【作者】 杨金玲；

【导师】 潘芳；

【作者基本信息】 山东大学 ， 医学心理学， 2017， 博士

【摘要】 肥胖是由于环境和遗传等因素造成的机体脂肪过度累积状态,易并发糖尿病、冠心病和呼吸窘迫综合征等多种生理疾病。随着社会经济的发展和生活方式的改变,世界范围内肥胖的发生率逐年上升,已经成为严重的公共卫生问题。抑郁是一种以显著而持续的情绪低落、思维迟缓和言语减少为主要特征的情绪障碍性疾病,伴有快感缺乏和认知功能损害。世界卫生组织(WHO)预测2020年抑郁症将在全球重大非致命性疾病中位居第二。抑郁情绪容易伴发糖尿病等代谢相关性疾病,危害患者的身心健康、生活及工作能力,给家庭和社会造成沉重的负担。流行病学调查发现,肥胖人群抑郁症发生的风险显著增加。由于抑郁情绪导致的社会功能下降伴随着运动量减少,抑郁患者的肥胖发生率也高于普通人群。另有研究提示抑郁症和肥胖存在相同的候选基因。抑郁和肥胖共病严重危害患者的健康状况,导致不良的结局。瘦素(Leptin)是一种由脂肪组织分泌的蛋白质类激素,发挥抑制食欲、降低能量摄取、增加能量消耗和抑制脂肪组织合成的作用,其生理功能主要是通过受体b(Leptin receptor b,LepRb)介导。肥胖人群由于中枢/血浆瘦素比例降低导致外周瘦素水平升高而中枢瘦素作用减弱称之为瘦素抵抗现象。近年有研究发现,抑郁症患者存在不同程度的瘦素水平的改变,如体重正常的抑郁症患者血浆瘦素水平升高,女性抑郁症患者的血浆瘦素降低,瘦素与抑郁症状的相互影响受腹部肥胖程度的调节。海马区域LepRb的缺失可引起抑郁样行为表现,而脑内注射瘦素具有抗抑郁的效果。这些研究提示瘦素作为一种内分泌激素,在调节能量摄入和代谢的同时,可能在抑郁、焦虑样行为的调节中发挥重要作用。但瘦素在肥胖与抑郁共病中的机制尚不清楚。肥胖导致机体长期处于低度炎症状态,伴有炎性细胞因子的分泌升高和脂肪组织巨噬细胞的浸润,而免疫介导的炎症状态是导致肥胖的重要原因之一。脑内细胞因子与神经元的完整性密切相关,在神经突触重塑方面具有重要作用,且影响神经环路和神经递质功能。研究发现,炎性细胞因子升高导致快感缺乏、厌食、发热、睡眠的变化和社会能力下降等抑郁样行为。炎性细胞因子的持续升高会影响神经递质的功能从而导致神经功能障碍如抑郁症。局部注射细胞因子可以使动物产生神经精神症状和与抑郁症行为改变。最近更有学者指出,肥胖时的游离脂肪酸可通过TLR4信号通路升高炎性细胞因子的水平。在炎性细胞因子中,IL-1β、IL-6和TNF-α被认为是介导抑郁样行为和认知功能损害的关键因子。NF-κB是TLR通路的关键调控因子,同时也是影响压力性精神失调和抑郁症的调节因子。但炎性细胞因子在肥胖和抑郁共病中的作用尚没有系统的研究。高能量食物的过量摄入是导致肥胖的重要因素,因此高脂饮食(High-fat-diet,HFD)饲养是常用的建立肥胖动物模型的方法。同时,慢性轻度不可预期性应激(Chronic unpredictable mild stress,CUMS)模式可导致动物出现快感缺失和对新奇环境的探索能力下降等抑郁焦虑样症状,是一种建立抑郁动物模型的有效方法。本研究旨在以高脂饮食诱导肥胖后,联合应用高脂饮食和慢性轻度不可预期性应激建立肥胖与抑郁症共病的动物模型,采用行为学测试评估动物模型的表观效度,进而检测血浆Leptin、中枢LepRb、中枢IL-1β、IL-6、TNF-α和NF-κB蛋白水平和mRNA水平,阐明Leptin/LepRb和炎性细胞因子在高脂饮食和慢性轻度不可预期性应激诱导的肥胖和抑郁共病中的作用。1.研究目的(1)高脂饮食与慢性轻度不可预期性应激构建的肥胖和抑郁共病大鼠的表观效度。(2)Leptin/LepRb在肥胖和抑郁共病大鼠的抑郁、焦虑样行为和认知功能损害中的作用。(3)下丘脑和前额叶IL-1β、IL-6、TNF-α和NF-κB在肥胖和抑郁共病大鼠抑郁、焦虑样行为和认知功能损害中的作用。2.材料和方法(1)实验分组68只Wistar大鼠适应性喂养7天,根据体重随机分为两组。正常对照组24只(Regulardiet,RD),高脂饲料组44只。第8周末,将高脂饲料组大鼠体重与对照组平均体重进行比较,其中26只高脂饲料组大鼠判定为饮食诱导肥胖(Diet-induced obesity,DIO)大鼠。第9周开始,24只RD组大鼠和26只DIO大鼠被各自随机分为两组,其中1组接受21天的慢性轻度不可预期性应激。因此,本研究包括四组:对照组(Ctr,n=12),CUMS组(CUMS,n=12),单纯肥胖组(Ob,n=13),肥胖合并CUMS组(Co,n=13)。(2)慢性轻度不可预期性应激每只大鼠均单笼孤养。刺激方式包括:禁食8h,禁水8h,45°斜笼,群养,昼夜节律颠倒,浸湿鼠笼,水平摇晃20min。每天随机采用一种方法刺激大鼠,每种方法不出现连续重复,整个过程持续3周。(3)行为学测试抑郁样行为如快感缺乏和探索能力的下降使用糖水偏好试验(Sucrose preference test,SPT)和旷场试验(Open field test,OPT)来评估,焦虑样行为通过高架十字迷宫测试(Elevated plus maze test,EPT)来评估,空间学习记忆损害采用Morris水迷宫试验(Morris water maze,MWM)进行检测。(4)体重和脂肪/体重比测定每周固定时间测量一次大鼠体重。脂肪/体重比(%)=[腹膜后脂肪(g)+网膜脂肪(g)+附睾脂肪(g)]/体重(g)×100%。(5)血生化检测使用LX-20型全自动生化分析仪测定大鼠总胆固醇、甘油三酯、高密度脂蛋白和低密度脂蛋白水平。(6)Elisa检测采用大鼠瘦素Elisa试剂盒测定血浆瘦素浓度,大鼠IL-1β、IL-6、TNF-α和NF-κB Elisa试剂盒测定海马和前额叶区域脑组织炎性细胞因子表达。(7)免疫组织化学检测LepRb表达将石蜡包埋脑组织冠状方向切成厚度为5μm薄片,采用免疫组化染色方法检测海马和下丘脑区域的LepRb表达,阳性细胞显示棕黄色颗粒,高倍镜下随机选择5个视野进行细胞计数。(8)免疫印迹检测LepRb表达分别取30mg海马和下丘脑冰上匀浆后离心,取上清液测定蛋白浓度,每个样品取等量的蛋白,用12%的SDS-PAGE分离并转移到PVDF膜,脱脂牛奶缓冲1小时,然后加入一抗4℃过夜。次日洗涤三次后,加入二抗室温条件下孵育1小时,随后加入增强发光液显影。条带的光密度使用c-digit记录和分析。(9)实时定量PCR检测海马、下丘脑LepRb和海马、前额叶IL-1β、IL-6、TNF-α和NF-κB表达用Trizol从组织中提取总RNA,用标准试剂和程序进行反转录和实时定量PCR。使用96孔板20μL反应体系,95℃/5min,以下条件运行 40 个周期:95℃/30s,60℃/30s,72℃/30s,终止在 72℃/5min。所有 PCR数据分析利用2-△△CT方法。3.结果(1)11周高脂喂养导致大鼠体重、体脂比和血脂增加,3周的慢性轻度不可预期性应激降低大鼠的体重和体脂比,肥胖合并CUMS组大鼠的体重和体脂比高于对照组和CUMS组而低于单纯肥胖组。(2)高脂饮食与慢性轻度不可预期性应激联合应用导致大鼠在糖水偏好试验、旷场试验和高架十字迷宫试验中表现出焦虑抑郁样行为,并在Morris水迷宫试验中表现出认知记忆功能障碍。(3)高脂饮食与慢性轻度不可预期性应激单独和联合应用均导致大鼠血浆Leptin升高,海马和下丘脑区域LepRb表达下降。糖水偏好,旷场试验跨格数目、高架十字迷宫开放臂时间百分比以及Morris水迷宫目标象限时间百分比与海马和下丘脑区域的LepRb蛋白水平均呈显著负相关。(4)高脂饮食与慢性轻度不可预期性应激单独和联合应用均导致大鼠下丘脑和前额叶IL-1β、IL-6、TNF-α和NF-κB升高,糖水偏好,旷场试验跨格数目、高架十字迷宫开放臂时间百分比以及Morris水迷宫目标象限时间百分比与海马和前额叶区域的IL-1β、IL-6、TNF-α和NF-κB蛋白水平均呈显著负相关。4.结论(1)慢性轻度不可预期性应激可能对高脂饮食诱导的肥胖有逆转作用。(2)高脂喂养和慢性轻度不可预期性应激联合应用可导致大鼠焦虑、抑郁样行为和认知记忆功能障碍,可作为肥胖和抑郁共病的动物模型。(3)外周血Leptin和中枢海马下丘脑区域的LepRb表达异常在肥胖和抑郁共病导致的焦虑、抑郁样行为和认知记忆功能障碍中发挥重要作用。(4)海马和前额叶IL-1β、IL-6、TNF-α和NF-κB表达异常在肥胖和抑郁共病导致的焦虑抑郁样行为和认知记忆功能障碍中发挥重要作用。更多还原

【Abstract】 Obesity is excessive accumulation of body fat state caused by environmental and genetic factors,which is easy to diabetes,coronary heart disease and respiratory distress syndrome.With the development of social economy and the change of life style,the obesity has become serious public health problem.Depression is one kind of emotional disorder which is characterized by slow thinking,reduced speech lack of pleasure and cognitive impairment.WHO predicts that depression will be the second biggest world’s major non fatal diseases in 2020.Depression is a heavy burden to the family and society which prone to combine with diabetes and other metabolic related diseases,endanger the physical and mental health of patients,living and working ability.Epidemiological survey found that the risk of depression in obese patientsincreased significantly,while the prevalence of obesity in patients with depression was higher than that of the general population.Other studies have shown that the same candidate genes for depression and obesity.Comorbidity of depression and obesity seriously affects the health of patients,resulting in adverse outcomes.Leptin is a secreted by adipose tissue protein hormones,inhibit appetite,reduce energy intake,increase energy consumption,inhibit fat synthesis,its physiological function is mainly mediated through the receptor B(LepRb).The increase of peripheral leptin level and the decrease of leptin in central nervous system are resulted from the decrease of central/plasma leptin ratio,which is called leptin resistance.In recent years,some scholars have found that patients with depression have different levels of leptin in normal weight change,such as depression and plasma leptin level in patients with elevated leptin interaction and depressive symptoms is regulated by the degree of abdominal obesity.The absence of LepRb in hippocampus can cause depressive like behavior and the effect of leptin injection in brain has antidepressant effect.These studies suggest that leptin may play an important role in the regulation of depression.However,the mechanism of leptin in obesity and depression is not clear.Obesity leads to chronic inflammation in the body for a long time,and immune mediated inflammation is one of the important reasons leading to obesity.The cytokines in the brain are closely related to the integrity of neurons,and play an important role in the synaptic remodeling.The study found that elevated inflammatory cytokines lead to depression,anorexia,fever,sleep changes and social decline and other depressive behavior.The persistent elevation of inflammatory cytokines may affect the function of neurotransmitters,leading to neurological disorders such as depression.Local injection of cytokines can induce the occurrence of neuropsychiatric symptoms and behavioral changes in patients with depression.More recently,scholars have pointed out that the free fatty acids can increase the level of inflammatory cytokines.IL-1β,IL-6 and TNF-a are considered to be the key factors mediating the depression like behavior and cognitive impairment in inflammatory cytokines.NF-κB is a key regulator of the TLR pathway and is also an important regulator of stress disorder and depression.However,there is no systematic study on the role of inflammatory cytokines in the comorbidity of obesity and depression.High energy food intake is an important factor leading to obesity,so that high-fat diet(HFD)feeding is commonly used to establish the animal model of obesity.At the same time,chronic unpredictable mild stress(CUMS)model is an effective method to establish the animal model of depression,which leads to the loss of appetite and the ability to explore the new environment,such as depression and anxiety symptoms.The purpose of this study was to study obesity induced by a high-fat diet combined with chronic unpredictable mild stress animal model of diet and high-fat establishment of obesity and depression,the concept of validity by behavioral test and evaluation of animal model,and plasma Leptin,LepRb,IL-1β,IL-6,TNF-a and NF-κιB protein level and mRNA level.To elucidate the role of Leptin/LepRb and inflammatory factors in comorbid obesity and depression induced by HFD and CUMS.1.Objectives(1)The apparent validity of an animal model of comorbid obesity and depression induced by high-fat diet and chronic unpredictable mild stress.(2)The role of Leptin/LepRb in depression and anxiety like behavior and cognitive impairment in rats withobesity and depression.(3)The role of IL-1,IL-6,TNF-a and NF-κB in hypothalamus and prefrontal cortex in depression and anxiety like behavior and cognitive impairment in rats with obesity and depression.2.Methods(1)Animals68 Wistar rats in experimental group were fed for 7 days.The rats were randomly divided into 2 groups according to the weight of the rats in the experiment group(n ＝68).The control group(Regulate diet,RD)was 24,and the high fat diet group was 44.At the end of the eighth week,the weight of rats in high fat diet group was compared with that of control group,of which 26 rats in high fat diet group were treated with diet induced obesity DIO).At the beginning of ninth week,the rats in the RD group and 26 DIO rats were randomly divided into 2 groups,and the rats in the 1 groups were treated with chronic unpredictable mild stress for 21 days.Therefore,there were four groups in the study:control group(Ctr,n=12),CUMS group(CUMS,n=12),obesity group(Ob,n=13),combined obesity and CUMS group(Co,n=13).(2)Chronic unpredictable mild stressExperimental rats were received chronic unpredictable mild stress(CUMS).Stimulation methods include:fasting 8h,water deprivation 8h,45 degree oblique cage,group feeding,circadian rhythm reversed,wet squirrel cage,shaking the 20min level.Every day,a method was used to stimulate rat,the process lasted 3 weeks.(3)The behavioral testsSucrose preference test,open field test,the elevated plus maze and Morris water maze test were used to evaluated depressive and anxiety like behaviors and memorydamage.(4)Weight and fat/body ratioWeight of all rats was measured once a week at a fixed time.Fat/body weight percentage(%)=([retroperitoneal fat(g)+ omental fat(g)+ epididymal fat(g)]/[weight](g))×100%.(5)Serum levels of total cholesterol,triglyceride,high-density lipoprotein and low-density lipoprotein were measured by LX-20 automatic biochemical analyzer.(6)Elisa assay was used to determine the concentration of plasma leptin in rats,and the expression of inflammatory factors in hippocampus and prefrontal cortex was determined by IL-1β,IL-6,TNF-a and NF-κB Elisa kit.(7)ImmunohistochemistryImmunohistochemistry to detect the expression of LepRb in paraffin embedded brain tissue into coronary direction of thickness of 5 mu m slice expression by immunohistochemistry staining detection of LepRb region of hippocampus and hypothalamus,randomly selected 5 high magnification view of cell counting and cells showed brown granules.(8)Western BlotThe expression of LepRb was detected by Western blot respectively ice hippocampus and hypothalamus 30mg homogenate after centrifugation,the supernatant protein concentration was measured in each sample,the same amount of 40ug protein,isolated by SDS-PAGE 12%and transferred to PVDF membrane,skim milk buffer for 1 hours,and then adding a resistance 4 degrees overnight.The next day after washing three times,add the resistance of the room temperature for 1 hours,then add to enhance the development of light emitting liquid.The optical density of the strip was recorded and analyzed using c-digit.(9)Real time quantitative PCRReal time quantitative PCR detection of LepRb in hypothalamus and hippocampus,hippocampus,prefrontal IL-1β,IL-6,TNF-a and NF-κB expression to extract total RNA from tissues using Trizol,reverse transcription and quantitative real-time PCR using standard procedures and reagents.Using 96 hole plate L reaction system of 95℃/5min,the following conditions run for 40 cycles:95℃/30s,60℃/30s,72℃/30s,terminated at 72℃/5min.All PCR data analysis using 2-Delta CT method.3.Results(1)11 weeks of high-fat feeding resulted in increased body weight,body fat ratio and blood lipid,3 weeks of chronic unpredictable mild stress decreased rat body weight and body fat ratio,the body weight and body fat ratio of comorbidity rats is higher than the control group and the depression group and lower than the obese group.(2)The combination of high fat diet and chronic unpredictable mild stress induced anxiety and depression like behavior of rats in the preference test,open field test and elevated plus maze test,and cognitive impairment of rats in the Morris water maze test.(3)The Leptin level was elevated in the plasma of rats and the expression of LepRb in hippocampus and hypothalamus decreased with the combination of high fat diet and chronic unpredictable mild stress.Sucrose preference,open field test cross grid number,the elevated plus maze of open arm time percentage and the percentage of Morris water maze and target quadrant time in hypothalamus and hippocampus regions of the LepRb protein level was negatively correlated.(4)The combination of high fat diet and chronic unpredictable mild stress caused increase of IL-1β,IL-6,TNF-a and NF-κB levels in hypothalamus and prefrontal cortex of rats.Sucrose preference,open field test cross grid number,the elevated plus maze of open arm time percentage and the percentage of Morris water maze and target quadrant time in hippocampus and prefrontal regions IL-1β,IL-6,TNF-a and NF-κB protein level was negatively correlated.4.Conclusion(1)Chronic unpredictable mild stress may reverse the obesity induced by high fat diet.(2)The combination of high fat diet and chronic unpredictable mild stress can lead to depression anxiety and cognitive impairment in rats.(3)The abnormal expression Leptin in the peripheral blood and LepRb in the hypothalamus and hippocampus may play an important role in the anxiety,depression and cognitive dysfunction in obesity and depression comorbidity.(4)The abnormal expression of IL-1β,IL-6,TNF-and NF-in the hippocampus and prefrontal cortex may play an important role in the anxiety,depression and cognitive dysfunction in obesity and depression comorbidity.更多还原