ABSTRACT
OBJECTIVE To evaluate the embryo toxicity of Shuanghuanglian (SHL) by the combination of a human placental barrier model and embryonic stem (ES) cell test model.METHODS A human placental barrier model was set up by placenta slice culture and Ussing chamber.SHL 0.2,0.4,0.8,1.6,3.2,6.4 and 12.8 g· L-1 was added into the maternal side of the human placental model,respectively.All the media was collected respectively from the matemal side and fetal side 60 min later and taken as the SHL containing medium.ES cells (D3 line) and embryonic fibroblast cells (BALB/c 3T3) were cultured with the SHL containing medium respectively from the maternal side and the fetal side for 10 d.Cell viability was detected by MTT assay,and 50％ survival inhibitory ratio of ES and 3T3 cells by SHL was calculated.ES cells were incubated with the SHL containing medium from the matemal side or fetal side when they differentiated to cardiac myoblasts using hanging drop-suspension-attachment method.Messenger RNA of myosin heavy chain genes (β-MHC) was detected by Q-PCR for differentiation ratio,and 50％ differentiation inhibitory ratio of ES cells by SHL was calculated.A statistics formula was used for prediction of SHL embryotoxicity potential.RESULTS The IC50 of SHL in the matemal side of the human placental model for 3T3 cell survival,ES cell survival and ES differentiation was 1.97,0.84 and 0.48 g· L-1,respectively.According to the criteria for embryo toxicity evaluation,SHL had weak embryo toxicity.However,the IC50 of SHL in the fetal side of the human placental model for 3T3 cell survival,ES cell survival and ES differentiation was 3.19,2.57 and 0.95 g· L-1,respectively.According to the criteria for embryo toxicity evaluation,the supernatant containing SHL that went through the placental barrier had no embryo toxicity.CONCLUSION SHL is safe in the test concentration range during pregnancy.It is more scientific to evaluate embryo toxicity of drugs by ES cell test with the samples obtained through the placental barrier during pregnancy.