CN117982468A - Application and medicine of an ERK agonist in promoting wound healing in diabetic foot - Google Patents

Abstract

The invention discloses an application of an ERK agonist in promoting the healing of a diabetic foot wound surface and a medicine thereof, which are characterized in that the difficult healing of the DFU wound surface is caused by crosstalk of keratinocytes and fibroblasts of skin tissues of the diabetic foot wound edge through bioinformatics analysis, and various embodiments are used for verifying that the fibroblast in the DFU wound surface presents aging characteristics due to induction of high sugar, and inhibiting the expression of pERK/ERK protein in keratinocytes, so that the keratinocytes in the DFU wound surface present low migration characteristics and are difficult to heal, and the migration of the keratinocytes in the DFU wound surface can be promoted by promoting the expression of pERK/ERK protein in the keratinocytes, so as to promote the healing of the DFU wound surface. And provides a medicament containing ERK agonist capable of promoting the healing of the DFU wound surface on the basis.

Description

Application and medicine of an ERK agonist in promoting diabetic foot wound healing

Technical Field

The present invention belongs to the field of biomedicine, and more specifically, relates to an application and medicine for promoting the healing of diabetic foot wounds by using an ERK agonist.

Background technique

Diabetic foot ulcer is a common complication of diabetes, which seriously affects the patient's ability to work and quality of life. Diabetic foot ulcer is the main clinical manifestation of diabetic foot. The clinical signs of diabetic foot ulcer caused by peripheral circulation disorder in the early stage are lower skin temperature of the lower limbs, dullness or loss of pain and pressure sensation. When inappropriate pressure is applied by the outside world, it is easy to cause foot trauma, tissue rupture, and eventually lead to the occurrence of ulcers (Risk Factors and Treatment Progress of Diabetic Foot Ulcers. Chinese General Practice, 2013, 16: 3159-3163).

The pathogenesis of DFU is mainly that with the continuous increase of blood sugar level, long-term poor blood sugar control in the human body leads to impaired islet function, reduced adenosine triphosphate (ATP) synthesis, reduced insulin secretion, and increased blood sugar and blood lipids in the human body. High concentrations of blood sugar and blood lipid levels inhibit the mitochondrial electron transport chain, leading to mitochondrial oxidative stress damage, vascular endothelial damage and vascular lumen stenosis, leading to ischemia and affecting new blood vessel formation, and decreased immunity to infection. This disease is easy to be uncured for a long time, infection occurs, and there are many influencing factors that cause osteomyelitis and bone destruction. Among them, diabetic nephropathy, peripheral neuropathy (DPN), autonomic neuropathy, and lower extremity arteriosclerosis (LEAD) are risk factors for diabetic foot. The patient's advanced age, long course of disease, poor blood sugar control, infection level, systemic nutritional status, DPN, lower extremity arterial ischemia, etc. are important causes of ulcer failure and amputation. Smoking, poor blood sugar control, DPN and LEAD with ankle joint reflex loss are important independent risk factors for foot ulcer recurrence. Dialysis treatment and poor blood sugar control are independent predictors of severe limb ischemia recurrence after endovascular treatment. The clinical recurrence of severe acute limb ischemia and the recurrence of foot ulcers, major amputations and even the increased incidence of death are closely related to the inadequate treatment, misdiagnosis and poor prognosis of diabetic foot.

The clinical manifestations of DFU are neuropathy and lower limb ischemia. Neuropathy is manifested as dry skin without sweating, tingling, burning pain, numbness, decreased or absent sensation in the extremities, sock-like changes, and a feeling of stepping on cotton wool when walking; lower limb ischemia is manifested as skin malnutrition, muscle atrophy, dry and poor skin elasticity, decreased skin temperature, pigmentation, weakened or absent arterial pulsation in the extremities, and patients may have symptoms of intermittent claudication in the lower limbs. As the disease progresses, rest pain, gangrene at the toe tips, ulcers at the pressure points of the heel or metatarsophalangeal joints, and some patients may have limb infections.

The occurrence and development of diabetic foot ulcer (DFU) is closely related to neuropathy, peripheral arterial disease and infection. It is a disease caused by the high sugar environment in the body and multiple biological factors, and its pathogenesis is very complex. DFU is a very difficult disease to treat clinically. Conventional treatment mainly treats various causes symptomatically. New biotechnology has been applied to the treatment of DFU by improving the local microenvironment, reducing inflammatory response, and increasing wound healing rate, but the popularity rate is low.

Summary of the invention

In view of the above defects or improvement needs of the prior art, the present invention provides a drug for promoting the healing of diabetic foot wounds by using an ERK agonist, which repairs the weakened keratinocyte migration ability caused by the crosstalk between keratinocytes and fibroblasts in diabetic foot tissue and enhances the inhibition of the pERK/ERK signaling pathway of the keratinocytes, thereby promoting the healing of diabetic foot wounds.

In order to achieve the above object, the present invention provides the following technical solutions:

On the one hand, the present invention provides an application of an ERK agonist to promote the healing of diabetic foot wounds, wherein the ERK agonist repairs the weakened keratinocyte migration ability caused by the inhibition of the pERK/ERK signaling pathway in diabetic foot tissue by upregulating the pERK/ERK signaling pathway, thereby promoting the healing of diabetic foot wounds;

Furthermore, the application includes promoting the healing of diabetic foot ulcer wounds;

Further, the ERK agonist includes tert-butylhydroquinone and a salt, solvate, hydrate or stereoisomer of tert-butylhydroquinone;

Furthermore, the drug further comprises a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises at least one of a flavoring agent, an osmotic pressure regulator, a filler, a lubricant, a preservative, a suspending agent, a food coloring, a diluent, an emulsifier, a disintegrant or a plasticizer;

Furthermore, the dosage form of the drug is an oral preparation or an injection; wherein the dosage form of the oral preparation is tablets, pills, oral liquid, capsules, syrups, pellets or granules; the injection includes an injection powder or injection solution.

In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

The inventors have discovered for the first time and verified through multiple embodiments that fibroblasts in DFU wounds exhibit aging characteristics due to high sugar induction, which inhibits the expression of pERK/ERK proteins in keratinocytes, so that keratinocytes in DFU wounds exhibit low migration characteristics and are therefore difficult to heal. By promoting the expression of pERK/ERK proteins in keratinocytes, the migration of keratinocytes in DFU wounds can be promoted, thereby promoting the healing of DFU wounds. On this basis, a drug containing an ERK agonist that can promote the healing of DFU wounds can be provided, which provides a new idea for the effective treatment of DFU wound healing in clinical practice, and has good prospects for clinical application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the top ten cell types in terms of the percentage of skin tissue within 2 cm of the wound edge on the dorsum of the foot of a male patient with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

FIG2 is a bar graph showing the percentage of the top ten cells in the skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

3 is a cluster diagram of the top ten differentially expressed genes in the skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

4 is a schematic diagram of keratinocyte subcluster types in skin tissue within 2 cm of the wound edge on the dorsum of the foot of a male patient with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

5 is a bar graph showing the percentage of keratinocyte subclusters in skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

6 is a cluster diagram of differentially expressed genes in subclusters of keratinocytes in skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

7 is a schematic diagram of the subcluster types of skin tissue fibroblasts within 2 cm of the wound edge on the dorsum of the foot of a male patient with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

FIG8 is a bar graph showing the percentage of fibroblast subclusters in skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

9 is a cluster diagram of differentially expressed genes in subclusters of skin tissue fibroblasts within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

10 is a schematic diagram of the sub-cluster types of vascular endothelial cells in the skin tissue within 2 cm of the wound edge on the dorsum of the foot of a male patient with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

FIG11 is a bar graph showing the percentage of vascular endothelial cell subclusters in the skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

12 is a cluster diagram of differentially expressed genes in subclusters of vascular endothelial cells in skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma according to an embodiment of the present invention;

13 is an analysis diagram of the interaction between subpopulations of fibroblasts, keratinocytes and vascular endothelial cells in skin tissue of acute dorsal wound of the foot according to an embodiment of the present invention;

FIG14 is an analysis diagram of the interaction between subpopulations of fibroblasts, keratinocytes and vascular endothelial cells in skin tissue at the edge of a DFU wound according to an embodiment of the present invention;

FIG15 is a comprehensive analysis diagram of changes in cell interactions among fibroblasts, keratinocytes and vascular endothelial cells in DFU wound edge skin tissue and acute dorsal wound edge skin tissue according to an embodiment of the present invention;

FIG16 is a HE staining image of a skin tissue section of an acute dorsum of the foot wound edge according to an embodiment of the present invention;

FIG17 is a HE staining image of a DFU wound edge skin tissue section according to an embodiment of the present invention;

18 is a slice diagram of immunohistochemical staining of VIM target object in skin tissue of acute dorsal wound of the foot according to an embodiment of the present invention;

FIG19 is a slice diagram of immunohistochemical staining of VIM target object of DFU wound edge skin tissue according to an embodiment of the present invention;

FIG20 is a slice diagram of immunohistochemical staining of Ki67 target in skin tissue at the edge of acute dorsal wound of the foot according to an embodiment of the present invention;

FIG21 is a slice diagram of immunohistochemical staining of Ki67 target object in DFU wound edge skin tissue according to an embodiment of the present invention;

FIG22 is a graph showing the effect of fibroblast culture supernatant on keratinocyte proliferation in an embodiment of the present invention;

FIG23 is a graph showing the effect of TBHQ on keratinocyte proliferation in an embodiment of the present invention;

FIG24 is a graph showing the effect of fibroblast culture supernatant on keratinocyte migration in an embodiment of the present invention;

FIG25 is a graph showing the effect of TBHQ on keratinocyte migration in an embodiment of the present invention;

FIG26 is a diagram showing the results of Western blot analysis according to an embodiment of the present invention;

Detailed ways

In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

If no specific techniques or conditions are specified in the following examples, the techniques or conditions described in the literature in the art or the product instructions shall be followed.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1

1. Experimental purpose: This example uses single-cell transcriptome sequencing technology to explore and analyze the heterogeneity of DFU peri-injury tissue cells and their cell-cell interactions.

2. Experimental materials: Skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma; Tissue preservation fluid, Tissue dissociation solution, Red blood cell lysis buffer, Single cell processing system,/> Single-cell RNA library kits were purchased from Xingeyuan Biotechnology; Illumina Novaseq 6000 desktop tester.

3. Experimental steps:

3.1 Tissue separation and dissociation

Skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma was obtained and the fresh tissue was preserved in Before dissociation, the specimens were taken out of the preservation solution, washed three times with Hanks balanced salt solution (HBSS) and chopped into small pieces. The tissue fragments were then washed with / > 3ml of tissue dissociation system/> The tissue dissociation solution was digested in a 15 ml centrifuge tube at 37°C with constant stirring for 15 minutes.

The cell suspension was further collected and filtered through a 40 μm sterile filter and centrifuged at 300 g for 5 min. Red blood cell lysis buffer, i.e., RCLB, was used to mix cells and RCLB at a volume ratio of cells:RCLB = 1:2, and the mixture was incubated at 37°C for 5-8 minutes to remove red blood cells. Further, the mixture was centrifuged at 300×g for 5 minutes at 4°C, and the supernatant was removed after centrifugation and suspended with PBS.

3.2 RT-PCR and library construction

use Single cell processing system A single cell suspension (2×105 cells/mL) containing PBS (HyClone) was loaded onto a microwell chip and a scRNA-seq library was constructed using the GEXSCOPE Single-Cell RNA Library Kit (Singleron Biotechnologies) according to the Singleron GEXSCOPE protocol, including cell lysis, mRNA capture, labeling of cells (barcodes) and mRNA (UMIs). The barcoded beads were then collected from the microwell chip, and the captured mRNA was reverse transcribed to obtain cDNA, which was then PCR amplified. The amplified cDNA was then fragmented and ligated to the sequencing adapter. According to/> scRNA-seq libraries were constructed using the Single Cell RNA Library Kit (Singleron). Individual libraries were diluted 4 nM, pooled, and sequenced on an Illumina novaseq 6000 with 150 bp paired end reads. Raw reads were processed using fastQC and fastp to remove low-quality reads. Poly-A tails and adapter sequences were removed by cutadapt. After quality control, reads were mapped to the reference genome GRCh38 using STAR. Gene expression and UMI counts were counted using the FeatureCounts function. Expression matrix files for subsequent analysis were generated based on gene expression and UMI counts. Raw reads were first processed with fastQC v0.11.4 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastp to remove low-quality reads, and poly-A tails and adapter sequences were trimmed using cutadapt. After extracting cell barcodes and UMIs, we mapped Resds to the reference genome GRCh38 using STAR v2.5.3a. FeatureCountsv1.6.2 software was used to obtain the number of UMIs and gene expression levels of each cell and to generate expression matrix files for subsequent analysis.

3.3 Bioinformatics Analysis

Before analysis, cells were filtered by UMI counts below 30,000 and gene counts between 200 and 5,000, and then cells with more than 20% mitochondrial content were removed. After filtering, functions in Seurat v2.3 were used for dimensionality reduction and clustering. We then normalized and scaled all gene expressions using the NormalizeData and ScaleData functions, and selected the top 2000 highly variable genes for PCA analysis using the FindVariableFeautres function. Using the top 20 principal components, we used FindClusters to separate the principal components into multiple clusters. Batch effects between samples were removed by the Harnomy algorithm. Finally, the UMAP algorithm was used for visualization in two-dimensional space.

To identify differentially expressed genes (DEGSs), a Wilcox nonparametric test-based method was used and the Seurat FindMarkers function was used with default parameters, and genes expressed in more than 10% of cells in a cluster were selected with an average log(Fold Change)>0.25 as DEGSs. For the cell type annotation of each cluster, the expression of the classic markers found in the DEGSs was combined with the literature, and the heat map, dot map, and violin map generated by Seurat were used to display the expression of markers for each cell type. Double cells identified as expressing markers of different cell types were manually filtered out.

To investigate the potential functions of DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using the “clusterProfiler” R package. Pathways with P < 0.05 were considered significantly enriched. Gene Ontology gene sets included molecular function (MF), biological process (BP), and cellular component (CC) categories as references. Protein-protein interactions (PPIs) of DEGs in each cluster were predicted based on known interactions of genes with relevant GO terms in StringDB v1.22.0.

CellPhoneDB performs cell-cell interaction analysis based on receptor-ligand interactions between two cell types/subtypes. The cluster labels of all cells were randomly permuted 1000 times to calculate the null distribution of the mean ligand-receptor expression levels of the interaction clusters. Individual ligand or receptor expression was thresholded using a cutoff value based on the mean log gene expression distribution of all genes in all cell types. Significant cell-cell interactions were defined as p < 0.05 and mean log expression > 0.1 and visualized using the circlize (0.4.10) R package.

4. Result analysis:

As shown in Figures 1-4, single-cell transcriptome sequencing data of skin tissue cells within 2 cm of the wound edge of the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma were used to analyze tissue cell heterogeneity by using "Wilcox" (likelihood ratio test) and the FindAllMarkers function in Seurat. The identification results are shown in Figure 1. It can be seen that the top ten cell types in the DFU wound edge skin tissue include keratinocytes, fibroblasts, vascular endothelial cells, melanocytes, macrophages, mast cells, dendritic cells, T cells, sweat gland cells and duct cells; Figure 2 is a bar graph of the top ten cell types, and the differential gene clustering diagram is shown in Figure 3.

Among them, unsupervised cluster analysis of keratinocytes was performed, and as shown in FIG4 , keratinocytes can be divided into 7 cell subpopulations; FIG5 is a bar graph of the proportion of keratinocyte subpopulations, and the differential gene clustering diagram thereof is shown in FIG6 ;

Unsupervised cluster analysis of fibroblasts was performed, and as shown in FIG7 , they can be divided into 6 cell subpopulations; FIG8 is a bar graph of the proportion of fibroblast subpopulations, and the differential gene clustering graph is shown in FIG9 ;

Unsupervised cluster analysis of vascular endothelial cells was performed. As shown in FIG10 , endothelial cells can be divided into five cell subpopulations including arterial endothelial cells (AECs), capillary endothelial cells (CapECs), venous endothelial cells (AECs), proliferating endothelial cells (proliferationECs) and lymphatic endothelial cells (LECs); FIG11 is a bar graph of the proportion of endothelial cell subpopulations, and the differential gene clustering diagram thereof is shown in FIG12 ;

Further, by analyzing the cell-cell interactions, the results are shown in Figures 13-15. As shown in Figure 13, in the acute dorsal wound edge skin (AWE) tissue, the interactions between fibroblast subclusters (FB1, FB4 and FB5) and endothelial cell subclusters (AECs and CapECs) are the most abundant; as shown in Figure 14, in the DFU wound edge skin (CWE) tissue, the interactions between fibroblast subclusters (FB1, FB2, FB3, FB4 and FB5) and endothelial cell subclusters (VECs and CapECs) are the most abundant. As shown in Figure 15, fibroblast subclusters are pan-activated, however, they have fewer interactions with keratinocyte subclusters, especially the interactions with KC2, KC4 and KC6 are significantly weakened, which shows that the communication between fibroblast subclusters and keratinocyte subclusters is significantly weakened, suggesting that crosstalk between fibroblasts and keratinocytes may be an important cause of keratinocyte migration disorders.

Example 2

1. Experimental purpose: In this example, the skin tissue within 2 cm of the wound edge of the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma was sliced and analyzed by immunohistochemistry to analyze its tissue cell characteristics, and to verify the results of the cell-cell interaction analysis in Example 1.

2. Experimental materials: Skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma.

3. Experimental steps:

3.1. Tissue sections:

Skin tissue within 2 cm of the wound edge on the dorsum of the foot of male patients with type 2 diabetic foot and foot trauma was fixed with 4% paraformaldehyde for 12 h. The fixed tissue was dehydrated with ethanol in a gradient manner and then embedded in paraffin. The embedded paraffin tissue was sliced on a microtome, keeping each slice at a thickness of 10-12 μm to facilitate subsequent immunofluorescence staining and hematoxylin and eosin staining.

3.2 Immunofluorescence staining

(1) Dewaxing of paraffin sections: Fix the sections on glass slides and soak the slides in xylene for 15 min, in a new xylene reagent that has been replaced for 15 min, in anhydrous ethanol for 5 min, in a new anhydrous ethanol reagent that has been replaced for 5 min, in 85% alcohol for 5 min, and in 75% alcohol for 5 min. Finally, wash the slides with distilled water.

(2) Antigen retrieval: Soak the slides in sodium citrate antigen retrieval solution at pH 6.0 and heat at 95-100°C for about 30 minutes. During this process, be careful to prevent excessive evaporation of the buffer and do not let the slides dry. After heating, let the slides cool naturally and place the sections in PBS at pH 7.4 and wash on a decolorizing shaker three times for 5 minutes each time.

(3) Circle serum blocking: After the slide is slightly shaken dry, use a histochemical pen to draw circles around the tissue to prevent the antibody from flowing away. Shake dry the PBS and select the blocking solution based on the primary antibody to be added later. The primary antibody is mouse anti-human vim monoclonal antibody blocking and rabbit anti-human ki67 monoclonal antibody. After adding the blocking solution to the slice, block for 30 minutes.

(4) Adding primary antibody: Gently shake off the blocking solution, add PBS and primary antibody prepared in a certain ratio on the slices, place the slices flat in a humidified box and incubate overnight at 4°C. Add a small amount of water in the humidified box to prevent the antibody from evaporating.

(5) Adding secondary antibody: Place the slide in PBS at pH 7.4 and shake on a decolorizing shaker to wash three times, each time for 5 minutes; after the slide is slightly dried, add the secondary antibody of the same species as the primary antibody in the circle drawn by the histochemical pen to cover the tissue, among which, add HRP-labeled goat anti-mouse IgG antibody to the primary antibody mouse anti-human vim monoclonal antibody, and add HRP-labeled goat anti-rabbit IgG antibody to the primary antibody rabbit anti-human ki67 monoclonal antibody. Incubate the slide at room temperature in the dark for 50 minutes.

(6) DAPI counterstaining of cell nuclei: Place the slide in PBS at pH 7.4 and wash on a decolorizing shaker for 3 times, 5 min each time. After the slide is dried slightly, add DAPI staining solution in the circle drawn by the histochemical pen and incubate the slide at room temperature in the dark for 10 min.

(7) Quenching tissue autofluorescence: Place the slide in PBS at pH 7.4 and wash on a decolorizing shaker for 3 times, 5 min each time. Add autofluorescence quencher within the circle drawn by the histochemical pen for 5 min, and rinse the slide under running water for 10 min.

(8) Shake the slides dry and seal with anti-fluorescence quenching sealing medium.

(9) Microscopic examination and photography: The slides are observed under a fluorescence microscope and images are collected.

3.3 Hematoxylin and eosin (H&E) staining:

(1) Dewaxing of paraffin sections: Fix the sections on a glass slide and soak them in xylene for 15 min, in fresh xylene for 15 min, in anhydrous ethanol for 5 min, in anhydrous ethanol for 5 min, in 85% alcohol for 5 min, in 75% alcohol for 5 min, and then wash with distilled water.

(2) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

(3) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin solution for 5 min.

(4) Dehydration and sealing: The sections were sequentially immersed in anhydrous ethanol for 5 min, immersed in anhydrous ethanol that had been replaced for 5 min, immersed in anhydrous ethanol that had been replaced again for 5 min, immersed in xylene for 5 min, immersed in xylene that had been replaced for 5 min, and finally sealed with neutral gum.

4. Result analysis:

The HE staining results are shown in Figures 16 and 17, where Figure 16 is HE staining of the skin tissue at the edge of the acute dorsum foot wound, and Figure 17 is HE staining of the skin tissue at the edge of the DFU wound. It can be seen that compared with the skin tissue at the edge of the acute dorsum foot wound, the epidermal cell layers of the skin tissue at the edge of the DFU wound are unclear, and the basal layer structure is destroyed.

The results of immunohistochemical staining are shown in Figures 18-21, wherein Figure 18 is a graph showing the immunohistochemical staining results of the skin tissue at the edge of the acute dorsal foot wound when the primary antibody selected mouse anti-human vim monoclonal antibody, Figure 19 is a graph showing the immunohistochemical staining results of the skin tissue at the edge of the DFU wound when the primary antibody selected mouse anti-human vim monoclonal antibody, Figure 20 is a graph showing the immunohistochemical staining results of the skin tissue at the edge of the acute dorsal foot wound when the primary antibody selected rabbit anti-human ki67 monoclonal antibody, and Figure 21 is a graph showing the immunohistochemical staining results of the skin tissue at the edge of the acute dorsal foot wound when the primary antibody selected rabbit anti-human ki67 monoclonal antibody;

Vimentin VIM is a marker of epithelial-mesenchymal transition and is involved in processes such as cell adhesion, migration and cell signaling pathways. By comparing the results of Figure 18 with Figure 20, it was found that the expression of VIM in keratinocytes in the DFU wound edge skin tissue was decreased; Ki67 is a nuclear protein that is related to ribosomal RNA transcription. As a cell nuclear proliferation-related antigen, it can indicate the degree of cell proliferation activity. By comparing the results of Figure 19 with Figure 21, it was found that the expression of Ki67 in keratinocytes in the DFU wound edge skin tissue was enhanced, which indicates that the DFU epidermal tissue presents the characteristics of high proliferation and low migration.

Example 3

1. Experimental purpose: In this example, a senescent fibroblast model was established by inducing fibroblasts with high glucose, and the supernatant was collected as a keratinocyte culture condition to explore the effect of the supernatant of senescent fibroblast culture on the proliferation ability of keratinocytes.

2. Experimental materials: Keratinocytes (HaTaC cells) and fibroblasts (HDF) were derived from normal human skin tissue and purchased from the Cell Bank of the Chinese Academy of Sciences.

3. Experimental steps:

3.1 Preparation of NFb-CM and HGFb-CM

Human fibroblasts (HDF) were cultured in DMEM/F12 medium (complete medium) containing 10% fetal bovine serum by volume. The supernatant of fibroblast culture after 7 days of culture was used as normal fibroblast culture medium (NFb-CM). When the cells grew to 80% confluence, they were cultured in a complete medium containing 30 mM glucose at 37°C, 5% CO2 incubator for 7 days. The supernatant was collected as high-glucose induced fibroblast conditioned medium (HGFb-CM).

3.1 Experimental Grouping:

The keratinocytes grown to the logarithmic phase were digested by trypsin to obtain a single cell suspension, and resuspended in DMEM medium containing 10% fetal bovine serum to adjust the cell concentration and inoculate in a 96-well culture plate at a seeding amount of 2×10 ³ /well per well. The 96-well culture plate was placed in a cell culture incubator under 5% CO2 and 37°C for 24 hours. Further, the cultured keratinocytes were intervened with different culture medium conditions and divided into 4 groups, namely, FM group, NFb-CM group, HGFb-CM group and NM group. The culture medium used in the FM group was free FBS medium.

3.2. Detection of keratinocyte proliferation activity using CCK8 method:

(1) 10 ul of CCK-8 solution was added to each well of the 96-well culture plate inoculated with keratinocytes, and the cells were further incubated in a cell culture incubator under CO 2 and 37° C. for 1 hour.

(2) Take the keratinocytes out of the cell culture incubator and observe their color development reaction. Suspension cells are more difficult to develop color than adherent cells. For suspension cells, if the suspension cells have difficulty in developing color, take them out of the incubator and visually observe the degree of staining or measure them with an ELISA reader and estimate the optimal incubation time for obvious color development. Put the 96-well culture plate back into the cell culture incubator and continue to culture under CO2 and 37°C for 1-3 hours before measuring again.

(3) After an obvious color reaction occurs, the absorbance of each well of the cell culture plate is measured at 450 nm using an ELISA reader.

4. Result analysis:

The absorbance measurement results of each group of cells are shown in Figure 22. The HGFb-CM group, i.e., the culture supernatant of senescent fibroblasts, did not have a significant effect on the proliferation ability of keratinocytes.

Example 4

1. Experimental purpose: In this example, cultured keratinocytes were intervened with TBHQ culture medium of different concentrations, and the effect of TBHQ on keratinocyte proliferation was explored by CCK8 method.

2. Experimental materials: Keratinocytes (HaTaC cells) and fibroblasts (HDF) were derived from normal human skin tissue and purchased from the Cell Bank of the Chinese Academy of Sciences.

3. Experimental steps:

3.1 Experimental Grouping:

Keratinocytes grown to the logarithmic phase were digested by trypsin to obtain single-cell suspensions, and resuspended in DMEM medium containing 10% fetal bovine serum to adjust the cell concentration to 2×103/well per well and inoculated into 96-well culture plates. The 96-well culture plates were placed in a cell culture incubator under 5% CO2 and 37°C for 24 h, and then intervened with different concentrations of TBHQ, with 3 wells in each group.

3.2. Detection of keratinocyte proliferation activity using CCK8 method:

(1) 10 ul of CCK-8 solution was added to each well of the 96-well culture plate inoculated with keratinocytes, and the cells were further incubated in a cell culture incubator at 5% CO ₂ and 37° C. for 1 hour.

(2) Take the keratinocytes out of the cell culture incubator and observe their color development reaction. Suspension cells are more difficult to develop color than adherent cells. For suspension cells, if the suspension cells have difficulty in developing color, take them out of the incubator and visually observe the degree of staining or measure with an ELISA reader and estimate the optimal incubation time for obvious color development. Put the 96-well culture plate back into the cell culture incubator and continue to culture at 5% CO ₂ and 37°C for 1-3 hours before measuring again.

(3) After an obvious color reaction occurs, the absorbance of each well of the cell culture plate is measured at 450 nm using an ELISA reader.

4. Experimental results:

The results of the absorbance measurement of each group of cells are shown in Figure 23, which shows that in each experimental group, a concentration greater than 50 μmol/L TBHQ significantly promoted the proliferation of keratinocytes, so 20 μmol/L TBHQ was selected in the subsequent intervention experiment to exclude the effect of proliferation on migration.

Example 5

1. Experimental purpose: In this example, a senescent fibroblast model was established by inducing fibroblasts with high glucose, and the supernatant was collected as a keratinocyte culture condition. The effect of senescent fibroblasts on keratinocyte migration was detected by a scratch test.

2. Experimental materials: Keratinocytes (HaTaC cells) and fibroblasts (HDF) were derived from normal human skin tissue and purchased from the Cell Bank of the Chinese Academy of Sciences.

3. Experimental steps:

3.1 Experimental Grouping

(1) Keratinocytes grown to the logarithmic phase were digested with trypsin to obtain a single cell suspension, and resuspended in DMEM medium containing 10% fetal bovine serum to adjust the cell concentration and inoculate the cells in a 12-well culture plate at a seeding amount of 2×10 ⁵ /well per well. The cells were cultured in a cell culture incubator at 5% CO ₂ and 37°C until the cells filled the culture dish. The tip of a 200 μL pipette was used to scrape the surface monolayer of cells perpendicularly to the culture dish, and the cells scraped by the tip were washed with PBS.

(2) Furthermore, 2 ml of culture medium was added to each well. According to the different culture media, there were FM group, NFb-CM group, HGFb-CM group and NM group, with 4 wells in each group.

3.2 Experimental Results

After the culture dish was scratched with the gun tip, the culture dish was cultured in a cell culture incubator at 5% CO ₂ and 37° C. The cell culture plate was taken out of the cell culture incubator at 0 h, 24 h, 48 h, and 72 h of culture, and the cell scratch healing was observed under a 100x optical microscope.

4. Results Analysis

The culture medium result graph is shown in FIG24 , and it can be seen that the HGFb-CM group can inhibit the migration of keratinocytes, indicating that the culture supernatant of high-glucose-induced senescent fibroblasts has the effect of inhibiting the migration of keratinocytes.

Example 6

1. Experimental purpose: Based on Example 5, this example adds TBHQ to the original experimental group for comparison, and conducts a scratch experiment to explore the effect of TBHQ on keratinocyte migration.

2. Experimental materials: Keratinocytes (HaTaC cells) and fibroblasts (HDF) were derived from normal human skin tissue and purchased from the Cell Bank of the Chinese Academy of Sciences.

3. Experimental steps:

3.1 Experimental Grouping

(1) Keratinocytes grown to the logarithmic phase were digested with trypsin to obtain a single cell suspension, and resuspended in DMEM medium containing 10% fetal bovine serum to adjust the cell concentration and inoculate the cells in a 12-well culture plate at a seeding amount of 2×10 ⁵ /well per well. The cells were cultured in a cell culture incubator at 5% CO ₂ and 37°C until the cells filled the culture dish. The tip of a 200 μL pipette was used to scrape the surface monolayer of cells perpendicularly to the culture dish, and the cells scraped by the tip were washed with PBS.

(2) Further, 2 ml of culture medium was added to each well. According to the different culture media, there were NFb-CM group, NFb-CM+20 μM TBHQ group, HGFb-CM group and HGFb-CM+20 μM TBHQ group, with 4 wells in each group.

3.2 Experimental Results

After the culture dish was scratched with the gun tip, the culture dish was cultured in a cell culture incubator under 5% CO2 and 37°C. The cell culture plate was taken out of the cell culture incubator at 0h, 24h, 48h and 72h of culture, and the cell scratch healing was observed under a 100x optical microscope.

4. Results Analysis

The culture medium result graph is shown in FIG25 , and it can be seen that TBHQ can induce keratinocyte migration under the effect of high glucose-induced senescent fibroblast culture supernatant inhibiting keratinocyte migration.

Example 7

1. Experimental purpose: In this example, the expression level of the target protein in each experimental group was extracted by Western blot to explore the pathway by which senescent fibroblasts inhibit keratinocyte migration and to verify whether TBHQ promotes keratinocyte migration by upregulating the pERK/ERK signaling pathway.

2. Experimental materials: Keratinocytes (HaTaC cells) and fibroblasts (HDF) were derived from normal human skin tissue and purchased from the Cell Bank of the Chinese Academy of Sciences.

3. Experimental steps:

3.1 Experimental Grouping:

Keratinocytes grown to the logarithmic phase were digested with trypsin to obtain single-cell suspensions, and resuspended in DMEM medium containing 10% fetal bovine serum to adjust the cell concentration and inoculate in a six-well culture plate at an inoculation size of 0.5×10 ⁶ /well. The 96-well culture plate was placed in a cell culture incubator under 5% CO2 and 37°C for 24 h, and then intervened with different culture media. According to the different culture media, they were NFb-CM group, NFb-CM+20μM TBHQ group, HGFb-CM group and HGFb-CM+20μM TBHQ group, respectively.

3.2 Western Blotting

(1) Protein extraction

Collect cells in good condition in the six-well plate, digest them with trypsin, collect the cell pellet in a 1.5ml centrifuge tube, wash twice with 1×PBS buffer, further, centrifuge at 1000rpm for 5min and discard the supernatant, add appropriate amount of cell lysis buffer according to the amount of cells, the ratio of protease lysis buffer to protease inhibitor is 100:1, place on ice and shake for 10-30min to fully lyse the cells. Then put the sample into a centrifuge pre-cooled to 4℃ and centrifuge at 12000rpm for 5min; the supernatant obtained by centrifugation is the total cell protein. Pipette the supernatant into a new EP tube and store at -80℃.

(2) Sample loading test

Prepare the stacking gel and separation gel according to the instructions of the kit and allow them to solidify at room temperature, then place the prepared gel plate in the electrophoresis tank, add the prepared 1X electrophoresis buffer to the indicated position, add the maker and each group of proteins to the gel wells, perform electrophoresis at 120V, and analyze the protein gel after electrophoresis using nanodrop2000 software.

4. Results Analysis

ERK is an extracellular regulated protein kinase, which is the key to transmitting signals from surface receptors to cells and plays an important role in tumor migration, including gastric cancer, glioblastoma, esophageal cancer, etc. As shown in Figure 26, the results indicate that the HGFb-CM group can inhibit the expression of pERK/ERK protein in keratinocytes, that is, senescent fibroblasts inhibit the expression of pERK/ERK protein in keratinocytes, and TBHQ can induce the expression of pERK/ERK protein in keratinocytes under the influence of senescent fibroblasts.

In summary, the inventors have discovered for the first time and verified through multiple embodiments that the fibroblasts in the DFU wound surface exhibit senescence characteristics due to high glucose induction, which inhibits the expression of pERK/ERK protein in keratinocytes, so that the keratinocytes in the DFU wound surface exhibit low migration characteristics and are difficult to heal. By promoting the expression of pERK/ERK protein in keratinocytes, the migration of keratinocytes in the DFU wound surface can be promoted, thereby promoting the healing of the DFU wound surface. On this basis, a drug containing an ERK agonist that can promote the healing of the DFU wound surface can be provided.

Based on the above embodiments, as an optional embodiment, the ERK agonist described in the present invention can be combined with any one or several medically acceptable pharmaceutical excipients to prepare a pharmaceutical composition for treating or preventing liver fibrosis or metabolic syndrome caused by liver fibrosis, including at least one of a flavoring agent, an osmotic pressure regulator, a filler, a lubricant, a preservative, a suspending agent, a food coloring, a diluent, an emulsifier, a disintegrant or a plasticizer.

Preferably, the dosage form of the drug is an oral preparation such as tablets, pills, oral liquid, capsules, syrups, pills and granules, or an injectable preparation such as injectable powder and injection.

It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. Use of an ERK agonist to promote healing of a diabetic foot wound, wherein the ERK agonist promotes healing of a diabetic foot wound by up-regulating a pERK/ERK signaling pathway, thereby repairing impaired keratinocyte migration due to cross-talk of keratinocytes and fibroblasts in diabetic foot tissue and enhancing the inhibition of the pERK/ERK signaling pathway in the keratinocytes.

2. The use of an ERK agonist to promote healing of a diabetic foot wound according to claim 1, wherein the use comprises promoting healing of a diabetic foot ulcer wound.

3. A medicament for promoting diabetic foot wound healing by an ERK agonist, which is characterized in that the ERK agonist comprises tert-butylhydroquinone and salts, solvates, hydrates or stereoisomers of the tert-butylhydroquinone.

4. A medicament for promoting healing of diabetic foot wound bed according to claim 3 wherein the medicament further comprises pharmaceutically acceptable excipients including at least one of flavoring agents, osmotic pressure modifiers, fillers, lubricants, preservatives, suspending agents, food colors, diluents, emulsifiers, disintegrants or plasticizers.

5. The medicament for promoting the healing of diabetic foot wound surface by using the ERK agonist according to claim 4, wherein the medicament is in the form of an oral preparation or an injection; wherein the oral preparation is in the form of tablet, pill, oral liquid, capsule, syrup, dripping pill or granule; the injection comprises injection powder injection or injection liquid.