【作者】 迟莉；

【导师】 陈蔚文；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 研究背景和目的健脾消食类中药是中药中较有特色的一类。该类中药对由食积停滞所致的脘腹胀满、嗳气泛酸、恶心呕吐、不思饮食、泄泻或便秘等症有较好的治疗效果。目前该类中药的药理学研究多是从其对胃肠运动和胃肠功能的影响方面着手,国内尚少见涉及调节中枢神经系统中药药理研究的文献报道。厌食症是较常见的食欲不振的疾病,本实验拟将厌食症作为研究对象。目前研究厌食的动物模型大都直接采用脾虚动物模型,临床上厌食症的中医证型不一,例如有脾失健运、脾胃虚弱、胃阴不足、虚实夹杂等证型,脾虚只是其中证型之一。因机体调节食欲的中枢在神经系统,厌食的根本原因在于中枢神经系统调控失常,故本研究拟建立符合致病原因的中枢抑制性厌食症动物模型,为厌食症的药理学研究提供参考依据。瘦素(Leptin,LP)是肥胖基因的产物,是白色脂肪细胞分泌的一种由167个氨基酸残基组成的蛋白质。Leptin可与下丘脑进食中枢的leptin受体相结合,抑制NPY及AgRP神经元的表达,抑制食欲,也可与饱食中枢的leptin受体结合,促使前鸦片黑皮质素原(POMC)神经元分泌前鸦片黑皮质素原,代谢为鸦片黑皮质素,作用于鸦片黑皮质素-4和鸦片黑皮质素-3受体,引起食欲降低及机体能量消耗增加。CCK参与胃肠功能的调节,并作为饱感信号起抑制食欲的作用,是外周摄食调节的主要生理因子;本文实验二研究发现多个剂量的CCK单独腹腔注射都没有抑制食欲和降低体重的作用。文献报道CCK短期抑制食欲的激素,可与长期影响脂肪沉积继而影响能量代谢的LP协同作用,共同参与中枢的摄食调节。CLAIRE报道LP50μg/kg单独腹腔注射可以降低大鼠进食量,LP 50μg/kg和8～16μg/kgCCK联合腹腔注射效果更佳,两者联合腹腔注射后与LP单独腹腔注射后大鼠食量比较差异有统计学意义,Scott等报道第三脑室注射LP 7μg/kg和腹腔注射CCK 2μg/kg都有降低食欲的作用。Kiely则发现给LP缺乏的肥胖大鼠腹腔注射LP 5μg/kg一个月,可增加大鼠十二指肠和结肠CCK表达,推测认为LP抑制食欲的作用可能通过作用于外周胃肠CCK起效。NPY是下丘脑最重要的食欲促进因子。CCK可能是通过作用于LP发挥抑制食欲的作用,而LP和CCK的抑制食欲的作用都要通过下丘脑NPY起效。脑室和腹腔注射瘦素能明显抑制下丘脑NPY的合成和释放。山楂麦芽颗粒是中山中智药业集团的产品,主要由消食和健脾两类中药组成,临床取得较好的疗效,本实验室药效学研究也表明该产品有较好的促进动物胃肠运动和提高动物进食量并促体重增长的作用。香砂六君子汤是著名古方,对脾虚气滞或痰瘀引起的食欲不振有较好效果。本课题拟建立中枢抑制性厌食症动物模型,用于观察山楂麦芽颗粒和香砂六君子汤的药理作用,从脑肠肽-食欲调节中枢角度探讨健脾消食类中药作用机制。研究方法1.筛选几种厌食症动物模型的造模方法,包括给幼龄大鼠喂养由鸡蛋、奶粉、五花肉、白糖等制成的高营养饲料模型,腹腔注射缩胆囊素模型,腹腔单用及联合注射缩胆囊素和瘦素模型,侧脑室预置套管注射瘦素模型等。2.在成功建立的中枢抑制性厌食症大鼠模型上,主要观察造模对大鼠整体状态、体重和进食量的影响,检测对进食起关键作用的下丘脑神经肽Y和血浆神经肽Y(NPY,一种脑肠肽)含量,探讨造模后对大鼠脑肠肽-食欲中枢的影响。观察山楂麦芽颗粒和香砂六君子汤水提液对模型大鼠的治疗作用,并检测下丘脑和血浆神经肽Y含量。3.建立山楂麦芽颗粒质量控制的定性定量检测指标;药效学实验观察山楂麦芽颗粒对动物胃肠运动和进食量等的影响。结果1.给幼龄大鼠喂养高营养饲料的造模方法可导致动物进食量降低,也能使动物体重增加非常缓慢,但停止造模并恢复给正常饲料后,大鼠体重和进食量都快速增加,造模期间大鼠厌食的原因在于这种由各类高脂肪高蛋白物质构成的食物不符合大鼠的进食习惯,并非食欲降低;缩胆囊素3个剂量5μg/kg、10μg/kg和20μg/kg腹腔注射均没有降低大鼠食欲的作用;缩胆囊素和瘦素单用及联合腹腔注射也没有降低动物食欲的作用;可能是由于动物吸收不好或药物作用时间过短等原因,致使造模药物不能抑制食欲。2.采用侧脑室注射瘦素的方法成功建立了厌食症动物模型。其方法为先给大鼠侧脑室置管(以300g左右大鼠为例,套管针埋在前囟后1.5cm,旁开1.5cm,深度4.0mm比较合适),大鼠置管并正常饲养1周后,给大鼠脑室注射瘦素每日1次共7天,剂量10μg/kg/20μL,注射速度约1μL/min。造模后大鼠平均每天进食13g左右饲料,体重平稳下降,平均每天6～10g左右。造模结束处死大鼠,放射免疫法检测NPY,结果造模大鼠下丘脑NPY含量显著降低(正常组下丘脑NPY平均含量为22.2ng/ml,模型组下丘脑NPY平均含量为14.8ng/ml,P＜0.05)。3.山楂麦芽颗粒8g/kg(药效学实验有效剂量,临床用量的9倍)和香砂六君子汤13.6g/kg(临床用量的15倍)对侧脑室注射瘦素致厌食症大鼠有一定治疗作用,虽没有提高动物进食量,但在一定程度提高动物体重,其中香砂六君子汤能提高大鼠下丘脑NPY含量(模型组下丘脑NPY平均含量为14.8ng/mL,香砂六君子汤组NPY平均含量为20.3ng/mL,P＜0.05)。4.建立了山楂麦芽颗粒的定性定量质量标准:高效液相法检测山楂麦芽颗粒中陈皮主要成份橙皮苷的含量,薄层色谱法定性鉴别山楂、麦芽和陈皮。药效学实验表明山楂麦芽颗粒8g/kg对动物胃肠运动及提高进食量和体重有效。结论侧脑室注射瘦素致中枢性厌食症大鼠模型,造成大鼠适度的厌食和体重降低,并能抑制下丘脑NPY表达,与机体食欲调节的机制原理相符,其致厌食的原理与厌食症发病机理类似,健脾消食中药山楂麦芽颗粒和香砂六君子汤对该模型大鼠有一定治疗作用,其中香砂六君子汤作用机制可能与下丘脑NPY调节有关,所以该模型可用于厌食症的治疗机理研究,也适合用于其它健脾消食中药的药理研究。更多还原

【Abstract】 Backgrounds and aimsInvigorating spleen Chinese medicines to promote digestion are of much more features in the whole of Chinese medicines,those Chinese medicines can obtain some good effects in treating such diseases as abdominal,belch,bepanthen,nausea and vomiting, anorexia,diarrhoea,adstricto,etc,resulting from fresh plot stagnation.At present, pharmacological researchs for those kinds of Chinese medicine almost began with studying the drug effect in stomach intestine & function,there is few literatures about pharmacological research for Chinese medicine in respect of adjusting central nervous system at home.We intend to take apositia as the object of study,which is a disease of losing appetite frequently occurred.At present,most of studies of apositia began at first to adopt animal model of splenic asthenia,there are many patterns of syndrome of apositia in the clinical practices,the model of splenic asthenia cannot represent all the models of apositia.The center of adjusting the appetite in the body activities lies in the nervous system,the root cause of apositia is controlling disorder of the central nervous system,this sdudy intends to establish an animal model of apositia with the function of central inhibition according to the cause of disease to give some reference for pharmacological research of apositia.Shanzha Maiya granula is the product of Zhongshan Zhongzhi Medical Industrial Group Coporation Ltd,mainly composed of two kinds of medicines promoting digestion and invigorating the spleen,with good effect in the clinical trial,this Laboratory’s pharmacodynamic research also illustrates that this product can play an important role in promoting stomach intestine and increasing the animal’s weight & food intake.Decoction of Xiangshasix Mild Drugs is a classical prescription of long history,which has good effect in treating the loss of appetite arising from Qi stagnant or sputum stasis.This topic intends to set up the animal model of apositia with the function of central inhibition to investigate the pharmacological effect of Shanzha Maiya granula and research the functional mechanism of the Chinese medicines for invigorating spleen to promote digestion in view of braingut petide-appestat.Methods of research1.screening some methods of setting up the animal model for children’s loss of appetite,including feeding the young mouse with the feeder of high nutrition made of eggs, milk,pork,sugar,etc;intraperitoneal injection of Cholecystokinin(CCK);intraperitoneal combined injection of Cholecystokinin(CCK) and Leptin(LP);pre-setting canula to inject LP in the lateral cerebral ventricle,etc.2.on the basis of successfully establishing the animal model of apositia,concentrates on investigating the model’s effect on the mouse’s whole pattern,weight and food intake, detecting the content of neuropeptide Y in the mouse’s hypothalamus & blood plasm,which plays key role in eating,and mastering the medicine’s effect on the mouse’s braingut petide-appestat after setting up the animal model.Investigate the medical effect of the water extract of Decoction of Xiangsha six Mild Drugs and Shanzha Maiya granula on the model mouse,and detect the content of NPY in hypothalamus & blood plasm.3.set up the indicators of qualitatively and quantitatively detecting Shanzha Maiya granula,and investigate the pharmacodynamics indicator of the Shanzha Maiya granula’s effect on the animal’s stomach intestine & food-intake,etc.Results1.the model method of feeding the young mouse with the feeder of hyperalimentation may make the mouse to eat less,and make the mouse’ weight to increase slowly, however,after stopping the model method and returning to feed the mouse normally,the mouse’ weight & food-intake increase quickly.During the time of making the model,the cause of the mouse’s apositia is the mouse disliking those foods composed of high-fat & high-protein substances,not loss of appetite;none of the 3 groups of intraperitoneal injection of 5μg/kg,10μg/kg,20μg/kg Cholecystokinin(CCK) can make the mouse’s appetite to lower;the intraperitoneal combined injection of Cholecystokinin(CCK) and Leptin(LP) cannot make the animal’s appetite to lower,it is likely that the medicine’s failure to inhibit the appetite results from the animal’s bad absorption or the time of the medicine taking effect being too short,etc.2.establishing successfully the animal mode of apositia through injecting Leptin (LP).This method can be illustrated as follows:at first,set canula in the lateral cerebral ventricle of mouse(take the mouse weighting about 300 grams as example,it is fit to bury the canula needle in 1.5cm after anterior fontanel,1.5cm left or right,4ram deep);after the mouse’s recovery for a week,inject in the every day the mouse with the dose of 10μg/20μl/kg leptin at speed of about 1 macro-litre as per minute.After making the model, the mouse eats about 13 gram of feeder everyday,its weight reduces steadily about 6-10 gram everyday on the average.After the termination of the model,kill the mouse,detect NPY and then see the content of NPY in the Hypothalamus of the mouse significantly lower.3.8g/kg(it is valid dose proved by pharmacodynamic experiment,about 9 times of human dose) of Shanzha Maiya granula and 13.6g/kg(15 times of human dose) of Decoction of Xiangsha six Mild Drugs can play a certain role in treating the animal model of child apositia resulting from leptin,fail to increase the animal’s food-intake,but can increase its weight to some extent,Decoction of Xiangsha six Mild Drugs can increase the content of NPY in the Hypothalamus of the mouse.4.set up quality standard of qualitatively and quantitatively detecting Shanzha Maiya granula:detecting the content of aurantiamarin as a main component of Chenpi in Shanzha Maiya granula through the method of high performance liquid phase,qualitatively identifying Shanzha,Maiya,Chenpi through the method of plate chromatography.The pharmacodynamic experiments illustrate that 8g/kg of Shanzha Maiya granula’s effect is the best in promoting stomach intestine,increasing food-intake and weight.ConclusionThe mouse model of intraperitoneal injection of leptin causing apositia which makes the mouse’s appetite & weight to lower,can inhibit expression of NPY in hypothalamus, conform to the principle about mechanism of body appetite’s adjustment,so the principle of the model causing apositia is similar with the incidence mechanism of apositia,Shanzha Maiya granula and Decoction of Xiangsha six Mild Drugs as Chinese medicines for invigorating spleen to promote digestion can play an important role in treating the model mouse,the functional mechanism of Decoction of Xiangsha six Mild Drugs is related to adjustment of NPY in hypothalamus,so this model which is used to researching the mechanism of treating child apositia,also may be used as pharmacological model fit for other Chinese medicines for invigorating spleen to promote Qi.更多还原