Effect Of Psychological Stress On Iron Absorption In The Small Intestine And Its Mechanisms In Rats

Iron is one of the most important elements inside an organism.It is present in several proteins,which play important roles in many physiological processed.Iron is necessary for oxygen and carbon dioxide transport,as well as for cellular metabolism.Interest in iron status has traditionally focused on iron deficiency because of its many negative effects on health,including changes in immune function,cognitive development, temperature regulation,energy metabolism and work performance.Proper balance of iron in an organism is necessary for good health,in terms of both psychological well-being and physical fitness.Our experiment studies had showed that serum iron decrease after psychological stress.The aim of the present study was to analyse iron absorption in rats after psychological stress.Our knowledge of the mechanisms that regulate iron absorption has recently advanced dramatically.Two iron transporters,divalent metal transporterl(DMT1) and ferroportin 1 (FPN1),are critical for intestinal iron absorption and are regulated by body iron stores.DMT1 is a transmembrane protein that transports ferrous iron across the apical membrane of intestinal epithelial cells,whereas FPN1 is an iron exporter located on the basolateral membrane.To date,all studies that have investigated the molecular mechanisms of the regulation of iron absorption have been conducted either in cell culture or in adult humans or animals.Little is known about the gene and protein expression of DMT1 and FPN1 or their role in the regulation of iron absorption during psychological stress.Although the pathway of iron absorption has been described,its regulation is not well understood.Recently,the hepatic antimicrobial peptide hepcidin has been identified as the putative central regulator of iron absorption.The regulation of hepcidin expression in response to the body's iron requirements is becoming well established.An intriguing feature of the hepcidin expression is its modulation by cytokines,especially IL-6(interleukin-6).In a recent elegant study,Nemeth et al.demonstrated that,in humans,IL-6 is able to induce hepcidin expression and significantly lower serum iron levels.This occurs probably by a combination of decreased small bowel iron absorption and increased sequestration of iron into the reticuloendothelial system.Psychological stress is anywhere in our soldiers,if iron deficiency happened in the army, there are very adverse effect in ensure the ability of army.So,it is important to know the iron metabolism during psychological stress.In the present study,we have used communication box models for psychological stress model,and used metabolism balance to investigate the iron absorption after psychological stress.ObjectiveTo study the characteristic effects of psychological stress on the small intestinal iron absorption and to establish a useful experimental basis for further study involving how stress changes cell normal iron homeostasis and the consequent effects on physiological function of the human body.Methods一,Effects of psychological stress on iron absorption in the rat(一) 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).After adaptation for 3 days,the rats were divided into the foot-shock group(FSG),psychological stress group(PSG) and the control group (CG).(二) 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.(三) Measurement of serum CORT and ACTH,hypothalamus NE levels in rats under stressThe concentrations of serum CORT and ACTH were measured by the enzyme-linked immunoassays(ELISAs) kit;the hypothalamus NE levels were measured by the radioimmunofocus assays(RI) kit.(四) 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.(五) The apparent absorption of iron under psychologicalWe 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) x100%/iron in diets. And at the day 1,the day 3 and the day 7 of psychological stress and the control group were calculated.(六) Peri's staining of duodenumParaffin sections were dewaxed and incubated in 1%HCl containing 1%ferrous cyanate for 20 min.Sections were washed and counterstained with Neutral Red before visualization.二,The regulation mechanism of iron absorption in the duodenum under psychological stress (一) 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±10℃,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).After adaptation for 3 days,the rats were divided into the foot-shock group(FSG),1 day of psychological stress group(PSG1),3 days of psychological stress group(PSG3),7 days of psychological stress group(PSG7) and the corresponding control groups(CG).(二) To build psychological stress model of SD ratsThe model of psychological stress is the same to the part 1.And PSG1 means 1 day under psychological stress,PSG3 means continuous 3 days under psychological stress,PSG7 means continuous 7 days under psychological stress.(三) Determination of IL-6 content in the liver under psychological stressThe concentrations of IL-6 in the liver were assessed using a commercially available ELISA kits with the absorbance read on a microplate reader at a wavelength of 450 nm.(四) Western blotting analysis of DMTI(+IRE/-IRE),FPN1 and Ferritin in the duodenum and Hepcidin in the liver.The duodenum and the liver were homogenized by a Membrane binding protein kits.The NC membranes were incubated overnight at 4℃with a primary antibody against DMT1(+IRE/-IRE,1:1000,ADI),FPNI(1:1000,ADI),Ferritin(1:5000,Sigma), Hepcidin(1:1000,ADI),orβ-actin(1:1000,Beyotime).The blots were developed by incubation in enhanced chemiluminescence(ECL) and subsequently exposed to BioMax Light Flim.三,Effect of psychological stress with IL-6 antibody treated on the mechanism of iron absorption in the duodenum (一) To divide experimental animals into groups40 male Sprague-Dawley rats were randomly into three groups with 8 rats each,the control group,the 7 days of psychological stress grout with PBS(we called 7PG) and the 7 days of psychological stress group with IL-6 Ab(we called antagonism-7PG).Before the psychological stress,the 7P were intraperitoneal injection 0.1 ml PBS,and the antagonism-7P were intraperitoneal injection 0.1 ml PBS contained 2μg IL-6 Ab.(二) To build psychological stress model of SD ratsThe model of psychological stress is the same to the part 1.(三) Perl's staining of duodenumParaffin sections were dewaxed and incubated in 1%HCl containing 1%ferrous cyanate for 20 min.Sections were washed and counterstained with Neutral Red before visualization.(四) Western blotting analysis of DMT1(+IRE/-IRE),FPN1 and Ferritin in the duodenum and Hepcidin in the liverThe duodenum and the liver were homogenized by a Membrane binding protein kits.The NC membranes were incubated overnight at 4℃with a primary antibody against DMT1(+IRE/-IRE,1:1000,ADI),FPN1(1:1000,ADI),Ferritin(1:5000,Sigma), Hepcidin(1:1000,ADI),orβ-actin(1:1000,Beyotime).The blots were developed by incubation in enhanced chemiluminescence(ECL) and subsequently exposed to BioMax Light Film.Results一,PS exposure reduced the iron absorption in the small intestine.(一) Determination of the PS model estabolishmentWe found that the levels of serum ACTH,corticosterone and hypothalamus noradrenalin in the model animals were significantly higher than those in the control animals(P<0.05), indicating the successful of model establishment.(二) 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.So there were no changed in the iron content in the diets between psychological stress and the control group.(三) 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 spectrophotorneter to measure the iron content of fecal.(四) The apparent absorption of iron under psychological stressWe found that there was no significantly changed between day 1 of psychological and the control group,and there were significantly decreased the apparent absorption of iron at day 3 and day 7 of psychological stress vs the control group.(五) PS exposure caused iron retention in the intestinal mucosaPrussian Blue staining was cleady observed in the apical poles of villous enterocytes after 3 days and 7 days of psychological stress,but there was no discernable staining both control group and 1 day of psychological stress.二,The regulation mechanism of iron in the duodenum changed under PS exposure(一) Effects of psychological stress on the IL-6 content in the liverThere were no significantly changed between 1 day of psychological stress and the control group.And there were significantly increased between PSG3 and the control group, PSG7 and the control group.(二) PS exposure caused changes in DMT1(+IRE),FPN1 and Ferritin levels in the duodenumThere were no significantly expression changed on DMT1(+IRE/-IRE),FPN1,Ferritin in duodenum between control group and 1 day of psychological stress.After 3 days and 7 days of psychological stress,there were an significantly decreased expression FPN1 and increased expression of Ferritin,.After 7 days of psychological stress,there was an significantly decreased expression of DMT1(+IRE).(三) PS exposure caused changes in Hepcidin in the liverThere were no significantly expression changed on Hepciidn in liver between control group and 1 day of psychological stress,there were an significantly increased expression of hepcidin after 3 days and 7 days of psychological stress.三Effect of psychological stress with IL-6 antibody treated on the mechanism of iron absorption in the duodenum in rats(一) Perl's staining in the small intestine with IL-6 treatedThe iron retention in the intestinal mucosa disappeared with IL-6 antibody treated on PS exposure and the ferritin expression reduced to the normal level(二) Western blot detected hepcidin changed with IL-6 treatedHepcidin expression in the liver reduced to the normal level with IL-6 antibody treated on PS exposure(三) Western blot detected FPN1,DMT1(+IER) changed with IL-6 treatedFPN1,DMT1(+IRE) expression in the duodenum increased to the normal level.ConclusionsThe Sprague-Dawley rats were used for animal models of psychological stress.And we used the modified communication box for psychological stress to study the iron absorption and the mechanism of regulation during psychological stress.Conclusions were made as follows:1,With the time prolonged of psychological stress,and no significantly changed of food intake,the iron absorption in rats during psychological stress can reduced.2,With the time prolonged of psychological stress,the hepatic IL-6 contents increased.3,Rat liver hepcidin protein expression increased with the development of psychological stress.In duodenum,The DMT1(+IRE) and FPN1 protein expression can reduced,and there were iron stagnation in the enterocytes of villous.4,The mechanism of regulation of iron absorption during psychological stress may be through IL-6-hepcidin axle.