| Background and Objectives:Acetaminophen (called paracetamol outside the United States) is a popular and widely used analgesic and antipyretic agent. Over300different preparations are now available in the United States with more than one billion pills sold annually. Although it is remarkably safe when used at usual therapeutic doses, it has a relatively narrow therapeutic window. Acetaminophen overdose is the leading cause for calls to Poison Control Centers (over100,000/yr), and accounts for more than56,000emergency room visits,2600hospitalizations and estimated458deaths due to acute liver failure (ALF) each year. Data from the U.S. Acute Liver Failure Study Group registry of more than1100patients with acute liver failure (ALF) from across the United States, suggests that acetaminophen poisoning alone currently constitutes nearly50%of all ALF. In January2011, the U.S. Food and Drug Administration (FDA) was asking manufacturers of prescription combination products that contain acetaminophen to limit the amount of acetaminophen to no more than325milligrams (mg) in each tablet or capsule. In February2014, FDA urged doctors to discontinue prescribing drugs that contain more than325milligrams of acetaminophen per tablet or capsule, to reduce the risk of liver injury. While the dose of acetaminophen ingested is clearly important in the development of hepatotoxicity, a number of other risk factors can severely aggravate liver injury induced by APAP, for example, chronic alcohol use, malnutrition and the concurrent intake of some medicinal agents. The effects of stress on APAP remain unexplored by now.Stress is an ever-present part of modern life. The general concept of "stress" describes the state of a living organism of the non-specific response of the body to any demand for change when, under the influence of internal or external stimuli or "stressors". Stressors can include physical or mental forces or combinations of both. The adaptive response to stressors comprises the activation of the hypothalamic-pituitary-adrenal (HPA) axis and components of the sympathoadreno-medullary (SAM) system, releasing the key peripheral mediators, i.e. glucocorticoids and catecholamines. For a long time, stress had been implicated as a cofactor in the severity and progression of a number of diseases. The current focus on the stress and disease phenomenon is directed towards the interactions of the immune system, the CNS, cardiovascular disease, liver injury, HIV/AIDS and carcinomas. Studies reveal that stress plays a role in suppressing functions of specific immunity, in triggering or worsening depression, cardiovascular disease, and liver injury and in speeding the progression of HIV/AIDS and carcinomas.Today, growing evidence has shown that stress can have an effect on liver disease and liver function, both in human and animal studies. Some early clinical reports suggested that psychosocial stress might affect the initiation, course and outcome of liver diseases. For example, Hirose et al revealed that emotional stress, such as that induced by hypnotic suggestion of’fear’ and ’anxiety’, significantly decreased hepatic blood flow (HBF). Fukudo et al demonstrated a significant positive correlation between the severity of psychosocial stress and the exaggeration of inflammatory and fibrosing changes in alcoholic hepatitis. A significant positive correlation between the alanine aminotransferase (ALT) level and the degree of depression, measured by the short form of the Beck Depression Inventory, was found in patients suffering from chronic hepatitis B. Moreover, the type I personality scales of Grossarth-Maticek have been associated with the severity of chronic hepatitis C, even after adjustment for the confounding factors of age, sex, education level, smoking, drinking, and duration of illness. In orthotropic liver transplantation, patients with acute rejection were significantly more likely to have the preoperative personality traits’submissive’,’relatively controlled’, and ’of indifferent mood’ than patients without acute rejection.Such a close interaction between stress and liver diseases is also suggested by basic research using several animal models. For example, electric foot-shock stress exacerbated liver injury in rats treated with carbon tetrachloride (CCl4), an animal model of drug induced liver injury. More recently, this stress paradigm has been reported to aggravate a-galactosylceramide-induced hepatitis, which is related to malaria and Salmonella infection induced liver injury and viral hepatitis B and C. After exposure to stress, electron microscopy of the liver revealed rough endoplasmic reticulum fragmentation and dilatation, glycogen depletion, and mitochondrial enlargement. The most striking change, however, was an increase in the number and size of autophagic vacuoles which were limited by single or multiple membranes. Interestingly, even in normal rodents, restraint and electric foot-shock stress triggered mild liver injury, which is defined by slightly elevated ALT levels. Moreover, social isolation stress elevated the incidence of spontaneous hepatocellular carcinoma in transforming growth factor-a transgenic mice and accelerated the development of liver metastasis in the colon of carcinoma cell-injected mice.Animal immobilization or restraint is known to be a straightforward, painless and convenient model to induce both psychological and physical stress. Immobilization or restraint can induce psychological escape reaction and physical muscle work. Compared to the stress induction achieved via drug administration, surgical interventions, extremes of temperature or acoustic stimulation, restraint allows for efficiently examining changes in levels of adrenocorticotropic hormones, corticosterone, fos-protein, desensitization of HPA response and expression of cytokines. A number of frank behavioral responses such as learning and conditioning, food intake or responses to acoustic startle and similar environmental challenges can readily be incorporated along with studies. In the recent15years,"restraint stress" continues to dominate stress-induced methodology.The aim of the present study was:①To assess the effects and the potential mechanisms of restraint stress on the progression of liver injury in mice treated with APAP.②To investigate the potential involvement of catecholamines and adrenoceptors in the regulation of acute restraint stress-induced liver injury.Methods and materialsAdult male BALB/c mice (8-10weeks old) were obtained from the Academy of Military Medical Science (Beijing, China). All animals were housed in an environmentally controlled room with a12h light/dark cycle and allowed free access to food and water. The temperature in the colony room was maintained at19-26℃. The mice were allowed to acclimatize themselves to the colony for3days before the experiments began. Animals were fasted overnight before the experiments. On the day of the experiment (starting at8:00AM), mice were introduced into a restraint tube for varying periods. The restraint tube was well ventilated and prevented animals from turning or ambulation, but it did not squeeze the mice. After restraint, mice were immediately sacrificed (see later) or returned to individual cages at room temperature for recovery. During the recovery period, food and water were restored for mice. Both control and restrained mice were euthanized with sodium pentobarbital either immediately after restraint or at specific time points after cessation of stress. Blood samples were collected from the abdominal aorta. The liver was removed and rinsed in saline. The left lateral Lobe of liver was fixed in10%phosphate-buffered formalin and embedded in paraffin for histological analyses. The remaining liver was snap-frozen in liquid nitrogen and stored at-80℃.For APAP administration experiments, animals were given intraperitoneally (i.p.)150mg/kg APAP or vehicle at specific time points after restraint stress. Four hours after drug administration, animals were euthanized for blood sampling. For pharmacological manipulations of catecholamines and adrenoceptors experiments, Prazosin (1mg/kg) was administered intraperitoneally30min before restraint stress to block α-1receptors. Yohimbine (2mg/kg) was administered intraperitoneally30min before restraint stress to block a-2receptors. Betaxolol (20mg/kg) was administered intraperitoneally30min before restraint stress to block β-1receptors. ICI118,551(5mg/kg) was administered intraperitoneally30min before restraint stress to block β-2receptors. Reserpine (2mg/kg) was subcutaneously injected24h prior to restraint stress in order to achieve a permanent depletion of catecholamines.Serum alanine transaminase (ALT) and aspartate transaminase (AST) were then determined using commercial test kits. Total soluble GSH and GSSG were measured in the liver homogenate using the enzymatic recycling method. Serum IL-6and TNF-a levels were measured by using an enzyme linked immunoassay kit. Sections of liver were stained with hematoxylin and eosin (H&E) for routine histological examinations and morphometric analyses. Apoptosis detection was performed using a TUNEL peroxidase apoptosis detection kit. Immunohistochemical detection of F4/80protein was performed in sections of formalin-fixed, paraffin embedded livers for the detection of Kupffer cells. The expression of caspse-3、Caspase-9、Bcl-2and Bax in the liver were detected by Western blot analysis.StatisticsThe data were analyzed with SPSS13.0. All results were expressed as mean±SD. Comparisons between multiple groups in single-factor experiment were performed with one-way ANOVA followed by a post hoc LSD test. If the data were not normally distributed, we used the Dunnett’S T3test for the comparison by nonparametric ANOVA. Two-way ANOVA was used to analyze two-factor interaction effects. Results were considered significant when p-values were less than0.05.Results:Experiment I:Establishment of restraint stress model in mice1.When mice were subjected to restrain for0.5,1.5or3h, serum ALT and AST activities continued to increase. However, extending restraint to6h started to decrease serum enzyme activities. In different restraint periods, both ALT and AST activities reached or exceeded3times the upper limit of normal after3h of restraint. According to "Hy’s Law" adopted by the U.S. Food and Drug Administration (FDA), severe hepatocellular injury has been defined as being cases where the serum ALT or AST is>3times the upper limit of normal.2. Serum ALT and AST reached maximum after3h of restraint. When the animals subjected to restrain3h were liberated, their ALT and AST activity progressively decreased. Significant differences between ALT and AST levels in the restraint group and those of the control group were also observed at6h after cessation of stress. ALT activity in the restraint3h group returned to control values24hours later, but AST activity remained elevated.3. Similar to plasma ALT and AST activities, restrained animals exhibited progressive changes in hepatic GSH (GSH+GSSG), GSSG and GSH/GSSG ratio. Hepatic GSH and GSH/GSSG content were significantly higher after1.5and3h of restraint in the restrained group than that of unrestrained animals (p<0.05or p<0.01). However, restrained animals exhibited significantly lower GSSG content after1.5and3h of restraint compared with unrestrained animals (p<0.01).4.Histopathological examinations revealed that there was no obvious liver lesion in the restraint mice compared to the control. Only mice exposure to3h restraint showed mild hepatic degeneration with the presence of slight cytoplasmic relaxation.ExperimentⅡ:Effect of restraint stress on APAP induced liver injury1.A dose of150mg/kg APAP was administered to mice subjected to restraint0.5,1.5or3h and recovery6or24h after restraint3h. Recovery6h after restraint3h significantly potentiated the APAP induced elevation of ALT and AST levels (P<0.05), and3h of restraint exhibited a higher ALT and AST levels, which was4times of that seen in mice receiving the same dose but without restraint. Animals restrained for other time points had a minimal effect on APAP induced change of ALT and AST levels, revealed that the enhancement of APAP induced liver injury was dependent on the duration of restraint.2.Histopathological examinations revealed that livers were normal in appearance with no change in the lobular architecture in the control. In the APAP group, limited hepatic necrosis was seen in the centrilobular regions with cytoplasmic vacuolization, but hepatocytes in the centrilobular regions showed significant and extensive centrilobular necrosis with vacuolization and congestion following restraint3h plus acetaminophen treatment.3. The mice restrained3h showed significantly higher hepatic GSSG content and GSH/GSSG than that of unrestrained animals, and GSH+GSSG content were significantly lower than that of unrestrained animals. The mice exposed to restraint3h exhibited a higher hepatic GSSG content and GSH/GSSG and lower GSH+GSSG content than that seen in mice receiving the same dose but without restraint (P<0.05or P<0.01)4. In restraint3h plus APAP group, there were no significant differences in serum IL-6and TNF-α levels compared to APAP group without restraint. Similarly, no significant differences were noted in the density of F4/80-positive Kupffer cells between them.5.IL-6and TNF-a activity showed different profiles after restraint3h. IL-6activity was statistically significantly increased after exposure to3h restraint stress, while no increase in TNF-a activity was found following restraint. Similarly, no significant differences were noted in the density of F4/80-positive Kupffer cells between control and restrained mice.6. As to both restraint3h plus APAP group and APAP group, none of them showed any processing of the procaspase-3to its active fragment. The level of active caspase-3was elevated in the liver tissue of mice subjected to3h of restraint compared to unrestraint.ExperimentⅢ:Potential mechanisms of restraint induced liver injury1. Treatment with reserpine, prazosin, yohimbine, betaxolol and ICI118551did not influence ALT and AST activities of unrestrained control mice compared with vehicle control group. In a state of a generalized catecholamine depletion produced by reserpine, the elevation of ALT or AST activities induced by restraint were significantly less than control animals (p<0.05). After blockade of α-1or a-2adrenoceptor by prazosin or yohimbine, the magnitude of the increase in ALT and AST activities induced by restraint had a similarly predominant drop compared with control animals (p<0.05), which were similar to the effects of reserpine. However, no changes were observed in ALT and AST activities induced by restraint stress after β-1or β-2adrenergic receptor antagonist treatment (betaxolol or ICI118,551). Regardless of drug treatment, ALT and AST activities of restrained groups were significantly higher than those of corresponding control groups (p<0.05or p<0.01).2. Treatment with reserpine, prazosin, yohimbine, betaxolol and ICI118,551did not affect hepatic GSH (GSH+GSSG), GSSG and GSH/GSSG ratio of unrestrained control mice compared with vehicle control group. After catecholamine depletion by reserpine or blockade of a-lor a-2adrenoceptor by prazosin or yohimbine, the magnitude of the increase in GSSG content and GSH/GSSG ratio and the decrease in GSH content induced by restraint were statistically attenuated compared with that of control animals (p<0.01), and these drugs had the same attenuating effect. However, hepatic total GSH, GSSG and GSH/GSSG ratio of restrained animals treated with drugs (reserpine, prazosin, or yohimbine) still significantly different from corresponding control animals (p<0.05or p<0.01). Changes of hepatic glutathione content induced by restraint were not affected by beta-1or beta-2adrenergic receptor antagonists (betaxolol or ICI118,551).3.Liver sections were stained with the TUNEL assay to evaluate the effects of prazosin and yohimbine on hepatocellular apoptosis. After restraint stress for3h, apoptotic hepatocytes were frequently observed, while no apparent apoptotic cell was found in the liver tissue of unrestrained mice. Quantitative analysis of TUNEL positive hepatocytes showed that the number of apoptotic hepatocyte in mice exposed to3h restraint was significantly higher than that in unrestrained control mice (p<0.01). Treatment with prazosin and yohimbine reduced the number of apoptotic cells in the liver and the two agents exhibited a similar inhibiting effect on the hepatocellular apoptosis.4. Restraint stress obviously increased the expression of cleaved forms of caspase-3, the proapoptotic factor Bax and the activated form of caspase-9, and decreased the expression of the antiapoptotic factors Bcl-2when compared with control group, leading to a high Bax/Bcl-2ratio. In the prazosin and yohimbine treatment group, the above factors induced by restraint were attenuated, but these factors were still unable to return to normal level of the unrestraint group.Conclusion1. Restraint stress can exacerbate acetaminophen-induced liver injury as indicated by an increase in necrotic hepatic tissue and serum aminotransferase activity. Reduction in hepatic GSH content and disruption of the balance between oxidants and antioxidants in live, but not releasing of inflammatory cytokines, are responsible for such stress induced aggravation of APAP induced liver injury. In addition, necrosis but not apoptosis is the principal mechanism of hepatocytes death after exacerbation of APAP induced liver injury by restraint stress.2. Restraint stress can induce reversible liver injury by reducing hepatic GSH content and promote hepatocytes apoptosis through caspase-9and Bcl-2family of apoptotic regulatory proteins.3.α-1and α-2adrenoceptors mediate restraint-induced liver. |
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