The inhibitory effects of Brusatol on mouse early embryonic development and its molecular mechanism / 医学研究生学报

ABSTRACT

Objective Up to now, the role of Brucea in early embryonic development of mice and its mechanism is still unclear.This paper aims to explore the role of Brusatol in mouse early embryonic development and its possible mechanism.Methods 100 kunming rats of clean grade(80 female rats and 20 male rats) were divided into 6 group negative control group(no DMSO)、blank control group(culture in fresh CM with equal DMSO)、20nmol/L brusatol treated group、50nmol/L brusatol treated group、100nmol/L brusatol treated group、200nmol/L brusatol treated group(A solution of Brusatol was diluted in CM to concentrations of 20, 50, 100 or 200nmol/L.).Each group used an average of 20 embryos each time, repeated 4 times.Fertilized eggs after cultured 24h, 48h,72h, 96h were respectively 2-cell stage, 4-cell stage,morula and blastocyst stage..The embryo development rate was observed in the culture medium and the optimal concentration was selected, the embryos were collected to analysis the subcellular localization of the Nrf2 by immunofluorescence.The mRNA expression level of Cyclin B, CDK1 and the protein expression of Nrf2 were detected by Q-PCR and western blot respectively.Results In 4-cell stage, the embryo development rates of 20、50、100nmol/L brusatol treated groups[(75.0±2.8)%、(30.4±7.5)%、(4.2±5.9)%] significantly reduced compared with the negative control group[(93.0±2.8)%]、blank control group[(90.9±1.2)%].In morula stage, compared with blastocyst rates of negative control group、blank control group [(83.5±2.1)%、(84.2±1.2)%], 50nmol/L brusatol treated group[(19.3±13.1)%] decreased obviously [(79.00±0.06)% vs 100%, P<0.05].In the cellular immunofluorescence assay, the expression of Nrf2 protein in 50nmol/L brusatol treated group was lower than blank control group(P<0.05).We further found that 50nmol/L brusatol treated group decreased more mRNA levels of Cyclin B[(59.5±9.2)%] and CDK1[(56.0±1.4)%] than blank control group(100%) in G2/M phase(P<0.05).Conclusion In this study, Brusatol mainly affects the cell cycle transformation from G2 to M phase dependent on Cyclin B-CDK1, further inhibiting the development of the embryo through down-regulating Nrf2.