發(fā)布時(shí)間：2018-05-09 12:58

  本文選題：慢性應(yīng)激 + 肝郁脾虛證�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:觀察慢性應(yīng)激肝郁脾虛模型大鼠下丘腦外側(cè)區(qū)(Lateral hypothalamic area,LHA)食欲素A(Orexin A,OXA)和食欲素受體1(Orexin receptor 1,OX1R)變化及逍遙散的干預(yù)效應(yīng),探討慢性應(yīng)激致機(jī)體出現(xiàn)食欲下降的中樞神經(jīng)內(nèi)分泌機(jī)制及逍遙散的作用機(jī)理。方法:72只健康雄性SD大鼠(體重180±20 g)隨機(jī)分為3組,即空白對(duì)照組(the control group)、模型組(the model group)和逍遙散組(the Xiaoyaosan group),每組24只。標(biāo)準(zhǔn)動(dòng)物房飼養(yǎng),室溫(21±1)℃,相對(duì)濕度40%-60%,光照明暗各12 h(明7:00-19:00,暗19:00-7:00)。各組大鼠均適應(yīng)性飼養(yǎng)1周,自由進(jìn)食水。模型組和逍遙散組大鼠以捆綁束縛方式(每天3 h連續(xù)21天)建立慢性應(yīng)激肝郁脾虛動(dòng)物模型。于每天束縛前30 min逍遙散組大鼠灌服逍遙散混懸液(3.85 g-1·kg-1·d-1),模型組大鼠灌服等體積生理鹽水�？瞻讓�(duì)照組常規(guī)飼養(yǎng),灌服等體積生理鹽水。根據(jù)大鼠體重調(diào)整用藥量。各組大鼠均于每日灌胃前稱取體重及攝食、飲水量,同時(shí)觀察大鼠皮毛、糞粒性狀、束縛反應(yīng)等外觀表現(xiàn);于實(shí)驗(yàn)第0、7、21天進(jìn)行曠場(chǎng)實(shí)驗(yàn)(open field text,OFT)和高架十字迷宮實(shí)驗(yàn)(the elevated plus maze,EPM)觀測(cè)各組大鼠行為學(xué)變化。實(shí)驗(yàn)結(jié)束,以2%戊巴比妥鈉(4 mg/100g體重)腹腔注射麻醉取材:每組隨機(jī)取6只大鼠行左心室升主動(dòng)脈灌注固定,斷頭取腦,以免疫熒光雙染法檢測(cè)LHA中OXA、OX1R與瘦素受體(Leptin receptor,ob-R)蛋白表達(dá)情況;其余大鼠斷頭取血,并剝離全腦,以間苯三酚法測(cè)定血清D-木糖含量,以酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay,ELISA)測(cè)定血清Leptin含量和腦組織LHA中OXA含量,實(shí)時(shí)熒光定量PCR(quantitative real-time reverse transcription PCR,q RT-PCR)方法檢測(cè)LHA中OXA、OX1R、ob-R m RNA表達(dá)情況。結(jié)果:1各組大鼠一般狀態(tài)情況實(shí)驗(yàn)期間,大鼠無(wú)死亡。空白對(duì)照組大鼠一般狀態(tài)無(wú)明顯變化,耳廓和鼻頭紅潤(rùn),眼角清潔,眼球運(yùn)動(dòng)靈活,皮毛色白光澤,大便成顆粒狀且干濕適中。束縛造模后,模型組大鼠在第1周表現(xiàn)出易激惹狀態(tài),提尾根部易反頸抓咬,在籠內(nèi)表現(xiàn)出弓背、毛發(fā)豎立的攻擊樣姿態(tài),束縛時(shí)易從束縛架上掙脫,或啃咬束縛帶或束縛架;束縛第2周模型大鼠警惕性強(qiáng),但束縛時(shí)反抗、撕咬和掙脫等表現(xiàn)稍減;第3周,捕捉時(shí)模型大鼠反抗減弱,蜷臥懶動(dòng),喜扎堆趴窩,毛發(fā)雜亂枯黃無(wú)光澤,大便時(shí)干時(shí)稀,眼角有分泌物,耳廓和鼻頭顏色變淡,束縛時(shí)幾無(wú)反抗,束縛過(guò)程中多嗜睡,但易驚醒,解除束縛放回籠中后多聚集蜷縮。逍遙散組大鼠在束縛之初與模型組大鼠表現(xiàn)基本無(wú)差異,束縛1周后激惹狀態(tài)較模型組有所緩解;束縛2周后,逍遙散大鼠動(dòng)作較模型大鼠靈活,大便成形且干濕適中,耳廓和鼻頭稍有色淡但亦顯紅潤(rùn),眼角較為清潔,偶有分泌物,皮毛稍有黯淡,但仍不失光澤整齊清潔。2各組大鼠體重、攝食量和攝水量變化情況與空白對(duì)照組大鼠比較,模型組大鼠體重增長(zhǎng)減緩,攝食量和攝水量總體呈減少趨勢(shì)(P0.05或P0.01);與模型組比較,逍遙散組大鼠體重、攝食量和攝水量均有所改善(P0.05或P0.01)。3各組大鼠行為學(xué)檢測(cè)結(jié)果與空白對(duì)照組比較,實(shí)驗(yàn)第7天和第21天,模型組大鼠在曠場(chǎng)試驗(yàn)中5 min移動(dòng)總距離,中央?yún)^(qū)移動(dòng)距離、停留時(shí)間及總穿格數(shù)均明顯減少(P0.01);高架十字迷宮實(shí)驗(yàn)?zāi)Ｐ徒M大鼠5 min開(kāi)放臂停留時(shí)間及進(jìn)入次數(shù),中央平臺(tái)停留時(shí)間及進(jìn)入次數(shù),以及進(jìn)入封閉臂次數(shù)亦明顯減少(P0.05或P0.01),但在封閉臂內(nèi)的停留時(shí)間增加(P0.01)。與模型組比較,逍遙散組上述指標(biāo)總體改善(P0.01)。4間苯三酚法檢測(cè)血清D-木糖含量結(jié)果與空白對(duì)照組比較,模型組大鼠血清D-木糖含量下降(P0.01);與模型組比較,逍遙散組大鼠血清D-木糖含量顯著升高(P0.01)。5 ELISA方法檢測(cè)各組大鼠血清Leptin和LHA中OXA含量結(jié)果與空白對(duì)照組比較,模型組大鼠血清Leptin含量顯著升高(P0.01),LHA中OXA含量減少(P0.01);與單純應(yīng)激模型大鼠比較,逍遙散組大鼠血清Leptin含量明顯下調(diào)(P0.05),LHA中OXA含量上調(diào)(P0.01)。6免疫熒光雙染法檢測(cè)LHA中OXA、OX1R與ob-R蛋白表達(dá)結(jié)果OXA與OX1R被標(biāo)記為紅色熒光,ob-R標(biāo)記為綠色熒光。統(tǒng)計(jì)結(jié)果顯示,與空白對(duì)照組比較,模型組大鼠LHA中OXA與OX1R的陽(yáng)性細(xì)胞表達(dá)數(shù)、陽(yáng)性表達(dá)面積和積分光密度(integrated optical density,IOD)均明顯下降(P0.01),但ob-R表達(dá)增加(P0.01)。與模型組比較,逍遙散組上述指標(biāo)明顯改善(P0.05或P0.01)。共表達(dá)結(jié)果顯示,模型組OXA與ob-R的相關(guān)系數(shù)(Pearson’s correlation)較空白對(duì)照組升高(P0.01);與模型組比較,逍遙散組OXA與ob-R的相關(guān)系數(shù)升高(P0.05)。與空白對(duì)照組比較,模型組OX1R與ob-R的相關(guān)系數(shù)較空白對(duì)照組下降(P0.01);模型組和逍遙散組之間比較無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。7 q RT-PCR方法檢測(cè)LHA中OXA m RNA、OX1R m RNA與ob-R m RNA表達(dá)結(jié)果與空白對(duì)照組比較,模型組OXA m RNA與OX1R m RNA表達(dá)水平明顯下降(P0.01),ob-R m RNA表達(dá)水平則明顯上升(P0.01);與模型組比較,逍遙散組大鼠OXA m RNA與OX1R m RNA表達(dá)水平明顯上調(diào)(P0.01),ob-R m RNA則下調(diào)(P0.01)。結(jié)論:1慢性應(yīng)激致肝郁脾虛樣模型大鼠出現(xiàn)食少便溏、體重增長(zhǎng)減緩等脾胃失于健運(yùn)的原因與LHA中OXA與OX1R表達(dá)下降,ob-R表達(dá)增高有關(guān)。其機(jī)制可能是外周過(guò)多的Leptin通過(guò)血腦屏障進(jìn)入LHA結(jié)合ob-R抑制了OXA與OX1R合成與分泌。2逍遙散可通過(guò)調(diào)節(jié)LHA中OXA、OX1R與ob-R的表達(dá)以及下調(diào)血清Leptin含量,以發(fā)揮疏肝健脾,調(diào)節(jié)脾胃功能的作用,其調(diào)節(jié)作用可能通過(guò)調(diào)控Leptin-ob-R-OXA/OX1R攝食通路實(shí)現(xiàn)。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李曉娟;馬慶宇;周巖;劉群;劉sボ，

本文編號(hào)：1866101