Research On The Technology And Methods Of Evaluating The Toxicity Of Traditional Chinese Medicine With Zebrafish Larva

Objective:Recently,the increasing popularity of Traditional Chinese Medicine(TCM)leads to frequent misuse and abuse.This triggers extensive discussion of the toxicity of TCM,and the subsequent negative influence obstructs the development of TCM.In this paper,we utilized zebrafish(Danio Rerio)as model animal,and established a method to evaluate TCM toxicity;we then assess the toxicity alteration of Kansui Radix before and after vinegar processing,and applied transcriptomics analysis to study the toxicity mechanism of Kansui Radix.Additionally,the applications of zebrafish enbryos on developmental toxicity of TCMs were also performed.Methods:1)To establish a method for evaluating toxicity of TCMs based on zebrafish larvae,we first stabilized the adult zebrafish husbandary system,and optimized the experimental conditions of zebrafish larvae.In order to establish the evaluation methods,we studied 4 toxic TCMs whose toxicity has been reported(Toosendan Fructus,Euodiae Fructus,Paridis Rhizoma and Genkwa Flos)and 2 non-toxic TCMs(Astragali Radix and Puerariae Lobatae Radix)on 72 hpf zebrafish larvae.As positive toxicity comparison,we applied Celebrex(for cardiac toxicity),Brefeldin A(for neural and hepatic toxicity),0.3%ethanol(for renal toxicity)and Domperidone(for Gastrointestinal irritation).We finally validated the applicability of the method by comparing the results with traditional animal experiments,clinical data and traditional records.2)Kansui Radix and Vinegar-processed Kansui Radix were sequential extract with petroleum ether,dichloromethane,ethyl acetate and 95%ethanol.The toxicity of separate extracts was assessed using the evaluation method based on zebrafish larvae that established in this paper,and the difference of Kansui Radix and vinegar-processed Kansui Radix were compared.We then selected the most toxic extract,and systematically evaluate its toxic effect and reversibility on zebrafish larvae.3)Taking advantage of Illumina sequencing platform and Paired-End technique,we record the transcriptome alteration of zebrafish larvae before and after the exposure of the most toxic Kansui Radix extract.We compared the transcription level of high(LC10),medium(LC1),low(1/3LC1)concentration groups with the control group.Then we screened the differentially expressed genes(DEGs)among the treatments and performed a Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of the DEGs.4)We studied the developmental toxicity of Polygoni Cuspidati Rhizoma Et Radix,Safflower and Carpesii Fructus on 6 hpf zebrafish embryos to 96 hpf.The analyses of mortality,malformations,spontaneous movement,heart rate,hatching rate,and body length were studied to examine the developmental toxicity of TCMs.Furthermore,activities of anti-oxidative enzymes,malondialdehyde(MDA)content,and caspase-3 activity were examined.In addition,the gene expression related to antioxidant proteins,the oxidative-DNA repair system,and apoptosis was tested to discuss the underlying toxicologic mechanism.Results:1)We optimized the fundamental experimental condition stated as follow.Normal developed 3 dpf larvae were selected under microscope,and placed into 12-wells plate with 20 larvea in each well.The larvae were presented to 3 ml exposure solution for three days with daily solution replacement.The mortility rate were recorded after 72 hrs drug administration and plotted into death curve.The LC50,LC10,and LC1 were calculated.Experiments were repeated with different concentration of exposure solution including 1/10 LC1,1/3 LC1,LC1 and LC10.The toxicity effects were observed on cardiac system,circulatory system,liver,kidney,nervous system,gastrointestinal tract,eyes,jaw,body pigmentation,muscle and skin.To access the reversibility of toxic effect,the larvae with three days drug administration were recovered for two days in incubation solution.The gastrointestinal irritation of drugs was detected by Nile red excretion test on 5 dpf larvae.The method is highly repeatable,and the results are highly consistent with that of rodent animal,clinical data and traditional records.Therefore,this method is capable to evaluate toxicity effect of TCMs.2)According to the LC50 of sequential extracts of Kansui and vinegar-processed Kansui,the toxicity order was petroleum ether extracs>dichloromethane extracs>ethyl acetate extracs>95%ethanol extracs.Converted to the original medicinal materials,petroleum ether extracs of Kansui was much more toxic than vinegar-processed Kansui,which suggested vinegar processing can decrease the toxicity of Kansui.Liver toxicity and gastrointestinal stimulation were observed in our studies.The enlarged liver was irreversibility,while liver degereration was reversibility in Kansui treated groups.The slight degree and reversibility of liver toxicity and gastrointestinal stimulation was also observed in vinegar-processed Kansui treated groups.3)The analysis of DEGs showed that 28 up-regulated genes and 1 down-regulated gene in low concertration,390 up-regulated genes and 32 down-regulated genes in medium concentrations,1651 up-regulated genes and 225 down-regulated genes in high concentrations were detected.According to GO and KEGG analysis,these genes were enrichment in Proteasome、Lysosome、Cytokine-cytokine receptor interaction and Apoptosis pathway.4)Heart rate,heart rhythm,hatching rate,hatching time,spontaneous movement and body length and malformations,such as pericardial edema,was detected in this study.Heart inhibition,hatching rate inhibition,body length inhibition,pericardial edema,enlarged yolk and yolk-sec edema was the most common endpoints observed in this study.The results also showed that TCMs exposure changed the activities of defense enzymes,increased MDA content,increased caspase-3 activity,and altered mRNA levels of related genesConclusion:In this paper,we established a method to evaluate Chinese medicine toxicity based on the zebrafish larvae.The method can be applied to evaluate the toxicity effect of TCM on multiple organ systems and its reversibility.The method is highly repeatable,and the results are highly consistent with that of rodent animal,clinical data and traditional records.Therefore,this method is capable to evaluate toxicity effect of TCM.Additionlly,zebrafish as a whole animal model demonstrates the advantages of low cost,low drug consumption and short experimental period,so it can be applied to TCM toxicity classification and toxicity screening.Our result shows that the toxicity of Kansui Radix is mainly derived from petroleum ether extracts,and characterized as hepatotoxicity and gastrointestinal irritation.The unprocessed drug introduces irreversible hepatomegaly and reversible hepatic degeneration,while the vinegar processed drug shows reduced toxicity and increase reversibility.The transcriptomics experiment demonstrates that the toxicity of Kansui Radix is relates to abnormal pathway of proteasome,lysosome,cytokine-cytokine receptor interaction,and apoptosis.The apoptosis is mainly mediated by external pathway.The expression of death ligand,TNFa and IL-1,was increased,and the membrane expression of Fas and TNF-R1 was upgraded,thus the expression of intracellular binding protein FADD and RIP1 was enhanced.These proteins can be cross-linked by homophilic aggregation of multiple caspase-8 molecules,and modify caspase-8 molecules from single to double chain protein with enzyme activity.The activated enzyme triggers caspases cascade,by activating Caspase-3,-6 and-7,and eventually apoptosis.The developmental toxicity experiment shows that Polygoni Cuspidati Rhizoma Et Radix,Safflower and Carpesii Fructus both have developmental toxicity on zebrafish larvae.Their sub-lethal concentration can generate a series of malformation,while imbalance of oxidative-reductive reaction and apoptosis is one of the mechanisms of the developmental toxicity.