Effect Of Psychological Stress On Iron Metabolism Of Hematopoietic System In Rats

Iron is an essential source of the production of hemoglobin. Many researches showed decreased serum iron would affect the production of hemoglobin, thus infulencing on the formation and maturation of erythroipiesis, even result in iron deficiency anemia (IDA) finally [1,2]. According to the WHO, there are about 1 billion people suffering from IDA all over the world. Also, there is a high prevalence of IDA in our country, among which the prevalance of IDA in children and aldolescents in Shanghai is up to 19.90% on the basis of the reports of Sun Jianqin, etc. IDA may influence many aspects of physiological functions and generativity ability. It is very important to prevent and treat IDA. The major causes of IDA are too less iron intake and too much iron loss. However, some reseaches showed there is still a high prevalance of IDA even though there is adequate iron in diets and no extra iron loss in body. Therefore, the high prevalance of IDA may be realated to some other reasons except for diet action. By far, researchers can not describe the cause exactly to explain the inconsistency between high-iron diet intake and high prevalance of IDA.From many previous researches we find that stress can result in greatly decrease of serum iron. For example, acceleration stimulation may result in serum iron decreased by 52.3%, exposure to hot can result in serum iron decreased by 31%. There are many reports of the same kind. For example, Navas FJ, etc, reported vigorous exercises could result in the decrease of serum iron in sporters; Smith SM, etc, found the flying of shuttle could lead to the decrease of seum iron in astronauts. Nikolova- Todorova Z, etc, pointed out injury of surgery could result in the decrease of serum iron in patients. It shows that stress can infuence the amount of serum iron by affecting the formation of erythrocyte.The changes of the serum iron,iron related protein and hematogenesis of SD rats were observed under different periods of stress in this study. All the findings are reported as following. ObjectiveTo study the characteristic effects of psychological stress on serum iron and erythropoiesis, and to establish a useful experimental basis for further study involving the relationship between psychological stress and erythropoiesis.Method1. To devide 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 (80±5 g body weight) fed with a standard diet were purchased from the Shanghai-BK Ltd. Co, and were housed individually in a cage in a temperature-controlled room (24±1℃, 55±5% humidity) with a 12-hour light and 12-hour dark cycle. 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 were divided into three sub-groups, i.e., 3-day group, 7-day group and 14-day group. Each rat was exposed to stress for 30 minutes every day.2. To build psychological stress model of SD ratsUsing a communication box system12, footshock 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. All rats were executed immediately after above administration, and rat blood and femoral bone marrow were collected for analysis. 3. Measurement of iron of serum, spleen and liver in rats under stressThe concentrations of serum iron (SI), spleen iron and liver iron were measured on the Z-2000 atomic absorption spectrophotometer (Hitachi, Japan). Standard iron solution of 100 u g/mL was provided by National Standard Subastance Center.4. Measurement of the iron of bone marrow in rats under stressThe fresh marrow bone of the femur heads of the rats were reserved to make up smear on the slide with previous deferric treatment. The smears were stained with acidic potassium ferrocyanide within 24 hours and observed on iron-stained particles under microscope to give semiquantitative and qualitative analysis.5. Measurement of the indexes of the red blood cell in rats under stressThe whole blood with anticoagulant was left to measure the indexes related to red blood cell. The blood autoanalyzer of type KX-21 (SYSMEX Company, Janan) was used to determine the indexes of red blood cell, including hemoglobin (Hb), red blood cell count (RBC), hematocrit (Hct), mean corpuscular volume (MCV) and red blood cell distribution width (RDW).6. The effect on the iron regulated proteinThe concentrations of serum ferritin (SF), serum transferrin receptor (sTfR) and erythropoietin (sEPO) were measured by the enzyme-linked immunoassays (ELISAs) kit (R&D Systems Inc, USA). The ELISA kit is composed of standard enzyme board with 96 bores, substrate A, substrate B, concentrated solution, sample diluent, stopping solution, enzyme-linked substance, and a set of standard solutions. Measuring instrument: automatic enzyme labeling instrument (SUNRISE company, U.S.A).7. Statistical methodAll the experimental datas were analyzed by one-way ANOVA with SPSS statistical package of 10.0 version. All the experimental datas were presented by average±standard deviation ( X±S). P lower than 0.05 refers to significant level and P lower than 0.01 referes to very significant level. Result1. The effect on the serum iron in rats under stressCompared with the control group, serum iron of the psychological stress group after 3d-, 7d- and 14d-period decreased by 28.4%, 27.7% and 34.0% respectivlely (P<0.05), serum irons of the foot-shock stress group after 3d-, 7d- and 14d-period decreased by 28.5%, 18.3% and 40.7% respectivlely (P<0.05). The tendency of both foot-shock stress group and psychological stress group are same (P<0.05).2. The effect on the iron of bone marrow in rats under stressCompared with control group, the result of the acidic potassium ferrocyanide staining showed the extracellular iron of bone marrow in rats of both foot-shock stress group and psychological stress group decreased significantly, even diappeared at all.3. The effect on the iron regulated protein in rats under stressCompared with control group, serum ferritin of psychological stress group increased significantly after 3d-period (P<0.05), there is no significant changes of sTfR of both experimental groups; serum ferritin in rats decreased significantly after continous psychological stress of 7d- and 14d- period, together with significantly increased serum transferrin receptor (P<0.05). The tendency of both foot-shock stress group and psychological stress group are same (P>0.05).4. The effect on the indexes of the red blood cell under stressCompared with the control group, hemoglobin of the psychological stress group after 7d- and 14d-period decreased by 10.0% and 12.8% respectivlely (P<0.05), RBC decreased by 4.7% and 9.8% respectivlely (P<0.05), hematocrit decreased by 7.0% and 12.9% respectivlely (P<0.05), MCV decreased 1.6% and 7.3% respectivlely (P<0.05), and RDW increased by 11.2% and 22.5% respectivlely (P<0.05); hemoglobin of the thenar electric shock stress group after 7d- and 14d-period decreased by 8.7% and 9.9% respectivlely (P<0.05), RBC decreased by 6.1% and 10.5%, hemotocrit decreased by 10.0% and 12.8% respectivlely (P<0.05), MCV decreased 3.6% and 1.5% respectivlely (P<0.05), and RDW increased by 3.6% and 6.1% respectivlely (P>0.05). The tendency of both foot-shock group and psychological stress group are same (P>0.05). 5. The effect on the changes of diet intake and weight of rats under stress Compared with the control group, there was no significant changes of diet intake and weight of rats in psychological stress group after 3d-, 7d- and 14d-period (P>0.05). The tendency of both foot-shock group and psychological stress group are same (P>0.05).6. The effect on the liver iron and spleen iron in rats under stress Compared with the control group, liver iron of the psychological stress group after 7d- and 14d-period decreased by 12.3% and 10.4% respectivlely (P<0.05), spleen iron decreased by 12.3% and 10.4% respectivlely (P<0.05); liver iron of the foot-shock stress group after 7d- and 14d-period decreased by 24.7% and 10.3% respectivlely (P<0.05), spleen irons decreased by 15.1% and 17.8% respectivlely (P<0.05). The tendency of both foot-shock stress group and psychological stress group are same (P>0.05).7. The effect on the serum erythropoietin in rats under stress Compared with the control group, serum erythropoietin of both psychological stress group and foot-shock stress group after 14d-period decreased by 12.3% and 35.8% respectivlely (P<0.05). The tendency of both foot-shock group and psychological stress group are same (P>0.05).Conclusion1. Continous 3-d period of psychological stress may result in significant decrease of serum iron in SD rats, and continous 14-d period of pychological stress may lead to further decrease of serum iron. The tendency of changes of serum iron between psychological stress and physical stress (foot-shock) is similar.2. Psychological stress may result in significant decrease of iron in bone marrow. The degree of decrease of extracellular iron staining of bone marrow cells in SD rats after 14-d period of stress is more serious than that of 7d-period. It shows that decrease of serum iron in rats of psychological stress group already affect the amount of iron in bone marrow gradually.3. Longer periods of continous psychological stress (more than 7-d period of stress ) may result in significant decrease of serum ferritin (SF) , as well as significant increase of serum transferrin receptor( sTfR). It shows that decreased serum iron due to psychological stress may lead to decreases of both storing iron in body and iron in bone marrow, which may affect erythropoiesis in bone marrow furthermore. As for the increase of SF after 3-d period of stress may be explained by the fact that SF is a kind of acute reactive protein and will increase significantly in the early stage of stress. However, serum iron and SF decrease gradually with the prolonging of the psychological stress. The exact cause by which psychological stress reduces serum iron is still to be discussed by future tests.4. Psychological stress may result in significant decrease of the indexes of red blood cell and the range of the changes increases with the prolonging of the periods of stress. Together with significantly decreased MCV and increased RDW indicate the presence of microcytic hypochromic anemia. Integrated the findings of this study, it shows that long periods of psychological stress may reduce the amount of iron in serum and in bone marrow, thus affecting the erythropoiesis in a certain degree.5. Compared with control group, there are no significant changes in diet intake and weight gain in rats of PSG after 3-day, 7-day and 14-day period of stress. The result indicates the decrease of serum iron may not be related to the diet iron intake in rats of psychological stress group. Certainly, whether psychological stress may affect the iron absorption in diet is still uncertain by far.6. Continous psychological stress may result in significant increase of iron in liver and in spleen. It indicates that the redistribution may be one of the reasons by which psychological stress reduces the level of serum iron.7. Generally, sEPO increases if erythropoiesis is not sufficient. But this study shows that sEPO decreses significantly after 14-d period of psychological stress. However, how psychological stress result in the decrease of sEPO and whether the decrease of sEPO is one of the reason by which psychological stress affects the erythropoietin both need to be discussed furthermore.