【作者】 谭勇；

【导师】 吕爱平；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2009， 博士

【摘要】 目的观察白附片对正常大鼠和肾阳虚证大鼠的毒性反应差异,探索基于中医证候动物模型评价中药毒性的科学、合理性。方法建立肾阳虚证大鼠模型,从生化、免疫、病理、代谢组学等方面评价白附片对正常大鼠和肾阳虚证大鼠的毒性反应并比较两者差异。1.动物SPF级雄性SD大鼠80只,体重200±20g。2.分组、造模及给药80只大鼠随机分为正常组和模型组。模型组连续9d肌注氢化可的松琥珀酸钠溶液35.715 mg/kg·d,制作肾阳虚证模型。造模结束后将两组大鼠分别随机分为低剂量、小剂量、中剂量和大剂量4个给药组和空白对照组,每组8只;给药浓度从低到高依次为:0.32 g/kg·d、0.64 g/kg·d、1.28g/kg·d和2.56 g/kg·d,连续给药14d。3.取血和取材给药结束后麻醉取血,制备血清、血浆;取脏器(心、肝、肾、肾上腺、脾);称取脏器湿重、计算脏器系数,4%的甲醛溶液固定组织,以及制备心肌组织匀浆。4.常规实验研究4.1观察指标观察大鼠行为形态及体重的变化。以Biopac MP多道生理记录仪描记Ⅱ导联心电图,记录心率(BPM)和峰-峰值(p-p),以及心律失常情况。全血检测:白细胞总数(WBC)、红细胞总数(RBC)、血红蛋白(HGB)和血小板总数(PLT)。血清生化检测:总胆固醇(CHO)、甘油三酯(TG)、总蛋白(TP)、血糖(GLU)、总胆红素(T-BIL)、尿素氮(BUN)、肌酐(CRE)、γ-谷氨酰转肽酶(GGT)、尿酸(UAC)、天门冬氨酸转换酶(AST)、丙氨酸氨基转换酶(ALT)、肌酸激酶(CK)和乳酸脱氢酶(LDH)。放射性免疫检测:促肾上腺皮质激素(ACTH),皮质醇(COR),环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)。免疫比浊检测:心肌琥珀酸脱氢酶(SDH)和心肌钠-钾ATP酶(Na~+-k~+-ATPase)。HE染色,光镜观察心、肝、肾、肾上腺和脾脏的病理改变情况。4.2统计分析计量资料采用单因素方差分析,广义线性模型两因素方差分析、协方差分析、球形检验、重复测量方差分析,以及直线回归分析;计数资料用Logistic回归分析。显著性水平取P<0.05。5.代谢组学研究采用液相色谱-质谱联用的代谢组学技术获取代谢物指纹图谱数据;用正交偏最小二乘法对数据进行挖掘。结果1.常规实验研究结果1.1大鼠行为形态及体重变化情况随着给药剂量的增大,正常大鼠的行为形态毒性表现比模型大鼠突出;在低剂量给药时,模型大鼠肾阳虚表现明显缓解,白附片发挥治疗效应。正常和模型大鼠体重均随天数增长,剂量越大增长的幅度越小,提示白附片的毒性与剂量相关,剂量越大则毒性越大;在低剂量给药时,模型大鼠体重随天数增长的幅度较正常大鼠大,白附片发挥治疗效应。1.2主要血常规指标变化情况氢化可的松造模导致WBC和PLT降低。给白附片后,正常大鼠WBC和PLT随剂量增加而降低,表现为白附片的毒性效应;而模型大鼠WBC和PLT随剂量增加而升高,表现为白附片的治疗效应。氢化可的松造模可导致RBC和HGB的降低,尤其以HGB降低更为明显。白附片对正常大鼠的RBC和HGB有一定的影响,主要表现为随着剂量的增加RBC和HGB有轻微降低的趋势。对模型大鼠,低剂量白附片能提高血液RBC和HGB的水平。1.3血清糖、脂、蛋白指标变化情况氢化可的松造模导致GLU降低。给白附片后,正常大鼠GLU随剂量增加而降低,表现为白附片对正常大鼠的毒性效应;而模型大鼠GLU随剂量增加而升高并趋于正常,表现为白附片提高正常大鼠GLU的治疗效应。氢化可的松造模导致TG的升高,而给白附片后,模型大鼠TG有降低的趋势。给予白附片能使正常大鼠和模型大鼠CHO和TP有升高的趋势。1.4反映心脏功能的指标变化情况白附片可使正常大鼠和模型大鼠心律失常。随着剂量的增加,心律失常发生率升高。白附片引起模型大鼠心律失常的发生率低于同剂量白附片引起正常大鼠心律失常的发生率,提示白附片对正常大鼠的心律影响更大。白附片可加快正常大鼠和模型大鼠的心率,随着剂量的增加心率加快,但模型大鼠心率加快的幅度较小。白附片引起正常大鼠和模型大鼠P-P增大,增幅一致。随着白附片剂量的增大,正常大鼠和模型大鼠的心脏系数和LDH有增大的趋势。正常大鼠LDH升高的更快,反映了白附片对正常大鼠周围心肌酶的异常影响。随着白附片剂量的增加,正常大鼠和模型大鼠的SDH和Na~+-K~+-ATP酶有下降的趋势,正常大鼠SDH下降的更快,提示白附片也对正常大鼠和模型大鼠的心肌组织线粒体标志酶有不同的影响。1.5反映肝肾功能的指标变化情况随着白附片剂量的增加,正常大鼠和模型大鼠的ALT有升高的趋势,正常大鼠升高的更快,提示白附片对正常大鼠的肝毒性更突出。随着白附片剂量的增加,正常大鼠BUN有升高的趋势,反映了白附片可能对正常大鼠有一定的肾毒性。1.6反映神经、内分泌功能的指标变化情况氢化可的松造模导致cAMP和COR降低而cGMP升高。随着白附片剂量增加,模型大鼠cAMP/cGMP和COR有升高的趋势。随着白附片剂量的增加,正常大鼠和模型大鼠的肾上腺系数有增大的趋势,而胸腺系数有减小的趋势。1.7病理组织形态学变化情况白附片可引起正常大鼠和模型大鼠心、肝、肾、肾上腺和脾脏的组织形态学改变,其改变程度随着剂量的增加而加重。同一剂量给药时,两种大鼠同一脏器的病理改变存在差异,模型大鼠病理改变相对较轻。2.代谢组学研究结果氢化可的松造模导致溶血磷脂酰乙醇胺(18:0LPE)和溶血磷脂酰胆碱(20:5LPC)下调。18:0LPE和20:5LPC是正常大鼠和模型大鼠给予白附片后两者血浆代谢不同变化的主要标志物。正常大鼠18:0LPE和20:5LPC随剂量的增加有下降的趋势,而模型大鼠变化不明显。结论本研究发现:白附片可导致正常大鼠和肾阳虚大鼠行为形态,体重,血细胞,糖、脂、蛋白代谢,心、肝、肾功能,神经、内分泌功能,组织形态学,以及血浆代谢物的异常。这些异常反映了白附片对正常大鼠和肾阳虚大鼠的毒性作用。两者异常的差异表现为所涉及的指标不同,并且同一指标的异常程度有差异,具体可见白附片导致肾阳虚大鼠的异常指标相对较少,异常程度较轻。而且,从某些指标来看,白附片对肾阳虚大鼠有明显的治疗作用。本研究为基于中医证候动物模型评价中药毒性的合理性提供了客观依据,是丰富和完善现代药物毒性评价理论和方法体系的有益探索。更多还原

【Abstract】 ObjectiveTo observe the difference of Toxic Reaction of white prepared lateral root to normal and kidney yang deficiency rats and explore the rationality of evaluating the toxicity of traditional Chinese herbs based upon traditional Chinese medicine(TCM) syndrome animal model.MethodsEighty Sprague-Dawley(SD) male rats were divided randomly into normal and kidney yang deficient model group,forty rats respectively.After producing kidney yang deficient model to rats in model group by injecting hydrocortisoni natrii succinas in intramuscular, normal and model rats were respectively and randomly divided into low,little,medium,great dose group and blank group,eight rats in each group.Those rats were administered orally with different concentrations of white prepared lateral root of aconite every day for two weeks. Taking blood and preparing serum and plasma after anaesthetizating rats.Taking the internal organs(heart,liver,kidney,adrenal gland and spleen),weighing wet heavy and calculating organs coefficient.Fixing the organ with 4%formaldehyde solution,as well as preparing the cardiac muscle organ admixture liquid.1.The Convention Experiment Study1.1 Observed TargetObserving the change of rats action,weight and morphology.TracingⅡcouplet electrocardiogram with Biopac multiple tracks physiology apparatus and recording heart rate(BPM) and peak-to-peak(P-P) as well as arrhythmia.Blood test:White blood cell total(WBC),red blood cell total(RBC),hemoglobin (HGB) and platelet total(PLT).Serum biochemistry test:Cholesterol(CHO),total glycerine esters(TG),total protein (TP),glucose(GLU),total bilirubin(T-BIL),urea nitrogen(BUN),urine creatinine(CRE), gamma glutamyl transferase(GGT),uric acid(UAC),glutamic oxaloacetic transaminase (AST),alanine aminotransferase(ALT),creatine Kinase(CK) and lactate dehydrogenase(LDH).Radioactivity immunity test:Adrenocorticotrophic hormone(ACTH),hydrocortisone (COR),cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP).Immunoturbidimetry test:Cardiac muscle Succinate dehydrogenase(SDH) and Na~+-k~+-ATPase enzyme(Na~+-k~+-ATPase).Dyeing with HE and observing the pathological change of heart,liver,kidney,adrenal gland and spleen with optical microscope.1.2 Statistical analysisQuantitative data:adopting one factor analysis of variance,general linear model two factors analysis of variance,covariance analysis,repeated measurement analysis,as well as the straight line regression analysis.Count data:Logistic’s regression analysis.P<0.05 was taken to the significance standard.2.Metabolomics studyCapturing metabolite fingerprint data with the liquid phase color spectrum-mass spectra (LC/MS) metabolomics technology and excavating data orthogonal partial least squares (OPLS).Results1.The Convention Experiment Results1.1 Rats Action,Weight and Morphology ChangeAs concentration increased,the Toxic Reaction due to action and morphology change of normal rats was more and more evident than model rats’.With low dose of administration, kidney yang deficient syndrome was clearly relieved.The weight of normal and model rats increased with extending time of medication.More dosage accompanied with slighter increase of weight.White prepared lateral root of aconite shown reating effect at low dosage.1.2 Major Blood Convention TargetHydrogenation cortisone caused WBC and PLT reduction.After taking white prepared lateral root of aconite,normal rats’WBC and PLT reduced with adding dose,which showed the toxicity effect of white prepared lateral root of aconite.However,model rats’WBC and PLT increased with adding dose,which showed the treating effect of white prepared lateral root of aconite.Hydrogenation cortisone leaded RBC and HGB to reduce,especially HGB reduce much more clearly.White prepared lateral root of aconite had definite influence on normal rats.Mostly representation was that RBC and HGB had the trend of light reduction with adding dose.As to model rats,low dose white prepared lateral root of aconite could raise blood RBC and HGB level.1.3 Serum Glucose,Ester and Protein LevelHydrogenation cortisone caused GLU reduce.After taking white prepared lateral root of aconite,normal rats’ GLU reduced with adding dose,which showed the toxicity effect of white prepared lateral root of aconite.However,model rats’ GLU increased with adding dose, which showed the treating effect of white prepared lateral root of aconite.Hydrogenation cortisone leaded TG to go up.Taking medicine made model rats’ TG have a trend of reduction with adding dose.Moreover,taking white prepared lateral root of aconite could make normal rats’ CHO and TP go up.1.4 Heart FunctionWhite prepared lateral root of aconite could cause normal rats and model rats arrhythmia. With the increase of dosage,the occurring rate of arrhythmia went up.The occurring rate of arrhythmia of model rats is lower than normal rats’,which pointed out that white prepared lateral root of aconite had more great infaust influence on normal rats.White prepared lateral root of aconite could quicken heart rate and P-P vaule of rats,and both were inclined to quicken with adding dose.The increasing extent of heart rate of model rats was lower than normal rats’,but it’s the increasing extent of P-P vaule was consistent to normal rats’.With adding dose,all rats heart coefficient and LDH squinted towards going up.Normal rats’LDH going up was quicker than model rats’,which indicated abnormal influence on surrounding cardiac muscle enzyme.With adding dose,all rats SDH and Na~+-K~+-ATPase had a trend of descending.Normal rats’ SDH descending was quicker than model rats’,which showed white prepared lateral root of aconite also impacted rats’cardiac muscle mitochondrion enzyme. 1.5 Liver and Kidney FunctionWith adding dose,all rats ALT squinted towards going up,but ALT of normal rats went up more quickly than model rats,which suggested white prepared lateral root of aconite caused more liver toxicity to normal rats.In addition,white prepared lateral root of aconite also produced kidney toxicity to normal rats.1.6 Nerve and Endocrine FunctionHydrogenation cortisone caused cAMP and COR descend,cGMP increase.With adding dose,cAMP/cGMP and COR of normal rats inceased,both adrenal gland coefficient auxed, but chest gland coefficient descend.1.7 Morphology Change of Pathology OrganWhite prepared lateral root of aconite caused both rats organ morphology change in heart, liver,kidney,adrenal gland and spleen.Trauma extent became serious gradually with adding dose.To same internal organ and same dose medicine,the pathology change of model rats was slighter than normal rats.2.Metabolomics Study ResultMetabolomics study indicated that Lysophosphatidyl choline(LPC) and lysophosphatidyl ethanolamine(LPE) were chief material symbols among various metabolite in normal and model rats caused by white prepared lateral root of aconite.LPC and LPE had different change trend between normal and model rats with dose change.ConclusionThis study showed that white prepared lateral root of aconite could cause abnormity in normal rats and kidney yang deficiency rats as following:action and morphology,weight, major blood convention target,serum glucose,serum lipid and protein level,heart function, liver and kidney function,nerve and endocrine function,pathology organ change,and plasma metabolite change.All abnormal changes reflected toxicity effect of white prepared lateral root of aconite to normal and kidney yang deficiency rats.The abnormal targets in kidney yang deficiency model rats were less than in normal rats both in quantity and degree. Moreover,white prepared lateral root of aconite shown treating effect to model rats by some abnormal targets.This study provided objective evidence for the theory of evaluating traditional Chinese herbs toxicity based on TCM syndrome animal model,which proved to be a beneficail exploration in enriching modern medicine toxicity evaluation system.更多还原