Effects of Moist Exposed Burn Ointment on Wound Neovascularization and Plasma Exosomal mir-31-5p in Diabetic Mice

Summary

This article describes how moist-exposed burn ointment promotes diabetic wound repair by dynamically regulating diabetic wound neovascularisation and the expression of plasma exosomal mir-31-5p.

Abstract

Moist-exposed burn ointment (MEBO) is a traditional Chinese medicine used to treat chronic diabetic wounds; however, the underlying mechanism is unclear. Recent evidence suggests the role of exosomes and functional miRNAs in wound neovascularization. Here, we aimed to investigate the effects of MEBO on wound neovascularization and plasma exosomal mir-31-5p (pExo-mir-31-5p) expression in diabetic mice. A total of 120 C57BL/6 mice were randomly divided into a non-diabetic control group (n = 24) and a diabetic group induced by streptozotocin (n = 96), which was further randomized into diabetic control, MEBO, beifuxin, and MHY1485 groups (n = 24 each). Five groups were established with full-thickness skin resection models, which were treated with physiologic saline, physiologic saline, MEBO, beifuxin, and MHY1485, respectively. On days 1, 7, 13, and 18, the wound closure rate, microvessel density, and pExo-mir-31-5p expression levels, together with VPS4a and Rab27a mRNA and protein levels in wound tissues, were analyzed using ImageJ, hematoxylin-eosin (HE) staining, quantitative real-time polymerase chain reaction (qPCR) and western blot (WB), respectively. Furthermore, correlations between the expression of VPS4a, Rab27a protein, pExo-mir-31-5p, and microvessel density were analyzed. We found that MEBO promoted wound repair in diabetic mice (p < 0.050). It enhanced wound neovascularization during the early and middle stages of wound healing but inhibited it during the later stage (p < 0.050). MEBO upregulated Vps4a and Rab27a mRNA and protein expression in wound tissues, as well as pExo-mir-31-5p during the early and intermediate stages of healing, while downregulating them in the later stage (p < 0.050). Moreover, VPS4a, Rab27a protein, pExo-mir-31-5p expression, and microvessel density were positively correlated in MEBO-treated mice (p < 0.050). In conclusion, MEBO dynamically regulates wound neovascularization and pExo-mir-31-5p expression in diabetic mice, providing new theoretical support for its clinical application.

Introduction

The prevalence of diabetes mellitus in older patients is high and increasing annually. The disease is associated with chronic diabetic wounds (CDWs), which have a detrimental effect on patient health, function, and overall quality of life¹. Typically, wound closure involves four distinct phases: hemostasis, inflammation, proliferation, and reepithelialization². Of these, the proliferative phase is the most important. During the early proliferative phase, capillary endothelial cells proliferate and migrate to the wound, sprouting new vessels, and forming an intricate network³. However, in the later stages of wound repair, most of the capillary lumina undergo occlusion, with a minority transitioning into small arteries and veins. In patients with CDWs, tissue loss, necrosis, and gangrene, and different levels of inflammation and hyperglycemia, disrupt the dynamic balance between angiogenesis and antiangiogenesis, delaying wound healing, especially when angiogenesis is impaired^4,5,6. Moist-exposed burn ointment (MEBO) is a traditional Chinese medicine used to treat CDWs, but its mechanism remains unclear. Recently, multiple studies have demonstrated that exosome-cargoed miRNAs could promote angiogenesis and thereby promote diabetic wound healing^7,8. However, few studies have investigated the association between MEBO and exosome-carried miRNAs. Therefore, there is a need to reveal the mechanism by which MEBO stimulates angiogenesis in CDWs from the exosome and functional miRNA perspective.

Exosomes are spherical membranous vesicles released by living cells, measuring 30-200 nm in size, 1.1-1.2 g/mL in density, and containing marker proteins like CD81 and tumor susceptibility 101 (TSG101)⁹. Exosome production and secretion are categorized into endosomal sorting complex required for transport (ESCRT)-dependent and non-ESCRT-dependent pathways. The most critical regulators within these pathways are vacuolar protein sorting 4 homolog a (VPS4a) and Ras-related protein Rab-27a (Rab27a)^10,11,12. Vps4a acts as an ATPase, catalyzing the ESCRT-dependent membrane remodeling process, which regulates the size of the exosome and rate of exosome formation^13,14. Rab27a is an essential small GTPase that regulates the docking of multivesicular bodies to the plasma membrane¹⁵. Exosome secretion is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1)¹⁶, which is activated by MHY1485 competitive binding to the ATP structural domain of rapamycin¹⁷. Overall, MHY1485 could indirectly inhibit the synthesis and secretion of exosomes. Exosomes can be detected in almost all bodily fluids. Plasma exosomes are more productive than exosomes derived from other body fluids¹⁸. Moreover, exosomes protect the nucleic acids they carry from degradation¹⁹.

MiRNAs are small non-coding RNA molecules that regulate cellular activity²⁰. Exosomes have been discovered abundantly in numerous miRNAs crucial for enhancing wound angiogenesis, specifically mir-126, mir-125a, and mir-31-5p. A previous study demonstrated that exosomes derived from mir-126 overexpressing bone marrow mesenchymal stem cells promoted the proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro by transferring mir-126²¹. Another study indicated that exosomes secreted by human adipose-derived mesenchymal stem cells transferred mir-125a to endothelial cells and promoted angiogenesis by repressing the angiogenic inhibitor delta-like 4 in wounds⁹. Yan et al. reported that mir-31-5p loaded into milk exosomes improved endothelial cell functions in vitro, promoted angiogenesis, and enhanced diabetic wound healing in vivo²². Among these miRNAs, mir-31-5p was the only miRNA that was significantly downregulated in diabetic mice wounds compared to that in normal mice wounds²².

MEBO is an oily ointment that can be used to treat refractory wounds such as CDWs^23,24. However, the mechanism of MEBO promoting wound healing has not been elucidated. Previous study have shown that MEBO promotes wound neovascularization by upregulating vascular endothelial growth factor expression in diabetic rats²⁵. However, only a few studies have addressed this issue from the perspective of exosome-carried miRNAs. In this study, to investigate the effects of MEBO on CDWs, we induced a full-thickness wound model in diabetic mice using streptozotocin (STZ) and a high-fat diet. We showed that MEBO significantly accelerated wound healing in diabetic mice. Moreover, we studied the effects of MEBO on microvessel density, expression levels of pExo-mir-31-5p, together with VPS4a and Rab27a mRNA and protein in wound tissues. Therefore, we hypothesized that MEBO promoted wound healing by regulating pExo-mir-31-5p to regulate wound angiogenesis in diabetic mice. A schematic overview of the study design is presented in Figure 1.

Protocol

All animal experimental protocols were approved by the Animal Care and Use Committee of Youjiang Medical University for Nationalities, Baise, China (2018051501). Specific pathogen-free (SPF) male C57BL/6 mice (7 weeks old) were housed in the SPF Laboratory Animal Center of Youjiang Medical College for Nationalities and fed an ordinary or high-fat diet and clean water. Throughout the study, the temperature of the animal feeding room was maintained at 23 °C to 25 °C, the humidity was 40% to 60%, and the light/dark cycle was 12 h. Animal care was provided in accordance with the guidelines of the US National Institutes of Health and the Chinese National Institute of Health (Beijing, China). All surgical interventions were performed under anesthesia with sodium pentobarbital (0.3%, 50 mg/kg) using a standardized protocol established in our laboratory. All efforts were made to reduce the number of animals used and to minimize animal discomfort. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research.

Mouse wound model and treatment
A total of 120 mice were randomly assigned to a non-diabetic control (NDB-CTR; n = 24) or diabetic (n = 96) group. To simulate human type 2 diabetes, mice in the diabetic group were fed a high-fat, refined sugar diet for 4 weeks and then intraperitoneally injected (ip) with STZ (100 mg/kg), once per day, for 2 consecutive days after fasting for 16 h. Fasting blood glucose levels were measured on the 5th and 6th day after STZ injection. Mice with fasting blood glucose values greater than 16.7 mmol/L on two occasions and with symptoms such as polyphagia, polydipsia, polyuria, and weight loss were considered successful diabetes models²⁶. After 1 week, the dorsal hair was shaved, and a round full-thickness cutaneous wound (diameter = 15 mm) was generated on the back of each mouse following intraperitoneal administration of sodium pentobarbital (0.3%, 50 mg/kg). The mice in the diabetic group were randomly assigned to the MEBO, BFX (beifuxin), DB-CTR (diabetic control), or MHY1485 (MHY) groups. Mice in the MEBO and BFX groups were treated with MEBO (0.2 g/cm²) and BFX gel (0.2 g/cm²) applied to the wound once a day, respectively. BFX gel, whose generic name is bovine basic fibroblast growth factor gel, is a topical medication used to treat chronic wounds²⁷. The mice in the NDB-CTR, DB-CTR, and MHY groups were treated with physiological saline. The MHY group was also treated with MHY1485 (10 mg/kg, ip) for 2 consecutive days. The diabetic mice were subcutaneously injected with insulin at a dosage of 2-4 IU/kg once per day to control the blood glucose level.

Specimen collection
On days 1, 7, 13, and 18, six mice in each group were randomly selected for euthanasia by cervical dislocation, and samples of ring-shaped epidermal tissues, including the wound, were taken at 0.5 cm from the wound edge with sterile tissue scissors. Then the wound tissues were collected for HE staining, qPCR analysis, and WB assays. Blood samples were harvested from mice in the DB-CTR and MEBO groups using the retro-orbital method under sodium pentobarbital anesthesia. Blood samples were mixed with heparin in anticoagulant tubes and centrifuged at 3,000 x g for 15 min to recover the plasma supernatant. All the samples were stored at -80 °C. To calculate the wound closure rate, a digital camera was used to capture pictures of wound healing at a fixed time point and at an equal distance with a graduated scale on the side. The wound area was measured using the ImageJ software. The wound closure rate was calculated as follows:

wound closure rate (%) = [W1-Wn] / W1 x 100%

where W1 and Wn refer to the wound areas on days 1 and n, respectively.

Histopathological assay
HE staining was performed to dynamically observe pathological wound healing. After fixation with a 4% paraformaldehyde solution for 16 h, the wound tissues were dehydrated, embedded in paraffin, and then sliced into 3 µm sections. HE staining was performed on tissue slides. To explore the microvessel density of the wound tissues, tissue slides were imaged using an optical microscope. To quantify the microvessel density of each sample, we selected the area with the highest capillary density under a 10x microscope, then selected and photographed three non-overlapping areas under a 20x microscope. The microvessel densities were calculated and averaged using ImageJ.

Isolation and characterization of plasma exosomes
Ultracentrifugation was performed to isolate plasma exosomes according to a published protocol with minor modifications²⁸. Briefly, 2 mL of plasma was centrifuged at 2,000 x g at 4 °C for 30 min. The supernatant was centrifuged again at 10,000 x g at 4 °C for 45 min to remove the larger vesicles. The supernatant was filtered through a 0.45 µm filter membrane, and the filtrate was collected. The filtrate was then centrifuged at 100,000 x g at 4 °C for 70 min. After removing the supernatant, the pellet was re-suspended with 10 mL of pre-cooled 1x PBS and centrifuged at 4 °C, 100,000 x g, for 70 min. The supernatant was removed, and the pellet was re-suspended with 150 µL of pre-cooled 1x PBS and stored at -80 °C. To confirm the plasma exosomes collected above, we used transmission electron microscopy (TEM) to determine the morphology of exosomes, a nano-flow cytometry system to analyze the size and concentration of exosomes, and WB to detect the expression levels of the exosome markers CD81 and TSG101.

RNA extraction, reverse transcribe (RT), and qPCR
qPCR was performed to quantify the mRNA expression levels of Vps4a, Rab27a, and pExo-mir-31-5p. Wound tissues were collected to extract total RNA using RNA extraction reagent. A total RNA concentration between 400-700 ng/µL was considered acceptable. The RNA samples were reverse-transcribed into cDNA using reverse transcriptase with dsDNase according to the manufacturer's instructions. qPCR was performed using the SYBR Green qPCR Mix (No ROX). As for mir-31-5p, RNA from plasma exosomes was extracted using the mini kit, reverse-transcribed using the First Strand cDNA Synthesis Kit, and amplified using SYBR Green qPCR Master Mix. The expressions of mRNAs and miRNAs were analyzed using the 2-ΔΔCt method relative to β-actin and cel-mir-39, respectively. The primers used are presented in Table 1.

Western blot
The WB assay was used to detect the expression levels of Vps4a and Rab27a proteins, as well as the levels of the exosome markers CD81 and TSG101. Total protein levels of the wound tissues and plasma exosomes were extracted by RIPA lysis buffer, and the protein concentration was determined by the Bicinchoninic Acid Protein Assay Kit. A total of 50 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide. After blotting onto polyvinylidene fluoride membranes, the membranes were blocked with bovine serum albumin, then incubated at 4 °C overnight with antibodies against Vps4a (1:3000), Rab27a (1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000), CD81 (1:3000), TSG101 (1:3000), then incubated with secondary antibodies, anti-rabbit IgG peroxidase conjugated (1:3000), at 37 °C for 1 h. The membranes were visualized using an enhanced chemiluminescence kit.

Statistical analysis
All data presented in this study were analyzed for statistical significance using SPSS software. All results obtained in these analyses were visualized using GraphPad Prism software. Differences between the two groups were examined using an independent sample t-test. For multiple group comparisons, one-way analysis of variance was used for homogeneous variance data, whereas Welch's test was applied to heterogeneous variance data. Furthermore, we used the least significant difference test for pairwise comparison of equal variance data and Tamhane's T2 test for unequal variance data. Quantitative data are presented as mean ± standard deviation. Correlations were analyzed using Spearman's correlation analysis for non-normally distributed variables and were described as correlation coefficients and 95% confidence intervals. P < 0.050 was considered statistically significant. All experiments were repeated at least three times.

Results

MEBO accelerates wound healing in diabetic mice
To validate the efficacy of MEBO in diabetic wounds, we established a diabetic wound model and took photographs on days 1, 7, 13, and 18. The wounds in each group healed gradually over time (Figure 2A). The wounds in the NDB-CTR group healed completely by day 18. The wounds in the MEBO group healed the most rapidly among the diabetic wounds, forming a few pink granulation particles on day 7, then growing a large area of bri...

Discussion

CDWs, a complication of diabetes, can lead to significant mortality and disability, and are often associated with vascular disorders²⁹. MEBO is effective in treating CDWs; however, its underlying mechanism is unclear³⁰. Therefore, we aimed to examine it from the perspective of exosomes and functional miRNAs. In this study, we found that the wound closure rate and microvessel density of the MEBO group were the highest at all time points in diabetic mice. qPCR and WB results ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1×PBS	Solarbio (Beijing, China)	#P1020	Buffered solution
4% paraformaldehyde solution	Solarbio (Beijing, China)	#P1110	Fix tissue
anti-rabbit IgG peroxidase conjugated	Affinity	#S0001	Secondary antibody
Beifuxin gel	Essexbio (Zhuhai, China)	#042404A06	An ointment to treat chronic wounds
CD81 antibody	Signalway Antibody	#41779	Primary antibody
enhanced chemiluminescence kit	Epizyme(China)	#SQ101	Chemiluminescence kit for protein band
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody	Affinity	#AF7021	Primary antibody
GraphPad Prism	https://www.graphpad.com/prism/		Visualization
ImageJ	https://imagej.net/ij/		Measure wound area
Instant Bicinchoninic Acid (BCA) Protein Assay Kit	Epizyme(China)	#ZJ102	Determine the protein concentration
LightCycler96 System	Roche		qPCR equipment
MHY1485	MedChemExpress (USA)	#326914-06-1	An activator of the mammalian target of rapamycin complex 1 (mTORC1)
miRNeasy Mini kit	Tiangen (China)	#217004	RNA extraction (miRNA)
Moist-exposed burn ointment	Mebo (Shantou, China)	#2403103B	An ointment to treat chronic wounds
MonAmp SYBR Green qPCR Mix (No ROX)	Monad (China)	#MQ10101S	qPCR kit (mRNA)
MonScript RTIII All-in-One Mix with dsDNase	Monad (China)	#MR05101S	Reverse transcribe kit (mRNA)
nano flow cytometry system	N30E (China)		An equipment to analyze the size and concentration of exosomes
optical microscope	Olympus (Tokyo, Japan)		Observe tissue pathomorphology
physiologic saline	Kelun (Sichuan, China)	#L24041709	Physiologic saline
polyvinylidene fluoride membranes	Millipore(USA)	#IPVH00010	Western blotting membrane
Primers	Generay Biotech Co., Ltd. (Shanghai, China)
Rab27a antibody	Cell Signaling Technology	#69295	Primary antibody
RevertAid First Strand cDNA Synthesis Kit	Thermo Fisher (USA)	#K1622	Reverse transcribe kit (miRNA)
RIPA lysis buffer	Solarbio (Beijing, China)	#R0010	Total protein extraction
sodium dodecyl sulfate–polyacrylamide	Epizyme(China)	#PG222	Electrophoresis buffer
sodium pentobarbital	Merck (Shanghai, China)	#P3761	Anesthetic
Specific pathogen-free male C57BL/6 mice	Guangdong Vital River Laboratory Animal Technology Co., Ltd (Guangdong, China)		Experimental animals
SPSS	https://www.ibm.com/spss		Statistical analysis
streptozotocin	Solarbio (Beijing, China)	#S8050	Induce diabetic mouse model
SYBR Green qPCR Master Mix	Roche (Switzerland)	#4943914001-SR	qPCR kit (miRNA)
transmission electron microscopy	Hitachi (Japan)	HT-7700	An equipment to determine the morphology of exosomes
TRIZOL	Invitrogen (USA)	#15596026CN	RNA extraction (mRNA)
TSG101 antibody	Abcam	# ab125011	Primary antibody
Vps4a antibody	Abcam	# ab197896	Primary antibody