Iron Supplementation In Psychological Stress: Effect On Iron Metabolism In Rats

Many studies have confirmed that psychological stress could provoke the occurrence of many diseases such as cardiovascular disease, diabetes, neuropsychiatric diseases and autoimmune diseases and aging. It is suggested that PS is associated with increased oxidant production and oxidative damage. The etiology of these diseases has been linked to the oxidative damages of DNA, proteins and lipids, which are catalyzed by reactive oxygen species. In the present study we investigated the influence of iron supplementation on brain oxidative stress in psychological stress rats. The results showed that iron overload significantly elevated both iron and MDA levels in rat brain from control and PS animals. The GSH contents decreased more significant in rats maintained on iron overload diet compared to those maintained on normal diet. Iron supplementation also significantly modulated activities of vital antioxidant enzymes in all the tissues. SOD activity decreased more significant in rats maintained on iron overload diet compared to those maintained on normal diet. Dietary iron decreased GSH-Px activity in cortex and cerebellum. This study demonstrated that iron overload, might act as a confounding factor in PS-induced oxidative stress. However, more studies may be needed to confirm effect of nutritional factors on psychological stress.ObjectiveIn this study, we examined different iron regimens to evaluate its effects on the course of oxidative stress and antioxidant status in PS rats. For this reason, we measured iron levels and status of oxidative stress in the organs in PS mice and compared these values to those in controls.Methods1. Effects of iron supplementation on iron absorption of psychological stress rats fed with different iron dietary1.1 To divide experimental animals into groupsAll experimental procedures involving animals received the approval from the Animal Care and Use Committee of the Second Military Medicine University. Guidelines and Policy on using and caring of the laboratory animals were followed at all time. Male SD rats (120±10 g body weight) were purchased from the Shanghai-BK Ltd. Co, and were housed individually in a stainless steel metabolism cage in a temperature-controlled room (24±1℃,55±5%humidity) with a 12-hour light and 12-hour dark cycle and given access to deionized water ad labium Animals were fed with a formula diet and the diet refer to American Institution of Nutrition-93 for rapid growth (AIN-93G). The diets were replete and same in all other nutrients except iron content was added as the requirement. After adaptation for 3 days, the rats were divided into the foot-shock group (FSG), psychological stress group (PSG) and the control group (CG), and then each group was divided into two subgroups except FSG, those were adequate iron (AFe,35 mg Fe/kg diet, n=8) group, high iron (HFe,245 mg Fe/kg diet, n= 8).1.2 To build psychological stress model of SD ratsUsing a communication box system, foot-shock stress (FS) and psychological stress (PS) were administered to the rats. The communication box was divided into two parts with a transparent acrylic board, i.e., Part A including ten rooms with a plastic board-covered floor for electric insulation and part B including ten rooms with a metal grid-exposed floor. Rats in part B were administered an electrical shock through the floor (90 V,0.8 mA for 1 second) randomly for 30 min,90 times in total, and then exhibited a nociceptive stimulation-evoked response such as jumping up, defecation and crying. Thus they were exposed to systemic (physical) stress. Rats in part A were not directly administered the electrical shock, but were exposed to psychological stress in response to the actions of the rats in Room B.1.3 Calculated the diet intake and collected completely the stool in rats under psychological stressWe used the Carmine red labelling the stools to insure collected completely and we calculated the food intake each day exactly.1.4 The iron apparent absorption and iron absorbed dose of psychological stress rats fed with different iron dietary We used the mineral balance study to determine the apparent absorption of iron during psychological stress. Fecal samples were collected, dried and weighted daily. Urine was not collected or analyzed for iron content because urinary excretion of iron was assumed to be negligible. The apparent absorption of iron= (Iron in diets-iron in fecal)×100%/iron in diets, the iron absorbed dose= Iron in diets-iron in fecal. And at the day 1, the day 3 and the day 7 of all psychological stress and the control groups were calculated.2. Effects of iron supplementation on tissues iron contents of psychological stress rats fed with different iron dietary2.1 To divide experimental animals into groupsAll experimental procedures involving animals received the approval from the Animal Care and Use Committee of the Second Military Medicine University. Guidelines and Policy on using and caring of the laboratory animals were followed at all time. Male SD rats (120±10 g body weight) were purchased from the Shanghai-BK Ltd. Co, and were housed individually in a stainless steel metabolism cage in a temperature-controlled room (24±1℃,55±5% humidity) with a 12-hour light and 12-hour dark cycle and given access to deionized water ad labium Animals were fed with a nature diet.The diets were replete and same in all other nutrients except iron content was added as the requirement. After adaptation for 3 days, the rats were divided into the foot-shock group (FSG), psychological stress group (PSG) and the control group (CG), and then each group was divided into three subgroups except FSG, those were low iron (LFe,80 mg Fe/kg diet, n=8) group, moderately iron (MFe,160 mg Fe/kg diet, n= 8) group, high iron (HFe,320 mg Fe/kg diet, n= 8).2.2 To build psychological stress model of SD ratsThe model of psychological stress is the same to the part 1.2.3 Determination of iron content in the liver, brian, spleen from psychological stress rats fed with different iron dietaryEach sample was wet-acid digested with concentrated nitric-perchloric acid mixture (4 to 1 ratio). An aliquot of each sample was analyzed by using iron flame or graphite atomic absorption spectrophotometer (Z-8100, Hitachi, Japan). 2.4 Determination of serum iron from psychological stress rats fed with different iron dietarySerum iron concentration was determined in nonhemolyzed serum samples by spectrophotometric analysis using using iron flame or graphite atomic absorption spectrophotometer (Z-8100, Hitachi, Japan).3. Effects of iron supplementation on oxidative stress of psychological stress rats fed with different iron dietary3.1 To divide experimental animals into groupsThe method of dividing groups is the same to the part 2.3.2 To build psychological stress model of SD ratsThe model of psychological stress is the same to the part 1.3.3 Effects of iron supplementation on oxidative stress status of PS rats brian and liverTissues were homogenized (Potter-Elvehjem homogenizer) in nine volumes of chilled 100 mM Tris-HCl buffer containing 0.1 mM EDTA and 0.1%(v/v) Triton X-100, pH 7.8. All procedures were performed on ice. Homogenates were centrifuged at 10,500 g for 30 min at 4℃and the resultant supernatants were kept in aliquots.3.3.1 Measurement of MDA in rat brain and liverMalondialdehyde (MDA) concentration was assessed using a MDA assay kit with the absorbance read on a microplate reader at a wavelength of 586 nm. MDA concentration of each region were normalized to wet tissue weight (mg) and expressed asμg/mg.3.3.2 Measurement of GSH in rat brain and liverGlutathione reduced (GSH) level was measured using kit with the absorbance read on a microplate reader at a wavelength of 490 nm. GSH concentration of each region were normalized to wet tissue weight (mg) and expressed asμmol/mg. 3.3.3 Measurement of SOD in rat brain and liverSuperoxide dismutase (SOD) activity was measured using WST-1 kit with the absorbance read on a microplate reader at a wavelength of 450 nm. SOD activity of each region were normalized to wet tissue weight (mg) and expressed asμmol/mg.3.3.4 Measurement of GSH-Px in rat brain and liverGlutathione peroxidase (GPX) activity was assayed using WST-1 kit with the absorbance read on a microplate reader at a wavelength of 340 nm. GPX activity of each region were normalized to wet tissue weight (mg) and expressed as unit of activity.4. Statistical analysisAll results were expressed as mean±SE. Statistical analysis was carried out by using SPSS 11.0. All values below the detection limits were set to zero and absolute values without correction for recovery rate were used in analyses. A P value less than 0.05 was considered statistically significant.Results1. Iron supplementation decrease the iron apparent digestibility as the PS treatment1.1 Effect of food intaked during psychological stressDiets were changed daily and data collection was made at day 1, day 3 and day 7, we found there were no significantly changed between psychological stress group and the control group. There were also no significantly changed among the groups fed with different iron dietary.1.2 Effect of the iron content of fecal under psychological stressWe collected the fecal of day 1, day 3 and day 7 with the carmine red labeled (we collected between the red stool but not included the second red stool)and used the atomic absorption spectrophotometer to measure the iron content of fecal. We found the iron contents of fecal increased significantly as the iron supplementation, and the increase was more obvious at PS group.1.3 Effect of iron supplementation during psychological stress on the iron apparent digestibilityIron supplementation decreased the iron apparent digestibility at day 1,3 and 7. We found that there was no significantly changed between day 1 of psychological and the control group at both doses of dietary iron, and there were significantly decreased the apparent absorption of iron at day 3 and day 7 of psychological stress vs the control group at doses of AFe and HFe. However, the absolute value of iron intake is increased at HFe group.2. Iron supplementation effect the iron content on the organs of PS rats2.1 Effect of iron supplementation on the iron content of PS rats liverIron supplementation induced a dose-dependent increase in iron content in rats liver (P＜0.05). At doses of MFe and HFe dietary, PS increased the iron content of liver (P＜0.05).2.2 Effect of iron supplementation on the serum iron of PS ratsIron supplementation increased the serum iron significantly (P＜0.05).At the same time, PS decreased the serum iron significantly (P＜0.05) at all doses of dietary iron.2.3 Effect of iron supplementation on the iron content of PS rats cortex, hippocampus, striatum, brain stem and cerebellumIron supplementation induced a dose-dependent increase in iron content in rats brain. When responses of control and PS treated animals were compared using t-test, iron content increase was significantly greater (P＜0.05) in the PS animals in cortex and cerebellum at three doses.3. Iron supplementation effect the oxidative stress status of PS rats3.1 Iron supplementation increased the MDA of PS ratsIron supplementation also induced a dose-dependent increase in MDA in rats'brain. MDA increased more significantly (P＜0.05) in most of the regions of brain in PS group than in control group. When the rats were fed with AFe diet, PS treatment did not have significant influence on MDA response. The change of liver MDA is similar to the brain.3.2 Iron supplementation changed the GSH of PS rats GSH concentration decreased as iron supplementation at control group. However, the GSH concentration didn't change in PS rats. Comparison between PS and control group by t-test revealed that GSH was seriously influenced by PS. The GSH level detected in PS rats is significantly (P＜0.5) lower than control rats. Iron supplementation induced a dose-dependent decrease in GSH in liver.3.3 Iron supplementation decreased the SOD of PS ratsAs the increasing of iron supplementation, the activity of SOD decreased in brain and liver. And this decrease was more obvious at PS group. The highest dose of dietary iron (320 mgFe/kgdiet) most significantly (P＜0.05) reduced the activities of SOD in PS rats than in control rats.3.4 Iron supplementation changed the GSH-Px of PS ratsAs the increasing of iron contents in diets, GSH-Px activities were decreased significantly in cortex and cerebellum in all groups, and this decrease was more obvious at PS group. The iron supplementation didn't change the GSH-Px activity of liver in PS and control groups.ConclusionsThe Sprague-Dawley rats were used for animal models of psychological stress. And we used the modified communication box for psychological stress to study how the iron supplementation affect iron metabolism in psychological stress rats. Conclusions were made as follows:1. With the time prolonged of psychological stress, there is no significantly changed of food intake among different iron supplementation, the iron absorption in rats during psychological stress can reduced. However, the absolute value of iron intake is increased at HFe group.2. Iron supplementation induced a dose-dependent increase in iron content in rats's brain, liver and serum. At the same time, the phenomenen of iron deposition in brain and liver and the decreasing of iron serum is still exist.3. In most of the brain regions and liver, oxidative stress status increased as iron supplementation, and this increase was more obvious in PS group.4. The antioxidant enzyme activities of brain and liver were decreased as iron supplementation, and the PS treatment aggravate the decrease.