Antiperoxidative properties of oil mixes of high ratio Omega-9 : Omega-6 and low ratio Omega-6 : Omega-3 after molar extraction in rats 1

PURPOSE: To evaluate the antioxidant and antiperoxidative effects of oil mixes of high ratio Omega-9:Omega-6 and low ratio Omega6:Omega-3 in the third day after tooth extraction in rats. METHODS: Thirty-two male Wistar rats (270-310g) were randomly distributed in two groups: Control (n=24) and Test (n=8). Control group was divided into three subgroups (n=8): G1: Sham-Saline; G2: Saline; G3: Isolipid. G1 and G2 animals received NaCl 0.9% while G3 rats were treated with an isolipid mixture (alpha-linolenic acid – ALA) containing -6/-3 oils (8:1 ratio) and-9/-6 (0.4:1 ratio). Test group animals (G4) received oily mixtures (alpha-linolenic acid – ALA, docosahexaenoic acid – DHA, eicosapentaenoic acid – EPA) of -6/-3 (1.4:1 ratio) and -9/-6 (3.4:1 ratio). Saline and oils were administered by gavage during four days before and three days after first mandibular molar extraction. Following, samples (arterial blood and alveolar mucosa) were collected for glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) assays. RESULTS: Oil mixes induced a significant decrease in GSH and TBARS tissue and plasma concentrations in the third day post-surgery. CONCLUSION: Gavage administration of oil mixes of high ratio Omega-9:Omega-6 and low ratio Omega-6:Omega-3 after molar extraction in rats induces a significant decrease in lipid peroxidation.


Introduction
Oxidative stress can be defined as imbalance between production and collection of reactive oxygen species (ROS).The oral supplementation of antioxidants is well indicated in several situations, such as cancer, sepsis, preoperative of major surgeries and malnourished or critically ill patients 1 .The omega-3 is one of the most used in research immunonutrients 2 .
Currently, the adequacy of the balance of dietary lipids has motivated numerous investigations.In patients with changes in metabolic responses, the balance between dietary lipids aims to control oxidative stress and increased inflammatory response, through the relationship between the types of polyunsaturated fatty acids ingested 3 , affecting the synthesis of eicosanoids that act as intermediate messengers of growth factors controlling the growth and differentiation of epithelial cells 4 .
Protective effects of oils mixes against ischemia/ reperfusion injury have been demonstrated.Pinheiro et al. 5 investigated the effects of pre-conditioning with mixtures of oils containing high and low ratio ω-6/ω-3 and ω-9/ω-6 relationship in an experimental model and concluded that these oil combinations protects brain neurons against ischemiareperfusion injury.The data found in the literature vary widely as to the best combination of antioxidants, the dosage, route of administration, the levels to be ideally achieved, the best time and how long they have to be administered in order to provide an effective protection against the oxidative injury.Despite the fact that there is no consensus regarding those topics, an early study has recommended that therapy should be instituted before the establishment of oxidative injury 6 .
The hypothesis of this study is that the use of combinations containing different proportions of omega 3, 6 and 9 amino acids may have a noticeable antioxidant effect during the inflammatory phase of the wound healing after dental extraction.

Composition
Source ω-3 Ratio  Samples (arterial blood and alveolar mucosa) were collected on the third post-operative and the animals were killed by cervical dislocation at the end of the experiment.

Laboratory parameters
Thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) concentrations were assayed to evaluate the oxidative stress, using methods described in the literature.Lipid peroxidation was assayed by measuring malondialdehyde as TBAreactive substances 8 .GSH levels were estimated by the method of Sedlak and Lindsay 9 .

Statistical analysis
Data distribution was analyzed by the Shapiro-Wilk test.All data were expressed as mean ± SEM.SPSS (Statistical Package for the Social Sciences) was used for statistical analysis.
One-way ANOVA was performed to determine differences among groups.A probability value of p<0.05 was considered to indicate statistical significance.

Discussion
Research studies concerning the relationship between the repair and the possible factors that affect the healing process after tooth extraction in rats were initiated in early 1923 [10][11] .The healing process post dental extraction has been studied in several animal models, and its sequence is relatively understood and divided into three phases: (a) initial phase: inflammatory between one and five days, (b) middle phase: during bone formation five to 20 days, (c) final phase: bone remodeling phase from 20 to 60 days 12 .Local and systemic factors may affect rat socket healing, including salivary gland hypofunction, osteoporosis, osteopetrosis, diabetes mellitus and treatment with calcitonin or cortisone [12][13][14][15][16][17] .In this work it was found that oil mixes administered by gavage before and after dental extraction may provide protection from lipid peroxidation in the third post-operative day.(Figure 1) Fatty acids are used in a selective synthesis of phospholipids of cell membranes and their organelles.Essential fatty acids can determine structural and functional alterations of membrane phospholipids, including cells of the immune system, modifying its permeability, activity of receptors and enzymes, transport, regulatory functions and cellular metabolism 18 .
Moreover, activate intracellular signaling routes for the formation of biologically active molecules that act as second messengers.Thus, may interfere with physiological events related to hemodynamic 19 , oxygenation 20 , inflammation 21 and organic defense 22 .
Omega-9 (oleic acid) confer protection against lipid peroxidation of different polyunsaturated fatty acids such as linolenic acid, EPA, DHA containing 4, 5 and 6 double bonds respectively and because that are much less stable.The use of compositions rich in monounsaturated lipids as compared to the use of polyunsaturated showed low inflammatory response and low production of free radicals rich in monounsaturated formula.
Membranes that are rich in monounsaturated fatty acids (MUFA) are less susceptible to oxidation by free radicals that membranes rich in saturated fatty acids, presumably because the greater number of unsaturations increase the likelihood of double bonds than in reactive oxygen species 23 .This may explain the antiperoxidative protection of oil mixes test shown in Figure 1A and 1B.
Olive oil is rich in ω-9 fatty acids and vitamin E and is known for its antioxidant properties.The fact that the molecular structure of oleic acid have only one double bond, together with the presence of vitamin E confers greater protection against lipid peroxidation.
Antioxidants decrease the accumulation of ROS 24 , which can reduce local tissue damage and accelerate the healing process 25 .
In this study, the use of an oil mix containing alpha-linolenic acid -ALA, docosahexaenoic acid -DHA, eicosapentaenoic acid -EPA of -6/-3 (1.4:1 ratio) and -9/-6 (3.4:1 ratio) promoted a decrease in GSH levels, an issue showing that GSH becomes "lesser necessary" and, in this way, an antioxidant action.Generally, cells react to oxidative stress with a increase in GSH pool as part of their adaptative answer to the potential oxidative lesion. 20Therefore, a lower GSH level indicates a less aggressive potential oxidative stress.

TABLE 1 -
Composition of isolipid and oil mixes preparations.