慢性应激肝郁脾虚模型大鼠下丘脑外侧区Orexin A与Orexin receptor 1变化及逍遥散调节作用

发布时间：2018-05-09 12:58

本文选题：慢性应激 + 肝郁脾虚证　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:观察慢性应激肝郁脾虚模型大鼠下丘脑外侧区(Lateral hypothalamic area,LHA)食欲素A(Orexin A,OXA)和食欲素受体1(Orexin receptor 1,OX1R)变化及逍遥散的干预效应,探讨慢性应激致机体出现食欲下降的中枢神经内分泌机制及逍遥散的作用机理。方法:72只健康雄性SD大鼠(体重180±20 g)随机分为3组,即空白对照组(the control group)、模型组(the model group)和逍遥散组(the Xiaoyaosan group),每组24只。标准动物房饲养,室温(21±1)℃,相对湿度40%-60%,光照明暗各12 h(明7:00-19:00,暗19:00-7:00)。各组大鼠均适应性饲养1周,自由进食水。模型组和逍遥散组大鼠以捆绑束缚方式(每天3 h连续21天)建立慢性应激肝郁脾虚动物模型。于每天束缚前30 min逍遥散组大鼠灌服逍遥散混悬液(3.85 g-1·kg-1·d-1),模型组大鼠灌服等体积生理盐水。空白对照组常规饲养,灌服等体积生理盐水。根据大鼠体重调整用药量。各组大鼠均于每日灌胃前称取体重及摄食、饮水量,同时观察大鼠皮毛、粪粒性状、束缚反应等外观表现;于实验第0、7、21天进行旷场实验(open field text,OFT)和高架十字迷宫实验(the elevated plus maze,EPM)观测各组大鼠行为学变化。实验结束,以2%戊巴比妥钠(4 mg/100g体重)腹腔注射麻醉取材:每组随机取6只大鼠行左心室升主动脉灌注固定,断头取脑,以免疫荧光双染法检测LHA中OXA、OX1R与瘦素受体(Leptin receptor,ob-R)蛋白表达情况;其余大鼠断头取血,并剥离全脑,以间苯三酚法测定血清D-木糖含量,以酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定血清Leptin含量和脑组织LHA中OXA含量,实时荧光定量PCR(quantitative real-time reverse transcription PCR,q RT-PCR)方法检测LHA中OXA、OX1R、ob-R m RNA表达情况。结果:1各组大鼠一般状态情况实验期间,大鼠无死亡。空白对照组大鼠一般状态无明显变化,耳廓和鼻头红润,眼角清洁,眼球运动灵活,皮毛色白光泽,大便成颗粒状且干湿适中。束缚造模后,模型组大鼠在第1周表现出易激惹状态,提尾根部易反颈抓咬,在笼内表现出弓背、毛发竖立的攻击样姿态,束缚时易从束缚架上挣脱,或啃咬束缚带或束缚架;束缚第2周模型大鼠警惕性强,但束缚时反抗、撕咬和挣脱等表现稍减;第3周,捕捉时模型大鼠反抗减弱,蜷卧懒动,喜扎堆趴窝,毛发杂乱枯黄无光泽,大便时干时稀,眼角有分泌物,耳廓和鼻头颜色变淡,束缚时几无反抗,束缚过程中多嗜睡,但易惊醒,解除束缚放回笼中后多聚集蜷缩。逍遥散组大鼠在束缚之初与模型组大鼠表现基本无差异,束缚1周后激惹状态较模型组有所缓解;束缚2周后,逍遥散大鼠动作较模型大鼠灵活,大便成形且干湿适中,耳廓和鼻头稍有色淡但亦显红润,眼角较为清洁,偶有分泌物,皮毛稍有黯淡,但仍不失光泽整齐清洁。2各组大鼠体重、摄食量和摄水量变化情况与空白对照组大鼠比较,模型组大鼠体重增长减缓,摄食量和摄水量总体呈减少趋势(P0.05或P0.01);与模型组比较,逍遥散组大鼠体重、摄食量和摄水量均有所改善(P0.05或P0.01)。3各组大鼠行为学检测结果与空白对照组比较,实验第7天和第21天,模型组大鼠在旷场试验中5 min移动总距离,中央区移动距离、停留时间及总穿格数均明显减少(P0.01);高架十字迷宫实验模型组大鼠5 min开放臂停留时间及进入次数,中央平台停留时间及进入次数,以及进入封闭臂次数亦明显减少(P0.05或P0.01),但在封闭臂内的停留时间增加(P0.01)。与模型组比较,逍遥散组上述指标总体改善(P0.01)。4间苯三酚法检测血清D-木糖含量结果与空白对照组比较,模型组大鼠血清D-木糖含量下降(P0.01);与模型组比较,逍遥散组大鼠血清D-木糖含量显著升高(P0.01)。5 ELISA方法检测各组大鼠血清Leptin和LHA中OXA含量结果与空白对照组比较,模型组大鼠血清Leptin含量显著升高(P0.01),LHA中OXA含量减少(P0.01);与单纯应激模型大鼠比较,逍遥散组大鼠血清Leptin含量明显下调(P0.05),LHA中OXA含量上调(P0.01)。6免疫荧光双染法检测LHA中OXA、OX1R与ob-R蛋白表达结果OXA与OX1R被标记为红色荧光,ob-R标记为绿色荧光。统计结果显示,与空白对照组比较,模型组大鼠LHA中OXA与OX1R的阳性细胞表达数、阳性表达面积和积分光密度(integrated optical density,IOD)均明显下降(P0.01),但ob-R表达增加(P0.01)。与模型组比较,逍遥散组上述指标明显改善(P0.05或P0.01)。共表达结果显示,模型组OXA与ob-R的相关系数(Pearson’s correlation)较空白对照组升高(P0.01);与模型组比较,逍遥散组OXA与ob-R的相关系数升高(P0.05)。与空白对照组比较,模型组OX1R与ob-R的相关系数较空白对照组下降(P0.01);模型组和逍遥散组之间比较无统计学差异(P0.05)。7 q RT-PCR方法检测LHA中OXA m RNA、OX1R m RNA与ob-R m RNA表达结果与空白对照组比较,模型组OXA m RNA与OX1R m RNA表达水平明显下降(P0.01),ob-R m RNA表达水平则明显上升(P0.01);与模型组比较,逍遥散组大鼠OXA m RNA与OX1R m RNA表达水平明显上调(P0.01),ob-R m RNA则下调(P0.01)。结论:1慢性应激致肝郁脾虚样模型大鼠出现食少便溏、体重增长减缓等脾胃失于健运的原因与LHA中OXA与OX1R表达下降,ob-R表达增高有关。其机制可能是外周过多的Leptin通过血脑屏障进入LHA结合ob-R抑制了OXA与OX1R合成与分泌。2逍遥散可通过调节LHA中OXA、OX1R与ob-R的表达以及下调血清Leptin含量,以发挥疏肝健脾,调节脾胃功能的作用,其调节作用可能通过调控Leptin-ob-R-OXA/OX1R摄食通路实现。
[Abstract]:Objective: To observe the changes of orexin A (Orexin A, OXA) and orexin receptor 1 (Orexin receptor 1, OX1R) in the lateral hypothalamus (Lateral hypothalamic area, OXA) and orexin receptor (Orexin receptor 1, OX1R) in rats with chronic stress liver depression and spleen deficiency, and to explore the central neuroendocrine mechanism of decreasing appetite and the action machine of Xiaoyao Powder caused by chronic stress. Methods: 72 healthy male SD rats (weight 180 + 20 g) were randomly divided into 3 groups, namely, the blank control group (the control group), the model group (the model group) and the Xiaoyao Powder group (the Xiaoyaosan group), 24 in each group. The standard animal room was raised, room temperature (21 + 1), relative humidity 40%-60%, and light illumination 12 (bright, dark). Rats were fed for 1 weeks and were free to eat water. The model group and Xiaoyao group rats established chronic stress liver depression and spleen deficiency animal model with binding binding mode (3 h days for 21 days). The rats were filled with Xiaoyao Powder (3.85 g-1. Kg-1. D-1) before shackles of 30 min every day, and the rats in the model group were filled with equal volume of saline. The control group was fed with the same volume of normal saline. According to the weight of the rats, the rats were given weight and feeding, the amount of drinking water, and the appearance of the fur, the fecal grain characters and the binding reaction of the rats. The open field experiment (open field text, OFT) and the elevated cross maze were carried out on the day 0,7,21 of the experiment. The elevated plus maze (EPM) was used to observe the behavioral changes of rats in each group. The experiment was completed by intraperitoneal injection of 2% pentobarbital sodium (4 mg/100g body weight). 6 rats in each group were randomly selected for the left ventricular ascending aorta perfusion fixation, the head taken out of the brain, and the LHA OXA, OX1R and leptin receptor (Leptin receptor, ob-R) eggs were detected by immunofluorescence double staining method. The white expression was found in the rest of the rats. The rest of the rats took the head and removed the whole brain. The content of D- xylose in serum was measured by the method of interphenyl three phenol. The content of serum Leptin and the content of OXA in the brain tissue were measured by enzyme-linked immunosorbent assay (ELISA). The real-time fluorescent quantitative PCR (quantitative real-time reverse) was used. Methods the expression of OXA, OX1R, ob-R m RNA in LHA was detected. Results: 1 the rats were not dead during the general state experiment of rats. The general state of the rats in the blank control group had no obvious change, the auricle and nose were ruddy, the angle of the eye was clean, the eye movement was flexible, the fur color Bai Guangze, the stool became granular and dry and wet. After the binding model, model group In the first week, the rats showed irritable state, the tail of the tail was easy to reverse the neck and bite, showing the bow back in the cage, the erect attack like posture in the cage, easy to break from the bondage, or bite the bondage or the bondage. The second week model rats were vigilant, but the restraint, tearing and breaking off were slightly reduced; third weeks, catching the time model. The type of rat's resistance weakened, crouched and lazy, like a heap of the nest, the hair disorderly and dull yellow, the stool dry when it was thin, the eye secretions, the auricle and nose color light, the bondage no resistance, and the sleepiness in the binding process, but it was easy to wake up, and relieve the shackles and back in the cage. The rats in the Xiaoyao group were at the beginning of the shackle and model group rats. There was no difference in performance. After 1 weeks of bondage, the state of irritability was more relieved than the model group. After 2 weeks, the action of Xiaoyao Powder rats was more flexible than the model rats. The stool was formed and dry and wet, the auricle and nose were slightly colored and red, the angle of the eye was more clean, occasionally secreted, and the fur was slightly bleak, but the shiny and neat.2 rats were still clean and clean. Weight, food intake and water intake in the blank control group, the weight growth of the model rats was slowed down, and the intake and water intake were decreased (P0.05 or P0.01). Compared with the model group, the weight, feeding and water intake of the rats in the Xiaoyao group were improved (P0.05 or P0.01), and the behavior test results and empty of the rats in each group.3 were empty. In the white control group, in the seventh and twenty-first days of the experiment, the total distance of the model group was 5 min in the open field test, the moving distance in the central area, the stay time and the total number of lattices were significantly reduced (P0.01). The residence time and entry times of the 5 min open arm in the model group of the elevated cross maze model group, the residence time and the entry times of the central platform, and the number of entry times, and the number of entry times, and the number of entry times of the central platform, and the number of entry times, and the number of entry times of the central platform, and the number of entry times, and the number of entry times, and the number of entry times, and the number of entry times, and the number of entry times, and the number of entry times and the number of entry times of the central platform in the model group were also compared. The number of entering the closed arm also decreased (P0.05 or P0.01), but the retention time in the closed arm increased (P0.01). Compared with the model group, the above indexes of the Xiaoyao group were improved (P0.01) the content of serum D- xylose was compared with the blank control group. The content of D- xylose in the serum of the model group decreased (P0.01), and the ratio of the model group to the model group was compared with the model group. The content of serum D- xylose in the serum of Xiaoyao group increased significantly (P0.01).5 ELISA method to detect the content of OXA in the serum Leptin and LHA of each group. The serum Leptin content in the model group increased significantly (P0.01) and OXA content in LHA decreased (P0.01), and the serum of Xiaoyao group rats was compared with the simple stress model rats. The content of N was significantly down (P0.05), and the content of OXA in LHA was up-regulated (P0.01).6 immunofluorescence staining method to detect OXA in LHA. The expression of OX1R and ob-R protein was OXA and OX1R was marked as red fluorescence. The product and integral optical density (integrated optical density, IOD) decreased significantly (P0.01), but the expression of ob-R increased (P0.01). Compared with the model group, the above indexes were obviously improved (P0.05 or P0.01). The co expression results showed that the correlation coefficient of OXA and ob-R in the model group was higher than that in the blank control group; and the model group was compared with the model group. Compared with the blank control group, the correlation coefficient of OXA and ob-R increased (P0.05). Compared with the blank control group, the correlation coefficient of OX1R and ob-R in the model group was lower than that in the blank control group (P0.01), and there was no statistical difference between the model group and the Xiaoyao group (P0.05).7 Q RT-PCR method detection LHA OXA. Compared with the model group, the expression level of OXA m RNA and OX1R m RNA decreased significantly (P0.01), and ob-R m RNA expression level increased significantly (P0.01). Compared with the model group, the expression level of the rats was significantly up-regulated. Conclusion: 1 chronic stress caused liver depression and spleen deficiency model rats to eat food. The reason for the loss of spleen and stomach, such as loose stool and slow weight growth, is related to the decrease of OXA and OX1R expression in LHA and the increase of ob-R expression. The mechanism may be that the excessive peripheral Leptin through the blood brain barrier into LHA binding ob-R inhibits OXA and OX1R synthesis and secretion of.2 Xiaoyao Powder by regulating LHA OXA, expression and down regulation of serum PTIN content can play a role in regulating liver and spleen, regulating spleen and stomach function, and its regulation may be achieved by regulating Leptin-ob-R-OXA/OX1R feeding pathway.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5;R-332

【参考文献】