the cells are attached, replacing the conditional medium in part of the holes with the conditional medium of the biguanide chitosan solution with the biguanide chitosan content of 1mg/mL in S4, and carrying out 5% CO at 37 DEG C ₂ Incubating for 24 hours under the culture condition;

s6.2, positive control group treatment: after cell attachment, the conditioned medium in part of the wells was replaced with 10ng/mL LPS and 2ng/mL IFN-. Gamma.cocktail and incubated at 37℃with 5% CO ₂ Incubating for 24 hours under the culture condition;

s6.3, negative control group: no treatment is carried out after the cells are attached, and the temperature is 37 ℃ and the concentration of CO is 5 percent ₂ Incubating for 24 hours under the culture condition;

s7, after the incubation of S6.1, S6.2 and S6.3, collecting the macrophages after the incubation, washing the macrophages once with PBS, staining the macrophages with FITC-CD86 fluorescent antibody, and then collecting 10,000 cells at medium speed by a flow cytometer for fluorescence intensity analysis, and the results are shown in FIG. 1, which show that the biguanide chitosan stimulated mononuclear macrophages are mostly polarized towards the M1 pro-inflammatory polarization phenotype.

Example 2

S1, dissolving 2.5g of chitosan with a molecular weight of 500,000Da into 250mL of hydrochloric acid solution with a mass concentration of 0.1mol/L to prepare chitosan hydrochloride solution;

s2, adding 2.35g of dicyandiamide into the chitosan hydrochloride solution in the step S1 under the stirring condition to obtain a mixture B2;

s3, reacting the mixture B2 at the reflux of 90 ℃ for 24 hours to obtain an intermediate reaction product A2;

s4, transferring the intermediate reaction product A2 into a dialysis bag with the molecular weight cutoff of 8,000-14,000Da, dialyzing in deionized water, changing water once every 8-12 hours, changing water for 8-10 times, and freeze-drying to obtain the biguanide chitosan.

S5, inoculating 2 mL/hole of macrophage cell suspension derived from mouse bone marrow into a 6-hole plate, and keeping the number of cells in each hole at 50,000-100,000;

s6.1, treatment of experimental group: after the cells are attached, replacing the conditional medium in part of the holes with the conditional medium of the biguanide chitosan solution with the content of 0.5mg/mL biguanide chitosan in S4, and carrying out 5% CO at 37 DEG C ₂ Incubating for 48h under the culture condition;

s6.2, positive control group treatment: after cell attachment, the conditioned medium in part of the wells was replaced with 10ng/mL LPS and 2ng/mL IFN-. Gamma.cocktail and incubated at 37℃with 5% CO ₂ Incubating for 48h under the culture condition;

s6.3, negative control group: no treatment is carried out after the cells are attached, and the temperature is 37 ℃ and the concentration of CO is 5 percent ₂ Incubating for 48h under the culture condition;

s7, after the incubation of S6.1, S6.2 and S6.3, the macrophages after the incubation were collected with a cell scraper, the macrophages were stained with FITC-CD86 after washing once with PBS, and 10,000 cells were collected by a flow cytometer at medium speed for fluorescence intensity analysis, respectively, and the results are shown in FIG. 2, indicating that the biguanide chitosan-stimulated mononuclear macrophages were mostly polarized to the M1 pro-inflammatory polarization phenotype.

Example 3

S1, primary macrophage (namely BMDM) treatment of mouse bone marrow source:

m1 polarization experimental group treatment: adding biguanide chitosan into primary macrophages derived from mouse bone marrow so that the concentration of biguanide chitosan in the primary macrophages is 1mg/mL; temperature 37 ℃, 5% CO ₂ Conventional cell culture under saturated humidity for 48 hours;

m2 polarization experimental group treatment: adding polyethylene glycol chitosan into primary macrophages derived from mouse bone marrow, so that the concentration of the polyethylene glycol chitosan in the primary macrophages is 1mg/mL; temperature 37 ℃, 5% CO ₂ Conventional cell culture under saturated humidity for 48 hours;

negative control group treatment: untreated primary macrophages derived from mouse bone marrow at 37℃and 5% CO ₂ Conventional cell culture under saturated humidity for 48 hours;

treatment of M1 type polarization positive control group: adding bacterial lipopolysaccharide (i.e., LPS) and IFN-gamma mixture to the primary macrophage derived from mouse bone marrow such that the concentration of bacterial Lipopolysaccharide (LPS) and IFN-gamma mixture in the primary macrophage is 1mg/mL; temperature 37 ℃, 5% CO ₂ Conventional cell culture under saturated humidity for 48 hours;

treatment of M2 polarized positive control group: adding interleukin-4 (i.e., IL-4) to a primary macrophage derived from mouse bone marrow such that the concentration of IL-4 in the primary macrophage is 1mg/mL; temperature 37 ℃, 5% CO ₂ Conventional cell culture under saturated humidity for 48 hours; s2, after the culture is finished, collecting macrophages treated by an M1 type polarization experimental group, an M2 type polarization experimental group, a negative control group, an M1 type polarization positive control group and an M2 type polarization positive control group by using a cell scraper; after one washing with PBS, it was stained with fluorescent-labeled anti-CD 86 and anti-CD 206;

s3, detecting the BMDM proportion of the positive CD86 to the positive CD206 after treatment in S2 by using a flow cytometry, and drawing into FIG. 3a and FIG. 3b;

as shown in FIG. 3a, the M1 polarization experimental group treatment was performed by using CD 86-positive M1 macrophages as the most BMDM stimulated by DICY-CS than the negative control group treatment;

as shown in FIG. 3b, the M2 type polarization experimental group treatment was compared with the negative control group treatment, and the BMDM cells stimulated with PEG-CS were mostly CD206 positive, i.e., M2 type macrophages.

Example 4

Collecting the cell culture supernatant obtained after culturing in the step S1 of the example 1 for 48 hours, and measuring tumor necrosis factor-alpha (namely TNF-alpha) and interleukin-6 (namely IL-6) in inflammatory cytokines in culture supernatants of a negative control group, an M1 type polarization positive control group, an M1 type polarization experimental group and an M2 type polarization experimental group by an enzyme-linked immunosorbent assay (namely ELISA) method; making figures 4a, 4b;

the same ELISA method is used for measuring the contents of Vascular Endothelial Growth Factor (VEGF) and interleukin-10 (IL-10) in anti-inflammatory cytokines in a group negative control group, an M2 type polarization positive control group, an M1 type polarization experimental group and an M2 type polarization experimental group; drawing figures 4c and 4d.

As shown in fig. 4a and 4b, BMDM stimulated with dic y-CS secreted more inflammatory cytokines in the M1 polarization experimental group treatment than in the negative control group treatment;

according to fig. 4c and 4d, the treatment of the M2 type polarization experimental group secreted more anti-inflammatory cytokines in BMDM cells stimulated with PEG-CS than the treatment of the negative control group.

Example 5

Collecting macrophages after culturing for 48 hours in the step S1 in the embodiment 3, extracting RNA of each group by adopting a centrifugal column method, and respectively measuring the expression conditions of TNF-alpha, IL-1 beta, IL-6 and iNOS in inflammatory genes of a negative control group, an M1 type polarization positive control group, an M1 type polarization experimental group and an M2 type polarization experimental group by adopting a fluorescent quantitative PCR method (namely a qRT-PCR method); making fig. 5a;

the same method is used for measuring the expression conditions of anti-inflammatory genes Stat6, mcr1, IL-10 and Arg1 in a negative control group, an M2 type polarization positive control group, an M1 type polarization experimental group and an M2 type polarization experimental group; making fig. 5b;

as shown in FIG. 5a, the levels of transcription of TNF-. Alpha., iNOS, IL-6 and IL-1. Beta. Genes were significantly up-regulated in the M1 type polarization experimental group treatment using DICY-CS stimulated BMDM cells as compared to the negative control group treatment;

as shown in FIG. 5b, the transcript levels of Stat6, mcr1 and IL-10 genes were significantly up-regulated in the M2-type polarization experimental group treatment compared to the negative control group treatment using the PEG-CS stimulated BMDM cells.

Example 6

S1, randomly selecting 15 rats with similar mass, using 4% volume fraction chloral hydrate to anesthetize the rats at a rate of 0.1mL/10g, using an electric shaver to shave the backs of the rats, and using tweezers and scissors to manufacture a circular wound with a diameter of about 25mm on the skin of the backs of the rats, wherein the circular wound is used for avoiding damaging muscle tissues;

s2, 100 mu L of drug-resistant staphylococcus aureus (namely MRSA bacterial liquid) cultured for 12 hours is dripped on the circular wound manufactured in the S1, and then the wound is put back into a cage for 72 hours.

S3, dividing rats into a blank control group, a commercial control group, a DICY-CS group, a PEG-CS group and a combination group, wherein each three rats are one group;

spraying normal saline on the wound of the blank control group, and changing the medicine once every other day;

the commercial control group wound is coated with sulfadiazine silver cream, and the dressing is changed once every other day;

DICY-CS group is sprayed with DICY-CS solution prepared by 5mg/mL physiological saline, and the dressing is changed once every other day;

PEG-CS group is sprayed with PEG-CS solution prepared by 5mg/mL physiological saline, and the medicine is changed once every other day;

the wound surface is firstly generated until the granulation tissue is formed in the first 7 days of the combined use, 5mg/mL of DICY-CS solution prepared by physiological saline is sprayed every other day, and then 5mg/mL of PEG-CS solution prepared by physiological saline is sprayed every other day until the skin granulation tissue of the wound surface begins to reconstruct the tissue proliferation period;

s4, observing the rat wound in the step S3 for 14 days, and photographing and recording the wound healing condition on the 0 th, 7 th, 10 th and 14 th days, as shown in FIG. 6;

s5, counting the wound areas at different times in S3, and calculating the percentage of the initial wound area occupied by different time points through image processing software, wherein the result is shown in figure 7.

According to FIGS. 6 and 7, the combination group used the fastest method of promoting wound healing by DICY-CS and PEG-CS sequentially compared with the control group, commercial control group, DICY-CS group and PEG-CS group.

Example 7

1 wound tissue section was prepared from rats of example 6, a hollow white control group, a commercial control group, a DICY-CS group, a PEG-CS group and a combination group, respectively, 7 days after injury;

1 wound tissue section was prepared from each of the rats in the control group, commercial control group, DICY-CS group, PEG-CS group and combination group of example 6 14 days after injury;

the method for preparing the wound tissue slice of the rat comprises the following steps: the wound tissue is obtained and fixed; tissue samples are taken to be 3mm thick, dehydrated for 30 minutes by 70%, 80%, 95% and 100% ethanol gradient, treated by 1L of dimethylbenzene for 20 minutes, embedded after being immersed in paraffin for 12 minutes in two cylinders, sliced for 4 mu m, and baked; 1.5L of xylene was then dewaxed in three portions for 8 minutes each; 1L absolute ethanol is treated twice for 8 minutes with 500mL each time; 90%, 80%, 60% ethanol for 8 minutes each. Hematoxylin staining for 4 minutes, and washing with running water; hydrochloric acid ethanol is differentiated for 2-3 seconds, and then the mixture is washed by running water; treating with 0.5% ammonia water for 20 seconds, and washing with running water; and (5) observing by an optical microscope. 0.5% eosin staining for 1 min; differentiation of 80% and 90% ethanol for 3-5 seconds respectively; treating with 95% ethanol for 5 min; 1.5L absolute ethanol is treated for three times, each time for 5 minutes; 1L of xylene is treated twice for 5 minutes each time to prepare wound tissue sections.

The wound tissue slice is washed by PBS, blocked by goat serum at room temperature, the CD68 and CD86 proteins are marked by immunofluorescence staining, meanwhile, the tablet sealing agent containing DAPI is used for sealing the tablet, and a laser confocal microscope is used for observing the expression condition of the CD68 and CD86 proteins in the wound tissue.

As shown in FIG. 8a (scale bar 50 μm), the fluorescence intensity of CD86 was higher in DICY-CS group and in the combination group in the first 7 days, compared to other groups, 7 days after injury;

as shown in FIG. 8b (scale bar 50 μm), significant fluorescence was observed only in DICY-CS group 14 days after injury, and the combined group showed a decrease in CD86 fluorescence intensity. It was demonstrated that CD86 in the combination group was mainly derived from the stimulatory effect of DICY-CS on macrophages.

Example 8

The concentration of inflammatory factor TNF-. Alpha.per unit amount of protein in rat tissue homogenates 7 days and 14 days after injury in example 7 was measured by ELISA kit, and the results are shown in FIGS. 8c and 8 d. The higher TNF-alpha concentration in the wound surface of the M1 type polarization experimental group shows that DICY-CS has great induction effect on the proinflammatory macrophages.

Example 9

In example 6, 7 days and 14 days after injury, the wound tissue sections were washed with PBS, blocked with goat serum at room temperature, immunofluorescent staining was performed to label CD68 and CD206 proteins, and meanwhile, DAPI-containing blocking tablets were used for blocking, and laser confocal microscopy was used to observe the expression of CD68 and CD206 proteins in the wound tissue.

The results are shown in FIGS. 9a, 9b (scale bar 50 μm), and the fluorescence intensity of CD206 was higher in PEG-CS group compared to other groups 7 days after injury; 14 days after injury, the fluorescence intensity of the PEG-CS group and the combined group CD206 is higher. The CD206 of the combination group is mainly derived from the stimulatory effect of PEG-CS on macrophages.

Example 10

The concentration of anti-inflammatory factor IL-10 per unit protein mass in rat tissue homogenates 7 days after injury in example 6 was determined by enzyme-linked immunosorbent assay, as shown in fig. 9c (ns p >0.05, p <0.01, p <0.001, p < 0.0001);

the concentration of anti-inflammatory factor IL-10 in the unit protein mass in rat tissue homogenate 14 days after injury in example 4 was determined by enzyme-linked immunosorbent assay kit and the results are shown in fig. 9d (ns p >0.05, p <0.01, p <0.001, p < 0.0001); the higher IL-10 concentration in the wound surface of the PEG-CS group 14 days after injury indicates the induction of the PEG-CS against macrophages.

Example 11

The rat wound tissue section in example 6 was sealed with a neutral resin gel, and the growth of wound granulation tissue, fibroblasts, and the like was observed under an optical microscope, and the results are shown in FIG. 10a (scale: 50 μm). The width of each wound was counted, and as shown in fig. 10d, the combined wound width was smaller, and no inflammatory cell infiltration was evident.

Example 12

After 21 days from injury, the remaining rats in example 4 were sectioned into tissues in the same manner as in example 7, paraffin sections were dewaxed to water, and then washed with tap water and distilled water in this order.

Staining nuclei with Regaud's hematoxylin staining solution or Weigert hematoxylin semen staining solution for 5-10 minutes; washing with distilled water was performed thoroughly.

Dissolving with Masson ponceau acid red compound red liquid staining solution for 5-10 min, soaking with 2% glacial acetic acid aqueous solution, differentiating with 1% phosphomolybdic acid aqueous solution for 3-5 min, directly staining with aniline blue or light green solution for 5 min without washing, soaking with 0.2% glacial acetic acid aqueous solution, treating with 95% alcohol, absolute alcohol and transparent xylene, and sealing with neutral gel. The obtained Masson-stained tissue sections were shown in FIG. 10b (scale: 100 μm). The percentage of collagen deposition area in each group of wound surfaces was counted.

As shown in fig. 10e, the combined group has rich collagen deposition and better wound healing.

Example 13

The sections in example 11 were washed with PBS, blocked with goat serum at room temperature, then incubated with rabbit CD31 polyclonal antibody for 3 hours, the sections were washed with PBS and the neovasculature was labeled with the corresponding fluorescent secondary antibody, while the sections were blocked with DAPI-containing blocking agents. Images were obtained by confocal laser microscopy, where blue fluorescence was the nucleus and red fluorescence was used to label the condition of the new blood vessel, as shown in FIG. 10c (scale: 100 μm). The percentage of CD31 positive area was counted as shown in fig. 10 f. Compared with the control group, the combined group has rich CD31 expression, which proves that the wound treatment mode adopted by the application has the promotion effect on the angiogenesis.

Example 14

In order to verify the effect of the application on promoting the healing of chronic difficult-to-heal wound surfaces of the diabetic body surfaces, the application adopts the following modes:

diabetic rats were weighed, anesthetized with 4% volume fraction chloral hydrate at 0.1mL/10g, shaved on the back with an electric shaver, and a circular wound of about 25mm diameter was made on the back skin of the rats using forceps and scissors, avoiding damage to muscle tissue. Rats were randomly grouped into a blank control group, an M2-polarization-promoting material application group, an M1-polarization-promoting material + M2-polarization-promoting material application group, each group being 3. The control group is sprayed with 1mL of physiological saline, and the M2 macrophage polarization group is sprayed with 1mL of PEG-CS solution with concentration of 8mg/mL prepared by 5mg/mL physiological saline, and the above groups are changed every other day. 1mL of DICY-CS solution prepared by 5mg/mL of physiological saline is sprayed every 7 days before the combined use, and 1mL of PEG-CS solution prepared by 5mg/mL of physiological saline is sprayed every other day. Rat wounds were observed and photographed for recording. The photographs of the wound at different times after injury are shown in fig. 11 (ns p >0.05, p <0.01, p <0.001, p < 0.0001), and the combination of the treatment methods of the present application using dic-CS and PEG-CS showed the fastest wound healing compared to the control groups.

From examples 1 to 5, it was revealed that the biguanide chitosan-stimulated mononuclear macrophages were mostly polarized toward the M1 pro-inflammatory polarized phenotype, and in the preparation method of the biguanide chitosan solution provided in the present application, when the biguanide chitosan prepared according to the method provided in example 1 was applied to the cell wall attachment test effect, it was found that only 11.9% of macrophages were polarized toward the M1 pro-inflammatory polarized phenotype in comparison with the classical LPS and IFN- γ mixed solution incubation, whereas 82.1% of macrophages were polarized toward the M1 pro-inflammatory polarized phenotype, which is far greater than the number of macrophages polarized in comparison with the LPS and IFN- γ mixed solution incubation; and the transcription level of inflammatory cytokines, TNF-alpha, iNOS, IL-6 and IL-1 beta genes is obviously up-regulated by the mononuclear macrophages stimulated by the biguanide chitosan, which is helpful for accelerating the healing of chronic wound surfaces difficult to heal on the body surface.

From the experimental data combined with examples 6-13, the material inducing macrophage M1 polarization and the material inducing macrophage M2 polarization are sequentially segmented and used for the tissue proliferation period from the initial generation of chronic wound healing to the initial reconstruction of granulation tissue formation stage and the initial reconstruction of skin granulation tissue; the effect of promoting wound healing is obviously better than that of continuously using one material only on the wound; proved by the application, the macrophage-promoting polarization material can restore the M1/M2 macrophage balance in the chronic difficult-to-heal wound surface, and the application of the macrophage-promoting polarization material in the chronic difficult-to-heal wound surface can obviously improve the healing speed of the surface wound surface.

From example 14, it can be seen that the present application has unexpected technical effects on promoting the healing of chronic difficult-to-heal wound surfaces of diabetic body surfaces, and on day 21, the wound of the combination group is completely closed, because the material of the polarization of the pro-situation cells M1 induces acute immune response to generate in the inflammatory phase, phagocytose pathogenic microorganisms, and promote smooth transition from the inflammatory phase to the proliferative phase; the material for inducing macrophage M2 polarization eliminates necrotic tissue in proliferation period, dissolves inflammation and promotes tissue repair and regeneration; thus providing the optimal wound immune microenvironment for different wound healing periods of diabetes mellitus, recovering the M1/M2 type macrophage balance in the chronic difficult-to-heal wound, and accelerating the healing of the chronic difficult-to-heal wound on the body surface.

The application is to be understood as being limited to the preferred embodiments thereof, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. The application of macrophage-promoting polarization material in chronic wound surface difficult to heal is characterized in that: the macrophage-stimulating polarization material comprises a macrophage-stimulating M1 polarization material and a macrophage-stimulating M2 polarization material.

2. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 1, wherein: the macrophage-promoting M1 polarization material is biguanide chitosan, and the biguanide chitosan is prepared by the following steps:

s1, dissolving chitosan with a molecular weight of 10,000-1,000,000Da raw material into hydrochloric acid solution with a mass concentration of 0.1mol/L to prepare chitosan hydrochloride solution;

s2, adding dicyandiamide which is 2-4 times of the amount of amino substances in the chitosan into the chitosan hydrochloride solution in the S1 under the stirring condition to obtain a mixture B;

s3, reacting the mixture B at the reflux of 90 ℃ for 6-48 hours to obtain an intermediate reaction product A;

s4, transferring the intermediate reaction product A into a dialysis bag with the molecular weight cutoff of 3,500-14,000Da, dialyzing in deionized water, changing water once every 6-12 hours, changing water for 6-10 times, and freeze-drying to obtain the biguanide chitosan.

3. The use of a macrophage-stimulating polar material in a chronic refractory wound surface according to claim 1, wherein the macrophages comprise macrophage line RAW264.7, macrophage line THP-1, macrophage line U937, bone marrow-induced macrophages, peripheral blood-derived mononuclear macrophage PBMCs, mammalian tissue-derived primary macrophages.

4. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 2, wherein: the biguanide chitosan is biguanide chitosan solution, biguanide chitosan spray and biguanide chitosan gel.

5. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 2, wherein: the biguanide substitution degree of the biguanide chitosan is 5-20%.

6. The use of macrophage-stimulating polar material according to claim 1 or 2 in chronic refractory wounds on the body surface, wherein the macrophage-stimulating M2 polar material is polyethylene glycol chitosan.

7. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 1, wherein: the macrophage-promoting M1 polarization material is applied to the initial generation of the chronic wound surface difficult to heal until the granulation tissue formation stage; the macrophage-promoting M2 polarization material is applied to the tissue proliferation period of the wound skin granulation tissue which is difficult to heal on the body surface and is reconstructed at the beginning.

8. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 7, wherein: after the macrophage-promoting M1 polarization material is used for the chronic wound surface difficult to heal of the body surface, the macrophages are expressed as A and/or B and/or C:

A. enhancing transcription of M1-type macrophage genes;

B. enhancing secretion of inflammatory factors;

C. the expression level of the co-stimulatory factor CD86 is increased.

9. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 8, wherein: the inflammatory factors in B include one or more of tumor necrosis factor alpha, interleukin-6 and interleukin-12.

10. The use of macrophage-stimulating polar material in chronic refractory wounds on a body surface according to claim 1, wherein: the chronic wound surface difficult to heal is a wound surface with damaged macrophage function, severely unbalanced macrophage phenotype and obviously insufficient inflammatory response in early stage of wound.