| 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.Tracingâ…¡couplet 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. |
