The Studies Of Nitidine Chloride’s Cardiovascular Effects Based On Zebrafish Embryos’ Evaluation System

OBJECTIVE:(1) To study the effects of nitidine chloride on themorphology and heart development of zebrafish embryos at different time points,and to provide the basis of drug safety evaluation with zebrafish embryos.(2) Toestablish the evaluation methods of zebrafish embryos’ angiogenesis, toevaluate the effect of nitidine chloride on angiogenesis during zebrafish embryosdevelopment, and to expore the underlying mechanism.METHODS:(1) Based on the preliminary experiment, the concentrationof NC was divided into5groups,6hpf chorion-intact group(8.53,9.48,10.53,11.70,13.00mg/L),24hpf chorion-intact group(19.68,21.87,24.30,27.00,30.00mg/L),24hpf dechorionated group(16.40,18.23,20.25,22.50,25.00mg/L), and Holt buffer as the control group, with0.5%DMSO as the solventcontrol group. Observed zebrafish embryos’ mortality and deformity at72hpf.(2) The groups and treatment were the same as the above, assessed the effects ofNC on the zebrafish embryos heart development at48hpf,54hpf,60hpf and72hpf.(3) Based on the preliminary experiments, the24hpf zebrafish embryoswere treated by NC (8.19,10.24,12.80,16.00mg/L), and Holt buffer as thecontrol group, with0.5%DMSO as the solvent control group.The effects of NC at different concentrations on zebrafish’s intersegmental blood vessels (ISVs)and subintestinal vessels (SIVs) were observed by NBT/BCIP vessel staining.(4)The24hpf zebrafish embryos induced by VEGF were treated by NC (8.19,10.24,12.80,16.00mg/L), and Holt buffer as the control group, with50μg/LVEGF as the VEGF group, with0.5%DMSO as the solvent control group. Theevaluation methods were the same as the above.(5) The24hpf zebrafishembryos were treated by NC (8.19,10.24,12.80,16.00mg/L) and blank controlgroup was treated by0.5%DMSO. The effects of NC at different concentrationson zebrafish’s the expression of angiogenesis gene was analyzed by RT-PCR.RESULTS:(1) The LD50for different time points:24hpf chorion-intactgroup(24.16mg/L)>24hpf dechorionated group(20.56mg/L)>6hpfchorion-intact group(10.50mg/L). But Therapeutic Index(TI) and SafetyIndex(SI) were similar.(2) Each group treated by NC showed heart deformity,NC dose-and time-dependently decreased the zebrafish embryos’ heart rate.(3) NC dose-dependently showed inhibition effect on ISVs and SIVs.(4) VEGFcould induce zebrafish embryos’ ISVs and SIVs development, and NC dose-dependently showed inhibition effect on ISVs and SIVs of zebrafish embryosinduced by VEGF. The angiogenesis inhibition rate slightly increase.(5) NCdown-regulated mRNA expressions of VEGF, FGF2, EGF and VEGFR inzebrafish embryos.CONCLUSION:(1)6hpf zebrafish embryos are more sensitive to NCthan24hpf. The chorion can protect embryos development.(2) NC can effectzebrafish embryos’ heart development, and more sensitive at6hpf. The chorioncan decrease the effects of NC.(3) NC can inhibit the zebrafish embryosangiogenesis.(4) NC can inhibit the zebrafish embryos angiogenesis, whichinduced by VEGF. VEGF can induce zebrafish embryos angiogenesis, but the angiogenesis inhibition rate isn’t increased significantly.(5) The mechanism ofNC inhibit angiogenesis is probably related to the inhibitory effect on mRNAexpressions of VEGF, VEGFR, FGF2and EGF.