The Effect Of Brucea Javanica On The Early Embryonic Development Of Mice And Its Mechanism

The molecular regulation of mammalian early embryonic development is one of the most important subjects in life science, it is a orderly complex regulation process,including the cleavage of zygote, formation of morula and blastocysts. It is very important that the development of preimplantation embryos on embryo implantation and produce a healthy offspring, embryonic protein regulated by many factors during embryonic development, any abnormal gene expression are likely to lead to the disorder of the zygote.Although there are a lot of studies on preimplantation embryo development, the underlying molecular mechanisms remain unclear. Therefore,the study of the factors that may affect the development are likely to provide a new theoretical basis and clinical evidence for revealling the reproductive regularity in mammals, improving the quality of embryos and preventing birth defects.Nuclear factor E2-related factor 2 (Nrf2), is an important transcription factor in the cell, and it is known to regulate many antioxidant responses in the somatic cells and tumor cells, it can enhance the resistance of cells to oxidative stress and pro-chemical substances.Under physiological conditions, Keap1 which containing Cul3 ubiquitin ligase activity can degrade the Nrf2 protein, and the Keap1-Nrf2 system enables the combination of inducible antioxidant protection and cellular bioenergetics. Brucea Javanica is a kind of herbal medicine used for antitumor, Brusatol is a natural inhibitor for Nrf2 and is the main component from Brucea Javanica. This experiment used preimplantation embryos of Kunming mouse as experimental materials, this experiment used Brusatol to inhibit the expression of Nrf2, to confirm the expression of Nrf2 in mouse preimplantation embryo development, to explore the impact of different concentrations of Brusatol on preimplantation embryo development, to evaluate the contribution of Nrf2 in maintaining the quality of embryos.This study used different concentrations of Brusatol in zygote cells of mouse preimplantation embryos, the development rates of cells were observed in different concentration and screen out the optimal concentration; The expression and location of Nrf2 was detected by immunofluorescence in different stages ( 2-cell, 4-cell, morula and blastocysts) of mouse preimplantation embryo; real-time fluorescence quantitative PCR method for detection mRNA levels of cell cycle related genes and their kinase in 2-cell embryos. EdU experiment for detection of cell cycle; Western blot to verify the influence of Brusatol on the expression of Nrf2 protein; through addition of Brusatol, Nrf2 actived plasmid was injected in zygophase at the same time proved Nrf2 indeed played a role in the embryonic development; the degree of DNA damage proved by gamma-H2AX antibody staining. These results showed that Brusatol could down-regulate the expression of Nrf2 protein. Immunofluorescence analysis showed that Nrf2 was mainly localized in cytoplasm and nucleus from 2-cell stage to blastocyst stage. After treated with different concentrations of Brusatol, the development rate of cells were negatively correlated with the increase of concentration. After treatment with 200 nM Brusatol, there was almost no cell developed to the 4 cell stage, and most of the cells were arrested in 2-cell phase. We further examined the decreased mRNA levels of Cyclin B and its kinase CDK1, Cyclin E and its kinase CDK2 as well as their downstream Rb levels in the Brusatol inhibitor group compared with the control group. After y-H2AX staining and fluorescence microscopy analysis, it was found that the nucleus was not stained. The results indicated that Brusatol treatment cells might not be able to play a role in DNA damage. At the same time after microinjection with Brusatol, the development rate of cell were observed and compared with the Brusatol group, which indicated that the growth rate were restored.So we can draw the conclusion that in mouse early embryo development, Brusatol can inhibit the expression of Nrf2 protein, and the results show that the inhibition expression of Nrf2 may affect the mitotic cell cycle, which may be one of the main reasons for obstacles of embryo development in vitro. Brusatol mainly inhibit the early embryonic development through down-regulated Nrf2, further adjust Cyclin B-CDK1 from G2 to M phase shift.