Restraint Stress Aggravates Rat Intestine Damage Caused By A Crush Injury And Changes Of Endoplasmic Reticulum Stress Related Proteins

Objective:In judicial practice,some litigants suffered from a variety of nonfatal mechanical damage,such as continuous binding or restriction position,etc,often turned out multiple organ dysfunction syndrome and even death.Owing to this kind of injury itself is not enough serious to cause death,it was used to cause the death becoming intangible and controversial,which delayed the case.Thereby,it is meaningful to investigate the impact of morphology and function on vital organs by this kind of damage and further to reveal the exact mechanism of the death.If we carefully analysis the context of the cases,one common feature will come out,namely there is an obvious psychological stress.Neuroendocrine responses to stress are mainly for the activation of locus ceruleus-norepinephrine system and hypothalamus-pituitary-adrenal cortex system.Subsequent increase of stress hormones in blood caused a series of functional metabolic changes,which has an important significance compensatory defense.However,excessive or persistent stress may cause dysfunction,metabolic disorders and tissue damage.The reserve capacity and defense mechanism gradually consumes,internal environment turns up serious disorder,and finally one or multiple organ failure or even death.At present,the restraint stress can commendably replicate the mental stress.Previous researches have shown that restraint stress could aggravate heart,liver and kidney damage in rats with crush injuryand further lead to MODS,which suggest that heavy psychological stress may play critical role in the process of death caused by non-lethal mechanical injury.With the continuous development of human technology and the increasing degree of social competition,the problem of stress influenced human health is becoming more and more serious.Thus intestinal disease induced by stress has been become a focus problem.Intestinal barrier is composed of intestinal epithelial cells and the close connection between these cells and others,which is a complex of various functions.The intestinal tract injures or comes out dysfunction in the stress,which will set off bacterial translocation and endotoxin and even systemic inflammatory response syndrome.Previous studies have shown that restraint stress could damage the intestinal barrier and lead to chronic colitis.The mechanism may have a variety of factors such as immune disorders,microbial environment changes,suggesting that the intestine is extremely sensitive to psychological stress.However,the specific mechanism is not clear.The endoplasmic reticulum is a major organelle for cell synthesis,processing proteins and storage of Ca²⁺.The function of endoplasmic reticulum can be interfered by many factors such as oxygen free radicals,stress hormones and cytokines,which resulted in not/misfolded protein accumulation,endoplasmic reticulum stress proteins such as glucose regulated protein 78,eukaryotic translation initiation factory 2 alpha and chop elevation,and finally activating ERS.Though ERS is an important component of cellular defense response,excessive or prolonged ERS can induce tissue and cell damage via apoptosis and inflammatory.Previous results indicated that ERS is associated with the development of inflammatory bowel disease.However,the change of ERS proteins such as GRP78,P-e IF2α and CHOPduring the intestinal injury induced by stress is not clear.In conclusion,the study was to simulate the actual cases consisted of nonfatal injury and psychological stress,establish the composite model of crush injury and restraint stress,and investigate the pathological changes as well as the expression of ERS proteins in intestinal tissue of rat.It is meaningful to provide a scientific basis for studying the mechanism of death induced by nonfatal injury and psychological stress.Methods:1 Healthy male Sprague Dawley（SD）rats（210 230g）,adaptively fed for 7 days,were randomly divided into four groups（n=24）.（1）control group（Con）: rats were fasted water at a fixed time every day（8h and 10 h alternately）activating and drinking freely the rest time.（2）restraint stress group（Rs）: rats were put at a fixed time every day（8h and 10 h alternately）into the restraint stress mold,fasting water.（3）crush injury group（CI）: rats anesthesiaed with anhydrous diethyl ether,were subjected to crush with24 kg weight on their double hind limbs for six hours,activating and drinking freely the rest time.（4）composite model group（Com）: rats were continuously subjected to crushed for six hours,and suffered to restraint stress at a fixed time every day（8h and 10 h alternately）.The weight before the experiment each day were recorded to evaluate whether to establish a model of restraint stress.The partial performance and station were observed to assay whether to successfully establish the crush injury model.After restraint stress continuous for 7 days,intraperitoneal injection of rats were anesthesiaed with 10% chloral hydrate ip.Blood was collected from abdominal aorta,and the supernatant was used to detect catecholamine concentrations.Ileum tissues were removed rapidly and put into 10% paraformaldehyde solution to fix.Tissues paraffin sections were prepared,and carried out HE staining for histopathological observation,immunohistochemical staining（IHC）for observation expression changes of GRP78 、CHOP 、P-eIF2αin tissues.2 The data were presented as Mean±SD and analyzed with one way ANOVA and least significant difference test（LSD）or Tamhane’s by SPSS 21.0 statistical program.A level of P<0.05 was supposed to be statistically significant.Results:1 The changes of rat body weightsThe weight of control group gradually increased.Compared with control group,the weight of restraint stress group showed a significant slowly increased（P<0.01）.The model of restraint stress was successfully established.2 NE、E levels changes in rat plasma samplesNo significant difference was noted in plasma concentration NE and E between Con group and Crush injury group.Compared with crush injury group,NEand E concentration increased significantly in the composite model group（P<0.01）.3 Results of HE stainingThe results of routine histopathological observation showed that the ileum of the control group was clear and the mucosa was orderly,inflammatory edema and other pathological changes were not found.Mild edema and the infiltration of inflammatory cells were found in the mucosa of the restraint stress group.In the crush injury group,mucosa was mild edema,the inflammatory cell infiltration was obvious,and the proliferation of mucosal epithelial cells was remarkable.The histopathological changes were most obvious in the composite group,the inflammatory cell infiltration was found in the mucosa and mucosal interstitial,and the proliferation of the epithelial cells was obvious in some areas.4 Immunohistochemical staining of GRP78,P-eIF2αand CHOPImmunohistochemical staining showed that the changes of these proteins were consistent.The positive expression of epithelial cells in the ileum mucosa was weak in the control group.The positive expression was tend to increase in the restraint stress group and the crush injury group.Moreover,the positive expression of composite model group was further increased.Conclusions:The model constituted ofrestraint stress and crush injury was successfully established.By observing histopathological changes in intestinal tissue,assaying the CA concentration and detecting the expression of GRP78、P-eIF2α、CHOPin intestinal tissue,the present study drew a conclusion: restraint stress aggravated the damage of ileum in rat with crush injury,and ERS was involved in the process.