Study On The Toxicity And Mechanism Of Panax Notoginseng Saponins Based On Larvae Zebrafish And Metabolomics

Panax notoginseng[Panax notoginseng(Burk.)F.H.Chen]is a rare traditional Chinese herbal medicine and is processed from the dry roots and rhizomes of Panax notoginseng.Its main active ingredient is PNS(panax notoginseng saponins),with a content of up to 12%.It has many functions,such as promoting blood circulation,inhibiting tumors,and is often used in the treatment of cardiovascular diseases.Review of the literature revealed that there were a few reports on its heart and hepatorenal toxicity.We can not ignore the report of its negative effects,and we should study it in depth.Therefore,this study,using the model of larvae zebrafish,aims to analyze the changes in endogenous metabolic profiles and biomarkers in the body induced by overdose of PNS,identify potential biomarkers associated with toxicity,establish its possible toxicity-related metabolic pathways,and elucidate the toxicity mechanism of overdose of PNS from a micro and holistic perspective,so as to provide a reference for clinical rational drug use and complete safety and toxicity evaluation of PNS.To improve the safety and effectiveness of Panax notoginseng and its preparations in clinical use.1.The PNS were determined by the content determination and acute toxicity test using the larvae zebrafish model.By observing the effects of short-term exposure to drugs on 2 dpf～6 dpf in healthy embryos and larvae of zebrafish,the behavior/morphological characteristics and organ changes of the fish were examined microscopically,and the number of fish deaths was counted every day to calculate the 50%lethal concentration LC50(6 dpf).It was analyzed that the LC50 of 6 dpf in larvae zebrafish treated with PNS was 110μg·mL-1,which had moderate toxicity to larvae zebrafish.2.The aim of this study was to investigate the effect of PNS by low dose non-toxicity on endogenous metabolites in larvae zebrafish and to search for metabolic biomarkers of PNS.The 5 dpf larvae zebrafish were treated at a low dose of PNS for 24 hours.With the help of UPLC-Q-TOF MS techniques,the groups of zebrafish samples were measured;in addition,multiple methods such as PCA and OPLS-DA were used for pattern recognition to study the endogenous metabolism of PNS in larvae zebrafish.The results showed that the QC sample group and the drug administration group were significantly different from the normal group,and gathered well within a certain range.Combining the variable importance projection VIP value of OPLS-DA model,one-way ANOVA is performed on the variable larger than 1 and the P value less than 0.05 is selected as the difference variable which contributes greatly to the classification between groups.A total of 26 potential biomarkers were identified.The PNS can change the metabolism of larvae zebrafish under low dose nontoxic characterization,which may be related to the regulation of 8 pathways such as glycerophospholipid metabolism,Alanine,aspartate and glutamate metabolism,aminoacyl-tRNA biosynthesis,Linoleic acid metabolism,etc.Compared with the metabolic markers and metabolic pathways of excessive toxic PNS in larvae zebrafish,to make the search result of the marker and pathways more reliable.3.Using metabolomics techniques to study the endogenous biomarkers and metabolic pathways associated with toxicity of PNS in larvae zebrafish.An experimental animal model of toxicity induced by PNS overdose was established by administering 1 dpf larvae zebrafish to 6 dpf at low,medium and high doses of PNS,respectively.Relying on UPLC-Q-TOF MS technology,the metabolite profile information of fish body and metabolic fluid of larvae zebrafish poisoned by PNS at different time points and doses was collected,in order to establish the metabolic profiles of larvae zebrafish about toxicity of PNS by the administration group and blank group.The differences in metabolic spectrum between the two groups were analyzed by means of principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).The results showed that in the administration group,both zebrafish body and metabolic fluid samples deviated from the blank control group,and increased with the extension of the number of administration days,showing a time-dependent metabolic trajectory until the end of the experiment.Specifically,the phenomenon that the metabolites in the drug-administered group "deviated"from those in the normal group,which was consistent with the expression of drug toxicity,suggesting that excessive administration of PNS could lead to metabolic disorders in larvae zebrafish,resulting in toxic damage,and the degree of damage is positively correlated with the dose of PNS.29 potential metabolites related to toxicity(VIP>1,P<0.05)were further screened and identified,mainly involving 6 metabolic pathways such as glycerophospholipid metabolism,arachidonic acid metabolism,biosynthesis of unsaturated fatty acids,valine,leucine and isoleucine,glycine,serine and threonine metabolism,etc.Through the attribution analysis of the metabolic pathways of the toxicity related biomarkers of PNS,the toxic targets and mechanism of PNS were searched from the perspective of metabolomics,and it was found that the excessive toxicity of PNS may be related to the disorder of fatty acid metabolism,amino acid metabolism,energy metabolism and other metabolic pathways.