Effect of Pistacia Atlantica Resin Oil on Anti-Oxidant, Hydroxyprolin and VEGF Changes in Experimentally-Induced Skin Burn in Rat

BACKGROUND Severe burn damage and its consequences are life threatening which can complicate patients’ health. Medicinal and traditional plants are considered as safe, natural and inexpensive source of treatment for wide variety of diseases. This study assessed beneficial effect of Pistacia atlantica oil on rats burn wound healing and its potential effects on malondialdehyde (MDA), vasculoendothelial growth factor (VEGF), hydroxyprolin and antioxidant status in wound area. METHODS Thirty male rats weighing 200±10 g were randomly divided into three groups (n=10) as follows. Group 1 underwent just burn injury, Group 2 underwent burn injury and received 150 mg/kg/day P. atlantica oil topically, and Group 3 underwent burn injury and received 150 mg/kg/day sulfadiazine cream topically. At the end of the study (day 14), wounded areas were measured and then skin in the burn damage were dissected and anti-oxidative parameter, MDA, VEGF and hydroxyprolin were evaluated. RESULTS P. Atlantica oil significantly increased antioxidant defense, VEGF, hydroxyprolin and reduced MDA levels. It could remarkably reduce wound size compared to burn control group. P. Atlantica oil showed more beneficial effects than sulfadiazine. CONCLUSION P. atlantica resin oil could be considered as a new therapeutic agent for treatment of injuries.


INTRODUCTION
strict regular care including wound healing care and treatment, nutritional supports and control of probable infection. 1 Pathophysiological changes in burned area caused by increased tissue temperature leads to inflammatory response and also thermal exposure can cause necrosis in burn area especially in middle parts. 2 Increased Reactive Oxygen Species (ROSs) lead to severe damage to the cells in burn area. 3 Wound healing is a dynamic process which consist three phases including inflammation, proliferation and maturation and cytokines and reduced local ischemia and ROSs have a pivotal role in this process. ROSs are one of the components which are participates in tissue damage. Also following thermal injury ROSs have been considered to participate in a number of pathophysiological steps. It has also been reported that ROSs are involved in burn shock and lung damage following thermal injury. 1,2 Sepsis is the leading cause of mortality in burn units and infection is one of the major problems correlated burn injuries. Nevertheless, antibacterial remedies which used in order to eliminate infection from burn area, the infection problem still is present and need more attention. 4 Vasculoendothelial Growth Factor (VEGF) is a multi-action growth factor that facilitates wound healing and helps tissue repair. VEGF increases inflammatory cells in damage area and also promotes migration and proliferation of endothelial cells. 4,5 Collagen is an extracellular matrix protein which is related to wound healing and strength. Hydroxyproline is an amino acid that especially presents in collagen structure and its levels in wound parts may be as a marker of wound healing rate. 6 Medicinal and traditional plants always considered as a good, safe and inexpensive source of remedy for many diseases. Therefore, a wide range of plants are used as pharmacological agent against diseases and improve body health. [7][8][9][10] It has been reported that Emu oil postpones the wound healing process at inflammatory process but on the other hand Emu oil showed beneficial wound healing effect on keratinization of epidermis. 8 Other study demonstrated that Capparis spinosa leaves hydro-alcoholic extract attenuates inflammation and also promoted wound healing process. 7 One of these traditional plants that used commonly is Pistacia atlantica which has been reported that have many potential beneficial effects. 10-15 P. atlantica is a plant which is widely distributed in Algeria, Iran, Iraq, Mediterranean and turkey. 9 Its resin used as chewing gum and mouth freshener. P. atlantica resin have used as a traditional treatment for peptic ulcer disease. 11 There are some studies about wound healing and anti-inflammatory properties of P. atlantica resin in animal models and also it used traditionally as a remedy for wound healing in some parts of Iran. 10 Peksel et al. (2013) showed that aqueous extract of Pistacia leaves has radical scavenging properties. 16 Also other studies were approved antioxidant activity of P. atlantica. 12,14 It have been proved that P. atlantica have used for treatment of digestive diseases and also traditionally used for treatment of disorders such as colitis, gastrointestinal problems, kidney, heart and liver complications. 9,10 Other studies reported antifungal, antiparasite and antibacterial activity of P. atlantica. [13][14][15] In present study we evaluated ameliorative effect of P. atlantica resin oil on wound healing in rat which burden experimentally burn wound on skin and also we assessed P. atlantica resin oil effects on VEGF, hydroxyproline, and antioxidant status in wound area.

MATERIALS AND METHODS
Thirty male Sprague-Dawley rats weighing 200±10 g were obtained from the animal care center of Kerman Neuroscience Research Center. The animals were maintained at controlled condition, 25±1 ºC and 12 h lightdark cycle and have access freely to standard chow diet and water. Our study was approved by the ethic committee of Kerman University of Medical Sciences, Kerman, Iran. P. atlantica resin oil which we have used in our study was prepared from Hakim-Tehrani Co. Kerman, Iran. Full compositions of P. atlantica oil have been reported previously. 17,18 The animals were anesthetized by i.p. injection of Ketamin and Xylazine (60 and 4 mg/ kg, respectively). The dorsal side of the animals were shaved and then induction of burn damage conducted by an aluminum plaque (2.5x2.5 cm) on the shaved skin of rats for 15-20 seconds which was heated to 100ºC to create a deep dermal burn wound. 19,20 Duration of study was 14 days after burn injury and animals were divided into three groups (n=10) randomly as follow: Group 1 underwent just burn injury, Group 2 underwent burn injury and received 200 mg/kg/ day of P. atlantica resin oil topically, and Group 3 underwent burn injury and received 200 mg/ kg/day of sulfadiazine cream topically.
At the end of the study animals were anesthetized and sacrificed then burned skin were incised and separated. Samples were homogenized by Ultrasonic Processor (Hielscher, UP200H) in cold phosphate buffered saline (PBS, pH=7.4) and then centrifuge at 4°C and 15000 rpm for 15 min. Supernatants were separated and aliquoted, then maintained at -80 until further experiments. Superoxide dismutase (SOD), Glutathione peroxidase (GPX), Total Antioxidant Status (TAS), Malondialdehyde (MDA), VEGF and hydroxyproline were measured in supernatant.
The wound contractions was reported as percent and were calculates by following formula: 21  MDA was measured as thiobarbituric acidreactive substances (TBARS) at 534 nm and 1,1,3,3-tetramethoxypropane was used to plot calibration curve. The data were expressed as mean±SEM. For comparison between groups One-way ANOVA test followed by post hoc Tukey's performed to compare mean differences between groups, and p<0.05 was considered as statistically significant.

RESULTS
Our results showed that P. atlantica resin oil significantly increase antioxidant defense, VEGF, hydroxyprolin and reduced MDA levels ( Table 1, Figures 1 and 2). Also P. atlantica compared to sulfadiazine significantly increased  Figures 1 and 2). P. atlantica remarkably reduced wound size compared to burn control group (Table1)

DISCUSSION
Our results showed that Pistacia atlantica resin oil has remarkable antioxidant properties in experimentally induced-burn wound. Also we found that its resin oil is capable of increase the VEGF and hydroxyproline levels in wound area. Bozorgi et al. have reported that resin of P. atlantica has been used as treatment for some diseases and complicated condition such as digestive, hepatic, and kidney diseases. Also it demonstrated that the gum resin of P. atlantica can be used for wound healing and treatment of gastrointestinal problems. 9 Previous studies have showed that P. atlantica resin oil have many compounds and α-pinene (about 45-70%) is the major components and it seems that α-pinene is the effective substance present in resin oil. 9,17,22,23 Also, Memariani et al. (2017) demonstrated that α-pinene is the main component of P. atlantica oil and 2g/kg of P. atlantica oil was not harmful or toxic in vivo. They reported that P. atlantica oil showed protective effect against experimentally ethanol induced gastric ulcer. 24 Also it has been reported that α-Pinene showed significant low toxicity. 18 P. atlantica oil antioxidative properties have been reported previously. 12,16,25 Rezaie et al. have reported that P. atlantica oil has remarkable antioxidant activities compared with positive control. 14 Koizumi et al. showed that severe burn damage cause promotion of free radicals which in turn result in vasodilatation and SOD can act as a protective factor against vasodilatation. 26 Here we proved that P. atlantica resin oil significantly compared with burn control group elevated SOD levels.
We also found that P. atlantica resin oil is a potent antioxidant ointment which significantly improved antioxidant status of wound area in rats. Also we found that P. atlantica resin oil is rather potent than sulfadiazine to fight against free radicals present in burn wound. Farahpour et al. showed that P. atlantica hydroalcoholic hull extract showed antioxidant activity even higher than ascorbic acid, their finding are consistent with our antioxidative data about P. atlantica resin oil. 22 It showed that P. atlantica resin oil has important antimicrobial activity against bacteria which showed resistance against some common antimicrobial drugs. 15 Also other studies reported antimicrobial activity of P. atlantica oil. 14,25 Therefore, P. atlantica resin oil in addition to increase antioxidants also has antimicrobial properties and it reduce the chance of infection and accelerate the process of wound healing.
Galiano et al. demonstrated that topical VEGF increased growth factor in damaged area and also recruits bone marrow derived cells which have remarkable role in wound repair. All together they showed that topical application of VEGF has wound healing properties. 5 Haghdoost Fig. 2: Hydroxyproline levels in burn wound area. Group 1, burn control; group 2, burn-treated with P. atlantica resin oil topically; group 3, burn-treated with sulfadiazine. *statistically significant compared to Burn control group, # statistically significant compared to burn-treated with sulfadiazine group.
et al. showed that P. atlantica increases bFGF and PDGF and therefore result in angiogenesis. 20 We showed that P. atlantica resin oil promotes VEGF levels in wound area in burn model injury in rats and this could explains its beneficial effects on wound contraction and repair in this study. Therefore, Galiano et al. and Haghdoost and colleagues reports are confirming our data regarding VEGF variations in wound area. 5,20 Collagen is an extracellular matrix protein which is related to wound contraction and strength. Collagen turnover directly related to free hydroxyproline and quantification of hydroxyproline can be considered as a good parameter to monitor collagen turnover. 22,27 Hamidi and colleagues showed that P. atlantica oil changed collagen pattern and caused organized collagen fiber after three weeks. 28 Farahpour et al. showed that P. atlantica hydroalcoholic hull extract as ointment increased hydroxyproline content and histological study showed that collagen score increased significantly. Also they found that P. atlantica hydroalcoholic hull extract promotes fibroblasts proliferation and therefore decline inflammation. 22 IIango et al. evaluated methanol extract of L. acidissima on SOD, catalase, hydroxyproline, and epithelialization. They found that methanol extract of L. acidissima increase antioxidant SOD and also promotes hydroxyproline and epithelialization, therefore showed significant dose dependent wound healing activity. 27 We showed that P. atlantica resin oil increased hydroxyproline levels in wound area and this could be considered as a collagen turnover marker. Therefore, P. atlantica resin oil wound healing and contraction activity can be related to elevation of collagen turnover and consequently hydroxyproline.
Epithelialization is considered as an important factor which serves as a defining parameter of affluent wound contraction. 28 Mehrabani et al. used P. atlantica oil in combination with three other oils from sesame (Sesamum indicum L.), hemp (Cannabis sativa L.) and walnut (Juglans regia L.) as a combined formula. This new formula increased wound contraction and accelerated epithelialization. 21 Hamidi et al. showed that P. atlantica oil as gels ameliorated epithelialization which is as a marker of wound contraction. Also they showed that topical P. atlantica oil in rat with experimental wound cause biochemical and morphological promotion compared with control group. 28 Therefore, according to these reports about P. atlantica oil we can anticipate that increased epithelialization and other beneficial histological changes can be considered as potential mechanism in addition to elevation of VEF and hydroxyproline and antioxidant status to increase. Finally according to previous studies P. atlantica resin oil has many beneficial properties and low toxicity, and our finding showed that Pistacia atlantica resin oil has antioxidant effects and increases VEGF and hydroxyproline, therefore, it seems that P. atlantica resin oil is a good, safe and potent candidate as a new therapeutic topical ointment for wound healing.

ACKNOWLEDGEMENT
The present article was financially supported by the Kerman University of Medical Sciences, Kerman, Iran.