【作者】 李云鹏；

【导师】 文小敏；

【作者基本信息】 南方医科大学 ， 中西医结合基础， 2010， 硕士

【摘要】 目的脾胃湿热证又名“中焦湿热证”,是感受湿热之邪；或过食辛热肥甘；或脾气虚弱,湿邪中阻,久之酿成湿热,内蕴脾胃而表现的一类病证,岭南地区常见多发。其临床症状复杂,病程较久,病势缠绵难愈,在疾病发生发展过程中,各种致病因素对人体的刺激,躯体将会做出应激反应,本实验拟通过复合因素(外部湿热环境、肥甘饮食及病原微生物)造成脾胃湿热证大鼠模型,并探讨三仁汤对脾胃湿热证大鼠的行为学及HPA轴引起的行为学改变,肾上腺指数的变化,血浆促肾上腺皮质激素释放激素(CRH)、促肾上腺皮质激素(ACTH)、β内啡肽(β-EP)、抗利尿激素(ADH)和血清皮质醇(Cor)、醛固酮(ALD)的含量的影响。以探讨亢进的HPA轴对脾胃湿热证大鼠神经内分泌的影响、行为学的改变及水盐代谢的机制,及三仁汤清热祛湿的作用机理。方法1.药物和饲料三仁汤药物组成：苦杏仁、法半夏、飞滑石、生薏苡仁、白通草、白蔻仁、淡竹叶、川厚朴。高糖高脂饲料：普通饲料中混入12%猪油,8%蜂蜜加工而成。2.分组将50只SPF级雄性Wistar大鼠随机分成5组,分别为正常对照组、脾胃湿热证组、三仁汤组(低剂量、中剂量、高剂量组),每组10只。3.造模本实验拟通过复合因素(外部湿热环境、肥甘饮食及病原微生物)造成脾胃湿热证大鼠模型：①正常对照组：在20-28℃温度,50-60%相对湿度的环境下,以普通混合饲料喂养,每日上午9时投饲料。②脾胃湿热证大鼠模型：肥甘饮食饲料+仿真模拟气候仓+大肠杆菌。③三仁汤低剂量组：肥甘饮食饲料+仿真模拟气候仓+大肠杆菌+三仁汤低剂量。④三仁汤中剂量组：肥甘饮食饲料+仿真模拟气候仓+大肠杆菌+三仁汤中剂量。⑤三仁汤高剂量组：肥甘饮食饲料+仿真模拟气候仓+大肠杆菌+三仁汤高剂量。4.观察及检测指标对比观察实验大鼠造模前后的一般状态、体重、肛温、饮食、饮水、大便性状、小便颜色及量的改变。用小动物行为学记录分析系统观察记录动物的行为学表现旷场试验,分别观察在造模成功后(给药前)、灌服三仁汤后(给药后)的差别；通过称量法计算。肾上腺质量,计算肾上腺指数；同时采用放射免疫法检测大鼠血浆CRH、ACTH、β-EP、ADH和血清Cor、ALD的水平。5.统计学方法采用SPSS软件(13.0版)进行数据处理,实验结果的数据类型为计量资料,各数据均用均数±标准差(x±s)表示。体重、体温采用重复测量的方差分析进行比较；旷场实验通过配对t检验进行比较；肾上腺指数、实验室指标CRH、ACTH、COR、β-EP、ALD、ADH均采用One-Way ANOVA对各组实验结果进行方差分析,组间多重比较用LSD法检验,以P<0.05为差异有显著性。结果1.大鼠一般状态实验前：所有动物状态良好,活动灵活,皮毛洁白有光泽,活动正常,摄食饮水正常,大便成形,小便、体温正常。实验后：正常组无明显异常表现。第1天各组大鼠表现一致,无差别。各造模组大鼠则随着造模时间的推移,大部分出现倦怠、嗜卧懒动,反应迟钝,毛发蓬松,颜色枯槁,饮食饮水减少,排便次数增多,从造模第10天开始,均出现大便溏泄或粘腻,体温升高较快,进入仿真气候仓后,大便溏泄或粘腻症状加重,渐见肛周污秽。三仁汤(高、中、低剂量)组灌服三仁汤后,活动逐渐增强,饮水、饮食量增加,大便渐渐恢复正常,体温恢复到正常水平。2.体重、肛温变化体重变化：第1天各组大鼠间体重差异无统计学意义(F=0.467,P=0.759)。前10天各组体重的增长趋势无明显改变,进入仿真气候仓后4个造模组的体重与正常组比较增长缓慢,第18天到20天,各造模组体重增长趋势基本一致,与正常组比较很缓慢,第20天以后,三仁汤各剂量组的体重增长恢复正常,但模型组无改变。在不同时间点均低于正常对照组,差异有显著性(P=0.000),提示体重下降,给予三仁汤低中高剂量灌胃后,均增长至正常增长水平；正常对照组的体重随时间一直是正常增长。肛温变化：第1天各组大鼠的肛温差异无统计学意义(F=0.665,P=0.602)前10天各组肛温无明显改变,进入仿真造模仓后4个模型组的肛温上升较快,灌服大肠杆菌(第20天)后肛温急剧上升；与正常对照组比较有显著性差异(P<0.01)。三仁汤各剂量组大鼠灌服三仁汤7天后肛温降低明显,而模型组肛温无明显下降；正常组的肛温随时间基本无变化。3.旷场实验大鼠中央格停留时间变化：治疗前,由于造模因素的影响,模型组、三仁汤高剂量组、三仁汤低剂量组与正常组比较,中央格停留时间均延长,差异有显著性(P<0.01),三仁汤中剂量组与正常组比较,停留时间延长,差异有统计学意义(P<0.05)；表明大鼠的空间认知能力下降。治疗后,上述各组停留时间仍然大于正常组,但三仁汤各剂量组与治疗前比较,均有不同程度的时间缩短,与模型组比较具有显著性差异(P<0.01或P<0.05),三仁汤各剂量组间,两两比较无显著性差异(P>0.05)。表明了三仁汤对大鼠的空间认知能力有改善作用。大鼠运动格数及直立次数变化：治疗前,在造模因素的影响下,各造模组与正常组比较,运动格数减少,差异有显著性(P<0.01或P<0.05),表明大鼠兴奋性降低,对新环境的适应能力减弱,探究行为减少；治疗后,三仁汤各剂量组与治疗前比较,运动格数均有不同程度的增加,与模型组比较具有显著性差异(P<0.01或P<0.05),三仁汤各剂量组间,两两比较无显著性差异(P>0.05),表明三仁汤对大鼠的探究行为和自主活动有改善作用,增加了大鼠的兴奋性。大鼠修饰次数变化：治疗前,在造模因素的影响下,造模各组与正常组比较,修饰次数减少,差异有显著性(P<0.05),表明大鼠对新环境的警觉性的降低,对环境的满意度下降；治疗后,三仁汤各剂量组与治疗前比较,运动格数均有不同程度的增加,与模型组比较具有显著性差异(P<0.05),三仁汤各剂量组间,两两比较无显著性差异(P>0.05),表明三仁汤能改善大鼠对环境的满意度。4.实验室指标检测造模成功后的模型组大鼠与正常对照组相比,模型组大鼠血浆CRH、ACTH、血清COR含量均增高,差异有显著性(P<0.01)；与模型组相比,三仁汤(高、中、低剂量)组大鼠血浆CRH、ACTH、血清Cor含量均降低,有显著性差异(P<0.01),尤以三仁汤中剂量组效果最好,其中对降低CRH,中剂量效果优于高、低剂量,而降低ACTH,中剂量优于低剂量(P=0.038),但与高剂量无显著性差异(P=0.091)。而降低Cor,则中剂量优于高剂量(P=0.034),与低剂量差异无显著性(P=0.334)模型组大鼠与正常组比较,肾上腺指数及血浆β-EP明显升高,差异显著(P<0.01)；三仁汤(高、中、低剂量)与模型组比较,肾上腺指数、血浆β-EP含量均明显降低,差异显著(P<0.01或P<0.05)；中剂量组与高、低剂量组相比,具有差异性(P<0.01或P<0.05),但高、低剂量组之间无显著性差异(P>0.05)。模型组大鼠与正常组比较,与正常对照组比较,模型组血清ALD、血浆ADH显著升高,差异有显著性意义(P<0.01),经灌服三仁汤一周后,与模型组比较,三仁汤(高、中、低剂量)组的ALD、ADH均有下降(P<0.05或P<0.01)；其中对ALD的影响,以中剂量效果最好,最接近正常值,中剂量与高、低剂量比较有显著性差异(P<0.05),而高剂量与低剂量间无显著性差异(P=0.534)。其中对ADH的影响,三仁汤(高、中、低剂量)组之间无显著性差异(P>0.05)。结论实验结果显示,通过复合因素(外部湿热环境+肥甘饮食+病原微生物)的应激因素造模,可以成功复制脾胃湿热证大鼠动物模型,同时会引起脾胃湿热证大鼠行为学及神经内分泌发生改变。模型组的中央格停留时间与对照组比较显著延长,说明模型组大鼠空间认知能力降低；运动格数、直立次数、修饰次数明显少于对照组,表明模型组大鼠兴奋性降低,对新环境的适应能力减弱,表明大鼠在陌生环境中的自主活动和探究行为减少。模型组血浆CRH、ACTH、血清Cor与对照组均有明显差别,显著升高,行为发生改变,说明这些指标升高与大鼠行为及症状体征有密切关系,大鼠处于一种应激状态,调动机体自我调节及自我保护。模型组肾上腺指数和β内啡呔的明显升高,表明大鼠在应激状态下发生了内部变化,以适应环境的改变；以上几个指标可以作为脾胃湿热证大鼠神经内分泌等变化的指标,外部反映为体重、体温、精神状态、饮食量、饮水量、大小便及行为学各种变化。脾胃湿热证大鼠ADH、ALD较正常组显著升高,表明湿热证大鼠远端肾小管和集合管水分重吸收加强,引起水湿停滞,水钠潴留,大鼠出现倦怠,嗜卧懒动,口渴不欲饮、小便短赤,饮食减少,大便溏泄,这可能是脾胃湿热证发生的机理之一,这两个指标可以作为脾胃湿热证水湿停聚的客观检测指标。三仁汤可能存在以下作用：1)能恢复由复合应激因素所致的脾胃湿热证的亢进的病理状态,如发热、倦怠、嗜卧懒动、纳呆、大便溏或粘腻、小便短赤等；2)可以调节HPA轴功能,具有抗应激的作用,能降低CRH、ACTH、Cor和β-EP等激素分泌,维持内环境的稳定及改善大鼠的行为学改变；3)可以改善脾胃湿热证大鼠的肾上腺肥大；4)调节ALD、ADH的释放水平,减少水湿停聚,恢复正常的水盐代谢。更多还原

【Abstract】 Obgective:the spleen and stomach Damp-heat syndrome is a type of diseases take place in Lingnan,caused by damp-heat,over-eating fatness and pungent,which may injure the spleen and stomach.It is marked with complicated clinical symptoms,longer history and lingering to heal,so in the occurrence and developent of disease,many factors may spur the organism and the body may in turn reflect for the stimulation.This study intend to fabricate the mode of spleen and stomach Damp-heat syndrome in rats by multiple factors including external damp-heat environment,fat and sweet diet and microorganism.We may probe into the behavior change of spleen and stomach damp-heat syndrome,and we also explore the impact on neuroendocrine systyme of hyperactive hypothalamus-pituitary-adrenal axis,the change of adrenal galnd index and the different secretion of CRH,ACTH,COR,β-EP,ALD and ADH.We may probe into mechanism of HPA axis and water-sodium metabolism.In addition,we will get to know the principle of Qingre Qushi of sanren decotion. Methods:1.chinese herbal medicine and foodstuffComposition of sanren decotion:bitter apricot seed,Rhizoma Pinellinae Praeparata,talc,crude semen coicis,ricepaperplant pith,amomum caramomum, bamboo leaf, officinal magnolia bark.High glucose and lipid foodstuff:the ordinary mix with 12% pork lard and 8% honey.2.Groupfifty male Wister rats were randomly divided into five groups:normal control group,model group,sanren decoction high and medium and low dosage groups. Each group has 10 rats.3.creation of modelsThis experiment intend to creat the model of spleen and stomach damp-heat syndrome by compositite factors including external damp-heat environment,fat and sweet diet and microorganism.①Dnorma control group:fed with ordinary foodstuff in 20-28℃and 50-70% relative humid environment,②model group:fed by high suger and fat diet and escherichia coli in simulation of climate warehouse.③feed the rats with low dosage sanren decotion based on the model of spleen and stomach damp-heat syndrome.④feed the rats with medium dosage sanren decotion based on the model of spleen and stomach damp-heat syndrome.⑤fed with high dosage sanren decotion based on the model of spleen and stomach damp-heat syndrome.4.indexs of observation and detectionObserve the change of general state,weight,rectal temperature,diet,drinking and shit character between before and after modeling.Observe the behabior of rats in open-field test by small animal behavior recording and analysis system.Observe respectively the difference of before and after administration.Figure out the index of adrenal gland by weighing method and simultaneously detect the concentration of CRH,ACTH,COR,β-EP,ALD and ADH of rats.5.stastistic methodAll the datas were processed by statistical software SPSS13.0.the data type is quantitative data,expressed by mean±standard deviation(x±s).The data of weight and rectal temperature are analyzed by variance of repeated measure.The data of open-field test is compared by paired t test.The index of adrenal gland and other indexs including CRH,ACTH,COR,β-EP,ALD,ADH are analysed by one-way ANOVA method.Multiple comparison is employed by LSD for different groups, p<0.05 is the significant level.Result:1.general condition of the rats Before the test,all the rats have good condition,active movements,glossy white fur,normal eating and drinking,shape stool and normal temperature.After the test,normal group have not abnormal performance.With the conduct of model establishing,most of rats appear as tired,lazy,dull,fluffy hair,haggard face,reduced diet,sticky stool.From the tenth day,groups employed in composite methods has some abnormal performance,stool is sticky,rectal temperature increases quickly.after entering simulation of climate warehouse,the symptoms are aggravated.the groups with different dose sanren decotion,the movements,diet and stool are improved to norma level.2.the changes of weight and temperatureThe change of weight:there is no statistical significance between every two groups on 1st day(F=0.467,P=0.759).the first ten days,the growth of weight in each group have no difference.Compared with norma group,weight of 4 model groups grow slowly when going into simulation of climate warehouse.The trend of weight gain in model groups keep consistent from 18th to 20th day,but slower than normal group.20 days later,weight growth of Sanren decotion with differtent dose groups return to normal level.It is lower than norma control group at different time point (P=0.000),which prompts weight losss.After giving Sanren decotion,all treatment groups keep grow normaly.Weight of normal control group increase normal all the time.Change of rectal temperature:there is no statistical significance between every two groups on 1st day(F=0.665,P=0.602).the first ten days,the rectal temperature in each group have no significant difference.Retal temperature of 4 model groups when going into simulation of climate warehouse increase quickly.The temperature of model groups go up obviously after giving enteroinvasive E.Coli.Compared with normal group,it has statistic significance(P<0.01).After giving every dosage sanren decotion for 7 days,the rectal temperature of rats decline largely,but model group has no obvious decline.Normal control group has no change at different time point.3.observation of open-field testChange of central cell residence time:befor the treatment,compared with normal group,central cell residence time of model group,sanren decotion high and low dose group prolong significantly(P<0.01),and so sanren decotion does (P<0.05).this reveals that spatial cognitive competenc of rats declines.After the treatment,time of sanren decotion group with different dose is less than that of before the treatment.And compared with model group,it has significant difference(P<0.01 or P<0.05), there is no significant difference between sanren decotion high dose group and medium dose group and low dose group(P>0.05).this shows that sanren decoton can improve the spatial cognitive competence of rats.Change of sports cell and vertical frequency:befor the treatment,compared with normal group,sports cell and vertical frequency of model group,sanren decotion high,medium and low dose group decrease significantly(P<0.01 or P<0.05),this reveals that excitability,capacity to adapt to new environment and competenc to explre of rats declines.After the treatment,sports cell and vertical frequency of sanren decotion group with different dose increase more than that of before the treatment.And compared with model group,it has significant difference(P<0.01 or P<0.05),there is no significant difference between sanren decotion high dose group and medium dose group and low dose group(P>0.05).this shows that sanren decoton can improve the explore behavior and independent activity,and increase excitability of rats.Change of modification frequency:befor the treatment,compared with normal group,modification frequency of model group,sanren decotion high,medium and low dose group decrease significantly(P<0.05),this reveals that awarness and satisfaction to new environment of rats decline.After the treatment,modification frequency of sanren decotion group with different dose increase differently.And compared with model group,it has significant difference(P<0.05),there is no significant difference between sanren decotion high dose group and medium dose group and low dose group(P<0.05),this shows that sanren decoton can improve the degree of satisfaction to new environment of rats.4.detection of experimental indexAfter the success of establishing model,the concentration of plasma CRH、ACTH、serum COR elevates obviously(P<0.01).compared with model group,the concentration of CRH,ACTH and COR in sanren decotion high,medium and low dose group decline obviously and have significant difference(P<0.01 or P<0.05), especially sanren decotion medium dose group has best effect.Sanren decotion medium dose group is better than high dose group to decline CRH,while it is better than low dose group to decline ACTH(P=0.038),while has no difference with high dose group(P=0.091). Medium dose group is better than high dose group to decline Cor(P=0.034),while has no difference with low dose group(P=0.034)Compared with normal group,adrenal gland index and plasmaβ-EP of normal group go up obviously and has significant difference(P<0.01).compared with model group,adrenal gland index and plasmaβ-EP of sanren decotion high,medium and low dose group decreases obviously and has significant difference (P<0.01 or P<0.05).Compared with medium dose group,high and low dose group has significant difference(P<0.01 or P<0.05),but there is no significant difference between high and low dose group(P>0.05).Compared with normal group,ALD and ADH of normal group go up obviously and has significant difference(P<0.01).Compared with model group,ALD and ADH of sanren decotion high,medium and low dose group decreases obviously and has significant difference after giving sanren decotion for 7 days(P<0.01 or P<0.05).Compared with medium dose group,high and low dose group has significant difference to decrease ALD(P<0.05),but there is no significant difference between high and low dose group(P=0.534).there is no significant difference between high,medium and low dose group(P<0.05).Conclusion:The experimental results reveal that under the composite factors of high glucose and lipid foodstuff, damp-heat environment and microorganism,the speen and stomach damp-heat syndrome animal model can be copied successfully and may result in the abnormal change of behavior and secretion of neuroendocrine system.compared with normal control group,the central cell residence time prolong obviously,this reveals that cognitive competence decrease,sports cells,vertical frequency and modification frequency of model group are less than control group,it shows that excitability and the capacity adapt to the new surrounding are lower than control group.And it also reveal that autonomous activities and exploratory behavior of rats in unfamiliar surrounding decrease.Compared with control group, the concentration of plasma CRH,ACTH,secrum Cor in the model rats have a significant difference and lead to the change of the behavior,this show that there are close relation among experimental indexs,behavior and neuroendocrine system,rats are in a stress state,promote to improve self-protection and self-regulation.Growth of adrenal gland index andβ-EP indicates pathological change to adapt to new enviroment in stress state.Several indicators all above may use as uch as indicators of rectal temperature,weight and behavior.The concentration of plasma ADH and secrem ALD increase obviously,reveals that distal renal tubular and collecting ducts reabsorb water enhancely,which may result in moisture stagnation and water-sodium retention.Sanren decotion may have the effects as follows:1)can recover the general condition of spleen and stomach damp-heat syndrome animal model result from composite factors.2) may decrease concentration of ACTH andβ-EP and improve the situation of rats.3)may improve adrenal gland hypertrophy.4)regulate the function of HPA axis and decrease the secretion of CRH,ACTH and Cor.5)regulate secretion of ALD and ADH to maintain constancy of the internal environment.更多还原