Area% of collagen fibers
There was a significant increase (P≤0.05) in the mean area% of collagen fibers in the propolis-treated subgroups compared with the other subgroups (Table).
Area% of vascular endothelial growth factor in epidermis
There was a significant increase (P≤0.05) in the mean area% of VEGF immunoexpression in the propolistreated subgroups compared with the other subgroups. A significant increase (P≤0.05) in the mean area% of VEGF in subgroup IIb compared with subgroup IIa was observed (Table 1).
Area% of vascular endothelial growth factor in dermis
There was a significant increase (P≤0.05) in the mean area% of VEGF immunoexpression in the propolis-treated subgroups compared with the other subgroups. A significant increase (P≤0.05) in the mean area% of VEGF in subgroup IIb compared with subgroup IIa was observed (Table).
Incidence of diabetes mellitus is increasing. It represents a major health problem in the 21st century. The total number of diabetic patients is expected to increase from 171 million in 2000 to 439 million in 2030, compromising around 7.7% of the world population 15.
Diabetes in human induces chronic complications such as cardiovascular damage, delayed wound healing, cataract, retinopathy, nephropathy, and polyneuropathy. The most common animal model of human diabetes is STZ-induced diabetes in rat 16. It has been commonly used to induce not only animal models of type I diabetes, but also type II diabetes 17.
In the present study, marked improvement in wound healing in the propolis-treated group was detected. This improvement was confirmed by histological, immunohistochemical, and morphometric results.
In the present study, diabetic control subgroups Ia and Ib showed nonhealed epidermis with deficient granulation tissue in the wound area. A mass of inflammatory cells under the nonhealed epidermis was also observed in subgroup Ia. These findings were correlated to a previous study 18 that revealed delayed wound healing in diabetic nontreated rats in the form of reduced wound vascularization and collagen synthesis and with an increased extent of inflammatory cells in wound tissues. Another study 13 showed that diabetes decreased epithelial closure and re-epithelialization. In contrast, results of the current study were inconsistent with that of Lin et al. 12, who demonstrated complete epithelialization of the wound area on day 10 but without all four strata structure.
Defective wound healing in diabetes might be associated with an abnormality in one or more phases of the healing process. In acute wound healing, the inflammatory response should occur rapidly and sustain for 3 days to permit the development of subsequent phases of wound healing. This required that inflammatory cells (such as neutrophils and macrophages) migrate to the wound area and phagocytose necrotic tissue and microorganisms. However, the inflammatory response in chronic wound healing, such as diabetes, will last for extended periods, affecting wound regeneration 2,3. Furthermore, diabetes mellitus has been shown to be associated with a decrease in number and function of circulating endothelial progenitor cells, leading to impaired angiogenesis, lymphangiogenesis, and wound healing 19.
Another possible theory of defective wound healing in diabetes was lack of oxygen. Oxygen is important for cell metabolism, especially energy production by means of ATP, and is necessary for all wound-healing processes. It prevents wounds from infection, induces angiogenesis, increases keratinocyte differentiation, migration, and re-epithelialization, enhances fibroblast proliferation and collagen synthesis, and promotes wound contraction. Furthermore, the level of superoxide production (a key factor for oxidative killing of pathogens) by polymorphonuclear leukocytes is critically dependent on the oxygen levels. Diabetes created impaired vascular flow, resulting in poor tissue oxygenation 20,21.
Propolis-treated subgroup IIa showed filling of the wound area with granulation tissue and inflammatory cells. Other sections revealed disorganized dermal pattern in the form of a gap between the epidermis and dermis. However, subgroup IIb revealed filling of the wound area with granulation tissue with a well-formed epithelium. These findings were in agreement to the findings of McLennan et al. 14, who demonstrated accelerated wound healing by propolis application in the diabetic rat models. A previous study conducted on diabetic human patients 22 has documented photographically that wounds have healed completely by topical application of propolis dressings. Other studies 23,24 demonstrated the powerful healing effect of propolis on normal surgical wounds.
Congested blood vessels were also noticed in subgroup IIa. This was concomitant with some investigators 14,24 who showed presence of microvessels. Dilated lymphatic vessels were also observed. This was in agreement to the study by Martínez-Corral et al. 25, who documented increased angiogenesis and lymphangiogenesis in normal wound healing process. Another study showed that topical application of simvastatin has promoted lymphangiogenesis and angiogenesis during wound healing in genetically diabetic mice 19.
The increased rate of wound closure in propolis-treated wounds might be attributed to increased proliferation and transformation of fibroblast cells into myofibroblasts. The early re-epithelialization and faster wound closure in propolis-treated wounds might also be associated with increased keratinocytes proliferation and their migration to the wound surface. Angiogenesis during wound repair serves the dual function of providing the required nutrients by supplying essential nutrients and oxygen to the wound site and promoting granulation tissue formation 6,26.
The anti-inflammatory effect of propolis has been attributed to various mechanisms such as inhibiting the production of eicosanoids and nitric oxide, antioxidant action, modulation of calcium ion mobilization, angiogenesis, and antileukocyte activity 27.
Angiogenesis and lymphangiogenesis are transient processes during wound healing. Reduction in exudate from the wound appears to be related to blood vessels. However, increasing lymphatics may play a role in the late phase of the wound-healing process. They are induced by VEGF release. Hence, propolis might induce angiogenesis and lymphangiogenesis by its modulatory effect on VEGF expression 28,29.
A significant decrease (P≤0.05) in collagen fibers deposition (represented by mean area%) in the diabetic control subgroups compared with other subgroups was observed. This was in accordance to previous study 13 that showed small number of collagen fibers and few reticular fibers under the wound. Another study demonstrated low hydroxyproline contents in the skin, 7 and 14 days after wounding 18.
The previous findings might be attributed to impaired fibroblast migration 30. This could result in reduction of collagen deposition and impairment of wound healing. High levels of metalloproteinases (MMPs) are a feature of diabetic foot ulcers, and the MMP levels in chronic wound fluid are almost 60 times higher than those in acute wounds. This increased protease activity supports tissue destruction and inhibits normal repair processes 31,32.
In propolis-treated subgroup IIa and IIb, there was a significant increase in the mean area% of collagen fibers compared with nontreated group. This was consistent with previous studies 14,23,24,27 that revealed increased collagen deposition and granulation tissue.
The propolis may potently inhibit MMP-3 activity, which occupies a central position in the collagenolytic and elastolytic cascades 33. It also may act by minimizing the acute inflammatory exudate as well as stimulating macrophages and T lymphocytes and consequently fibroblast activity 34. De Moura et al. 35 found that propolis had the capacity to hinder the deposition rate of type I collagen only in the earliest phases of the healing process (days 4–7 of the postoperative period), with collagen deposition reaching the normal degree in 2 weeks. According to the authors, it is likely that the attenuation of the recruitment of cells by propolis accelerated the proliferative phase of the healing process, promoting the rapid transformation of type III collagen into type I and modulating the inflammation process.
The onset of the maturation phase may vary extensively, depending on the size of the wound and whether it was initially closed or left open. It ranges from ∼3 days to 3 weeks. The maturation phase can last for a year or longer, depending on wound type 36,37. The previous facts might explain delayed remodeling effect of propolis after 2 weeks.
The present study demonstrated a significant decrease in dermal and epidermal VEGF immunoexpression (represented by mean area%) in the diabetic control subgroups compared with the other subgroups. This finding was in accordance to some investigators 18,38 who revealed decreased numbers of cells expressing VEGF in vehicle-treated diabetic rats.
It is known that cytokines, especially various growth factors, provide the cellular and molecular signals necessary for the normal healing process but are deficient in diabetic wounds 38. One of these growth factors is VEGF. It is secreted by many cell types that participate in wound healing, such as endothelial cells, fibroblasts, smooth muscle cells, platelets, neutrophils, and macrophages, 3 and 7 days after the injury. Platelet is the first vascular component that appears in the wound site followed by neutrophils and then macrophages. Activated platelets release VEGF, particularly after thrombin stimulation. Monocytes express the VEGF receptor and induce angiogenesis by releasing tumor necrosis factor α, which may in turn induce VEGF expression in the keratinocytes and fibroblasts. Cells involved in releasing cytokines and growth factors may act as paracrine factors for further VEGF expression 39.
A significant increase in immunoexpression of VEGF was revealed in propolis-treated diabetic rats as compared with the vehicle-treated diabetic group. This might be due to the modulatory effect of propolis on VEGF secretion 29. In addition, there is evidence suggesting that oxygen free radicals play an important role in diabetic wounds. These molecules cause failure of healing of diabetic wound. Therefore, antioxidants might improve healing of diabetic wounds 18.
There was also a significant increase in dermal and epidermal VEGF immunoexpression in the propolis-treated subgroup IIb compared with the subgroup IIa. This was inconsistent with a previous study 38 that revealed reduced expression of VEGF in bioactive glass-treated diabetic wound after 2 weeks. It explained this reduction because of the entry in the maturation phase of wound healing. In the present study, this continued increase in VEGF expression might be attributed to delayed entry into the remodeling phase of wound healing. As VEGF directly increases endothelial cell secretion of interstitial collagenase (MMP-1), which breaks down collagen types I–III, this may spot light on the role of VEGF not only in the early stage of healing, but also in the remodeling (maturation) stage 39.
In conclusion, the findings of the present study reinforced the significant role of VEGF in the wound healing process. It also revealed the powerful healing effect of propolis on diabetic wounds. This could be an effective strategy for managing patients with diabetic foot.
Further studies are required on propolis before clinical application. Another experimental study may be designed to evaluate the effect of orally administered propolis on diabetic wound to assess its systemic effect. Another study may be designed for complete understanding of the role of different cytokines and growth factors in wound healing. As a trial to assess the healing effect of propolis on normal wounds, another study may be performed.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2013 The Egyptian Journal of Histology
diabetic foot; propolis; streptozotocin; vascular endothelial growth factor