Effect of restraint stress on depression-like behaviors in rats after transient focal cerebral ischemic injury

BACKGROUND： Restraint stress is a typical psychophysiological stressor. Simulating the early passion and difficulty in walking of patients after attack of stroke meets onset features.OBJECTIVE： To evaluate the effect of restraint stress on depression-like behaviors in rats after transient focal cerebral ischemic injury, and to investigate the feasibility for its being as modeling method of depression model after stroke.DESIGN： A randomized controlled animal experiment.SETTING： Department of Clinical Medicine, Faculty of Aerospace Medicine of the Fourth Military Medical University of Chinese PLA.MATERIALS： Forty-eight male Sprague-Dawley rats, weighing 240 - 270 g, provided by the Experimental Animal Center of the Fourth Military Medical University of Chinese PLA were used in the current study.METHODS： The experiments were carried out in the Faculty of Aerospace Medicine of the Fourth Military Medical University of Chinese PLA between August 2005 and August 2006. ①Experiment intervention： The rats were randomized into middle cerebral artery occlusion-reperfusion （MCAO） ＋stress group, simple MCAO group, sham-operation ＋ stress group and simple sham-operation group, with 12 rats in each group.Rats in the first two groups were developed into cerebral ischemia/reperfusion models by suture-occluded method. Rats in the MCAO＋stress group were modeled and restraint stress scheme was performed. At week 5 after modeling, the rats were placed in self-made restraining tubes, 2 hours/time, once a day, for 2 successive weeks. The common carotid artery, external and internal carotid arteries of rats in the latter two groups were exposed. The stress way of sham-operation＋ stress group was the same as that of MCAO＋ stress group. ②The neurological status grading and motor performance evaluation （screen test, rota-rod test and balance beam test） were conducted in rats with simple sham-operation group and MCAO group before, 1st and 28^th days after modeling. Depression-like behavior test was performed in the rats of each group by sucrose preference test and open field test at the end of the experiment.MAIN OUTCOME MEASURES： Changes of depression-like behaviors of rats in each group.RESULTS： Forty-eight rats were involved in the experiment. Two rats with meningeal irritation sign were excluded from simple MCAO group, and one rat in the MCAO＋stress group died of some unclear causes during the experiments. The other 45 rats entered the stage of finial analysis. ① Depression-like behavior assessment results： The rats in the MCAO＋ stress group had a significantly decreased preference for sucrose solution, crossing and rearing scores, and increased immobility duration after the 14-day restraint stress,compared with those in other three groups （all P〈0.05）. ②The neurological status grading and motor performance evaluation： There were significant differences in the two indexes of rats in the simple MCAO group before, 1^st and 28^th days after modeling （P〈0.01）, while there was no significant difference before and 28^th days after modeling （P〉0.05）. There were no significant changes in sham-operation group at each time point （P〉0.05）.CONCLUSION： After being exerted restraint stress, the rats with transient focal ischemic injury may show obvious depression-like behaviors. Therefore, restraint stress can be used as a novel animal modeling method for further studying biological mechanism in central nervous system of post-stroke depression animals.

Electroacupuncture pretreatment exhibits antidepressive effects by regulating hippocampal proteomics in rats with chronic restraint stress

The clinical effect of electroacupuncture on depression is widely recognized. However, the signal transduction pathways and target proteins involved remain unclear. In the present study, rat models of chronic restraint stress were used to explore the mechanism by which electroacupuncture alleviates depression. Rats were randomly divided into control, model, and electroacupuncture groups. Chronic restraint stress was induced in the model and electroacupuncture groups by restraining rats for 28 days. In the electroacupuncture group, electroacupuncture pretreatment at Baihui（GV20） and Yintang（GV29） acupoints was performed daily（1 m A, 2 Hz, discontinuous wave, 20 minutes） prior to restraint for 28 days. Open field tests and body weight measurements were carried out to evaluate the depressive symptoms at specific time points. On day 28, the crossing number, rearing number, and body weights of the model group were significantly lower than those in the control group. Behavior test results indicated that rat models of depressive-like symptoms were successfully established by chronic restraint stress combined with solitary raising. On day 28, an isobaric tag for a relative and absolute quantitation-based quantitative proteomic approach was performed to identify differentially expressed proteins in hippocampal samples obtained from the model and electroacupuncture groups. The potential function of these differential proteins was predicted through the use of the Cluster of Orthologous Groups of proteins（COG） database. Twenty-seven differential proteins（uncharacteristic proteins expected） were selected from the model and electroacupuncture groups. In addition to unknown protein functions, COG are mainly concentrated in general prediction function, mechanism of signal transduction, amino acid transport and metabolism groups. This suggests that electroacupuncture improved depressive-like symptoms by regulating differential proteins, and most of these related proteins exist in nerve cells.

Antidepressant-like Effects of Ginsenoside Rg1 in the Chronic Restraint Stress-induced Rat Model

Objective To investigate the ameliorating effect of ginsenoside Rg1 on the depression-like behaviors induced by chronic restraint stress(CRS)in rats and the underlying mechanisms.Methods Forty male Wistar rats were divided into 4 groups according to their baseline sucrose preference:control group,model group,and Rg1-treated groups(5 and 10 mg/kg).Except for control group,the groups were exposed to CRS(6 h/day)for 28 days.All drugs were intraperitoneally administered once daily to CRS rats after restraint stress for 14 days.The behavioral tests were carried out via the open field test(OFT),sucrose preference test(SPT),forced swim test(FST),and the Morris water maze(MWM)4 weeks following CRS induction.The levels of serum corticosterone(CORT)and the activities of the antioxidant defense biomarkers(SOD,MDA and GSH-x)in the prefrontal cortex(PFC)were analyzed using commercial ELISA kits.The levels of the neurotransmitter(5-HT,5-HIAA,Ach,NE,GABA and Glu)in the PFC were measured by ultra-performance liquid chromatography tandem mass spectrometry.The protein expression of BDNF,Trkb,Bax and Bcl-2 in the PFC was detected by western blotting.Results Owing to increased sucrose consumption in the SPT,decreased immobility time in the FST,and the improved cognitive performance in MWM,chronic treatment with Ginsenoside Rg1 was found to significantly attenuate depressionlike behaviors(anhedonia,behavioral despair and poor spatial memory)in rats.Moreover,CRS exposure caused evident alterations in the levels of the neurotransmitters(5-HT,5-HIAA,Ach,GABA and Glu)and the activities of the antioxidant defense biomarkers(SOD,MDA and GSH-x)in the PFC and the levels of corticosterone in serum.However,Ginsenoside Rg1 treatment could restore these levels to normal values.Additionally,Ginsenoside Rg1 treatment significantly reverted the decreased expression of BDNF,Trkb and Bcl-2 and the increased expression of Bax in the PFC of CRS rats.Conclusions Ginsenoside Rg1 could attenuate the CRS-induced depression-like behaviors,in part,by regulating neurotransmitter levels and HPA function,antagonizing oxidative stress and apoptosis,and restoring BDNF-TrkB signaling in PFC.Altogether,our results provide a novel basis regarding the potential therapeutic effects of Rg1 on depression.

Interleukin-18 levels in the hippocampus and behavior of adult rat offspring exposed to prenatal restraint stress during early and late pregnancy

Exposure to maternal stress during prenatal life is associated with an increased risk of neuropsychiatric disorders, such as depression and anxiety, in offspring. It has also been increasingly observed that prenatal stress alters the phenotype of offspring via immunological mechanisms and that immunological dysfunction, such as elevated interleukin-18 levels, has been reported in cultures of microglia. Prenatal restraint stress(PRS) in rats permits direct experimental investigation of the link between prenatal stress and adverse outcomes. However, the majority of studies have focused on the consequences of PRS delivered in the second half of pregnancy, while the effects of early prenatal stress have rarely been examined. Therefore, pregnant rats were subjected to PRS during early/middle and late gestation(days 8–14 and 15–21, respectively). PRS comprised restraint in a round plastic transparent cylinder under bright light(6500 lx) three times per day for 45 minutes. Differences in interleukin-18 expression in the hippocampus and in behavior were compared between offspring rats and control rats on postnatal day 75. We found that adult male offspring exposed to PRS during their late prenatal periods had higher levels of anxiety-related behavior and depression than control rats, and both male and female offspring exhibited higher levels of depression-related behavior, impaired recognition memory and diminished exploration of novel objects. Moreover, an elevated level of interleukin-18 was observed in the dorsal and ventral hippocampus of male and female early-and late-PRS offspring rats. The results indicate that PRS can cause anxiety and depression-related behaviors in adult offspring and affect the expression of interleukin-18 in the hippocampus. Thus, behavior and the molecular biology of the brain are affected by the timing of PRS exposure and the sex of the offspring. All experiments were approved by the Animal Experimentation Ethics Committee at Kunming Medical University, China(approval No. KMMU2019074) in January 2019.

Inhibition of T Cell and Stimulation of B Cell Proliferation by Restraint Stress Mediated by Voltage-Gated Potassium Channel 1.3 Expression

Our previous study has showed that restraint stress inhibits T cell proliferation. Kv1.3 plays a key role in the lymphocyte activation process. Here, we investigate the effects of restraint stress on murine splenic T and B cell proliferation and the role of Kv1.3 in the process. 3H-TdR incorporation is used to determine changes in splenocyte proliferation stimulated by Con A or LPS between control and restraint stress groups. The data shows that restraint stress inhibits T cell and enhanced B cell proliferation. Data from RT-PCR and Western blotting shows that Kv1.3 gene and protein levels are downregulated in T cells and upregulated in B cells in stressed mice. To examine a possible cause-and-effect relationship between Kv1.3 and stress-affected lymphocyte proliferation, we employ various Kv1.3 specific blockers (quinine, 4-AP and TEA) to determine K+ channel function under restraint stress. The data shows that Kv1.3 blockers reverse the decreased T cell proliferation and increase B cell proliferation induced by restraint stress. These results indicate that Kv1.3 mediates restraint stress-induced modulation of T/B lymphocyte proliferation.

A Case of Multiple Hemorrhagic Gastric Ulcers Developed via a Mechanism Similar to Water-Immersion Restraint Stress

In rats, water-immersion restraint stress is a model of experimental ulceration. We encountered a case in which multiple hemorrhagic gastric ulcers formed in the stomach in a setting similar to water-immersion restraint stress. The patient was a 54-year-old man who was found wet on a riverbank and transported by ambulance. Because of hypothermia and renal failure, hemodialysis was performed. Tarry stools were noted and endoscopy revealed the presence of multiple hemorrhagic gastric ulcers;thus, hemostasis was performed end oscopically. During the course, pseudo membranous colitis also developed and was ameliorated with vancomycin. Further, the renal failure and gastric ulcers improved, and the patient was discharged from hospital 25 days later. The reason why he survived more than 2 weeks was the hot summer season and he was not soaked in the river water throughout.

Dip2a regulates stress susceptibility in the basolateral amygdala

Dysregulation of neurotransmitter metabolism in the central nervous system contributes to mood disorders such as depression, anxiety, and post–traumatic stress disorder. Monoamines and amino acids are important types of neurotransmitters. Our previous results have shown that disco-interacting protein 2 homolog A(Dip2a) knockout mice exhibit brain development disorders and abnormal amino acid metabolism in serum. This suggests that DIP2A is involved in the metabolism of amino acid–associated neurotransmitters. Therefore, we performed targeted neurotransmitter metabolomics analysis and found that Dip2a deficiency caused abnormal metabolism of tryptophan and thyroxine in the basolateral amygdala and medial prefrontal cortex. In addition, acute restraint stress induced a decrease in 5-hydroxytryptamine in the basolateral amygdala. Additionally, Dip2a was abundantly expressed in excitatory neurons of the basolateral amygdala, and deletion of Dip2a in these neurons resulted in hopelessness-like behavior in the tail suspension test. Altogether, these findings demonstrate that DIP2A in the basolateral amygdala may be involved in the regulation of stress susceptibility. This provides critical evidence implicating a role of DIP2A in affective disorders.

Preventive effect of estrogen on depression-like behavior induced by chronic restraint stress

Objective To investigate the roles of estrogen and kalirin-7 in chronic restraint stress （CRS）-induced depression and the pathophysiological mechanism of depression. Methods Healthy female mice from Institute of Cancer Research （ICR） were randomly divided into 3 groups： control group, CRS group, and estrogen ＋ CRS group. CRS was used to establish the animal model of depression. Forced swimming test and immunohistochemistry method were utilized to investigate the animal behavior and kalirin-7 expression in the hippocampus, respectively. Results Compared with the control group, the CRS mice displayed depression-like behaviors, including a significant reduction in body weight, a significant increase in immobility time in forced swimming test, and a dramatic decrease in kalirin-7 expression in the hippocampus. However, administration of estrogen attenuated the CRS-induced negative behaviors, and simultaneously increased kalirin-7 expression. Conclusion Estrogen replacement therapy （ERT） could prevent CRS-induced depression-like behaviors in female ICR mice. Besides, kalirin-7 also plays a role in preventing CRS-induced depression-like behaviors.

Antidepressant-like effects of albiflorin involved the NO signaling pathway in rats model of chronic restraint stress

The depressant-like effects of albiflorin(AF) were studied on stressed chronic restraint stress(CRS) rats. Experimental rats were subjected to immobilization stress for a daily 6 h-restraining in a plastic restrainer for continuous 21 d and were treated with 30 or 15 mg·kg-1 of AF for 21 d. Control rats were maintained in completely non stressed conditions. Behavioral tests and biochemical analysis were applied to investigating a regulatory mechanism of anti-stress of AF. Treatment with AF significantly restored the depressant-like behaviors. Besides, AF increased the levels of 5-hydroxytryptophan(5-HT), 5-hydroxyindoleacetic acid(5-HIAA),noradrenaline(NE) and dopamine(DA) in the hippocampus and increased the level of brain-derived neurotrophic factor(BDNF) in serum and protein expression in hippocampus. In addition, AF decreased the levels of hypothalamo-pituitary-adrenal(HPA) cascade,reduced the level of NO and c GMP in serum and inhibited the overexpression of 5-HT2AR m RNA and protein expression. Taken together, AF can modulate the NO-mediated network pathway in the hippocampus against stress-induced depressive-like behaviors.These physiological and behavioral changes allow rats to avoid potential deleterious effects of stress that may result from chronically elevated levels of glucocorticosteroids over days.

Correlation between changes of central neurotransmitter expression and stress response in mice A restraint time-course analysis

BACKGROUND： Changes in central neurotransmitter expression play an important role in stress response and forms the basis for stress-induced psychological and behavior changes. OBJECTIVE： To observe the effects of different restraint stress intervals on brain monoamine neurotransmitter expression, and to investigate the correlation between stress response and neurotransmitter levels. DESIGN： Randomized controlled animal study. SETTING： Chinese Herb and Natural Medicine Institute, Pharmacological College of Jinan University. MATERIALS： Sixty 7-week-old male Kunming mice of clean grade, weighing 18-22 g, were provided by the Guangdong Medical Experimental Animal Center. The experiment was in accordance with animal ethics standards. METHODS： This study was performed at the Chinese Herb and Natural Medicine Institute, Pharmacological College of Jinan University from June 2006 to May 2007. A restraint device for mice was constructed according to published reports. Experimental mice were adaptively fed for 1 week and randomly divided into a control group （n = 10） and an experimental group （n = 50）. The experimental group was sub-divided into five restraint intervals： 4, 8, 12, 18, and 24 hours （n = 10 mice per time point）. Animals in the experimental group were not allowed to eat or drink during the restraint period. Mice in the control group did not undergo restraint, but had identical food and water restrictions. Cerebral cortex and hypothalamus were separated based on observational times and protein was extracted using perchloric acid. Central monoamine neurotransmitter levels were measured using high performance liquid chromatography with electrochemical detection. MAIN OUTCOME MEASURES： Levels of norepinephrine （NE）, dopamine hydrochloride （DA）, 3,4-dihydroxyphen-ylanetic acid （DOPAC）, homovanillic acid （HVA）, 5-hydroxytryptamine （5-HT）, and 5-hydroxyindoleac-etic acid （5-HIAA） in the cerebral cortex and hypothalamus of mice. RESULTS： Sixty mice were included in the final analysis. ① NE levels in the cerebral cortex, hypothalamus, and plasma： four hours after restraint, NE levels in the cerebral cortex and hypothalamus ere significantly lower than control levels （P 〈 0.05）. After 12 hours of restraint, NE levels in the experimental group were significantly higher than in the control group （P 〈 0.05）. At 18 hours of restraint, there was no significant difference in NE levels in the cerebral cortex between the experimental group and the control group （P 〉 0.05）. In addition, NE levels in the plasma gradually increased with longer restraint time, which was significant between experimental groups and the control group （P 〈 0.05-0.01）. ② Levels of DA, DOPAC, and HVA in the cerebral cortex and hypothalamus： there were significant differences in DA levels in the cerebral cortex and hypothalamus after 18 and 24 hours of restraint compared to control animals （P 〈 0.05）. DOPAC and HVA levels in the cerebral cortex were enhanced with longer restraint time, and there was significant difference in all restraint groups compared to control levels （P 〈 0.01）, except for DOPAC levels after 4 hours of restraint. Moreover, DOPAC and HVA levels in the hypothalamus were enhanced with increasing restraint time. Levels of 5-HT and 5-HIAA in the cerebral cortex and hypothalamus： after short restraint periods and in the control group, 5-HT was not detectable. However, it was quantitatively detected at 12 hours after restraint. The 5-HT levels in the cerebral cortex and hypothalamus reached peak levels at 12 and 18 hours of restraint. 5-HIAA levels in the cerebral cortex and hypothalamus showed a similar tendency to increase with restraint time- 5-HIAA levels at 4-8 hours after restraint were significantly higher than control levels （P 〈 0.01）. The 5-HIAA levels decreased at 12 hours after restraint, but remained significantly higher than the control group （P 〈 0.05）. CONCLUSION： Restraint stress affects the hypothalamic-pituitary-adrenal （HPA） axis and causes changes in monoamine neurotransmitters in brain tissues, which suggests stress status could be improved by adjusting HPA axis and neurotransmitter levels in the brain.

Proteomic Analysis of Chronic Restraint Stress-Induced Gan (肝)-Stagnancy Syndrome in Rats

Objective： To analyze the proteomic characteristics of Gan （肝））--ssttaaggnnaannccyy syndrome （（GGSSSS）） by seeking the differential protein in blood and tissues of GSS model rats.Methods： GSS model rats were established by chronic restraint stress,keeping rats in restrain chamber for 6 h every day for 21 successive days.Their blood and liver samples were collected at the end of experiment for differential protein detection with methods of isoelectrofocusing and polyacrylamide SDS-PAGE,silver staining,and scanning.The gel images were analyzed with Imagemaster 2D Elite software,and the excavated differential protein spots were identified with matrix assistant laser resolving TOF mass spectrometry,Western blot,ELISA,and RT-PCR,respectively.Results： A method for isolating the protein in blood serum and tissues by two-dimensional gel electrophoresis was established and optimized.Six serum proteins and three liver proteins that differentially expressed were identified.The down-regulated differential proteins in serum of GSS model rats were serum albumin precursor,beta 1 globin,antibody against muscle acetylcholine receptor,Ig lambda-2 C region,and transthyretin （TTR）,and those in liver tissue were aryl sulfotransferase,enoyl-CoA hydratase,and TTR.TTR down-regulation was found in both serum and liver.Preliminary biological information analysis showed that these differential proteins involved in immune,neuroendocrine,nutrition,and substance metabolism.Conclusion： Proteomic analysis of differential proteins showed that TTR,aryl sulfotransferase,and enoyl-CoA hydratase expressions are downregulated in the GSS model rats,suggesting that the susceptibility of cancer could be enhanced by chronic stress.

Acute Restraint Stress Augments 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Neurotoxicity via Increased Toxin Uptake into the Brain in C57BL/6 Mice

As an environmental risk factor, psychological stress may trigger the onset or accelerate the progression of Parkinson＇s disease （PD）. Here, we evaluated the effects of acute restraint stress on striatal dopaminergic terminals and the brain metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahy- dropyridine （MPTP）, which has been widely used for creating a mouse model of PD. Exposure to 2 h of restraint stress immediately after injection of a low dose of MPTP caused a severe loss of striatal dopaminergic terminals as indicated by decreases in the dopamine transporter protein and dopamine levels compared with MPTP administration alone. Both striatal 1-methyl-4-phenylpyridinium ion （MPP＋） and MPTP concentrations were significantly increased by the application of restraint stress. Striatal monoamine oxidase-B, which catalyzes the oxidation of MPTP to MPP＋, was not changed by the restraint stress. Our results indicate that the enhanced striatal dopaminergic terminal loss in the stressed mice is associated with an increase in the transport of neurotoxin into the brain.

Ela tablets improve sexual performance in mice with erectile dysfunction caused by repeated restraint stress

Ela tablets contain an optimized extract of a Chinese herbal prescription. The original prescription has been used to treat erectile dysfunction(ED), while the detailed indications and specific treatment mechanisms of Ela tablets remain unclear. In the present study, we aimed to investigate whether Ela tablets could protect mice from ED caused by repeated restraint stress and to explore the possible mechanisms. Mice were restrained daily in centrifuge tubes with venting holes for 14 consecutive days and administered Ela tablets at 1 h before restraining. On the 14 th night, mating experiments were conducted. Thereafter, blood, testicular, and penile samples were collected. Serum testosterone levels were measured using a kit. Testicular tissue morphology was observed by H&E staining. The PDE5 expression in the corpus cavernosum was measured by Western blotting analysis. The results indicated that Ela tablets significantly shortened the sexual arousal time, increased the number of sexual encounters, maintained the normal morphology of testicular tissue, increased the serum testosterone levels, and decreased the PDE5 expression in the corpus cavernosum. Our data showed that Ela tablets elicited protective effects on the sexual ability of mice exposed to repeated restraint stress. Moreover, the underlying mechanism was related to the preservation of testicular tissue morphology, maintenance of testosterone levels, and inhibition of penile PDE5 expression.

Hydrogen sulfide attenuates gastric mucosal injury induced by restraint water-immersion stress via activation of KATPchannel and NF-κB dependent pathway

AIMTo explore the effect of hydrogen sulfide (H2S) on restraint water-immersion stress (RWIS)-induced gastric lesions in rats and the influence of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway on such an effect.METHODSMale Wistar rats were randomly divided into a control group, a physiological saline (PS) group, a sodium hydrosulfide (NaHS) group, a glibenclamide (Gl) group, Gl plus NaHS group, a pyrrolidine dithiocarbamate (PDTC) group, and a PDTC plus NaHS group. Gastric mucosal injury was induced by RWIS for 3 h in rats, and gastric mucosal damage was analyzed after that. The PS, NaHS (100 μmol/kg body weight), Gl (100 μmol/kg body weight), Gl (100 μmol/kg or 150 μmol/kg body weight) plus NaHS (100 μmol/kg body weight), PDTC (100 μmol/kg body weight), and PDTC (100 μmol/kg body weight) plus NaHS (100 μmol/kg body weight) were respectively injected intravenously before RWIS.RESULTSRWIS induced serious gastric lesions in the rats in the PS pretreatment group. The pretreatment of NaHS (a H2S donor) significantly reduced the damage induced by RWIS. The gastric protective effect of the NaHS during RWIS was attenuated by PDTC, an NF-κB inhibitor, and also by glibenclamide, an ATP-sensitive potassium channel blocker, in a dose-dependent manner.CONCLUSIONThese results suggest that exogenous H2S plays a protective role against RWIS injury in rats, possibly through modulation of KATP channel opening and the NF-κB dependent pathway.

Nervous mechanisms of restraint water-immersion stress-induced gastric mucosal lesion

Stress-induced gastric mucosal lesion(SGML)is one of the most common visceral complications after trauma.Exploring the nervous mechanisms of SGML has become a research hotspot.Restraint water-immersion stress(RWIS)can induce GML and has been widely used to elucidate the nervous mechanisms of SGML.It is believed that RWIS-induced GML is mainly caused by the enhanced activity of vagal parasympathetic nerves.Many central nuclei,such as the dorsal motor nucleus of the vagus,nucleus of the solitary tract,supraoptic nucleus and paraventricular nucleus of the hypothalamus,mediodorsal nucleus of the thalamus,central nucleus of the amygdala and medial prefrontal cortex,are involved in the formation of SGML in varying degrees.Neurotransmitters/neuromodulators,such as nitric oxide,hydrogen sulfide,vasoactive intestinal peptide,calcitonin gene-related peptide,substance P,enkephalin,5-hydroxytryptamine,acetylcholine,catecholamine,glutamate,γ-aminobutyric acid,oxytocin and arginine vasopressin,can participate in the regulation of stress.However,inconsistent and even contradictory results have been obtained regarding the actual roles of each nucleus in the nervous mechanism of RWIS-induced GML,such as the involvement of different nuclei with the time of RWIS,the different levels of involvement of the sub-regions of the same nucleus,and the diverse signalling molecules,remain to be further elucidated.

Restraint-Induced Expression of Endoplasmic Reticulum Stress-Related Genes in the Mouse Brain

Depression is a significant public health concern but its pathology remains unclear. Previously, increases in an endoplasmic reticulum (ER) stress-related protein were reported in the temporal cortex of subjects with major depressive disorder who had died by suicide. This finding suggests an association between depression and ER stress. The present study was designed to investigate whether acute stress could affect the ER stress response. Mice were immobilized for a period of 6 hr and then expression of ER stress response-related genes was measured by real-time PCR. We also used enzyme-linked immunosorbent assay for concomitant measurement of the plasma corticosterone levels in the mice. The effect of corticosterone on ER stress proteins was further investigated by treating mice with corticosterone for 2 weeks and then measuring ER protein expression by Western blotting. After a 6 hr restraint stress, mRNA levels of ER stress-related genes, such as the 78-kilodalton glucose regulated protein (GRP78), the 94-kilodalton glucose regulated protein (GRP94), and calreticulin, were increased in the cortex, hippocampus, and striatum of mouse brain. Blood plasma corticosterone level was also increased. In the corticosterone-treated mouse model, the expression of GRP78 and GRP94 was significantly increased in the hippocampus. These results suggest that acute stress may affect ER function and that ER stress may be involved in the pathogenesis of restraint stress, including the development of depression.

4T1乳腺癌细胞株接种联合慢性束缚应激诱导乳腺癌合并抑郁小鼠模型构建探索

目的研究4T1乳腺癌细胞株接种联合慢性束缚应激方法构建乳腺癌合并抑郁症小鼠模型核心行为症状、生物学指标、病理学等改变。方法BABL/c小鼠随机分为正常(Control)组、束缚(Stress)组、移植瘤(Tumor)组和束缚移植瘤(Stress+Tumor,S+T)组,将4T1细胞株接种于Tumor和S+T组小鼠腋下,待成瘤后,给予Stress和S+T组小鼠束缚应激21 d。造模期间监测各组小鼠体重、摄食量。实验结束后,采用糖水偏好、旷场、高架十字迷宫、强迫游泳等试验评价各组小鼠抑郁样行为。小鼠处死后,测量小鼠瘤体重量和体积;采用ELISA方法检测各组小鼠血清肿瘤标志物糖类抗原(CA199)、癌胚抗原(CEA)、血管内皮生长因子(VEGF)以及相关神经递质5-羟色胺(5-HT)、去甲肾上腺素(NE)、皮质酮(CORT)的含量;运用HE染色法观察小鼠肿瘤和海马组织病理学改变。结果S+T组小鼠体重、摄食量显著降低,瘤体重量和体积明显增大,血清肿瘤标志物(CA199、CEA、VEGF)水平显著升高,快感和对新环境的探索欲望减弱,紧张和绝望行为显著增强,血清神经递质5-HT和NE水平显著降低,CORT水平显著升高;另外肿瘤组织细胞排列松散,间质减少,病理性核分裂象增多,海马CA3区神经元细胞排列和形态紊乱,核内空泡化样改变明显。结论4T1乳腺癌细胞株接种联合慢性束缚应激诱导的乳腺癌合并抑郁小鼠模型出现了乳腺癌和抑郁症双重典型症状和生物学指标改变,可为乳腺癌合并抑郁实验研究提供较好的模型参考。

基于TGF-β1/CD147信号探讨慢性束缚应激促小鼠乳腺癌进展及逍遥散调节机制研究

目的基于TGF-β1/CD147信号探讨慢性束缚应激促小鼠乳腺癌进展及逍遥散调节机制。方法40只BABL/c小鼠随机分为移植瘤组(Tumor)、模型组(Model)、逍遥散组(Xiaoyaosan)和米非司酮组(Mifepristone),将4T1细胞株接种于各组小鼠腋下,待成瘤后,除Tumor组外其余各组小鼠均进行慢性束缚应激21天,同时Xiaoyaosan组和Mifepristone组小鼠给予相对应的药物灌胃,Tumor组和Model组小鼠灌胃生理盐水。造模结束后,小鼠麻醉断头处死,测量小鼠瘤体重量和体积、内脏指数;采用ELISA方法检测各组小鼠血清肿瘤标志物糖类抗原ca199(Carbohydrate antigen199,CA199)、癌胚抗原(Carcino-embryonic antigen,CEA)、血管内皮生长因子(Vascular endothelial growth factor,VEGF)的含量,血清多巴胺(Dopamine,DA)和皮质酮(Corticosterone,CORT)的含量,以及肿瘤组织转化生长因子β1(Transforming growth factor-β1,TGF-β1)和白介素10(Interleukin 10,IL-10)的含量。采用免疫组化和Western blot方法检测各组小鼠肿瘤组织巨噬细胞极化标志物诱导型一氧化氮合酶(Inducible nitric oxide synthase,iNOS)和精氨酸酶1(Arginase-1,Arg-1)的表达,以及肿瘤组织中细胞外基质金属蛋白酶诱导剂(Extracellular matrix metalloproteinase inducer,EMMPRIN,CD147)及其下游信号分子基质金属蛋白酶2(Matrix metalloproteinases 2),MMP2、基质金属蛋白酶9(MMP9)和VEGF的表达。结果与Tumor组比较,Model组小鼠肿瘤重量和体积,血清CA199、CEA、VEGF、CORT含量,肿瘤TGF-β1和IL-10含量均显著增加;内脏指数和血清DA含量显著减少;肿瘤巨噬细胞M2型极化标志物Arg-1的表达显著增加,M1型极化标志物iNOS的表达显著降低;肿瘤CD147及其下游信号分子MMP2、MMP9和VEGF蛋白表达显著增加,逍遥散和米非司酮均可有效逆转以上改变。结论慢性束缚应激促小鼠乳腺癌进展的机制与肿瘤相关巨噬细胞M2型极化释放TGF-β1增加、激活CD147及其下游相关信号有关,而逍遥散可缓解应激条件下皮质酮增高引起的巨噬细胞M2型极化,减少TGF-β1的生成,抑制CD147及其下游信号,从而抑制慢性应激引起的小鼠乳腺癌进展。

滋水清肝饮对慢性束缚应激抑郁小鼠海马ERK、GSK3β、CREB、BDNF蛋白表达的影响

目的基于ERK/GSK-3β/CREB/BDNF信号通路探究滋水清肝饮对慢性束缚应激(CRS)抑郁小鼠的作用。方法除空白组外,其余小鼠建立CRS抑郁模型,造模成功后分为模型组、盐酸氟西汀组(10 mg/kg)和滋水清肝饮低、中、高剂量组(8.835、17.670、35.340 g/kg),给予相应剂量药物干预,同时继续进行CRS处理。给药7、14 d进行糖水偏好实验及行为学实验。给药14 d后,HE染色观察小鼠海马组织形态学改变,采用试剂盒检测血清超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,ELISA法检测血清5-羟色胺(5-HT)、肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)水平,RT-qPCR法检测海马组织BDNF、TNF-α、IL-1βmRNA表达,Western blot法检测海马组织ERK1/2、p-ERK1/2、GSK3β、p-GSK3β、CREB、BDNF蛋白表达。结果与模型组比较,给药14 d后,盐酸氟西汀组和滋水清肝饮中剂量组小鼠糖水偏好率升高(P<0.01),悬尾不动时间和强迫游泳不动时间缩短(P<0.01),海马组织神经细胞损伤有所改善,血清MDA、TNF-α、IL-1β水平降低(P<0.05,P<0.01),SOD活性和5-HT水平升高(P<0.05,P<0.01),海马组织TNF-α、IL-1βmRNA表达降低(P<0.01),BDNF mRNA和p-ERK1/2、p-GSK3β、CREB、BDNF蛋白表达升高(P<0.05,P<0.01)。结论滋水清肝饮可改善慢性束缚应激小鼠抑郁样行为,其机制可能与调节小鼠海马ERK/GSK3β/CREB/BDNF信号通路有关。

束缚应激致高脂血症ApoE^(-/-)小鼠心肌损伤的分子靶标筛选与机制分析

目的采用蛋白质组学技术筛选慢性束缚应激致高脂血症小鼠心肌损伤的标志物及其潜在机制。方法通过束缚ApoE-/-小鼠建立高脂血症合并慢性应激模型,运用蛋白质组学与生物信息学等技术描绘慢性应激对高脂血症小鼠心肌损伤的特征性分子改变及相关调控机制,并从中探索潜在的诊断标志物。结果蛋白质组学分析结果显示,与高脂血症组相比,高脂血症合并束缚应激组小鼠共有43个显著上调与58个显著下调的差异表达蛋白质。其中,GBP2、TAOK3、TFR1、UCP1是极具诊断潜力的分子标志物。KEGG通路富集分析结果表明,铁死亡是加剧高脂血症合并束缚应激模型心肌损伤的重要机制通路。mmu_circ_0001567/miR-7a/Tfr-1和mmu_circ_0001042/miR-7a/Tfr-1可能是该模型中与铁死亡相关的重要circRNA-miRNA-mRNA调控网络。结论慢性束缚应激可能通过铁死亡加剧高脂血症小鼠的心肌损伤,本研究筛选出4个具有潜在应用价值的心肌损伤分子诊断标志物,为高脂血症合并应激致心脏性猝死的研究提供了新的方向。