1. Background
The hip joint dislocation, trauma and drug injection may lead to ischemia-reperfusion injury which will have adverse effects on the lower limb especially sciatic nerve and branches. Ischemia plays an important role in the production and development of pathological changes in various neuropathies including peripheral neuropathy particularly sciatic nerve [1-3]. Pathophysiology ischemia-reperfusion is platelet aggregation, oxygen free radicals and the interaction between endothelial cells and leukocytes [1-3]. This phenomenon leads to endothelial damage, capillary occlusion and deficiency of oxygen reaching to neural tissue [1-3]. Many efforts have occurred to reduce the effects of ischemia-reperfusion injury. effects of antioxidants such as lipoic acid [4] and melatonin [5] and some medications such as statins [6-8] and cooling [9] has been studied to reduce reperfusion injury of nerve damage which this material did not provide objectives of the study. Today, the emphasis is on the proper use of antioxidants to reduce ischemia-reperfusion injury. Antioxidant is a molecule that prevents the oxidation of other molecules. Antioxidants protect cells from the dangers of free radicals by destroy free radicals and inhibit other oxidation reactions [5, 10-13]. The key enzymes defense system include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), nitric oxide (NO) and paraoxonase (POX) [5, 10-13]. These enzymes are necessary for cell viability even in normal circumstances. These enzymes can be induced, an important feature, under conditions of oxidative stress. Each enzyme has a unique function. Superoxide anions, first derivative of free radicals, convert by SOD into water and oxygen [5, 10-13]. Glutathione, the major nonprotein thiol in aerobic organisms, is abundant intracellular non-enzymatic antioxidant. Malondialdehyde (MDA) is one of frequently used markers of lipid peroxidation [5, 10-13]. Superoxide dismutase-catalase enzyme complex are the first line of cellular defense against free radical toxicity [14]. Exposure of cells to oxidative stress can induce this complex. These enzymes act synergistically with each other. Recently, selenium has consideration to reduce damage caused by ischemia-reperfusion. The effect of antioxidants on antioxidant enzymes to reduce cell damage caused by ischemia-reperfusion is one of the most important debates. Selenium, a trace element, has antioxidant and neuroprotective effects [14]. Selenium protects DNA, lipids and proteins against free radicals [14]. Selenium reduces lipoproxidase and hydroxides through a similar activity of glutathione peroxidase [15]. The protective effect of selenium against ischemia-reperfusion has been shown in several studies [15]. Researcher show beneficial effects of selenium on ischemia-reperfusion of brain [12, 16].
2. Objectives
The main goal of the present study was to examine if selenium induced serum antioxidant enzymes such as GPX, CAT, NO, POX and MDA in ischemia-reperfusion to the sciatic nerve at reperfusion phase.
3. Materials and Methods
This experimental study was performed at Razi herbal medicine center of Lorestan University of Medical Sciences. All experiments were performed in accordance with principles of laboratory animal care. All animal were obtained from Razi herbal medicine center of Lorestan University of Medical Sciences. Every effort was made to minimize the number of animals used and their suffering. Eighty adult male Wistar rats weighing 250 - 300 g were used. The animals were acclimatized for one week to the condition of our laboratory before the commencement of the experiment. The animals were exposed to 12 hours light and 12 hours dark cycle at a room temperature of 22°C. The animals had free access to standard laboratory chow and water ad libitum. The rats were divided into ten groups (n = 8 per group) (Table 1). The first group received sham surgery only (Table 1). The second group received 0.2 mg/kg selenium without ischemia-reperfusion surgery and eight remaining groups were divided to four placebo groups and four experimental groups which the latter groups received 0.2 mg/kg selenium (Table 1). The rats were anesthetized once intraperitoneally with ketamine HCl (50 mg/kg) and xylazine (5 mg/kg) [17] in accordance with the protocol approved by the animal care and use committee.
| Groups (N = 8) | Selenium Administration | Ischemia | Reperfusion Times, d |
|---|---|---|---|
| - | - | - | |
| + | - | - | |
| + | + | 3 | |
| - | + | 3 | |
| + | + | 7 | |
| - | + | 7 | |
| + | + | 14 | |
| - | + | 14 | |
| + | + | 28 | |
| - | + | 28 |
Features of Study Groups
The animals were placed in supine position on a heated mat during the operation and recovery. Right femoral vessels were exposed through an inguinal incision and were dissected free the femoral nerve under operating microscope. The trifurcation of the sciatic nerve into peroneal, tibial and sural branch were rendered near-completely ischemic by occluding the femoral artery and vein with a silk suture 6 - 0 using slipknot technique [13, 18] for rapid release and reperfusion was achieved by release of these ligature. In all groups the vascular ligature was removed after 3 hours of ischemia [13, 18] and the sciatic nerve was allowed to reperfusion for 3, 7, 14 and 28 days in both the control and experimental selenium treatment groups. After ischemia and release of ligature, inguinal incision was sutured with silk 0 - 2. Selenium was administered in all experimental selenium treatment groups with a single dose of 0.2 mg/kg prior to vascular ligature via intraperitoneal. Selenium (0.2 mg/kg) or the same volume of distilled water was injected via intraperitoneal for selenium and vehicle-injected groups, respectively. The animals were placed under heating lamps until they recovered from anesthesia. According to a schedule for each group of mice blood samples were obtained from hearts and allowed to clot for 20 minutes in laboratory temperature and then centrifuged at 3000 rpm for 10 minutes for serum separation.
3.1. Biochemical Study
3.1.1. Levels of Nitrite
Serum levels of nitrite were measured by measuring accumulation of nitrite in serum using the Griess reaction with sodium nitrate as the standard. Briefly, 50 μL samples of serum were mixed with equal volumes of 1% sulphanilamide and 0.1% N-1-naphthylethylene diaminedihydrochloride in 0.5% H3PO4. After 10 minutes at room temperature, the absorbance at 540 nm was measured in a microplate reader. Nitrite concentrations were calculated by comparison with a standard calibration curve with sodium nitrite (NaNO2: 0 - 110 µmol/L).
3.1.2. Levels of Malondialdehyde (MDA)
The serum levels of MDA as a product of lipid peroxidation which reacts with thiobarbituric acid (TBA) analyzed using Ohkawa et al. method [19]. Absorbance was measured spectrophotometrically at 532 μm and MDA concentration was expressed as nmol/mg pr [19].
3.1.3. Activity of Catalase (CAT)
CAT activity was estimated following the method of Sinha et al. [20]. The reaction was started by the addition of 20 μL of sample in 2 mL of 30 mmol/L hydrogen peroxide (H2O2) in 50 mmol/L potassium phosphate buffer (pH = 7.0). Enzyme units are expressed as mmol/L of consumed H2O2 per minute g or mL.
3.1.4. Activity of Glutathione Peroxidase (GPx)
The GPX activity in the serum was determined using a GPX assay kit (Randox Lab., Ltd., UK) according to the manufacturer’s protocol.
3.1.5. Determination of Paraoxonase (PON) Activity
PON activity was determined using paraoxon as a substrate and measured by increases in the absorbance at 412 nm due to the formation of 4-nitrophenol as already described [21]. The activity was measured at 25°C by adding 50 mL of serum to 1 mL tris-HCl buffer (100 mM at pH = 8.0) containing 2 mM CaCl2 and 5 mM of paraoxon. The rate of generation of 4-nitrophenol was determined at 412 nm. Enzymatic activity was calculated using molar extinction coefficient 17100/Mcm.
Kurskal-Wallis test, Mann-Whitney U test and Dunn’s multiple comparison tests were chosen for analysis variables (SPSS-22). Results are presented as mean ± SD. Results considered significant at P ≤ 0.05.
4. Results
4.1. Determination of GPX Activity
Comparison of serum GPX levels in the groups received reperfusion showed that in the group 5, selenium received with 7 days reperfusion, significantly higher than the other groups (P = 0.041) (Table 2). Comparison of serum GPX levels in the groups received reperfusion showed that in the group 4, 3 days reperfusion without selenium injection, significantly lower than the other groups (P = 0.041) (Table 2). Comparison of the serum GPX levels in the groups that received selenium showed that levels of GPX increased until days 14 and then decreased. Comparison of serum GPX levels between groups selenium treated with the control corresponding groups suggests that the only difference between group 5, selenium received with 7 days reperfusion, and group 6, 7 days reperfusion without selenium injection, are statistically significant (P = 0.029) (Table 3).
| Study Groups | Concentration of GPX | |
|---|---|---|
| Mean of Ranks | Mean ± SD | |
| 2.75 | 912.7 ± 123.9 | |
| 11.75 | 1439.5 ± 206.1 | |
| 9.5 | 1342.6 ± 209.4 | |
| 10 | 1311.7 ± 196.4 | |
Plasma Concentrations of GPX Compared at Different Times of Reperfusion With Injection of Selenium Which Groups Compared With Each Other’s Using Kurskal-Wallis Testa
| Study Groups | Concentration of GPX | P Value | |
|---|---|---|---|
| Mean of Ranks | Mean ± SD | ||
| 0.34 | |||
| With selenium injection | 3.5 | 979.1 ± 93.4 | |
| Without selenium injection | 5.5 | 912.7 ± 123.9 | |
| 0.029 | |||
| With selenium injection | 2.5 | 1017.5 ± 124.3 | |
| Without selenium injection | 6.5 | 1439.5 ± 206.1 | |
| 0.68 | |||
| With selenium injection | 4 | 1303.5 ± 271.2 | |
| Without selenium injection | 5 | 1342.5 ± 209.4 | |
| 0.88 | |||
| With selenium injection | 4.75 | 1368.2 ± 260.1 | |
| Without selenium injection | 4.25 | 1311.7 ± 196.4 | |
Plasma Concentrations of GPX Compared at Different Times of Ischemia-Reperfusion With and Without Injection of Selenium Which Each Group Compared With Placebo Using Mann-Whitney U Test
4.2. Results of Serum Nitrite Levels
Comparison of serum NO levels in the groups received reperfusion showed serum NO levels is lower in the group 7, 14 days reperfusion with selenium treated, than the other groups (P = 0.047) (Table 4). Comparison of serum NO levels between groups selenium treated with the untreated corresponding groups suggests that difference between group 7, selenium received with 14 days reperfusion, and group 8, 14 days reperfusion without selenium injection, (P = 0.006) and also between group 9, 28 days reperfusion with selenium treated, and group 10, 28 days reperfusion with selenium untreated, are statistically significant (P = 0.002) (Table 5).
| Study Groups | Concentration of NO, nmol/min/mL | |
|---|---|---|
| Mean of Ranks | Mean ± SD | |
| 12.5 | 1.26 ± 0.37 | |
| 18.6 | 1.47 ± 0.79 | |
| 8 | 1.86 ± 0.34 | |
| 9.8 | 1.88 ± 0.22 | |
Plasma Concentrations of NO Compared at Different Times of Reperfusion With Injection of Selenium Which Groups Compared With Each Other’s Using Kurskal-Wallis Testa
| Study Groups | Concentration of NO | P Value | |
|---|---|---|---|
| Mean of Ranks | Mean ± SD | ||
| 0.81 | |||
| 6.83 | 1.36 ± 0.42 | ||
| 6.18 | 1.26 ± 0.37 | ||
| 0.95 | |||
| 7.38 | 1.63 ± 0.34 | ||
| 7.67 | 1.47 ± 0.79 | ||
| 0.006 | |||
| 3.67 | 1.12 ± 0.33 | ||
| 9.33 | 1.86 ± 0.34 | ||
| 0.002 | |||
| 8 | 1.18 ± 0.25 | ||
| 15 | 1.88 ± 0.22 | ||
Plasma Concentrations of NO Compared at Different Times of Ischemia-Reperfusion With and Without Injection of Selenium Which Each Group Compared With Placebo Using Mann-Whitney U Test
4.3. Results of Serum POX Levels
Comparison of serum POX levels in the groups received reperfusion showed serum POX levels is lower in the group 3, 3 days reperfusion with selenium treated, than the other groups (P = 0.001) (Table 6). Comparison of serum POX levels in the groups received reperfusion showed that in the group 9, selenium received with 28 days reperfusion, significantly higher than the other groups (P = 0.001) (Table 6).
| Study Groups | Concentration of POX, nmol/min/mL | |
|---|---|---|
| Mean of Ranks | Mean ± SD | |
| 5.5 | 75.7 ± 8.4 | |
| 17.6 | 106.1 ± 14.6 | |
| 26.6 | 120.5 ± 20.8 | |
| 32.2 | 125.2 ± 16.5 | |
Plasma Concentrations of POX Compared at Different Times of Reperfusion With Injection of Selenium Which Groups Compared With Each Other’s Using Kurskal-Wallis Testa
Comparison of serum POX levels between groups selenium treated with the untreated corresponding groups suggests that difference between group 3, selenium received with 3 days reperfusion, and group 4, 3 days reperfusion without selenium injection, (P = 0.001) and also between group 5, 7 days reperfusion with selenium treated, and group 6, 7 days reperfusion with selenium untreated, are statistically significant (P = 0.029) (Table 7).
| Study Groups | Concentration of POX, nmol/min/mL | P Value | |
|---|---|---|---|
| Mean of Ranks | Mean ± SD | ||
| 0.001 | |||
| With selenium injection | 5.6 | 16.9 ± 7.1 | |
| Without selenium injection | 15.4 | 75.7 ± 8.4 | |
| 0.029 | |||
| With selenium injection | 7.6 | 92.3 ± 19.3 | |
| Without selenium injection | 13.4 | 106.1 ± 14.6 | |
| 0.79 | |||
| With selenium injection | 10.1 | 120.5 ± 20.1 | |
| Without selenium injection | 10.9 | 118.03 ± 20.9 | |
| 0.14 | |||
| With selenium injection | 12.5 | 137.6 ± 7 | |
| Without selenium injection | 8.5 | 125.2 ± 16.6 | |
Plasma Concentrations of POX Compared at Different Times of Ischemia-Reperfusion With and Without Injection of Selenium Which Each Group Compared With Placebo Using Mann-Whitney U Test
4.4. Results of Serum MDA and CAT Levels
Comparison of serum MDA levels in the treated groups with selenium and corresponding untreated groups do not statistically significant. Comparison of serum CAT levels in the treated groups with selenium and corresponding untreated groups do not statistically significant.
5. Discussion
Sciatic nerve ischemia led to nerve damage but the injury caused by reperfusion is more severe and induced nerve fibers degeneration. Previous studies have shown that nerve fibers degeneration reaches its maximum at 14 days reperfusion. Studies showed that nerve fibers regenerated until 28 days of reperfusion. Use of various antioxidants such as simvastatin reduces the damage caused by reperfusion. Use of antioxidants against oxidative stress induced by ischemia-reperfusion injury changes in the antioxidant activity. Selenium is an antioxidant that can help prevent damage induced by ischemia-reperfusion. In the present study observed that selenium with reduce serum level of NO and increase serum level of GPX and POX can reduce damage induced by oxidative stress. Ansari et al. demonstrated that selenium can prevent the degeneration of neurons in the brain caused by ischemia [22]. They also showed that selenium can influence the activity levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and monoamine oxidase (MAO-A and MAO-B) have a significant impact on the brain [22].
Results of present study showed that selenium cannot influence the activity levels CAT but can influence the activity levels of GPX significantly. Venardos et al. demonstrated that selenium can increase the activity of thioredoxin reductase and glutathione peroxidase, which leads to reduced myocardial injury induced by ischemia-reperfusion [23]. Results of their study were consistent with the results of present study because both studies showed that selenium can influence the activity levels of GPX. Heyland et al. demonstrated that the use of selenium, because of antioxidants properties, in chronic patients who are susceptible to damage by free radicals, reduce the mortality and morbidity while others oral antioxidants and vitamins have no such effects [24]. Tinggi et al. demonstrated that selenium can prevent the damage induced by ROS and NOS [25].
Turan et al. showed that selenium can reduce ischemia-reperfusion injury in the heart by increase GPX activity levels and reduce MDA [15, 26]. The above-mentioned outcomes are consistent with results obtained in this study. We suggest that cellular and molecular studies be done about the effect of selenium on the damage induced by sciatic nerve ischemia-reperfusion.
Taken together, it can be said that selenium can increased plasma activity levels of GPX, POX and decreased activity levels of NO. It is concluded that selenium, which is an essential part of our diet, might be helpful in protection against damage in lower limb ischemia.
