Tetramethylpyrazine On Early Embryo Development And Implantation Of Il-6 Expression

Object: In order to evaluate the safty of Ligustrazine Hydrochloride in the early pregnancy, estrogen and progesterone in mice serum as well as interleukine-6(IL-6) in uterus and embryos,which treat with Ligustrazine Hydrochloride were investigated. The effect of Ligustrazine Hydrochloride on the development of early embryos was also observed in vitro and in vivo.Methods: The pregnant mice were treated intragastrically with Ligustrazine Hydrochloride (10mg/d.ml,2mg/d.ml,0.4mg/d.ml) for 4 days from the first day of pregnancy (0.067mg/g), and the control group was treated with acidic saline. The content of progesterone and estradiol in serum were measured by RIA(radioimmunoassay) at 4th and 5th day of pregnancy, the expression of IL-6 at the protein and mRNA level were detected by immunohistochemistry and RT-PCR(reverse transcription-polymerase chain reaction).The number of embryos in each uterus were counted at 6th day of pregnancy. For investigation of the direct effect of Ligustrazine Hydrochloride, the two-cell four-cell and eight-cell of mouse embryos were cultured in the M16medium and treated with different concentrations (20ug/ml,2ug/ml,0.2ug/ml) of Ligustrazine Hydrochloride in for three days, the morphology of embryos was observed and took pictures under microscope.Result: The level of the serum estradiol in the 3 experimental groups is similar to the control group at 4th day of pregnancy (P>0.05) ,while the level of progesterone in 0.4mg/d.ml 2mg/d.ml and 10mg/d.ml groups is distinctly increased dependent with the dose of Ligustrazine Hydrochloride (P<0.05-P<0.01) .Compared with the control group,the level of serum estradiol distinctly increased in 10mg/d.ml group (P<0.05) . The level of serum progesterone in 0.4mg/d.ml 2mg/d.ml and 10mg/d.ml groups distinctly increased compared with the dose of Ligustrazine Hydrochloride (P<0.05-P<0.01) at 5th day of pregnancy. The number of embryos in 2mg/d.ml group is much less than that of the control group at 6th day of pregnancy (P<0.05), The number of embryos in 0.4mg/d.ml and 10mg/d.ml groups are similar to the control group (P>0.05); the development of embryos in 2mg/d.ml and 10mg/d.ml groups are slower than the control group.Compared with the control group,the development of 2-cell embryos in 20ug/ml is significantly inhibited in vitro. During the period of later development of 4-cell, 20ug/ml and 2ug/ml groups could significantly inhibitor development of 8-cell compared with the control group. There are not significantly difference among four groups. The results also show that the expression of IL-6 protein but not the IL-6 mRNA in uterus and embryos could be inhibited by 10mg/d and 2mg/d of Ligustrazine Hydrochloride.Conclusion: The Ligustrazine Hydrochloride could inhibitors the development of embryos through decrease the level of the serum estradiol and increase the level of serum progesterone,and inhibitors the circulation of uterus blood through decrease the expression of IL-6 protein in uterus epitheliumin in vivo; inhibitors the development of 2-cell 4-cell and 8-cell in vitro.