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....

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 Institut....

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