Therapeutic Effects of Oleuropein on Wounded Skin in Young Male Balb/c Mice

Introduction. Oleuropein is generally the most abundant phenolic compound in olive leaves. In this study, therapeutic effects of oleuropein were studied on wounded skin in young male Balb/c mice. Methods. Four-month-old male Balb/c mice were randomized into 2 groups, a control and an experimental group. Under ether anesthesia, hair on the neck of mice in both groups was shaved and 1-cm long full-thickness incisions were made and left unsutured. The experimental group was injected intradermally with a daily single dose of oleuropein (50 mg/kg) for a total of 7 days. The control group received only distilled water. On days 3 and 7 post-incision, mice were sacrificed and skin around the area of the incisions were dissected and processed for hematoxylin and eosin and Van Gieson’s staining. Portions of dissected tissues were also lysed and used for western blot analysis to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. Results. The analyses showed oleuropein reduced cell infiltration into the wound sites on day 3 and 7 post-incision; however, it significantly increased collagen fiber deposition and caused faster reepithelialization when compared to the control group (P < 0.05). Furthermore, western blot analysis showed a significant increase in VEGF protein level compared to the control group (P < 0.05). Conclusion. In summary, oleuropein showed healing effects on wounded skin by accelerating the reepithelialization process, enhancing collagen fiber generation, and increasing the blood supply to the wounded area by upregulation of VEGF protein expression.

  Wound repair is a complex process comprised of different phases: inflammation, proliferation, and cellular remodeling. One of the causes of delay in the wound healing process is a long duration of the inflammatory phase.¹ Hence, anti-inflammatory treatments are most often necessary for shortening the healing period.¹ Ancora and colleagues² have shown that compounds containing phenols have anti-inflammatory and antioxidant effects on the skin and act as free radical scavengers. It is also well known that most polyphenolic compounds have cyclooxygenase inhibitory properties.³ Reactive Oxygen Species (ROS) that include free radicals are produced in wounded tissue.⁴ Under normal circumstances free radical production is balanced by anti-oxidative defense mechanisms.⁵ The pharmacological properties of olive fruit and its oil and leaves, because of their phenolic content, have been recognized as important compounds for treatment of wounds.⁶Phenolic compounds are found in all parts of the olive plant, but their nature and concentration varies greatly. Biochemists isolated a crude powder extract from olive leaves that contained oleuropein, the most prominent phenolic compound in the olive leaves,⁷ which has antioxidant properties.⁸ Few studies have been published regarding the effects of this compound on skin wound repair. This study examined the effects of oleuropein on wounded skin in young male Balb/c mice.

  Reagents. Oleuropein was provided by Razi Herbal Medicines Research Center (Lorestan, Iran); vascular endothelial growth factor (VEGF) antibody and alkaline phosphatase - conjugated secondary antibody were supplied by AbCam, Cambridge, MA; and the mouse monoclonal antibody against β-actin and NBT/BCIP tablets were provided by Roche Diagnostics, Mannheim, Germany.

  Animals. Male Balb/c were purchased at 4 months of age from Pasteur Institute of Iran, Tehran, Iran, and housed in a temperature-controlled room at 23°C ± 2°C. All animal works were approved by the ethical guidelines for the care of laboratory animals of the Research Center of Iran University of Medical Sciences, Tehran, Iran.

  Experimental design. Mice were randomized into control and experimental groups. Under ether anesthesia, hair on the neck of both groups was shaved and 1-cm long full-thickness incisions were made and left unsutured. The incisions were washed and cleaned with physiological saline. A single daily dose of 50 mg/kg 9 of oleuropein dissolved in distilled water was injected slowly into incised edges of the wound of the experimental group for a period of 7 days.¹⁰ The control group received only distilled water for the same time period. On days 3 and 7 after post-incision and injection, mice from each group were sacrificed and full-thickness skin around the incision area was carefully dissected and divided into 2 parts. One part was fixed in 10% formalin; the other was used for western blot analysis.

  The formalin-fixed skin tissues were dehydrated in a graded concentration of alcohol, then cleared in xylene, infiltrated with paraffin and, finally, embedded in paraffin. Paraffin blocks were cut into 5µm thickness and stained with hematoxylin and eosin to evaluate the epithelialization.

  Measurement of new epithelium formation. Neo-epithelial formation was scored on a scale of 0 to 3, where “0” indicated no formation and “3” represented the maximum epithelial formation. A picture of each section was taken by microscope and digital camera (AX70 Research System Microscope, Olympus Optical Co, Ltd, Tokyo, Japan) with magnification at x400. To count the number of infiltrated cells, the pictures were transferred to the computer using OLYSIA autobioreport software (Olympus Optical Co, Ltd, Tokyo, Japan). A grid was superimposed on the pictures, and cells with obvious nuclei in 5 separate microscopic fields were counted. A cross was marked on the counted cells to prevent recounting. Van Gieson’s staining was used for evaluation of collagen fibers. Red Van Gieson’s stains were indicative of collagen fibers and scored on a scale of 0 to 3 for collagen fiber deposition, where 0 indicated no collagen fiber deposition and 3 is the maximum of collagen fiber deposition.

  The skin tissue that was not fixed in formalin on days 3 and 7 post-incision was cut into 100 mg sections, homogenized in lysis buffer, and then incubated on ice for 30 minutes. The lysates were centrifuged at 1300 rpm for 20 minutes at 4°C. Protein concentration of the cleared lysates were determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). The samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Millipore Corp, Billerica, MA). The membranes were blocked by 5% milk protein for 1 hour, incubated overnight with primary antibody for VEGF (1:400 [AbCam, Cambridge, MA]), washed 3 times with tris-buffered saline and Tween-20 buffer solution, and then incubated for 1 hour with a secondary antibody (alkaline phosphatase-conjugated goat anti rabbit IgG, 1:5000 [AbCam, Cambridge, MA]). Bands were detected using 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium tablets (SigmaFastBCIP/NBT, Sigma-Aldrich, St. Louis, MO). The density of bands were scanned and quantified, and the membranes were probed with β-actin to account for equal loading.

  Statistical analysis was performed using statistical analysis software (SPSS software, IBM, Armonk, NY). The t test was applied to assess the significance of changes between control and experimental groups.

  Photomicrographs of skin wound tissues in both control and experimental groups are shown in Figures 1 and 2. Hematoxylin and eosin staining of sections in the control group on day 3 showed infiltrated cells in the wound site (Figure 1A, Table 1) while reduced infiltration content was observed in the experimental group (Figure 1B, Table 1) (P < 0.05). On day 7 after oleuropein treatment, more advanced epidermal and dermal regeneration were observed in the experimental group compared to the control group and histological analysis demonstrated a faster epithelialization in the experimental group (Figure 1C,1D, Table 1)(P < 0.05). Van Geison’s staining showed higher rates of collagen fibers on day 3 in the experimental group compared to the control group (PP < 0.05). Figure 2D shows collagen fibers in the experimental group that are more compact and regular than those of the control group.

  The VEGF expression levels were examined by western blot analysis on days 3 and 7. The VEGF level in the experimental group on day 3 showed a significantly higher up-regulation than in the control group (P < 0.05). The highest up-regulation level of VEGF protein was detected on day 3 as levels peaked in the experimental group. The VEGF protein expression on day 7 was less than on day 3, but even at this time it was significantly higher than the control group (P < 0.05) (Figure 3).

  The search for natural remedies that aid in wound healing has drawn attention toward plant extracts. About 60% of the world population depends on traditional medicine for their primary health care needs, especially for wound treatment.11,12 Olive leaf extract contains 98 phytochemicals, one of the most important being oleuropein, a bitter tasting phenolic glycoside present in the leaves of the olive tree8 that has anti-inflammatory and antioxidant properties.6

  The results of the current study indicated that the treatment of wounds with oleuropein (experimental group) reduced infiltration of cells into incision sites, as well as helped wounds heal more quickly, when compared to the control group on day 3 post-incision. The histological examinations showed advanced reepithelialization and regeneration in the experimental group on day 7 post-incision. On day 7, the progression of wound healing was obvious when compared to the control group. Furthermore, Van Geison’s staining showed more collagen fiber formation in the wound sites of the experimental group on post-incision days 3 and 7, whereas the control group had less and irregularly arranged collagen fibers.

  Enhancement in the collagen content deposition provides stabilization and contributes to wound strength, which in turn, helps to repair wound tissue.13,14 Oleuropein has been demonstrated to stimulate proteasome function and fibroblast formation for synthesizing new collagen fibers and influence collagen metabolism and support wound healing. The increase in wound healing activity was also evident by the decrease of infiltrated cells in the wound site.15 Inhibition of inflammation and stimulation of collagen synthesis are advantages of oleuropein in the process of wound repair. In addition, oleuropein is an antioxidant compound that scavenges free radicals and helps heal wounds.16

  Induction of angiogenesis by VEGF can be considered as a factor to improve wound healing. Vascular endothelial growth factor is a pre-angiogenesis factor17,18 that promotes angiogenesis to supply nutrients for skin regeneration.19,20 Western blot analysis of tissues in the experimental group showed up-regulation of VEGF expression compared to the control group. The VEGF protein expression peaked on day 3 post-incision, although lesser level of VEGF expression was evident on day 7. This variation of VEGF level on day 3 and 7 might be related to the acceleration of wound healing on day 3, as the proliferative phase of wound healing is maximized on day 3. Vascular endothelial growth factor not only stimulates wound healing through angiogenesis, but also increases collagen deposition and epithelialization.17

  Oleuropein showed wound-healing effects by improving reepithelialization and enhancing the formation of collagen fibers and the up-regulation of VEGF expression. Much remains to be learned about the effects of oleuropein on wound healing and its mechanism of action. Based on the findings of this study, one possible mechanism of action may well be the anti-inflammatory and antioxidative properties of this phenolic compound.

  This research was supported by the Minimally Invasive Surgery Research Center at Iran University of Medical Sciences. The authors would like to thank Ms. Parisa Hayat and the Farzan Institute for Research and Technology, Tehran, Iran for technical assistance.

Fereshteh Mehraein, PhD is from the Anatomy Department, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran. Maryam Sarbishegi; MS is from Anatomy Department, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.