ARTIGO SOBRE DIMINUIÇÃO DA CICATRIZ

Prévia do material em texto

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/262143824
Epicatechin gallate improves healing and reduces scar formation of incisional
wounds in a rat type II diabetes mellitus model
Article  in  Wounds: a compendium of clinical research and practice · March 2012
CITATIONS
4
READS
51
2 authors, including:
Some of the authors of this publication are also working on these related projects:
Unravelling the tumour microenviroment in brain cancer View project
Inflammatory skin wound healing and novel therapeutics View project
Kelly Mckelvey
The University of Sydney
35 PUBLICATIONS   351 CITATIONS   
SEE PROFILE
All content following this page was uploaded by Kelly Mckelvey on 01 January 2015.
The user has requested enhancement of the downloaded file.
https://www.researchgate.net/publication/262143824_Epicatechin_gallate_improves_healing_and_reduces_scar_formation_of_incisional_wounds_in_a_rat_type_II_diabetes_mellitus_model?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_2&_esc=publicationCoverPdf
https://www.researchgate.net/publication/262143824_Epicatechin_gallate_improves_healing_and_reduces_scar_formation_of_incisional_wounds_in_a_rat_type_II_diabetes_mellitus_model?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_3&_esc=publicationCoverPdf
https://www.researchgate.net/project/Unravelling-the-tumour-microenviroment-in-brain-cancer?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_9&_esc=publicationCoverPdf
https://www.researchgate.net/project/Inflammatory-skin-wound-healing-and-novel-therapeutics?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_9&_esc=publicationCoverPdf
https://www.researchgate.net/?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_1&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Kelly_Mckelvey?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_4&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Kelly_Mckelvey?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_5&_esc=publicationCoverPdf
https://www.researchgate.net/institution/The_University_of_Sydney?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_6&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Kelly_Mckelvey?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_7&_esc=publicationCoverPdf
https://www.researchgate.net/profile/Kelly_Mckelvey?enrichId=rgreq-5d19a6d98447673f8d65fbffe324e61e-XXX&enrichSource=Y292ZXJQYWdlOzI2MjE0MzgyNDtBUzoxODA2NzA2NjgyMjI0NjRAMTQyMDA4NjY1MjU0NQ%3D%3D&el=1_x_10&_esc=publicationCoverPdf
1 
 
 
Epicatechin Gallate Improves Healing and Reduces Scar Formation of Incisional 
Wounds in a Rat Type II Diabetes Mellitus Model. 
 
Kelly J. McKelvey (PhD)a and Ian Appleton (PhD)a 
aDepartment of Pharmacology and Toxicology, School of Medical Sciences, University of 
Otago, Dunedin PO Box 9054, New Zealand. 
 
Corresponding Author and Request for Reprints to: 
Dr Kelly McKelvey 
Sutton Arthritis Research Laboratory, Kolling Institute of Medical Research, University of 
Sydney, St Leonards, NSW 2065, Australia. 
Email: kelly.mckelvey@sydney.edu.au 
Tel: +61 2 9926 4848 
Fax: +61 2 9926 6269 
 
Running Head: Effects of ECG on Impaired Wound Healing 
 
Abstract Word Count: 160 
Word Count: 832 excl. references 
No. Figures: 2 
No. Tables: 0 
 
 
mailto:kelly.mckelvey@sydney.edu.au�
2 
 
 
ABSTRACT 
Diabetic foot ulcers are the most severe clinical manifestation of diabetes-related impaired 
wound healing. Current standard and experimental treatments for these ulcers are largely 
ineffective. Epicatechin gallate (ECG) is a non-toxic flavonoid previously shown to improve 
normal wound healing and scar formation. In this study, the neonatal streptozotocin- induced 
diabetes mellitus (nSTZ-DM) type II model in rats was used to investigate the effects of ECG 
on impaired wound healing and scar formation. Administration of 100 mg/kg STZ induced a 
significant (P<0.05) state of mild hyperglycaemia in nSTZ-DM type II rats, compared to non-
diabetic controls. The effects of 0.8 mg/ml ECG on wound healing were then investigated 
using the full thickness incisional wound healing model. ECG significantly improves healing 
and reduces scar formation in nSTZ-DM type II rats (P<0.05). Biochemical improvements 
were also found including, significantly increased total nitric oxide synthase activity (NOS; 
P<0.001) and inducible NOS (iNOS) activity (P<0.01). This work highlights ECG as a 
potential treatment for DM-impaired wound healing. 
 
3 
 
 
INTRODUCTION 
By the year 2030 an estimated 366 million people will be diagnosed with diabetes mellitus 
(DM). Of those approximately 80% will be diagnosed with DM type II. Diabetic foot ulcers 
(DFUs) are the most severe clinical manifestation of DM-impaired wound healing and 
require long-term treatment. The DM type II epidemic has generated an increasing need for 
effective treatment of impaired wound healing. We have previously shown that epicatechin 
gallate (ECG), a polyphenolic flavonoid with antioxidant [1] and anti- inflammatory activity 
[2], improves wound healing and scar formation of full thickness incisional wounds in 
normal healthy rats [3]. Herein we utilise the neonatal streptozotocin-diabetes mellitus 
(nSTZ-DM) type II rat model to investigate the effects of ECG on DM-impaired incisional 
wound healing and scar formation. 
 
METHODS 
Neonatal streptozotocin-induced diabetes mellitus type II: Diabetes mellitus type II was 
induced in male neonatal Wistar rats on the day of birth (n0) by intraperitoneal (i.p.) injection 
of 100 mg/kg STZ in 0.9% saline (Sigma, USA; n=30). Non-diabetic controls (NDC) were 
administered an equal volume of vehicle (0.9% saline; n=13). Animals with blood glucose 
levels <7mM at the time of wounding were excluded from the study. All experimental 
procedures were performed in accordance with the University of Otago Animal Ethics 
Committee guidelines. 
 
Full thickness incisional wound induction and ECG dosing: Incisional wounds were 
induced as previously described [3] and left uncovered and unsutured. Non-diabetic control 
(n=13), STZ-vehicle (n=15) and STZ-treatment animals (n=15) were treated with 50 μL of 
0.9% saline and 0.8 mg/ml of ECG (in 0.9% saline; Sigma, USA) intradermally (i.d.), 
4 
 
 
respectively. A pre-dose of saline or ECG was administered 1 day prior to wounding (day –
1), the day of wounding (day 0) and each day for 6 days post-wounding (PW). 
 
Assessment of Scarring and biochemical analysis: Rats were sacrificed at day 14 PW by 
CO2 exposure followed by cervical dislocation. Ten µm sections of two wounds were 
examined for collagen using Van Gieson’s stain and assessed for scarring by six blinded 
observers, as previously described [3]. Micrographs were taken using a Zeiss Axioplan 
Microscope fitted with an AxioCam HR colour digital CCD camera (Carl Ziess Ltd., 
Germany). For biochemical analysis, two wounds were homogenised and nitrite level 
determination, nitric oxide synthase and cyclooxygenase activity performed, as previouslydescribed [3]. Protein concentrations were determined by Bradford assay [4]. 
 
Statistical Analysis: Results are expressed as mean ± standard error of the mean (SEM). In 
blinded histological assessment the data was normalised (+3 = 100%, -3 + 0%), then 
transformed (multiplied by 3) before being expressed as mean ± SEM relative to NDC. 
Statistical differences among the three treatment groups were analysed by Student T-test or 
one-way Analysis of Variance (ANOVA) followed by Tukey’s Post-Hoc test where 
appropriate. Statistical analyses were performed using GraphPad Prism version 4.00 for 
Windows (GraphPad Software, USA). 
 
RESULTS 
ECG improves healing and reduces scarring in nSTZ-DM type II diabetic rats: Van 
Gieson’s collagen stain was used to demonstrate the quality of healing and the amount of scar 
formation in the wounds of treatment groups at day 14 PW. NDC and STZ-treatment wounds 
showed a high proportion of mature (red) collagen fibres that were orientated parallel to the 
5 
 
 
epidermis (Figures 1A and C). The parallel orientation of collagen fibers indicates a normal 
non-scarring healing pattern. Additionally, there was complete wound contraction and the 
scar tracts were virtually indistinguishable from the surrounding tissue. In contrast, the STZ-
vehicle wounds demonstrated high proportions of immature (pink) collagen fibres that were 
largely disorientated and wide, clearly visible wound tracts indicating poor wound healing or 
scar formation (Figure 1B). Blinded histological assessment of the wounds identified 
significantly better healing and less scar formation in STZ-treatment wounds compared with 
STZ-vehicle wounds at day 14 PW (P<0.05; Figure 1D). Thus, ECG treatment improves scar 
formation of nSTZ-DM type II impaired wounds. 
 
Biochemical effects of ECG on nSTZ-DM type II scar formation: Dysregulation of 
arginine metabolism by the NOS and COX enzyme systems contributes to impaired wound 
healing [5]. We assessed the nitrite levels, NOS and COX activity in the wounds. No 
significant difference in nitrite levels was found between the treatment groups (Figure 2A). 
Total and iNOS activity was significantly higher (P<0.05) in the wounds of NDC and STZ-
treatment rats, compared with STZ-vehicle rats where NOS activity was undetectable. No 
significant differences in COX activity were found among the treatment groups (Figure 2C). 
Therefore, ECG treatment attenuates the reduced iNOS and total NOS activity in nSTZ-DM 
type II impaired wound healing. 
 
DISCUSSION 
The DFU wound environment has altered enzymatic activity which contributes to impaired 
wound healing and scar formation [6]. We show that treatment with ECG attenuates impaired 
scar formation of full thickness incisional wounds in nSTZ-DM type II rats. Specifically, 
ECG treatment improves collagen maturation and increases iNOS and total NOS activity in 
6 
 
 
nSTZ-DM type II impaired wounds at day 14 PW, consistent with ECG treatment of normal 
incisional wound healing in rats [3]. Our results suggest that ECG is a potential treatment for 
wounds in patients with DM type II. 
 
REFERENCES 
1. Caturla, N., et al., The relationship between the antioxidant and the antibacterial 
properties of galloylated catechins and the structure of phospholipid model 
membranes. Free Radic. Biol. Med., 2003. 34: p. 648-662. 
2. Frampton, L., et al., The effects of catechins on a murine model of chronic 
granulomatous inflammation. Proceedings of the Australian Health Medical Research 
Congress, Melbourne, National Health and Medical Research Council, November 25-
29, 2002: p. 2030. 
3. Kapoor, M., et al., Effects of epicatechin gallate on wound healing and scar formation 
in a full thickness incisional wound healing model in rats. AJCP, 2004. 165(1): p. 
299-307. 
4. Bradford, M.M., A rapid and sensitive method for quantitation of microgram 
quantities of protein utilizing the principle of protein-dye-binding. Anal. Biochem., 
1976. 72: p. 248-254. 
5. Gould, A., C. Naidoo, and G. Candy, Arginine metabolism and wound healing. 
Wound Healing Southern Africa, 2008. 1(1): p. 48-50. 
6. Jude, E.B., et al., The role of nitric oxide synthase isoforms and arginase in the 
pathogenesis of diabetic foot ulcers: possible modulatory effects by transforming 
growth factor beta 1. Diabetologia, 1999. 42: p. 748-757. 
 
7 
 
 
 
ACKNOWLEDGEMENTS 
We are grateful to Dr Kamali Pugazhenthi and Ms Irene Hall for their assistance in the care 
of animals and technical advice. 
 
This work was supported by funding from a New Zealand Lottery Board Health Grant (IA). 
 
 
8 
 
 
CONFLICT OF INTEREST STATEMENT 
 
There are no potential conflicts of interest with either author in this work. 
 
 
9 
 
 
FIGURE CAPTIONS 
 
Figure 1 Representative photomicrographs of van Gieson’s collagen staining depicting 
wound healing and scar formation in NDC (A), STZ-vehicle (B) and STZ-treatment (C) 
wounds. Arrowheads indicate the wound tract. *P<0.05, STZ-vehicle vs. STZ-treatment 
wounds. D, Blinded histological assessment improved healing and scar formation. The data is 
expressed as the mean ± SEM relative to NDC. N = 6 separate observations. 
 
Figure 2 Quantification of nitrite accumulation (A), total NOS and iNOS (B) and 
cyclooxygenase (C) in the wound environment. N = at least 4 samples per treatment. 
View publication statsView publication stats
https://www.researchgate.net/publication/262143824