Proteomics Analysis Of Embryonic Developmental Toxicity Of Raw Pinellia Ternata And Its Compound Compatibility

Pinellia ternata(Thunb.)Breit is a commonly prescribed Chinese medicinal herb which prevents vomiting."Gan-jiang-ren-shen-ban-xia pill," a classic and famous prescription of Synopsis of Golden Chamber,contains P.ternata.It is commonly used to treat vomiting during pregnancy.Although raw Pinellia was thought to be toxic since ancient times,it is unclear whether it is toxic to developing embryos;in addition,its compound compatibility is also unclear.Therefore,we carried out the following three parts of experiments to elucidate the effect of raw Pinellia.First,we used the subacute toxicity test and sensitive period to teratogenic agent toxicity test(STATT)to examine the toxic effect of raw Pinellia powder(BXp)and the attenuation effect of Gan-jiang-ren-shen-ban-xia powder(GJRSBXp)on embryonic development in mice.The results showed that the body weight of non-pregnant mice decreased after continuous administration of BXp(2.275g/kg/d)for 14 days;they also showed significant kidney disease and significant changes in the proportion of T lymphocyte subsets in the blood and spleen.The results of STATT showed significant changes in the heart and kidney pathology of pregnant mice after administration of BXp for 10 days from gestation day(GD)6.5 to GD 15.5.The weight on GD 18.5 and extrauterine weight gain of pregnant mice in BXp group were significantly lower than that in the control group(P<0.05).The body length of the mice fetus in BXp group was also significantly reduced than that in the control group(P<0.05).No significant differences were observed between the GJRSBXp group with the corresponding dose of BXp group and the normal control group(P>0.05).These results suggest that BXp shows maternal and embryonic toxicity and GJRSBXp exhibits a compatible detoxification effect.Based on the above findings,we further explored the mechanism of embryonic toxicity of raw Pinellia by using quantitative proteomics technology based on isobaric tags for relative and absolute quantification(iTRAQ).Pregnant mice were fed BXp from GD 6.5 to GD 8.5 and the gastrulae were collected on GD 9.5.Differentially significant proteins(DAPs)were identified by iTRAQ,and the bioinformatics analysis of 153 DAPs was performed using OmicsBean(http://www.omicsbean.cn).Then,the expression level of the DAPs was verified by real-time fluorescence quantitative PCR and western blotting.The results showed that 47.4%of the DAPs were associated with neurological development and were significantly enriched in the development of the substantia nigra and multiple degenerative disease pathways.These results suggest that the abnormal expression of neurological developmental proteins may be a potential mechanism of BXp involved in its toxic effect on embryonic development.Finally,we used label-free proteomics techniques to study the differential proteomes of mouse gastrulae before and after BXp or GJRSBXp intervention as well as the differential proteomes of the mouse embryonic stem cells(mESC)before and after treatment with the corresponding medicated serum.We identified 163 and 197 DAPs in embryonic tissue and mESC,respectively.The results of bioinformatics analysis of the differential proteomes in vivo or in vitro were consistent.The proportion of DAPs involved in the development of the nervous system significantly decreased after GJRSBXp intervention.The DAPs were scattered in the developmental processes related to the respiratory system,ventricular system,nervous system,bone,endothelium,eye,and others and significantly enriched in development-related Wnt and Hippo signal pathways.These results suggest that GJRSBXp may reduce the neurotoxicity of BXp by regulating the expression of proteins involved in developmental signaling pathways and exerted this attenuation effect via compatibility.In summary,we confirmed the toxic effect of raw Pinellia on embryonic development and the corresponding attenuation effect of its compound compatibility.Through the proteomics analyses,we found for the first time that BXp may affect the expression of proteins involved in the development of the nervous system of mice fetus and lead to embryonic developmental toxicity,whereas GJRSBXp along with ginger(Gan jiang)and ginseng(Ren shen)may play an important role in the attenuation of BXp’s toxic effects by regulating the expression of proteins involved in developmental pathways such as Wnt and Hippo signaling pathways.These results can provide a theoretical basis for the rational application of Pinellia and its compounds,and promote the modernization of Chinese medicine and international exchange of traditional medical knowledge.