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Analysis of miR-203a-3p/SOCS3-mediated induction of M2 macrophage polarization to promote diabetic wound healing based on epidermal stem cell-derived exosomes
Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
The development of therapeutic strategies to improve wound healing in individual diabetic patients remains challenging. Stem cell-derived exosomes represent a promising nanomaterial, and microRNAs (miRNAs) can be isolated from them. It is important to identify the potential therapeutic role of specific miRNAs, given that miRNAs can play a therapeutic role.
Methods
qPCR, flow cytometry, and western blotting were used to verify the effect of epidermal stem cell-derived exosomes (EpiSC-EXOs) on M2 macrophage polarization and SOCS3 expression. By screening key miRNAs targeting SOCS3 in EpiSC-EXOs by high-throughput sequencing, we verified the mechanism in vitro. Finally, an animal model was used to verify the effect of promoting healing.
Results
The use of EpiSC-EXOs reduced SOCS3 expression and promoted M2 macrophage polarization. The abundant miR-203a-3p present in the EpiSC-EXOs specifically bound to SOCS3 and activated the JAK2/STAT3 signaling pathway to induce M2 macrophage polarization. Treatment of the db/db mouse wound model with miR-203a-3p agomir exerted a pro-healing effect.
Conclusions
Our results demonstrated that the abundant miR-203a-3p present in EpiSC-EXOs can promote M2 macrophage polarization by downregulating SOCS3 and suggested that diabetic wounds can obtain better healing effects through this mechanism.
Diabetic wounds are the most common chronic and refractory wounds in the clinic, and they are one of the most common and serious complications in diabetic patients. Immune dysfunction, infection and vascular problems are important factors leading to poor healing outcomes of diabetic wounds [
]. Although the pathogenesis and treatment of diabetic wounds have been extensively studied over the years, there is no clear and effective treatment plan at present. Therefore, it is important to explore new methods for diabetic wound treatment, deeply study the effective mechanisms and reveal the key factors to overcome the current bottleneck of diabetic wound treatment.
Previous studies showed that the polarization disorder of macrophages (Mφs) in diabetic wounds caused the wound to remain in the inflammatory reaction stage and prevented the proliferation stage, which was an important factor leading to refractory diabetic wounds [
]. Therefore, effective induction of Mφ to M2 polarization in the wound is the key to promoting diabetic wound healing. JAK2/STAT3 signaling has been demonstrated to be a key factor regulating the polarization of Mφs to the M2 type [
]. This molecule blocks the JAK2/STAT3 pathway mainly by inhibiting JAK activation or uses the SH2 domain in its structure similar to STAT3 to compete for phosphorylation sites in the cytosolic region of the receptor, thus preventing STAT3 activation [
] and is a potential important target for diabetic wound treatment.
In recent years, studies have shown that stem cells mainly regulate the microenvironment by secreting exosomes (EXOs) to deliver proteins, nucleic acids and other biologically active substances [
]. Due to phospholipid bilayer membrane protection, the microRNAs (miRNAs) in exosomes can be stably transmitted from RNA-dependent enzyme degradation, exerting a major role in information transfer and interaction among cells [
]. Related studies have shown that exosomes from human cord blood via miR-21-3p can accelerate skin wound healing by promoting angiogenesis and fibroblast function [
Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function.
]. These findings indicated that exosome-derived miRNAs play an important role in wound healing.
Our team confirmed in previous studies that epidermal basal cells (EBCs) could effectively promote diabetic foot ulcer healing and that epidermal stem cells (EpiSCs) played a leading role [
], but the specific regulatory mechanism is unclear. Therefore, in this study, we explored the specific mechanism by which miR-203a-3p enriched in EpiSC-EXOs can inhibit SOCS3 in Mφs and activate the JAK2/STAT3 signaling pathway, thus inducing Mφ-to-M2-type polarization to ultimately promote diabetic wound healing. We hope these findings will provide more evidence for epidermal stem cell therapy and may help identify a novel treatment for chronic wounds.
2. Materials and methods
2.1 Applications of bioinformatics
The gene dataset GSE80178 was obtained by searching the Comprehensive Gene Expression Database. The GSE80178 expression profile consists of diabetic foot ulcers and nondiabetic foot skin tissues. Ulcers did not have any clinical signs of infection. The R package limma [
] was used to identify differentially expressed genes (DEGs). These DEGs were filtered with a fold expression greater than 2.0 and an adjusted p < 0.05. The results are shown by volcano plots and heatmaps. The scores for immune cells were calculated using the ESTIMATE algorithm [
]. Terms with a P value < 0.05 were selected and displayed.
2.3 Cell extraction and culture
After obtaining the informed consent of the patients, human prepuce tissues were collected from the First Affiliated Hospital of Sun Yat-sen University. The skin without subcutaneous fascia was cut to a size of 1 × 1 cm2 and then digested in MYSEED for 30 min. The epidermis was scraped off and then digested in 0.25 % trypsin-EDTA solution for 5 min. After the digested cell suspension was filtered and centrifuged, these cells were incubated in T25 culture flasks that were precoated with fibronectin (FN; ∼5 µg/cm2; Shanghai Fibronectin Biotechnology, China) solution for 20 min. The supernatant was aspirated, and the cells adhered to the bottom were regarded as EpiSCs. EpiSCs were then cultured in keratinocyte serum-free medium (K-SFM; 17005042; Gibco, USA) at 37 °C.
The human monocytic cell line (THP-1) was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). For differentiation into macrophages, THP-1 cells (1 × 106) were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; P1585; Abcam, UK) for 48 h. All cells were cultured in DMEM (C11995; Thermo Fisher Scientific, USA) containing 10 % fetal bovine serum and maintained at 37 ℃.
2.4 Fluorescence microscopy
Cell samples were fixed with 4 % paraformaldehyde (P0099; Beyotime Biotechnology, China) and then incubated with 0.5 % Triton X-100 (P0096; Beyotime Biotechnology, China). After blocking with 5 % goat serum (SL038; Solarbio, China) solution, the cells were incubated with antibodies against K15 (1:200; ab52816; Abcam, UK) and ITGα6 (1:200; ab181551; Abcam, UK) overnight. Then, the cells were incubated with secondary antibodies (SA00009-1/SA00009-2, SA00013-1/SA00013-2; Proteintech, USA). DAPI (C0065; Solarbio, China) was used for nuclear counterstaining. After all the incubation, the samples were observed using a fluorescence microscope.
2.5 Cell transfection
Restoration or inhibition of miR-203a-3p expression was achieved by transfecting cells with miR-203a-3p mimics or anti-miR-203a-3p vectors, which were purchased from RiboBio (Guangzhou, China). Overexpression or silencing of suppressor of cytokine signaling (SOCS3) was achieved by transfecting cells with Lv-SOCS3 or shSOCS3 (GeneChem, China). Empty mimics (miR-NC), anti-control (anti-NC), and empty vector (vector) were used as controls for miR-203a-3p mimics, anti-miR-203a-3p, Lv-SOCS3 and shSOCS3. Liposome ®3000 (L3000015-1.5ML; Invitrogen, USA) was used to perform cell transfection based on the manufacturer's instructions.
2.6 Isolation and analysis of exosomes
The collected supernatant was first centrifuged at 500 × g for 10 min to remove cell contamination. Next, the upper supernatant was centrifuged at 12,000 × g for 20 min to remove apoptotic bodies and large cell debris. Finally, exosomes were collected by ultracentrifugation at 100,000 × g for 70 min. The number and morphology (cup-shaped) of exosomes were examined using NanoSight and transmission electron microscopy, respectively. Sequencing of the exosomes was assisted by the Gene Denovo Company.
2.7 Western blot
RIPA lysis buffer (P0013B; Beyotime, China) was used for cell lysis and protein extraction. Protease (1 %) and phosphatase inhibitors (1 %; A8260; Solarbio, China) were used to inhibit protein degradation. Total proteins were isolated by 12.5 % SDS-PAGE (1610320; Bio-Rad, USA). All antibodies used in this study are summarized in Table S1. GAPDH was used as an internal control.
To transfer the samples to PVDF membranes, protein samples (30 µg) were first electrophoretically separated on SDS-PAGE gels. After the PVDF membrane was blocked, the membrane was treated with primary antibodies (Table S1) overnight and incubated with the secondary antibody for 1 h (Table S1). Protein bands were detected by ECL western blotting Detection Reagent (GE Healthcare).
2.8 Qrt-PCR
TRIzol reagent (15596026; Invitrogen, USA) was used to extract total RNA. The HiScript-TS 5/3′ RACE Kit (RA101-01; Vazyme, China) was used for reverse transcription of protein-coding genes. ChamQ SYBR Color qPCR Master Mix (Q421-02; Vazyme, China) was used for protein-coding gene quantification. GAPDH was used as an internal control for protein-coding genes. All sequences of the primers used are listed in Table S2.
2.9 Flow cytometry
After THP-1 cells were harvested and washed, these cells were resuspended in flow cytometry buffer. Then, the cells were stained with anti-CD163 and anti-CD11b antibodies (562643, 562399; BD Biosciences, USA) for 30 min. After washing and resuspension, flow cytometry (BD Biosciences, USA) was used to analyze these cells according to the manufacturer’s instructions.
2.10 Enzyme-linked immunosorbent assay (ELISA)
To determine the effect of exosomes or miR-203a-3p mimics on VEGF, TGF-β, bFGF, IL1β and TNF-α expression in M0 macrophages, ELISAs were performed using a Human ELISA Kit (MM-0151H2, MM-0115H2, MM-1774H2, MM-3351H2, MM-0122H2; Meimian, China). After the reaction was stopped, microplate reader was used to analyze the absorbance of the samples at 450 nm within 15 min.
2.11 Luciferase reporter assay
For generation of the luciferase construct, the SOCS3 gene, which contains miR-203a-3p binding sites, was synthesized and cloned into GV272 vectors by GeneChem (China). Next, 293 T cells were cotransfected with SOCS–3′-UTR–Luc firefly luciferase constructs (wild-type or mutant) and miR-934 mimics using liposome ®3000 (L3000015-1.5ML; Invitrogen, USA). Finally, cell lysates were harvested after 48 h of transfection, and the firefly/Renilla luciferase activities were analyzed by a substrate dispenser according to the Dual-Luciferase Reporter Assay Kit protocol (E1910; Promega, USA).
2.12 Immunohistochemistry
Tissue samples were embedded in paraffin. H&E and Masson’s trichrome were used to stain sections. After the slides were incubated with 5 % BSA solution for 60 min, CD68 (universal macrophage maker) antibody solution (1:200, ab283654; Abcam, UK) and CD163 (M2 phenotype of macrophage marker) antibody solution (1:300, ab182422; Abcam, UK) were incubated with the sections. CD31 staining (1:300, ab182981; Abcam, UK) was used to evaluate angiogenesis changes. SOCS3 staining (1:500, ab280884; Abcam, UK) was used to evaluate expression changes.
2.13 Wound model and treatment
The db/db mice were obtained from the center of the experimental animal. Mice were anesthetized by inhalation of 3 % isoflurane, and dorsal hair was shaved before excision for wounds. After disinfecting the skin with alcohol, an 8 mm hole punch was used to create two full-thickness wounds on the back. Animals were randomized into two groups, and miR-203a-3p agomirs (40 nmol/kg) were injected intradermally into the wound edges of the mice immediately after wounding. The control group was treated with the negative agomir injection. After injection treatment, the wound was covered with 3 M transparent films. The residual wound area rate was calculated as [(day n area)/(day 0 area)] × 100 % (n = 0, 3, 7, 10 or 14). Wound closure was assessed on different days with ImageJ software. After the mice were euthanized, wound samples were embedded in paraffin and subjected to staining.
2.14 Statistical analysis
Differences between treated and control groups were analyzed using t test or one-way ANOVA. All data were analyzed using Prism software. All in vitro experiments were performed in triplicate. When the P value ≤ 0.05, the difference between groups was considered to be statistically significant.
3. Results
3.1 M2 macrophage polarization disorder and SOCS3 overexpression in diabetic wounds
To explore the molecular mechanism of diabetic wound healing, we obtained the relevant chip GSE80178 of diabetic wounds from the GEO database. First, we performed immune cell infiltration analysis on diabetic foot ulcer (DFU) and foot skin (FS) group data using the CIBERSORT algorithm. As presented in Fig. 1A, the proportion of immune cells differed significantly between the two wounds. Compared with that of the FS group, the infiltration degree of M0 macrophages and M1 macrophages in the DFUs was significantly reduced, but the proportion of M2 macrophages was not significantly increased (Fig. 1B). The whole diabetic wound immune microenvironment was in the middle zone of the transformation to the anti-inflammatory and prohealing stage. Next, to distinguish the two transcriptomic differences, we used the R language “limma” software package to analyze the samples. A total of 3402 significantly differentially expressed genes were identified, of which 1321 were upregulated and 2081 were downregulated. Among the significantly upregulated genes, the target gene SOCS3 was identified. The results are shown in the volcano plot in Fig. 1C. We also showed the expression patterns of SOCS family members (Fig. 1D) and found that SOCS3 was indeed significantly highly expressed in the DFU group. To verify the expression pattern of SOCS3 in diabetic wounds, we tested clinically collected tissue samples of diabetic foot ulcers (DFUs) and normal skin (NS) and found that SOCS3 was significantly higher in diabetic wounds than in normal wounds (Fig. 1E). The infiltration of M2 macrophages in diabetic wounds was significantly less than that in normal wounds (Fig. 1F).
Fig. 1The differences in M2 macrophage infiltration and SOCS3 expression in diabetic wounds. A The fraction of 22 subsets of immune cells from DFU and FS tissues. B Box plot showing the difference in macrophage infiltration between DFU and FS tissues. C Volcano plot comparing the data between the DFU and FS groups. D Heatmap of SOCS family members. E Representative immunohistochemical images for the detection of SOCS3 in DFU and NS tissues. F Representative immunohistochemical images showing the infiltration of M2 macrophages in DFU and NS tissues. (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001).
], we further explored the polarization effect of epidermal stem cell-derived exosomes (EpiSC-EXOs) on M2 macrophages. We first extracted and characterized epidermal stem cells and their exosomes. Epidermal stem cells were identified by flow cytology and immunofluorescence, and the results showed high expression of the epidermal stem cell-related markers ITGα6 and K15, while the keratinocyte terminal differentiation marker K10 was poorly expressed (Fig. 2A, B). Human epidermal stem cell supernatants were collected, and EpiSC-EXOs were collected by ultracentrifugation and verified by western blotting, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The NTA showed particle sizes of exosomes between 30 and 200 nm (Fig. 2C), consistent with previous reports of exosome size [
]. Western blotting also confirmed that exosome surface markers (CD81 and TSG101) were highly expressed in these particles (Fig. 2E). These results all suggested that the exosomes were indeed extracted successfully.
Fig. 2EpiSC-EXOs induce M2 polarization of macrophages. A Flow cytometry to detect EpiSCs via K10 and ITGα6. B Immunofluorescence identification of EpiSCs via ITGα6 and K15. C The average particle size distribution of EpiSC-EXOs was measured by NTA. D Representative image of the ultrastructure of the exosomes observed by TEM. E The marker protein levels of CD 81 and TSG 101 in the exosomes. F Representative image of macrophages derived from THP-1 cells treated with PMA. G qPCR analysis of the expression of the macrophage marker CD68 was performed. H Immunofluorescence identification of EpiSCs via CD11b. I Representative immunofluorescence image showing the internalization of PHK26-labeled EpiSC-EXOs (green) by M0 macrophages. J qPCR analysis of the expression of typical M2 markers (CD206 and ARG-1) in M0 macrophages treated with EpiSC-EXOs or PBS (control). K Flow cytometry was performed to analyze the effect of EpiSC-EXOs on the expression of the typical M2 marker CD163. L, M The effect of EpiSC-EXOs on SOCS3 levels assessed by qPCR and western blotting. N The expression levels of cytokines (TGF-β, bFGF, VEGF, TNF-α and IL1β) in macrophages treated with EpiSC-EXOs were detected by ELISA. (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Human THP-1 cells (a normal monocyte macrophage cell line) were incubated with PMA to induce the differentiation of M0 macrophages. M0 macrophages exhibited adhesion morphology and elevated expression of the macrophage markers CD68 and CD11b (Fig. 2F, G, H). We next assessed the ability of M0 macrophages to absorb exosomes in vitro by labeling EpiSC-EXOs with PKH67 dye and then adding them to M0 macrophages. Based on the green fluorescent staining in the cells, these results showed that the M0 macrophages were able to absorb the exosomes within 24 h (Fig. 2I). Next, to verify the effect of epidermal stem cell-derived exosomes on M2 macrophage polarization, we added EpiSC-EXOs to the medium for 48 h, and then the M2 marker (ARG-1, CD206) was significantly increased in macrophages (Fig. 2J). Flow cytometry was used to measure the expression of the M2 macrophage marker CD163 in macrophages (Fig. 2K), and the results were consistent with the above results. Moreover, western blot analysis showed that EpiSC-EXOs inhibited SOCS3 expression during induction (Fig. 2L, M). EpiSC-EXOs enhanced the secretion of several specific cytokines (TGF-β, VEGF, and bFGF) in macrophages, and the release of inflammatory factors (TNF-α and IL1β) decreased. (Fig. 2N).
3.3 Screening of miRNAs within epidermal stem cell-derived exosomes
miRNAs that negatively regulate the target genes were enriched in the exosomes, so we performed miRNA sequencing analysis of the exosomes extracted in the previous step. We chose fibroblast exosomes (FB-EXOs) as a comparison because they are an important cell type in wound healing [
]. Differential analysis revealed 76 significantly different miRNAs (Fig. 3A), and seven of the miRNAs identified by target gene prediction could be targeted for binding to SOCS3 (Fig. 3B), in which miR-203a-3p was significantly highly expressed in epidermal stem cell-derived exosomes. According to the biological process analysis, miR-203a-3p was associated with cell polarity and inflammation (Fig. 3C), so we chose it for subsequent studies. We further investigated miR-200b-3p expression in the culture medium (CM) of EpiSCs. The level of miR-200b-3p in the CM of EpiSCs remained unchanged upon RNase A treatment, while it signifcantly decreased following treatment with RNase A plus Triton X-100 (Fig. S1A). It is suggested that extracellular miR-200b-3p was encased in a membrane and not secreted directly. We also investigated miR-200b-3p expression in EpiSCs derived exosomes and found that the patterns of miR-200b-3p expression in exosomes were consistent with miRNA-sequencing analysis (Fig. S1B), indicating that miR-200b-3p was mainly encapsulated in exosomes derived from EpiSCs. After treatment of macrophages with epidermal stem cell-derived exosome, we can found miR-200b-3p levels within macrophages increased significantly (Fig. S1C).
Fig. 3Screening miRNAs in EpiSC-EXOs. A Heatmap of the differentially expressed miRNAs between EpiSC-EXOs and FB-EXOs. B Volcano plot comparing the data between EpiSC-EXOs and FB-EXOs. C Biological process analysis of miR-203a-3p. D Sequences of the predicted binding sites between miR-203a-3p and the 3′-UTR of the wild-type (WT)/mutant (MUT) SOCS3 gene. E A luciferase reporter gene activity assay was performed to determine the effects of miR-203a-3p mimics on the luciferase activity of the 3′-UTR of the WT/MUT SOCS3 gene. F KEGG pathway enrichment analysis of the miR-203a-3p/SOCS3 axis. (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001).
Data from the TargetScan Human 7.2 database showed an alignment of the miR-203a-3p sequence to the 3′-UTR sequence of SOCS3, indicating that miR-203a-3p may target SOCS3 to induce M2 macrophage polarization (Fig. 3D). To verify whether SOCS3 is a target gene of miR-203a-3p, we transfected the miR-203a-3p mimics and its control vector into 293 T cells; either the wild-type or mutant luciferase vector was used for further validation. As presented in Fig. 3E, the luciferase activity of the SOCS3 wild-type binding site vector was significantly reduced in the group cotransfected with the SOCS3 wild-type binding site vector construct. However, no significant changes were detected in the SOCS3 mutant binding site group. We explored the pathways through which the miR-203a-3p/SOCS3 axis mainly functions, and clear enrichment in JAK/STAT signaling was found using KEGG pathway analysis (Fig. 3F).
3.4 miR-203a-3p induces M2 macrophage polarization via the SOCS3/JAK2/STAT3 signaling pathway
To explore the mechanism of miR-203a-3p-induced M2 macrophage polarization in vivo, we transfected macrophages with miR-203a-3p mimics or anti-miR-203a-3p (Fig. S2). The results showed that the mRNA and protein levels of SOCS3 were downregulated or upregulated compared to those in the respective control cells (Fig. 4A). We further investigated the effect of miR-203a-3p on M2 macrophage polarization and found significantly enhanced expression of M2 markers (CD206, ARG1) by the transfection of miR-203a-3p mimics into M0 macrophages (Fig. 4B). Since IL-4 can induce polarization in M2 macrophages [
], we treated macrophages with 50 ng/mL IL-4 3 days earlier and validated the inhibitory effect of anti-miR-203a-3p on M2 marker expression by transfection with anti-miR-203a-3p (Fig. 4B). In addition, flow cytometry was used to measure the expression of the M2 macrophage marker CD163 in macrophages, which was consistent with the above results (Fig. 4C, D). In summary, our results demonstrate that either miR-203a-3p mimics or anti-miR-203a-3p influences M2 macrophage polarization. miR-203a-3p increased the release of some specific cytokines (TGF-β, VEGF, and bFGF) in M2 macrophages and reduced the secretion of inflammatory factors (TNF-α and IL1β) (Fig. 4E). The JAK2/STAT3 signaling pathway has been widely reported as an important pathway involved in macrophage polarization. To test the activation of the JAK2/STAT3 pathway, we transfected M0 macrophages with a miR-203a-3p mimic or anti-miR-203a-3p and found that the miR-203a-3p mimics could be significantly activated and that anti-miR-203a-3p attenuated activation (Fig. 4F).
Fig. 4Mir-203a-3p induces m2 macrophage polarization via downregulation of socs3 expression and activation of the jak2/stat3 signaling pathway. A The effects of miR-203a-3p mimics and anti-miR-203a-3p on the expression of SOCS3 in M0 macrophages were tested using qPCR and western blotting. B, C, D M0 macrophages were transfected with miR-203a-3p mimics and IL4–anti-miR-934. The effects on the expression of typical M2 markers (CD206, ARG-1 and CD163) were determined using qPCR and flow cytometry. E The expression levels of cytokines (TGF-β, bFGF, VEGF, TNF-α and IL1β) in macrophages treated with miR-203a-3p mimics were detected by ELISA. F The effects of miR-203a-3p mimics or IL4–anti-miR-934 on the expression of JAK2, pJAK2, STAT and pSTAT3 in M0 macrophages were determined using western blotting. (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001).
3.5 Overexpression or knockdown of SOCS3 affects miR-203a-3p-mediated M2 macrophage polarization
We transfected upregulated or downregulated SOCS3 plasmids into M0 macrophages to investigate the effect of SOCS3 on miR-203a-3p-mediated M2 macrophage polarization (Fig. S3). Overexpression of SOCS3 attenuated the miR-203a-3p-mediated enhancement of the expression of M2 markers (CD206, ARG1), while silencing of SOCS3 proportionately reversed the inhibitory effect of anti-miR-203a-3p on the expression of M2 markers (Fig. 5A, B). The results of the M2 macrophage marker CD163 in M0 macrophages were measured using flow cytometry and were consistent with the above results (Fig. 5C, D). The western blot results showed that the overexpression of SOCS3 attenuated the miR-203a-3p-mediated enhancement of JAK2/STAT3 signaling pathway expression, while the silencing of SOCS3 showed the opposite effect (Fig. 5E).
Fig. 5SOCS3 affects miR-203a-3p-mediated M2 macrophage polarization. A, B, C, D M0 macrophages were transfected with miR-203a-3p mimics or IL4–anti-miR-203a-3p and further cocultured with LV-SOCS3 or shSOCS3. The combined effects of exogenous miR-203a-3p/LV-SOCS3 and anti-miR-203a-3p/shSOCS3 on the expression of typical M2 markers (CD206 and ARG-1) were determined using qPCR and flow cytometry. E western blot analysis was performed to detect the expression of JAK2/STAT3 pathway components in M0 macrophages transfected with miR-203a-3p mimics or IL4–anti-miR-203a-3p. (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001).
3.6 miR-203a-3p promotes wound healing and macrophage M2 polarization in db/db mice
To test whether miR-203a-3p has the same effect in vivo, we created 8 mm round wounds on the dorsal side of db/db mice, followed by intradermal injection of miR-203a-3p agomir or NC into the wound edges (Fig. 6A). Analysis of wound healing showed that the closure rate in the miR-203a-3p-treated group was significantly higher on days 7, 10, and 14 than that in the control group (Fig. 6B). Moreover, on day 3, SOCS3 expression was significantly reduced in the wounds of the mice injected with miR-203a-3p agomir compared with the controls (Fig. 6C). To estimate the change in macrophages in the wounds, total macrophages and M2 macrophages were labeled. Fig. 6D shows the CD68-labeled total macrophages in the wound at day 7, which were significantly less abundant in the control group of mice than in the miR-203a-3p agomir group. Fig. 6D also shows the CD163-labeled M2 macrophages in the wound site of the miR-203a-3p agomir and NC group mice. M2 macrophages were significantly less abundant in the NC group than in the miR-203a-3p agomir group, which was similar to the results of total macrophage labeling. This result indicates that miR-203a-3p agomir injection is effective and can strengthen macrophage recruitment and M2 macrophage polarization.
Fig. 6miR-203a-3p promotes diabetic wound healing in vivo. A We generated full-thickness cutaneous wounds on the backs of db/db mice and animals, which were randomized into two groups with miR-203a-3p agomir or negative control (NC) treatments, n = 6, per group. B Gross view of wounds among the two groups at days 0, 3, 7, 10, and 14 postwounding. The rate of wound closure among the two groups was quantified. C Representative immunohistochemical image for the detection of SOCS3 in wounds at day 3. D Representative immunohistochemical images of the infiltration of macrophages in wounds on day 7. (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001).
Wound healing in the two mouse groups was further assessed by H&E staining. As shown in Fig. 7A, thicker granulation tissue was observed in the miR-203a-3p agomir group of mice but was rarely observed in the control mice after 7 days. At day 10, we observed that more granulation tissue occurred in the miR-203a-3p agomir group mouse wound surface. By day 14, an intact neoepidermal layer was formed in the miR-203a-3p agomir group and was thicker and more complete in the miR-203a-3p agomir group than in the control group. In contrast, the control mice had only very thin new epidermal layers. Therefore, the miR-203a-3p agomir group had a better tissue regenerative effect. Collagen from the wounds of the two groups was stained by Masson staining. The collagen staining deposited in Fig. 7B is colored blue, which provides information not only on the collagen deposition but also on the deposited collagen structure. Collagen fiber dispersion in wounds was more uniform and denser in the miR-203a-3p agomir group, with significantly more blue collagen staining in the miR-203a-3p agomir group on days 7 and 14. Wound angiogenesis was visualized by IH staining with CD31. Fig. 7C shows that the number of CD31-positive staining structures present in the miR-203a-3p agomir group mice significantly exceeded that in the control group after 7 days. However, at 14 days, we found a decrease in the capillary number in the miR-203a-3p agomir group, but it was increased in the control mice. Overall, there were fewer capillaries in the miR-203a-3p agomir group, which may be due to the successful regeneration of the wound.
Fig. 7miR-203a-3p improved diabetic wound healing quality in vivo. A Images of H&E-stained wounds treated with miR-203a-3p agomir or NC at Day 7, Day 10 and Day 14. B Masson staining of wounds treated with miR-203a-3p agomir or NC at Day 7 and Day 14. C CD31 immunohistochemistry staining in wounds treated with miR-203a-3p agomir or NC at Day 7 and Day 14. (**p < 0.01).
The main finding of this study is that miR-203a-3p enriched in EpiSC-EXOs can reduce the expression of SOCS3 in macrophages, activating the JAK2/STAT3 signaling pathway, inducing polarization of M2 macrophages and releasing anti-inflammatory factors to promote diabetic wound healing (Fig. 8). Our study provides a strong basis for EpiSC-EXOs as potential therapeutic drugs, and engineered exosomes or small-molecule nucleic acid drugs based on miR-203a-3p can be a potential treatment for diabetic wounds in the future.
Fig. 8miR-203a-3p enriched in EpiSC-EXOs regulates wound healing (BioRender).
Salidroside-Pretreated Mesenchymal Stem Cells Enhance Diabetic Wound Healing by Promoting Paracrine Function and Survival of Mesenchymal Stem Cells Under Hyperglycemia.
] cells, can help restore the orderly process of wounds and accelerate closure in recent animal studies. EpiSCs, as an important component attached to the basal membrane of the skin, mainly play a role in promoting the regeneration of the epidermal layer [
]. Our previous study on epidermal stem cells found that the clinical application of epidermal stem cells can significantly promote chronic wound healing and reduce scar formation [
Given the limitations of stem cell therapy in its clinical applications, exosome therapy has been investigated. Compared with stem cells, stem cell-derived exosomes have the advantages of easy preservation and minimal immunogenicity. Exosomes, which can protect their payloads from chemical and enzymatic degradation and evade recognition and clearance by the immune system, are promising new drug delivery vectors [
]. Our previous related experiments verified that the rich exosomes of epidermal stem cells can promote angiogenesis, regulate macrophage polarization, and promote wound healing [
], but corresponding mechanistic studies are lacking. We therefore treated macrophages with epidermal stem cell-derived exosomes in vitro and found that they could induce macrophage M2 polarization by reducing SOCS3 expression.
Exosomes can protect their contents through their natural membrane structure and transport them into target cells. These contents include miRNAs, lncRNAs, proteins and other substances that can regulate gene expression and cell function in target cells. Therefore, exosomes are considered ideal nanomaterials [
]. Among them, miRNAs are essential mediators for exosomes to perform their biological functions. These small RNA molecules that cannot encode proteins, miRNAs, promote the degradation rate of the target mRNA by interacting directly with the complementary region of the 3′ untranslated region of the target mRNA, thereby affecting the upstream and downstream pathways associated with the target genes and thus playing an important role in cancer diseases, immune disorders, and inflammatory diseases [
Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function.
]. Therefore, in this study, we mainly focused on miRNAs within epidermal stem cell-derived exosomes.
Through high-throughput sequencing and differential expression analysis, we found that epidermal stem cell-derived exosomes were rich in miR-203a-3p targeting SOCS3. Pathway analysis found that miR-203a-3p was related to cell polarity and inflammation. miR-203a-3p was reported to be a positive regulator of epidermal differentiation and induced the differentiation of hair follicle stem cells [
]. To further clarify whether miR-203a-3p can function through the miR-203a-3p/SOCS3 axis, we demonstrated by transfection that miR-203a-3p induces the polarization of M2 macrophages by downregulating SOCS3 expression and activating JAK/STAT3 signaling. These findings are consistent with previous reports that exosomal miRNAs can be effectively delivered to target cells to regulate biological properties [
Macrophages can secrete a large number of proregenerative cytokines and chemokines after being induced to polarize into M2 macrophages. Epidermal stem cell-derived exosomes and miR-203a-3p can enhance the polarization of M2 macrophages and the secretion of specific cytokines, including VEGF, bFGF and TGF-β. These cytokines can perform multiple functions throughout the process of tissue regeneration [
]. The use of specific miRNA mimics or inhibitors as drugs to regulate mRNA expression may have significant therapeutic potential for nonhealing wounds [
Therefore, in this study, SOCS3 expression in wounds was reduced by intradermal injection of miR-203a-3p agomir into mouse skin wounds, and M2 macrophages were polarized by miR-203a-3p agomir. We also found that M2 macrophages exert an important role in the proliferative phase of wound healing compared to that of the control group. In animal models of diabetic wounds, administration of miR-203a-3p agomir significantly enhanced keratinocyte migration and improved skin thickness. At the same time, collagen deposition and angiogenesis in granulation tissue were promoted, and diabetic wound healing was accelerated. To support this phenomenon, chemokines and cytokines are necessary in the recruitment of repair cells, and wound healing has been proven [
]. TGF-β is a positive regulator of fibroblast proliferation and extracellular matrix (ECM) synthesis. This molecule is an important cytokine that functions at the various stages of wound healing and supports temporary granulation tissue formation at the wound site [
]. When fibroblasts are recruited to the wound site by macrophages activated by the miR-203a-3p agomir, they are further influenced by the cytokines secreted by M2 macrophages and begin ECM synthesis. The synthesis of ECM is an important basis for connective tissue reconstruction [
]. In addition, VEGF promotes the formation of capillaries as an angiogenic growth factor but also promotes the migration of vascular endothelial cells [
]. Taken together, our results suggest that macrophages play an important role in the recruitment of repair cells and regeneration of tissue components. The miR-203a-3p agomir significantly improved the regeneration process of various tissue cells in db/db mouse wounds by inducing the polarization of M2 macrophages.
Our results demonstrate that the abundant miR-203a-3p present in epidermal stem cell-derived exosomes can promote M2 macrophage polarization by downregulating SOCS3 and can be applied to promote diabetic wound healing in vivo. In addition, this study also has the limitation of only exploring miRNAs of exosomes without exploring other components. However, our findings demonstrate a new concept of miRNA regulation in diabetic wound healing and provide novel strategies and therapeutic targets for improving diabetic wound complications. More importantly, our study not only adds to the understanding of the role of epidermal stem cell-derived exosomes in treating diabetic wounds but also expands the development of epidermal stem cell derivative therapy.
Declarations
Ethics approval and consent to participate
This study was approved by ICE for Clinical Research and Animal Trials for the First Affiliated Hospital of Sun Yat-sen University. Approval No: [2022] 238.
Consent for publication
All authors have approved the enclosed manuscript and agreed to its publication.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Funding
This work was supported in part by the National Natural Science Foundation of China (82172949, 81972569, 91772925, 82072180) and the Sun Yat-sen University Clinical Research 5010 Program (2018003).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The present work is the result of all joint efforts. We wish to express our sincere appreciation to all those who have offered invaluable help during this study.
Appendix A. Supplementary material
The following are the Supplementary data to this article:
Supplementary figure 1miR-200b-3p is encapsulated within ESCs-derived exosomes. A qPCR analysis of the expression levels of miR-200b-3p in the nucleus, cytoplasm, and CM of ESCs. B qPCR analysis of the expression levels of miR-200b-3p in the ESCs treated with control medium or RNase A (2 mg/mL) alone or in combination with Triton X-100 (0.1%) for 30 min. C qPCR analysis of the expression levels of miR-200b-3p in ESCs-derived exosomes (*p<0.05; **p<0.01; ***p<0.005).
Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function.
Salidroside-Pretreated Mesenchymal Stem Cells Enhance Diabetic Wound Healing by Promoting Paracrine Function and Survival of Mesenchymal Stem Cells Under Hyperglycemia.
Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function.
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