Effect Of Restrain Stress On The Damage Of Liver In Rats With Crush Injury And Its Mechanism Of Endoplasmic Reticulum Stress

Objective: In forensic practice, the conditions that all kinds of nonfatalmechanical damage cause MODS even death are common. This situation oftenhappens in fighting, traffic accident, abuse and non-violence forced confession,etc. This kind of injury itself is not serious enough to kill a man, so the causesof death often become intangible or controversial, which leads to the casedragged on and does harm to our society. Moreover, in clinical practice, somepatients’ conditions worsened during treatment of their minor injury, which isdifficult to deal with. Therefore, the scientific problem urgently to be solved isrevealing the exact death mechanisms of nonfatal mechanical damages incritical care medicine and forensic medicine.The effects of this kind of injury on the organism include: firstly, onceunfastened, the crushed limbs suffer ischemia reperfusion injury, release lotsof free radicals, and then cause lipid peroxidation, intracellular calciumoverload, and neutrophil infiltration that release a large number ofinflammatory cytokines and lead inflammatory response in local tissue andremote organs; the second, the stress response induced by tissue injury asphysical stressor; and the last, the stress induced by negative feelings, such as,ache, anxiety, nervous, arised from tissue injury as psychological stressor.Thus, we conclude that the impact of such damage on the body is caused byinjury and stress together. When suffered stress, the body activate thehypothalamic-pituitary-adrenal (HPA) axis and the locus ceruleusnorepinephrine axis (LC/NE) as response to stimulate. The HPA axis andLC/NE axis, as the neuroendocrine systerm, stimulate the secretion of CA andcortisol (corticosterone in rats) into the blood stream, which plays major rolesin stress to protect organism from injury. Though its defense function remainswhen the stress was too strong or lasting too long, its main effect turns to cause disorder of body function and metabolic, and tissue damage. At present,the mechanism of injury caused by stress is unclear.Numerous studies have confirmed that, Endoplasmic reticulum stress(ERS) is one of the important cell responses. The endoplasmic reticulum isresponsible for much of a cell’s protein synthesis and folding, and storage ofthe Ca2+. Stress hormones, oxygen free radical and cytokine can interfere withthe function of endoplasmic reticulum, lead to not/misfolded proteinaccumulation and markable protein of ERS increased, such as GRP78, thusactivating the ERS. ERS is an important part of the adaptive reaction, butwhen the stress is prolonged and excessive, its response turn to cell damage byinducing apoptosis and inflammation tissueLiver is responsible for protein synthesis, biological transformation andimmunity, et al. And liver dysfunction can cause bleeding, infection, renaldysfunction and hepatic encephalopathy and other clinical syndrome.Hepatocytes has a active metabolism function and contains abundantendoplasmic reticulum(ER) which is essential for protein metabolism andstess signal, thus make it to be one of the most vulnerable organs during stress.Lots of studies have shown that ERS may be related to various of liver injury,and our previous studies have demonstrated that restraint stress can aggravatethe liver injury of rats with crush injury, but weather the mechanism isassociated with the ERS is not clear.The crush injury model was recognized commonly as the animal modelto simulate the massive mechanical soft tissue injury; restraint stress modelwas recognized as animal model to simulate the psychological stress. Toexplore the effects of injury and stress on liver injury in case, we will establishthe compound model of restraint stress and crush injury. And on this basic westudy the role of ERS in liver injury to proclaim the mechanism of deathinduced by non-lethal mechanical damage.Methods: Thirty-six male Sprague-Dawley (SD) rats (200g~220g) weredivided into six groups randomly after seven days of adaptive feed undercontrol condition: normal group, fasting water group, restraint stress group, crush group, the compound model group, the compound model+4-PBAgroup.Normal control group rats gave no disposal; Fasting water group ratswere forbiddened to eat and drinking for eight hours in a fix time every day(08:30~16:30); The rats in restraint stress group awere restrained for8h at adefined time period every day (08:30~16:30), without water and food, for7consecutive days; In crush group, the hindlimbs of rats were compressed with24kg standard heavy object for6h; Rats in the compound model group wererestrainted for8h every day for7d, and then at the8thday their hindlimbs ofrats were compressed with24kg standard heavy object for6h; In thecompound model+4-PBA group, the rats were treated with4-PBAadministration by intraperitoneal injection before restraint stress and crush.The liver tissue of rats in normal group, fasting water group and restraintgroup were separated at the end of required processing, and the other threegroup at the3thday after the end of required processing. Part of the liver tissuewere fixed and sections4μm thick were cut and stained with HE and IHC toobserve the morphological change and the expression of GRP78and CHOP.The other part of the liver tissue were saved in ice box of–80℃, thenthrough tissue homogenate and protein quantitative to detect the expressionsof GRP78, CHOP and Caspase3with Western blotting and the changes ofIL-1with ELISA.The data were presented as Mean±SD and analyzed with one wayANOVA and least significant difference test (LSD) by SPSS16.0statisticalprogram. A level of p＜0.05was supposed to be statistically significant.Results：1The pathomorphological changes of liverNo obvious pathological change was observed in normal group and thefasting water group. HE stains showed that a small amount of swollenhepatocytes and inflammatory cells in restrain stress group. In crush group,hepatic cords malalinement with liver cell edema, neutrophils, necrosis ofhepatocytes and fatty change was showed. In the compound group, the most serious pathological changes were showed，including inflammatory cellsinfiltration, hepatic sinusoid hypertrophy and hyperemia. But treatment with4-PBA alleviated the liver injury in rats of the compound model group.2Immunohistochemical stain images of GRP78and CHOP in liver tissueUnder the optical microscope, the positive expression is in the cytoplasm.Apoptosis morphological changes occurred to part of the positive cells: cellshrinkage, nuclear fragmentation and dissolution.Rare positive expression of GRP78and CHOP in normal group and thefasting water group. Positive expression in restraint group and crush groupincreased. Then the expression in the compound model group increasedevidently, compared with crush group. And the expression in rats with4-PBAby intraperitoneal injection decreased, compared with the compound modelgroup.3Dective results of GRP78, CHOP, Caspase3in liver by Western blottingSimilarly, the protein levels of GRP78and CHOP in normal group andfasting water group was few；and their expression were also elevated inrestraint group and crush group; the expression of these proteins increasedobviously (P<0.05); but their expression decreased after4-PBA throughintraperitoneal injection, compared with the compound model group (P<0.05).4The level of IL-1β in liver tissueThere was low expression of IL-1β in normal and fasting water group;the expression in restraint and crush group increased; compared with the crushgroup, the expression of IL-1β in the compound model group increasedevidently (P<0.05); the IL-1β expression decreased in rats with4-PBA byintraperitoneal injection compared with the compound model group (p＜0.05Conclusion:This study successfully established the rat model of restraint stress andcrush injury and its compound model, and then give the rats in compoundmodel group4-PBA to inhibit the ERS. And through a variety of experimentto detect the expression of GRP78、CHOP、Caspase3and il-1，we finallydraw a conclusion: Restraint stress can aggravate liver injury of rats caused by crush injury,whose mechanism may be related to cell apoptosis and the inflammatoryresponse induced by endoplasmic reticulum stress.