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.

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

AIM To 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.METHODS Male Wistar rats were randomly divided into a control group,a physiological saline(PS)group,a sodium hydrosulfide(Na HS)group,a glibenclamide(Gl)group,Gl plus Na HS group,a pyrrolidine dithiocarbamate(PDTC)group,and a PDTC plus Na HS group.Gastric mucosal injury was induced by RWIS for 3 h in rats,and gastric mucosal damage was analyzed after that.The PS,Na HS(100μmol/kg body weight),Gl(100μmol/kg body weight),Gl(100μmol/kg or 150μmol/kg body weight)plus Na HS(100μmol/kg body weight),PDTC(100μmol/kg body weight),and PDTC(100μmol/kg body weight)plus Na HS(100μmol/kg bodyweight)were respectively injected intravenously before RWIS.RESULTS RWIS induced serious gastric lesions in the rats in the PS pretreatment group.The pretreatment of Na HS(a H2S donor)significantly reduced the damage induced by RWIS.The gastric protective effect of the Na HS 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.CONCLUSION These 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.

Altered Neuronal Activity in the Central Nucleus of the Amygdala Induced by Restraint Water-Immersion Stress in Rats

Restraint water-immersion stress(RWIS), a compound stress model, has been widely used to induce acute gastric ulceration in rats. A wealth of evidence suggests that the central nucleus of the amygdala(CEA) is a focal region for mediating the biological response to stress. Different stressors induce distinct alterations of neuronal activity in the CEA; however, few studies have reported the characteristics of CEA neuronal activity induced by RWIS. Therefore, we explored this issue using immunohistochemistry and in vivo extracellular single-unit recording. Our results showed that RWIS and restraint stress(RS) differentially changed the c-Fos expression and firing properties of neurons in the medial CEA. In addition,RWIS, but not RS, induced the activation of corticotropinreleasing hormone neurons in the CEA. These findings suggested that specific neuronal activation in the CEA is involved in the formation of RWIS-induced gastric ulcers.This study also provides a possible theoretical explanation for the different gastric dysfunctions induced by different stressors.

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.

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.

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.

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.

Retigabine alleviates chronic restraint stress -induced memory retrieval impairment in male mice

Aim To investigate whether Kv7 channels opener retigabine could alleviate memory impairment induced by chronic restraint stress （CRS） and the underlying neuroprotective mechanisms. Methods Adult male Kunming （KM） mice, weighing 20 - 25 g, were restrained in well-ventilated Plexiglass tubes for 6 h daily beginning from 10 ： 00 to 16 ： 00 for 21 consecutive days. Mice were injected with retigabine （ 10 mg · kg^-1） or vehicle （ 10% DM- SO） 30 rain before restraint stress for 21 days. After stressor cessation, the spatial learning and memory was deter- mined by Morris water maze test, the levels of p-Akt, p-GSK-3β and p-Erkl/2 of hippocampal tissues were exam- ined by western blot. Results Compared with control group, CRS mice exhibited significantly longer escape laten- cies on day 2, 3 and 4 （P 〈 0.05, P 〈 0. 01, P 〈 0.01 ） respectively, but retigabine （ 10 mg · kg^-1） treatment had no influences on escape latencies compared with CRS group. During the probe test, CRS mice spent significant less time in target quadrant than control group （P 〈 0.01 ）. Compared with CRS group, retigabine （ 10 mg · kg^-1 ） treatment increased the time spent in target quadrant （P 〈 0.01 ）. Additionally, the swimming speed showed no significant differences among groups. Western blot results showed that the levels of p-Akt, p-GSK-3β and p-Erkl/ 2 in the hippocampus of CRS mice were significantly decreased compared with control group. Compared with CRS group, retigabine （ 10 mg· kg^-1） treatment strongly prevented the reduction of p-Akt and p-GSK-3 β （P 〈 0.01 ）, but had no effect on the reduction of p-Erkl/2. Conclusion Retigabine protected against CRS-induced spatial memory retrieval impairment partly via activation of Akt/GSK-3β signaling pathway.

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.

Chronic restraint stress impairs ischemia-induced neovascularization through DPP-4-dependent pathway

Background and Objective The morbidity of lower extremity atherosclerotic disease(LEAD)has increased year by year.Chronic stress attenuates the ability of angiogenesis and deteriorates the prognosis of LEAD.Chronic stress increases plasma and tissue DPP-4 activities in mice.DPP-4 plays an important role in angiogenesis.Therefore,we hypothesized that chronic stress exerted its cardiovascular effects by increasing DPP-4 activation.We investigated the role of DPP-4/GLP-1 axis in ischemiainduced neovascularization in mice under restraint stress.

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。

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。

岩体地基抗剪刚度系数试验研究

地基抗剪刚度系数反映了地基对上部结构在水平方向的约束强弱程度,直接影响地基上浇筑的混凝土结构温度约束应力的大小,是控制混凝土底板温度裂缝的关键参数之一。为完善温度约束应力计算中地基抗剪刚度系数的取值,基于岩体地基防水层底板温度应力监测现场试验,计算得到岩体地基防水层构造条件下地基抗剪刚度系数取值;而后进行了岩体地基上的防水层、垫层、滑动层3种构造条件的动力特性测试试验,基于量纲分析计算出了垫层和滑动层构造条件下的地基抗剪刚度系数取值。

慢性束缚应激对小鼠小肠屏障的损伤研究

肠屏障由机械屏障、免疫屏障、化学屏障和生物屏障共同组成,在维持肠腔内环境稳态和肠上皮结构完整性等方面发挥重要作用.首先建构小鼠慢性束缚应激模型并进行血浆激素水平检测和小鼠旷场实验,验证模型是否建构成功.随后通过常规组织学染色、免疫组化、 TUNEL免疫荧光、 ELISA等方法研究慢性束缚应激对小鼠小肠的损伤作用.结果显示,慢性束缚应激显著提高小鼠血浆NE和CORT水平(p<0.05),降低小鼠小肠绒毛高度(V,p<0.05)、增加隐窝深度(C,p<0.05),降低V/C比值(p<0.05)及紧密连接蛋白ZO-1表达量(p<0.01);显著减少小肠杯状细胞和内皮单核淋巴细胞数量(p<0.01),提示慢性束缚应激造成了小鼠肠道损伤和免疫功能抑制.进一步研究发现,慢性应激小鼠肠道内PCNA阳性表达显著减少(p<0.01)、凋亡细胞显著增多(p<0.01);血浆中TNF-α,IL-1β,IL-18含量显著升高(p<0.01),而IL-10显著降低(p<0.01).血浆氧化-抗氧化指标结果显示,慢性束缚应激显著增加小鼠血浆MDA含量(p<0.05),降低T-AOC,SOD,CAT,GSH-Px(p<0.05)活性,提示慢性束缚应激诱导机体氧化应激.该研究表明慢性束缚应激通过引发机体氧化应激,造成肠上皮细胞更新抑制和肠道屏障结构损伤,加重机体炎症反应.

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。

辅酶Q10通过下调焦亡信号通路缓解抑郁小鼠的抑郁样行为

目的探讨辅酶Q10对慢性应激束缚模型(CRS)抑郁小鼠的神经保护作用及其机制。方法采用慢性应激束缚模型制备抑郁小鼠。将小鼠分为空白组(CON)、CRS组、单用辅酶Q10高剂量组(Q10-H,200 mg/kg)、模型组+辅酶Q10低剂量组(CRS+Q10-L,50 mg/kg)、模型组+辅酶Q10中剂量组(CRS+Q10-M,100 mg/kg)、模型组+辅酶Q10高剂量组(CRS+Q10-H,200 mg/kg)、模型组+caspase-1特异性抑制剂VX765组(CRS+VX765,50 mg/kg)、模型组+氟西汀组(CRS+FLX,10 mg/kg)(n=8)。应用糖水偏好实验、强迫游泳实验、悬尾实验分析小鼠抑郁样行为;采用免疫组化检测胶质纤维酸性蛋白(GFAP)阳性率;高尔基染色检测神经元突触棘数量;免疫印迹实验检测海马脑区GFAP及焦亡相关蛋白的变化;免疫荧光检测神经元与caspase-1 p10的共定位情况。结果与对照组比较,CRS模型组糖水偏好率显著降低、强迫游泳、悬尾不动时间显著增加(P<0.05),辅酶Q10、VX765及FLX可改善上述情况;与对照组比较,CRS模型组海马脑区GFAP阳性率与蛋白表达量显著减少(P<0.05),神经元突触棘显著减少(P<0.001),GSDMD-N、caspase-1、IL-1β表达显著增加(P<0.05),神经元与caspase-1 p10共标显著增加(P<0.001),辅酶Q10可改善上述情况。结论辅酶Q10通过下调焦亡信号通路缓解抑郁小鼠抑郁样行为。

慢性束缚肠道应激损伤小鼠模型的构建与评价

目的结合社会心理应激导致炎症性肠病、肠易激综合征等一系列疾病的特点,建立实验性慢性束缚小鼠肠道应激损伤模型,为探讨慢性束缚应激诱导胃肠病的致病机制以及防治措施奠定基础。方法18只雄性SPF级BALB/c小鼠,适应7 d后,根据体重按随机数字表法分为2组,对照组及慢性束缚应激组。对小鼠进行束缚应激,每天3 h,连续束缚14 d,建立肠道损伤模型。通过观察体重、肠道组织病理形态变化、检测紧密连接蛋白表达、肠上皮细胞凋亡以及炎症细胞因子mRNA表达水平等指标对模型进行评价。结果慢性束缚应激14 d,小鼠表现为体重减轻,十二指肠绒毛高度变短、隐窝结构异常、绒毛/隐窝比下降;结肠黏膜炎性细胞浸润、隐窝结构不规则。蛋白免疫印迹、免疫组化和免疫荧光染色结果显示,慢性束缚应激后小鼠十二指肠和结肠紧密连接蛋白Occludin、Claudin-1表达显著下降(P<0.05);肠上皮细胞凋亡蛋白Cleaved-Caspase-3的表达显著增加(P<0.05)。此外,肠道炎症因子和趋化因子mRNA表达结果显示,慢性束缚应激14 d显著提高了小鼠十二指肠IL-1β、IL-6、MCP-1、TNF-α、IL-10基因的表达(P<0.05);慢性束缚应激14 d显著提高了小鼠结肠IL-1β、IL-6、MCP-1的表达(P<0.001)。结论利用小鼠行为限制装置对小鼠连续束缚14 d,导致应激小鼠肠道组织结构异常、肠屏障功能障碍、肠上皮细胞凋亡,引发肠道炎症反应,表明慢性束缚应激肠道损伤小鼠模型构建成功。

束缚应激通过激活Rho/ROCK通路诱导大鼠杏仁核血脑屏障的损伤

目的探讨Rho/ROCK信号通路在束缚应激诱导大鼠杏仁核血脑屏障损伤过程中的作用及机制。方法选取60只SPF级雄性SD大鼠建立束缚应激模型,将大鼠分为4组:Control组(n=15,每日禁食水6 h);Stress组(n=15,每日束缚6 h);Stress+Fasudil组(n=15,每日束缚6 h,束缚前0.5 h给予腹腔注射1 mg/100 g Fasudil溶液);Fasudil组(n=15,每日给予腹腔注射1 mg/100 g Fasudil溶液)。用高架十字迷宫实验(EPM)检测各组大鼠行为学变化,ELISA检测血清CORT和S100B水平,检测脑组织伊文思蓝(EB)的渗漏情况以评估渗透性的改变,免疫荧光法和Western blotting方法检测紧密连接蛋白Claudin-5、Occludin、ZO-1的表达变化,Pull-down实验和Western blotting检测Rho/ROCK通路的激活情况,透射电镜观察脑微血管内皮细胞超微结构的形态变化。结果与Control组相比,Stress组和Stress+Fasudil组大鼠表现出了明显的焦虑样行为;Stress组和Stress+Fasudil组血清CORT含量均显著高于Control组(P<0.001);与Control组和Stress+Fasudil组相比,Stress组EB渗漏含量和S100B含量均显著升高(P<0.05);Stress组紧密连接蛋白的表达显著低于Control组和Stress+Fasudil组(P<0.05);Pull-down实验和Western blot分析证实,Stress组RhoA-GTP(P<0.001)、ROCK2(P<0.001)、p-MLC2(P<0.05)的表达显著高于Control组和Stress+Fasudil组;脑微血管内皮细胞超微结构显示,Stress组呈现显著的形态学改变。结论束缚应激能够通过激活Rho/ROCK信号通路诱导大鼠杏仁核血脑屏障的损伤。