Pharmaceutical composition for reprogramming repair type macrophages and reprogramming method thereof

Abstract

The invention provides a pharmaceutical composition for reprogramming repair type macrophages and a reprogramming method thereof, the pharmaceutical composition comprises four micromolecular drugs of fenofibrate, dexamethasone, rosaxostat and calcitriol, and the four micromolecular drugs FDRC are combined in a certain dosage and applied to the macrophages to reprogram the macrophages with repair function phenotypes. The macrophages are reprogrammed into a repair-promoting functional phenotype, and the macrophages can promote the proliferation and repair effects of epithelial cells and vascular endothelial cells by secreting a large number of repair factors, such as Vegfa, Gdf15, Hbegf, Ndrg1, Tamp3, Ccn1 and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a drug combination for reprogramming reparative macrophages and a reprogramming method thereof. Background Art

[0002] Macrophages have phenotypic and functional diversity. Their sources mainly include the yolk sac and liver during embryonic development. In addition, monocytes derived from the bone marrow can also be transformed into tissue macrophages and participate in physiological and pathological processes. Inside tissues, macrophages play important roles. They can provide growth factors for local cells and play an indispensable promoting role in aspects such as cell development, nutrient circulation, and waste clearance, and are a key force in maintaining tissue growth, homeostasis, and repair. Therefore, the inhibition of macrophages may disrupt the homeostasis of the organ immune environment and cause adverse effects.

[0003] Multiple studies have confirmed that there are reparative macrophages in the body. They contribute to organogenesis, actively play a role in the regeneration process after tissue damage, and help restore the homeostatic tissue environment in the body. For example, renal medullary macrophages can effectively clear luminal particulate matter by extending transcellular protrusions across the renal tubular epithelial monolayer; the MARCO-positive macrophage subset has the ability to promote inflammation resolution and inhibit tissue fibrosis damage. The heterogeneity and functional plasticity of macrophages endow them with complex and diverse regulatory functions during tissue repair and regeneration. In previous studies, renal tissues at different injury stages of mice with unilateral ischemic reperfusion injury were collected for single-cell sequencing (GEO: GSE267242). The analysis results showed that kidney macrophages were divided into 7 subpopulations with different characteristics, including a pro-repair macrophage functional subpopulation, which was mainly characterized by the high expression of repair factors such as Vegfa, Gdf15, Hbegf, Ndrg1, Timp3, and Ccn1.

[0004] Although the importance of macrophages in tissue damage and repair has become increasingly clear, however, there is still a lack of methods for regulating the phenotype and function of reparative macrophages. Therefore, it is necessary to specifically develop new drug combinations to intervene in macrophages, which can reprogram macrophages to have a pro-repair functional phenotype. Summary of the Invention

[0005] Objective of the invention: Aiming at the deficiencies and defects of the prior art, the present invention provides a drug combination for reprogramming repair-type macrophages and a reprogramming method thereof. Four small molecule drugs, namely fenofibrate, dexamethasone, roxadustat, and calcitriol (FDRC), are applied to macrophages in a certain dosage combination to reprogram macrophages with a repair-functional phenotype, intervene in macrophages, be able to reprogram macrophages to have a pro-repair functional phenotype, promote macrophages to secrete repair factors, and have a potential repair effect on epithelial cells and vascular endothelial cells.

[0006] Technical solution: A drug combination for reprogramming repair-type macrophages of the present invention is characterized in that: the drug combination includes four small molecule drugs, namely fenofibrate, dexamethasone, roxadustat, and calcitriol. After the four small molecule drugs are combined, they are used to engineer and reprogram macrophages, and the reprogrammed macrophages are of a pro-repair functional phenotype.

[0007] The method for reprogramming macrophages with the drug combination of the present invention is characterized in that: the macrophages include in vitro macrophages and macrophages in vivo. After reprogramming with the drug combination, a phenotypic characteristic of high expression of repair factors is obtained to form repair-type macrophages. The repair factors secreted by the repair-type macrophages include Vegfa, Gdf15, Hbegf, Ndrg1, Timp3, and Ccn1.

[0008] Among them, the method for reprogramming the in vitro macrophages includes the following steps:

[0009] 1) Culture the RAW264.7 macrophage cell line or extract bone marrow primary macrophages BMDM;

[0010] 2) Reprogram the macrophages obtained in step 1) with a drug combination of fenofibrate, dexamethasone, roxadustat, and calcitriol; the concentrations of the four small molecule drugs, namely fenofibrate, dexamethasone, roxadustat, and calcitriol, are 1 μM - 100 μM, 0.1 μM - 10 μM, 1 μM - 100 μM, and 0.1 μM - 100 μM respectively; the obtained reprogrammed macrophages have the characteristics of high expression of repair factors and the function of promoting the proliferation and repair of epithelial cells or endothelial cells.

[0011] Further, the specific process of step 2) is as follows:

[0012] 1) Add the following preferred concentration drugs to the macrophages in sequence for intervention: fenofibrate 10 μM, dexamethasone 5 μM, roxadustat 30 μM, calcitriol 0.1 μM, mix evenly, and culture for 24 h;

[0013] 2) Detect the expression levels of pro-repair factors Vegfa, Gdf15, Hbegf, Ndrg1, Timp3, and Ccn1 before and after the drug combination intervention;

[0014] 3) Use the culture medium supernatant of macrophages reprogrammed with a drug combination to intervene in epithelial or endothelial cell lines to observe the effect of reprogrammed macrophages on promoting epithelial or endothelial proliferation and repair.

[0015] Among them, the method for reprogramming the in-vivo macrophages is: administering a drug combination of fenofibrate, dexamethasone, roxadustat, and calcitriol to an organism for macrophage reprogramming; the dosages of the four small molecule drugs, fenofibrate, dexamethasone, roxadustat, and calcitriol, are 10 mg / kg / d - 1000 mg / kg / d, 0.1 mg / kg / d - 10 mg / kg / d, 1 mg / kg / d - 100 mg / kg / d, and 0.1 μg / kg / d - 100 μg / kg / d, respectively.

[0016] Further, the process of the in-vivo macrophage reprogramming method is as follows:

[0017] 1) Intraperitoneally inject a drug combination for macrophage reprogramming into mice. The dosages of the four small molecule drugs, fenofibrate, dexamethasone, roxadustat, and calcitriol, are 100 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d, and 0.5 μg / kg / d, respectively, and administer continuously for 7 days;

[0018] 2) Immunofluorescence detection of the proportion of macrophages in mouse tissues reprogrammed into Vegfa+Gdf15+ reparative macrophages;

[0019] 3) Observe the effect of the drug combination on reprogramming mouse macrophages on the regeneration and repair of renal tissue epithelial or endothelial cells.

[0020] The drug combination of the present invention is made into a pharmaceutical preparation form.

[0021] Application of the drug combination for reprogramming reparative macrophages of the present invention.

[0022] Application of the drug combination for reprogramming reparative macrophages of the present invention in regeneration and repair.

[0023] Application of the drug combination for reprogramming reparative macrophages of the present invention in endothelial and epithelial repair.

[0024] Principle analysis: The principle by which the drug combination of the present invention successfully reprograms reparative macrophages is that, according to single-cell sequencing transcription factor analysis, the expression of repair factors in the reparative macrophage subset is regulated by the core transcription factors Egr1, Ppara, and Cebpb. Fenofibrate has a direct agonistic effect on Ppara, and existing literature also indicates that dexamethasone may have an inducing effect on Cebpb, and calcitriol has a tendency to promote the expression of Egr1. In addition, Roxadustat has a significant inducing effect on the expression of repair factors. Therefore, the FDRC reprogramming method can promote the transcription of macrophage repair factors Vegfa, Gdf15, Hbegf, Ndrg1, Timp3, and Ccn1, and stably reprogram macrophages into reparative macrophages.

[0025] Compared with the prior art, the present invention has the following remarkable advantages:

[0026] Beneficial effect 1: The present invention analyzes the characteristics and gene regulatory network of this group of reparative macrophages, and proposes a novel small molecule drug combination to intervene in macrophages, which can reprogram macrophages to have a reparative function phenotype. This precise intervention method is different from the relatively broad reprogramming methods in previous studies, and obtains a reparative macrophage phenotype with more definite functions than the classical subtype M2, providing a more targeted strategy for the functional regulation of macrophages.

[0027] Beneficial effect 2: The drug combination proposed by the present invention can effectively promote macrophages to secrete repair factors, and these repair factors play a key role in the process of tissue repair. Compared with the previous method of supplementing a single repair factor to damaged tissues for regenerative repair, or the situation where macrophages cannot be effectively stimulated to secrete sufficient amounts of repair factors, the drug combination of the present invention significantly increases the expression levels of multiple repair factors, providing a better regenerative microenvironment for tissue repair. Moreover, the combination of multiple small molecule drugs can act on multiple targets and signaling pathways simultaneously, realizing multi-dimensional regulation of the reprogramming process, and can more comprehensively regulate the functions and phenotypes of cells, improving the effect and specificity of reprogramming.

[0028] Beneficial effect 3: The small molecule drug combination FDRC has a stable macrophage reprogramming effect, and can continuously reprogram macrophages into a phenotype with reparative functions, ensuring the stability and reliability of the treatment effect. In previous studies, the method of genetically reprogramming macrophage phenotypes was cumbersome to operate, and there may be problems such as immunogenic risk and genetic mutation risk. The drug combination of the present invention effectively overcomes this shortcoming, and at the same time provides a more solid foundation for clinical application.

[0029] The drug combination (FDRC) of the present invention intervenes in macrophages and has the ability to reprogram macrophages to transform into a pro-repair phenotype. In in vitro studies, the reprogrammed repair macrophages can promote the proliferation and repair of epithelial cells and endothelial cells. In in vivo experiments, FDRC reprogrammed macrophages showed a good effect in improving tubular epithelial cell injury and had a good pro-repair effect. The present invention provides a new strategy for macrophage reprogramming, promotes the development of macrophage phenotype research, and provides new ideas for clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the dimensionality reduction map of macrophages in mice with renal ischemia-reperfusion; (A) Schematic diagram of the mouse model of renal ischemia-reperfusion; (B) TSNE dimensionality reduction map of renal macrophages subclustered into 7 cell subsets.

[0031] Figure 2 It is the process map of drug discovery for macrophage reprogramming; (A) Using Regulon, ChEA3, TRRUST, and Lisa2 transcription factor prediction tools to predict that the key transcription factors for gene regulation of pro-repair macrophages are Cebpb, Ppara, and Egr1; (B) Network diagram of the regulatory effects of key transcription factors on genes related to kidney development, angiogenesis, epithelial repair, and energy homeostasis; (C) Predicting drugs to promote key transcription factors and related repair factors: fenofibrate, dexamethasone, roxadustat, calcitriol.

[0032] Figure 3 It is the effect diagram of FDRC drug combination reprogramming pro-repair macrophages; (A) Transcription levels of key transcription factors and related repair factors of macrophages before and after FDRC drug intervention; (B) Flow cytometry diagram showing the proportion of pro-repair macrophages before and after FDRC drug intervention; (C) Heat map of differential gene clustering analysis of transcriptome sequencing of FDRC-intervened macrophages, M0, and M2 macrophage phenotypes.

[0033] Figure 4 It is the repair effect diagram of FDRC reprogrammed macrophages; (A) Schematic diagram and statistical chart of the scratch healing of HUVEC cells intervened by the supernatant of FDRC reprogrammed macrophages; (B) Statistical chart of the number of HK-2 cells intervened by the supernatant of FDRC reprogrammed macrophages; (C) Immunofluorescence diagram showing the change in the proportion of pro-repair macrophages after FDRC drug administration to AKI mice; (D) PAS and MASSON pathological diagrams showing the effect of FDRC drug administration to AKI mice on kidney injury.

[0034] Figure 5 It is the schematic diagram of FDRC drug combination reprogramming macrophages to promote regeneration and repair. DETAILED DESCRIPTION OF THE INVENTION

[0035] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0036] The research process of the drug combination for reprogramming and repairing macrophages of the present invention includes the following four parts:

[0037] The first part: Observation of macrophage phenotypes in vivo and discovery of pro-repair macrophages:

[0038] 1) Establishment of the AKI mouse model: A unilateral 35-minute ischemia / reperfusion injury model was established in 6-week-old C57BL / 6J mice (body weight 20 - 22 g). The mice were fasted for 12 h before surgery. After anesthesia, the body temperature of the mice was controlled at 36.8°C to 37.2°C using an animal temperature controller before surgery. During the operation, the bilateral renal pedicles were isolated and clamped with a non-invasive micro arterial clamp. After clamping, the color of the kidneys changed, and returned to normal after removing the arterial clamp. After the operation, the abdomen was sutured and closed. The sham operation group was operated in the same way but without clamping the renal pedicle. Four mouse models were constructed at each of the five time points of 0, 1, 3, 14, and 28 days after the operation, as Figure 1 shown in

[0039] 2) Sample collection: Renal tissue samples were collected from AKI mice at different injury stages, and single-cell samples were prepared through steps such as tissue shredding, enzymatic digestion, tissue dissociation, and red blood cell lysis.

[0040] 3) Single-cell quality control and sequencing: The quality control standards included a cell viability > 85%, a cell aggregation rate < 15%, a viable cell concentration range of 300 - 2000 cells / μl, a nucleated rate > 70%, a cell diameter range of 5 - 30 μm, and a cell suspension volume > 60 μl. After passing the quality inspection, the 10×Genomics library construction operation was immediately carried out. The cell suspension was added to the water-in-oil system, and after forming GEMs, steps such as reverse transcription were carried out, and finally the cDNA was sent to construct a sequencing library for sequencing.

[0041] 4) Macrophage sub-clustering: The RunTSNE function in Seurat 4.0.4 was used for dimensionality reduction analysis, and the FindAllMarker function was used for differential gene analysis. Functional analysis and cell annotation were carried out according to the highly expressed differential genes in each cell subpopulation.

[0042] The present invention successfully prepared an AKI chronic progression mouse model, obtained qualified single-cell samples of the kidneys at different stages of AKI chronic progression, and a total of 2241 renal macrophages were obtained by single-cell sequencing and clustered into 7 cell subpopulations, including repair macrophages, pro-inflammatory macrophages, extracellular matrix remodeling macrophages, etc. The present invention focused on the pro-repair macrophage subpopulation characterized by the high expression of repair factors Vegfa, Gdf15, Hbegf, Ndrg1, Timp3, and Ccn1, as Figure 1 shown in

[0043] Part II: Drug discovery for pro-repair reprogramming of macrophages:

[0044] 1) Prediction of key transcription factors for pro-repair macrophages: Using Regulon, ChEA3, TRRUST, and Lisa2 transcription factor prediction tools, we predicted that the key transcription factors for pro-repair macrophage gene regulation are Cebpb, Ppara, and Egr1. Figure 2 As shown in A;

[0045] 2) Establish a key transcription factor-repair factor regulatory network diagram: perform pathway enrichment analysis on genes highly expressed in pro-repair macrophages, obtain gene entry information and related factors related to repair, and use transcriptional regulatory information to draw a network diagram of the regulatory effects of key transcription factors on genes highly expressed in repair macrophages, including genes related to kidney development, angiogenesis, epithelial repair, and energy homeostasis, such as Figure 2 As shown in B;

[0046] 3) Predict drugs that promote key transcription factors and related repair factors: fenofibrate, dexamethasone, rosuvastatin, calcitriol, e.g. Figure 2 As shown in C.

[0047] The key transcription factors Cebpb, Ppara and Egr1 of the present invention are involved in the transcriptional regulation of the repair factors of the pro-repair macrophage subpopulation, and the small molecule drugs fenofibrate, dexamethasone, rosuvastatin and calcitriol that promote the key transcription factors and the repair factors are predicted.

[0048] Part III: Fenofibrate, dexamethasone, rosuvastatin, and calcitriol drug combination (FDRC) reprograms pro-repair macrophages:

[0049] 1) Macrophages were counted at 5×10 5 100 μM fenofibrate, 5 μM dexamethasone, 30 μM rosuvastatin, and 0.1 μM calcitriol were added in sequence, mixed evenly, and cultured for 24 h. The cells were discarded from the supernatant, washed twice with PBS, and lysed with Freezol reagent to extract RNA. After obtaining total RNA, the nucleic acid concentration was detected and reverse transcribed into cDNA. The expression levels of macrophage repair factors and repair-related transcription factors were detected using RT-PCR technology, such as Figure 3 As shown in A;

[0050] 2) After the macrophages were treated with the FDRC drug combination, the cell supernatant was discarded, the cells were collected and counted, and an equal number of cells (1×10 6(Add 250 μl of fixation and permeabilization solution and incubate for 10 min. Add 1 ml of wash buffer, centrifuge at 300 g for 3 min, resuspend with 100 μl of wash buffer. Add 1 μg of FC blocking solution to each sample and incubate for 15 min. Add 1 μl of PE-GDF15 and 1 μl of CL488-VEGFA, incubate at room temperature for 30 min. Add 1 ml of PBS, centrifuge at 300 g for 3 min, resuspend with 200 μl of PBS, and immediately analyze by flow cytometry. The flow cytometry plots show the changes in the proportion of Vegfa+Gdf15+ pro-repair macrophages before and after FDRC drug intervention, as Figure 3 shown in

[0051] 3) Macrophages were treated with the FDRC drug combination to reprogram the pro-repair cell phenotype, or induced to differentiate into the M2 phenotype with 20 ng / ml of IL-4. The control group was not treated with drugs. Total RNA was extracted for transcriptome sequencing. The differentially upregulated genes in each group were subjected to cluster analysis and the main enriched pathways of each cluster were shown, as Figure 3 shown in

[0052] The combination of fenofibrate, dexamethasone, roxadustat, and calcitriol at a certain concentration (FDRC) of the present invention promoted the expression of repair factors and key transcription factors in macrophages. Moreover, FDRC intervention in macrophages increased the proportion of pro-repair macrophages, and the pro-repair macrophages reprogrammed by FDRC were significantly different from M0 and M2 phenotype macrophages and had unique repair characteristics.

[0053] Part 4: Repair effect of FDRC-reprogrammed macrophages:

[0054] 1) Seed HUVEC (Human Umbilical Vein Endothelial Cells) in 6-well plates. After cell confluence, use a 200-μl pipette tip to make a cell scratch. Wash away the floating cells with PBS and take a picture of the cell scratch width at 0 h. Add the cell supernatant of FDRC-reprogrammed macrophages or the control supernatant, and take a picture of the cell scratch width at 24 h to observe the effect of the FDRC macrophage supernatant on the wound healing of HUVEC, as Figure 4 shown in

[0055] 2) Seed HK-2 (Human Kidney-2) in 6-well plates. Add the cell supernatant of FDRC-reprogrammed macrophages or the control supernatant. After 24 h, count the number of HK-2 cells using a cell counting plate and draw a statistical graph, as Figure 4 shown in

[0056] 3) A unilateral 35-min ischemia / reperfusion injury model was established in 6-week-old C57BL / 6J mice. After the operation, an FDRC drug mixture was intraperitoneally injected on the 7th day, once a day for a total of 7 days. Kidney tissues were obtained, and the macrophage marker F4 / 80 and the pro-repair macrophage marker Gdf15 were immunofluorescently stained to observe the effect of the FDRC drug on the proportion of pro-repair macrophages, as Figure 4 shown in C;

[0057] 4) PAS and MASSON pathological diagrams show the effect of the FDRC drug on kidney injury in AKI mice, as Figure 4 shown in D.

[0058] The FDRC reprogrammed macrophages of the present invention have the effects of promoting vascular endothelial cell wound healing and promoting the proliferation of tubular epithelial cells. In addition, the FDRC drug combination also has the effect of reprogramming macrophages in vivo, significantly increasing the proportion of pro-repair macrophages and improving kidney injury. (Statistical data are given in the form of mean ± standard error. One-way ANOVA was used for inter-group comparison, and the t-test was used for comparison between two groups. p < 0.05 was considered to have a significant difference. The experimental results were repeated more than 3 times)

[0059] The FDRC reprogramming method of the present invention can promote the transcription of macrophage repair factors Vegfa, Gdf15, Hbegf, and Ndrg1, and successfully increase the proportion of Vegfa+Gdf15+ reparative macrophages in vivo and in vitro. The reprogramming method of the present invention successfully upregulates the key transcription factors Egr1, Ppara, and Cebpb involved in the regulation of repair genes, and has a stable reprogramming effect. Compared with classical M2 macrophages reprogrammed by the method of the present invention, IL-4 intervention has no significant effect on the levels of Vegfa, Gdf15, and Ndrg1 in macrophages, while FDRC reprogrammed macrophages can significantly increase the levels of the above pro-repair factors. The genes upregulated by FDRC reprogrammed macrophages show repair-related signaling pathways such as angiogenesis, wound healing, and epithelial cell migration, and avoid the adverse effects such as T cell recruitment and fibrosis promotion of the M2 cell phenotype.

[0060] The FDRC drug combination of the present invention can significantly promote macrophages to express repair factors Vegfa, Gdf15, Hbegf, and Ndrg1. The reprogrammed macrophages have the effects of promoting endothelial cell wound healing and promoting the proliferation and regeneration of tubular cells. The FDRC drug combination preparation of the present invention shows the characteristic of high expression of repair factors in vivo or in vitro. The present invention can promote the regenerative repair reaction of endothelial cells or epithelial cells.

Claims

1. A drug combination for reprogramming and repairing macrophages, characterized in that: The described drug combination includes four small molecule drugs, namely fenofibrate, dexamethasone, roxadustat, and calcitriol. After the combination of the four small molecule drugs, macrophages are engineered and reprogrammed into a pro-repair functional phenotype.

2. The method for reprogramming macrophages by the drug combination according to claim 1, wherein: The described macrophages include in vitro macrophages and macrophages in vivo. Through drug combination reprogramming, a phenotypic characteristic of high expression of repair factors is obtained to form repair macrophages. The repair factors secreted by the repair macrophages include Vegfa, Gdf15, Hbegf, Ndrg1, Timp3, and Ccn1.

3. The method for reprogramming macrophages by the drug combination according to claim 1 or 2, characterized in that: The method for reprogramming the in vitro macrophages includes the following steps: 1) Cultivate the RAW264.7 macrophage cell line or extract bone marrow primary macrophages BMDM; 2) Reprogram the macrophages obtained in step 1) with a drug combination of fenofibrate, dexamethasone, roxadustat, and calcitriol; the concentrations of the four small molecule drugs, fenofibrate, dexamethasone, roxadustat, and calcitriol, are 1 μM - 100 μM, 0.1 μM - 10 μM, 1 μM - 100 μM, and 0.1 μM - 100 μM respectively; the obtained reprogrammed macrophages have the characteristics of high expression of repair factors and the function of promoting the proliferation and repair of epithelial cells or endothelial cells.

4. The method for reprogramming macrophages using the pharmaceutical combination according to claim 3, characterized in that: The specific process of step 2) is as follows: The macrophages are sequentially intervened with the following preferred concentration drugs: fenofibrate 10 μM, dexamethasone 5 μM, roxadustat 30 μM, and calcitriol 0.1 μM. Mix evenly and culture for 24 h.

5. The method for reprogramming macrophages by the drug combination according to claim 1 or 2, characterized in that: The method for reprogramming the in vivo macrophages is: Administer a drug combination of fenofibrate, dexamethasone, roxadustat, and calcitriol to an organism for macrophage reprogramming; the doses of the four small molecule drugs, fenofibrate, dexamethasone, roxadustat, and calcitriol, are 10 mg / kg / d - 1000 mg / kg / d, 0.1 mg / kg / d - 10 mg / kg / d, 1 mg / kg / d - 100 mg / kg / d, and 0.1 μg / kg / d - 100 μg / kg / d respectively.

6. The method for reprogramming macrophages by the drug combination according to claim 5, characterized in that: The method for reprogramming the in vivo macrophages is: Intraperitoneally inject a drug combination for macrophage reprogramming to mice. The administration doses of the four small molecule drugs, fenofibrate, dexamethasone, roxadustat, and calcitriol, are 100 mg / kg / d, 1 mg / kg / d, 10 mg / kg / d, and 0.5 μg / kg / d respectively. Administer continuously for 7 days; the tissue macrophages of the mice are reprogrammed into repair macrophages and promote the regeneration and repair of epithelial cells or endothelial cells of the mice.

7. The pharmaceutical combination of reprogrammed and repaired macrophages according to claim 1 or 2, characterized in that: The described drug combination is made into a pharmaceutical preparation form.

8. Application of the drug combination for reprogramming repair macrophages according to claim 1 or 2.

9. Application of the drug combination for reprogramming repair macrophages according to claim 8 in regenerative repair.

10. Application of the drug combination for reprogramming repair macrophages according to claim 8 in endothelial and epithelial repair.

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