Developmental Toxicity Of Five Dual-use Food And Drug Substances Evaluated By Embryoid Bodies And Zebrafish Models

PURPOSE:With the promotion of the strategy of"Healthy China"and the improvement of people’s awareness of health management,people’s pursuit of food is gradually changing from"satiety"in the past to"nourishment"now,and from the pursuit of From the pursuit of"spicy stimulation"to the pursuit of"mild nourishment".Statistics show that the market share of dual-use substances is increasing year by year,and the industrial application of dual-use raw materials is also very common in China.With the widespread use of dual-use substances,among the 110 varieties of dual-use Chinese medicines released by the National Health and Welfare Commission（hereinafter referred to as the"National Health and Planning Commission"）in 2022,46 dual-use substances were found to cause adverse reactions,which may be related to individual differences,consumption patterns,and consumption amounts.Because the public generally believes that dual-use food and drug substances are calm and safe to consume,they ignore the risk of possible interactions between dual-use food and drug substances and other drugs or Chinese medicines.Therefore,the rational and scientific use of dual-use substances has become a key issue in the public’s daily health care and wellness,and a prerequisite for the development of wellness and health industry.As dual-use herbal medicines are widely used on a daily basis,the safety of dual-use substances for pregnant women and women of childbearing age should be of sufficient concern.On the one hand,they can have potential negative effects on themselves,and on the other hand,there is a risk to the health and life of the unborn child,and once the presence of adverse reactions is detected,it may not be recovered after stopping the drug.Therefore,it is particularly important to protect the health of the fetus and mother and to improve eugenics and population quality.In this study,we investigated the effects of five dual-use substances（licorice,poria,lotus seed,fritillary and wolfberry）on embryonic development through in vitro experimental embryoid bodies（EBs）and in vivo experimental model organisms,zebrafish embryos,in order to provide clues for long-term consumption safety assessment and to provide reference for the scientific and rational use of dual-use substances by the public.METHODS:（1）The embryonic safety of five dual-use substances（Glycyrrhiza uralensis Fisch,Poria cocos,lotus seeds,Houttuynia cordata and Fructus Lycii）was evaluated using zebrafish embryo hatching rate,mortality and target organ toxicity and proposed embryo growth kinetic parameters.（2）A network toxicology approach was used to predict the target proteins and mechanisms of embryotoxicity production by Fritillaria spp.and then the core targets were verified by molecular docking,immunohistochemistry and RT-q PCR.（3）The effects of lotus seeds on the endo-and meso-epiblast and Bcl2/Bax/Caspase-3 pathway expression in mimic embryos were examined by immunofluorescence,ELISA and RT-q PCR.RESULTS:（1）The LC₅₀of Glycyrrhiza uralensis Fisch,lotus seed,Poria cocos and Houttuynia cordata extracts at 120 hpf were 413μg/m L,1508μg/m L,1161μg/m L and 2052μg/m L,respectively,and the LC₅₀of Fructus Lycii could not be calculated.Houttuynia cordata and lotus seeds had significant target organ toxicity to zebrafish.Glycyrrhiza uralensis Fisch,Poria cocos and Fructus Lycii did not show significant organ effects on zebrafish.Poria cocos,Glycyrrhiza uralensis Fisch and Houttuynia cordata reduced the hatching rate of zebrafish,while lotus seeds and Fructus Lycii had no effect on the hatching rate of zebrafish.Glycyrrhiza uralensis Fisch,Poria cocos,lotus seeds,Houttuynia cordata and Fructus Lycii had different degrees of effect on the diameter of mimic embryos,among which Houttuynia cordata.showed strong inhibition of the diameter of mimic embryos.（2）The important active components of Houttuynia cordata.producing embryonic effects were screened by network toxicology,including oleanolic acid,lignans and aristolochic acid lactam AII,etc.The key core targets were AKT1,EGFR,Caspase-3and IGF-1,etc.141 KEGG pathways were analyzed,mainly PI3K-Akt,MAPK and Ras signaling pathways.The molecular docking results showed that all the important active ingredients were successfully docked with the core targets.the results of RT-q PCR and immunohistochemistry showed that the expression of AKT1 and PI3K was significantly reduced in embryoid bodies after drug administration（P<0.05）.（3）In the effect of lotus seeds on embryo development,Bcl-2 was found to decrease with lotus seeds drug dose dependence and Bax and Caspase-3 increased with dose dependence by immunofluorescence,ELISA and RT-q PCR in this study.Also,it was found that lotus seeds had no effect on mimicry pluripotency factor,elevated exo-medioblast gene expression and mimicry mesoderm inhibition,suggesting that possibly lotus seeds have an effect on mimicry through the Bcl2/Bax/Caspase-3pathway and elevated exo-medioblast gene expression and mimicry mesoderm inhibition.CONCLUSION:In this study,we initially investigated the effects of aqueous extracts of Glycyrrhiza uralensis Fisch,Poria cocos,lotus seeds,Houttuynia cordata and Fructus Lycii on zebrafish and mimic embryos.It was found that Houttuynia cordata may produce anthropomorphic effects through AKT-PI3K,and lotus seeds may have effects on anthropomorphic embryos through Bcl2/Bax/Caspase-3 pathway.Meanwhile,screening the effects of dual-use food and drug substances on embryonic development based on 3D suspension technology,embryonic stem cells and network toxicology will help to solve the hidden toxicity which is difficult to be detected by conventional toxicology,and provide support to construct a suitable molecular-cell-whole toxicity evaluation system of dual-use food and drug substances.