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Evaluation of the effect of bee propolis cream on wound healing in experimentally induced type I diabetes mellitus: a histological and immunohistochemical study

Ibrahim, Noha A.

The Egyptian Journal of Histology: December 2013 - Volume 36 - Issue 4 - p 847–856
doi: 10.1097/01.EHX.0000437148.20026.7d
Original articles

Background Diabetic foot and poor wound healing are serious problems in diabetic patients. Propolis is a honeybee product. It can improve wound healing and has free radical scavenging activity.

Aim The aim of the study was to evaluate the possible wound-healing effect of bee propolis cream on streptozotocin-induced type I diabetes mellitus in adult male albino rats using histological, immunohistochemical, and morphometric studies.

Materials and methods Twenty adult male albino rats were used in the study. Diabetes mellitus was induced and two cutaneous wounds were created at the dorsal region of the rats. The rats were randomly divided into two equal groups: the control group, which was subdivided into two subgroups – five rats each; subgroup Ia received vehicle cream once daily for 1 week and subgroup Ib received vehicle cream once daily for 2 weeks and the propolis group, which was subdivided into two subgroups – five rats each; subgroup IIa received propolis cream once daily for 1 week and subgroup IIb received propolis cream once daily for 2 weeks. Histological (using H&E and Masson’s trichrome stains) and immunohistochemical [using vascular endothelial growth factor (VEGF)] studies were performed. Morphometric measurement of area% of collagen fibers and VEGF were carried out followed by statistical analysis.

Results There was a marked improvement in wound healing with a significant increase in collagen deposition in the propolis group. Increased area% of collagen fibers and VEGF immunoexpression were found in the propolis group.

Conclusion The present study reinforced the significant role of VEGF in the wound healing process. The powerful healing effect of propolis on diabetic wounds was also revealed. This could be an effective strategy for managing patients with diabetic foot.

Histology Department, Faculty of Medicine, Fayoum University, Al Fayoum, Egypt

Correspondence to Noha A. Ibrahim, MD, Histology Department, Faculty of Medicine, Fayoum University, Al Fayoum, Egypt Tel: + 01149939637; e-mail: nh_ebrahim@yahoo.com

Received April 30, 2013

Accepted October 9, 2013

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Introduction

Wound healing is a complex phenomenon characterized by a sequence of independent and overlapped events such as exudative/inflammatory, proliferative, and remodeling phases. Within these three broad phases, there are a complex and coordinated series of events that include chemotaxis, phagocytosis, neocollagenesis, collagen degradation, and collagen remodeling in addition to angiogenesis, epithelization, and the production of new glycosaminoglycans and proteoglycans 1,2.

Wounds that exhibit impaired healing, including delayed acute wounds and chronic wounds, generally have failed to progress through the normal stages of healing. Such wounds frequently enter a state of pathologic inflammation due to a postponed, incomplete, or uncoordinated healing process 3.

In all over the world, 15% of 200 million diabetic people suffer from diabetic foot disease. Foot ulcers and poor wound healing are serious problems in diabetic patients. Healing impairment in diabetes is characterized by delayed cellular infiltration, granulation, tissue formation, decreased collagen organization, diminished blood flow, increased blood viscosity, and reduced angiogenesis 4,5.

Propolis is a natural product from the honeybee (bee glue). The extract is an extremely complicated mixture of natural substances. It contains amino acids, phenolic acids, phenolic acid esters, flavonoids, cinnamic acid, terpenoids, and caffeic acid. Many biological and pharmacological properties of propolis have been noted. They include antibacterial, antifungal, anti-inflammatory, antioxidant, immunomodulatory, antiviral, and anticarcinogenic properties 6,7.

Vascular endothelial growth factor (VEGF) is a homodimeric, heparin-binding glycoprotein that promotes endothelial cell proliferation and migration. It binds to VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGFR-2 (also known as KDR/Flk-1) 8. VEGF exists in many organs in the body. It is an essential inducible factor in physiological and pathological angiogenesis and plays a local regulatory role 9. Furthermore, it is a key factor in influencing diverse cell functions, including cell survival, proliferation, tissue repair, and generation of nitric oxide and prostacyclin 10.

The aim of this study was to evaluate the possible healing effect of propolis cream on diabetic wound in adult male albino rats using histological and immunohistochemical techniques.

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Materials and methods

Animals

Twenty adult male albino rats, locally bred at the Animal House of Kasr El Aini, with an average weight of 200–250 g were used in the present study. The animals were housed at ordinary room temperature, exposed to natural daily light–dark cycles, and had access to food and water ad libitum according to the ethics of animal researches.

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Chemicals

Streptozotocin (STZ; Sigma, Munich, Germany): it is a chemical substance used in experimental studies.

Raw propolis: it was purchased from beekeeper, who gathers these resinous materials from bee hives by scraping. They put them in the fridge until they become hard enough to be ground into powder. Extract of propolis sample was prepared and used throughout this study as described by Bogdanov 11. The propolis powder was extracted with 80% ethanol (30% propolis solution was used). This mixture was allowed to stand for 10 days at room temperature, with periodic agitation several times each day. At the end of 10 days, the mixture was filtered through filter paper. For the base of the cream, Vaseline and lanolin in a proportion of 9:1 was used. For 100 g of this base, 20 ml of 30% propolis ethanol extract was used for preparation of 6% propolis cream. Then, warming up of the base in a water bath (at about 40–50°C) and adding propolis extract were performed. While stirring, evaporation of ethanol was performed by boiling. Thereafter, the warm cream was sieved and packed in a dark, tightly closed cream box.

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Diabetes induction

STZ was dissolved in normal saline. Following this, 45 mg/kg dose of STZ was injected to the overnight-fasted rats through the tail vein. After 3 days, blood samples were drawn from the tail of these rats to determine fasting blood glucose level using blood glucose tests strips and meter (Accu-Check; Roche Diagnostics, Benzberg, Germany). The rats with fasting blood glucose levels more than 144 mg/dl were labeled diabetic and were included for the experiment 12,13.

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Rat excision wound model and treatment

The fur on the back of the anesthetized rats was shaved with hair removal cream and cleaned with alcohol swab. Two full-thickness 1.4cm diameter excision wounds were made on the dorsum of each rat, using dissecting scissors and forceps under ketamine anesthesia. All wounds were cleaned daily with normal saline. The wounds were left undressed and were treated with vehicle or propolis cream according to their respective groups, topically once daily after cleaning for a period of 7 and 14 days.

Wound-size measurements: all wounds area was measured by using a ruler.

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Experimental groups

After 6 weeks of induction of diabetes 14, the diabetic rats were randomly divided into two groups, with 10 rats in each group.

  • Group I (the control group): this was considered as the diabetic control group and subdivided into the following:
    • Subgroup Ia: this included five rats that received vehicle cream once daily for 1 week.
    • Subgroup Ib: this included five rats that received vehicle cream once daily for 2 weeks.
  • Group II (the propolis group): this served as the diabetic treatment group and was subdivided into the following:
    • Subgroup IIa: this included five rats that were treated with propolis cream once daily for 1 week.
    • Subgroup IIb: this included five rats that were treated with propolis cream once daily for 2 weeks.
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Sample collection

The rats were sacrificed using overdose of ether on the 7th and 15th days from the start of the application of topical cream in the different groups. The wounds’ sites were excised with a rim of 5 mm of normal surrounding skin. Tissue specimens were fixed in 10% neutral buffered formalin solution and processed for paraffin sections. Five micrometer sections were cut and stained with H&E and Masson’s trichrome (using 2% methyl blue in 2% acetic acid) stains. Immunohistochemical staining was carried out for detection of expression of VEGF (Labvision, Thermoscientific, CA, USA) rabbit polyclonal antibody, antirat (code no. RB-9031-R7). The reaction takes place in the cytoplasm, cell surface, and extracellular matrix. The positive control was angiosarcoma. An additional slide of skin specimen was treated with buffer solution instead of the same concentration of primary antibody in every run as the negative control.

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Morphometric study

The data were obtained using the image analyzer computer system (Leica Qwin 500C; Leica, England, UK). The mean area% of collagen fibers was measured in 10 low-power nonoverlapping fields. The mean area% of VEGF immunoexpression was measured in 10 high-power nonoverlapping fields.

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Statistical analysis

Data were expressed as mean±SD. The statistical analysis was carried out using one-way analysis of variance, with SPSS (version 10; SPSS Inc., Chicago, Illinois, USA). All results were compared one by one with the other groups and control group results using post-hoc analysis and Tukey’s range test. A P value of 0.05 or less was considered as statistically significant.

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Results

Wound size measurements

Wound sizes from group I (control group) on days 0, 8, 15 were 1.4 cm2, 1.37 cm2 and 1.3 cm2 respectively. While wound sizes from group II (propolis treated group) on days 0, 8, 15 were 1.4 cm2, 0.65 cm2 and complete closure respectively.

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Histological results

H&E stain

Subgroup Ia (diabetic control rats for 1 week) revealed nonhealed epidermis with deficient granulation tissue in the wound area. A mass of inflammatory cells under the nonhealed epidermis was observed (Figs 1 and 2). Subgroup Ib (diabetic control rats for 2 weeks) showed nonhealed epidermis with deficient granulation tissue in the wound area (Fig. 3).

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Subgroup IIa (propolis treated rats for 1 week) demonstrated filling of the wound area with cellular granulation tissue. Congested blood vessels, dilated lymphatic vessels, and inflammatory cells were also observed (Figs 4 and 5). Other sections revealed disorganized dermal pattern in the form of separation between the epidermis and dermis (Fig. 6). However, subgroup IIb (propolis treated rats for 2 weeks) showed filling of the wound area with granulation tissue with a well-formed epithelium (Fig. 7).

Figure 4

Figure 4

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

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Masson’s trichrome stain

Subgroups Ia and Ib (diabetic control rats for 1 week and 2 weeks) revealed deficient collagen deposition in the wound area (Figs 8 and 9). However, subgroup IIa (propolis treated rats for 1 week) demonstrated loosely arranged collagen in the wound area (Fig. 10), whereas subgroup IIb (propolis treated rats for 2 weeks) showed a well-organized microarchitecture with abundant collagen deposition (Fig. 11).

Figure 8

Figure 8

Figure 9

Figure 9

Figure 10

Figure 10

Figure 11

Figure 11

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Immunohistochemical results

Subgroups Ia and Ib (diabetic control rats for 1 week and 2 weeks) revealed negative cytoplasmic immunoexpression of VEGF in the keratinocytes. Positive immunoreactive cells in the dermis were noticed (Figs 12a and b and 13a and b). Positive immunoreaction was detected in the external root sheath of hair follicles in subgroup Ib (Fig. 13a). However, in subgroups IIa and IIb (propolis treated rats for 1 week and 2 weeks), there was intense cytoplasmic immunoexpression of VEGF in the keratinocytes. Positive immunoreactive cells in the dermis were also observed (Figs 14a and b and 15a and b). External root sheath of hair follicles showed positive immunoreactivity in subgroup IIb (Fig. 15a).

Figure 12

Figure 12

Figure 13

Figure 13

Figure 14

Figure 14

Figure 15

Figure 15

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Morphometric results

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).

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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).

Table 1

Table 1

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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).

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Discussion

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.

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conclusion

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.

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Recommendations

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.

Table

Table

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

diabetic foot; propolis; streptozotocin; vascular endothelial growth factor

© 2013 The Egyptian Journal of Histology