A Study On Saffron Steep About The Expression Of LIF In Endometrium Of Early Pregnant Mice

Embryo implantation is the interaction, inter-recognition between embryo and endometriun, and the key point is synchronism of them, which is regulated precisely by many factors, besides estrogen and progestogen, the endometrial cells secret many cytokines, such as epidermal growth factor (EGF),insulin-like growth factor-2 (IGF-2),hepatocyte growth factor (HGF),interleukin-1 (IL-1),colony-stimulating factor (CSF),leukemia inhibitory factor (LIF) and so on, affecting physiological condition and local surrounding during implantation between mother and fetus, to guarantee synchronism development between them, fusion and implantation. Known from references, LIF is a kind of multifunctional cytokines, in endometrium, LIF is one of imperative factors during implantation and plays a key role, the accident of abortion or failure of implantation will happen if LIF is deficient. The research of Bhatt have shown that expression of LIF is the highest in glandular epithelium in endometrium of mice, especially on the 4^th day after fertilization. Zhang Songling has reported that it can be observed with hybridization in situ and immunohistochemistry in endometrium of mice: LIF mRNA and LIF protein were located in glandular and luminal epithelium of endometrium; and two peaks were observed in oestrus and pre-nidation respectively; on the other hand, for human, it was detected with immunohistochemistry that the expression of LIF in luminal epithelium was the highest, with glandular epithelium next, and matrix cells lowest; deficiency of LIF may cause infertility. Saffron has effect on activating blood to promote menstruation and removing blood stasis to relieve pain, so it was used to cure gynecological disease in clinic, for instance, dysmenorrhoea and menostasia. Saffron apozema can make the uteri of mice excite ex vivo or in vivo; lower dose can make uteri contraction tensely or rhythmically, whereas higher dose can increase the uteri' s tonicity and excitability, even to spasm, as is more obvious to conceived. We had taken saffron as major prescription to study the effect on antiearly pregnancy, the conclusion was : There is no difference among FSH,LH,E₂ and P between saffron group and control group, as proved that the effect of saffron on antiearly pregnancy is not through the hypophysis-ovary axis hormone, and the mechanism is still not known. Thus, we take the 2^nd pregnant mice as subject in this paper, through intragastric administration with saffron solution in different doses or physiological saline, to observe fertility inhibition after giving drug and recovery of fertility after stopping drug, and approach the effect of saffron steep on fertility in mice; to observe the morph of endometrium,expression of LIF protein and LIF mRNA, and approach the mechanism. The results showed, saffron steep may have effect on antiearly pregnancy through inhibiting the expression of LIF protein and LIF mRNA, most of all, the effect is reversible. The results provide important reference and theoretic proof for studying the mechanism deeply and clinic usage which targets saffron. Abstract as follow:An experimental study of saffron steep on fertility inhibition and recovery of fertilityWe take ripe, nulliparous female mice as subject, totally 40, matched 1:1, if seeing vagina embolus on the 2^nd morning, then we definite it as the 1^st day of pregnancy, and divide the mice into 4 groups at random, 10 respectively: one is given physiological saline as the normal control group; the other is given the saffron steep in different doses of 10.5 g/kg,15.5 g/kg,20.5 g/kg respectively, as experimental groupâ… ,â…¡,â…¢. Intragastric administration on the 2nd, 3rd and 4th morning after fertilization respectively to observe the effect of saffron steep on fertility inhibition and recovery of fertility.We found these results:(1) The breeding conditions were inhibited in different extent in experimental groups after administration, the fertility inhibition was more obvious along with the increasing of doses, 5 were inhibited in experimental groupâ… , 8 were inhibited in experimental groupâ…¡, and all were inhibited in experimental groupâ…¢; whereas the normal control group is not affected.(2) The fertilities recovered gradually after drug withdrawal, and the time of recovery of fertility delayed gradually along with the increasing of doses; on the 6^th day after drug withdrawal, all fertilities were recovered in experimental groupâ… , 9 recovered on the 8^th day after drug withdrawal in experimental groupâ…¡, and fertilities of 9 recovered on the 10^th day after drug withdrawal; whereas fertilities of control groups is normal.These results indicate that saffron steep has effect on antiearly pregnancy and the effect is reversible, and the fertilities can recover completely; It is evident that the dose-dependent of fertility inhibition and recovery of fertility after drug withdrawal.An experimental study of saffron steep on expression of LIF protein and LIF mRNA in the endometriumWe take ripe, nulliparous female mice as subject, totally 24, matched 1:1, if seeing vagina embolus on the 2^nd morning, then we definite it as the 1^st day of pregnancy, and divide the mice into 4 groups at random, 6 respectively: one is given physiological saline as the normal control group; the other is given the saffron steep in different doses of 10.5 g/kg,15.5 g/kg,20.5 g/kg respectively, as experimental groupâ… ,â…¡,â…¢. Intragastric administration on the 2nd, 3rd and 4th morning after fertilization respectively. The mice were put to death through cervical vertebrae luxation 24h later, then the uteri were removed, sectioned routinely, stoved, then treated as follow: (1)To observe the morph of endometrium with HE stain; (2) to observe the expression of LIF protein in the endometrium of the mice with immunohistochemistry stain; (3) to observe the expression of LIF mRNA in the endometrium with hybridization in situ stain. According to the morph and detected indexes, to analyze the effect of saffron steep on the expression of LIF in the endometrium of early pregnancy.The results are as follows:(1) HE stain showed: The morph of endometrium changed gradually among experimental groups after using saffron: The endometrium showed congestion and epithelial cells were in disorder, with the increasing doses of saffron, changes of morph were more obvious; while the endometrium were in a good norm in control group.(2) Expression of LIF with immunohistochemistry stain: Results analyzed by Quantimet, in normal control group, expression of LIF protein showed positive strongly staining in the cytoplasm of glandular and free surface of luminal epithelium in endometrial cells, the mean optical density (OD) is 0.3 702 201±0.0 390 275; in experimental groupâ… , it was positive staining, the mean OD is 0.3 368 947±0.0 421 678; in experimental groupâ…¡, it was positive weakly staining, the mean OD is 0.2 498 949±0.0 466 385; and in experimental groupâ…¢, it was negative staining, the mean OD is 0.1 738 566±0.0 081 749.It was significant for experimental groups to be compared with control group (P <0.05, P<0.01 ), and the expression of LIF decreased gradually along with the increasing doses, and It was significant among every two experimental groups (P <0.01).(3) Expression of LIF mRNA with hybridization in situ stain: Results analyzed by Quantimet, in normal control group, expression of LIF mRNA showed positive strongly staining, the mean OD is 0.4 476 607±0.0 449 072, mainly in glandular epithelial cytoplasm, cytoplasm of some matrix cells expressed either, while not in luminal epithelium ; in experimental groupâ… , it was positive staining, the mean OD is 0.3 549 777±0.0 263 848 ; in experimental groupâ…¡, it was positive weakly staining, the mean OD is 0.2 640 995±0.0 213 754.It was significant for experimental groups to be compared with control group ( P<0.01 ), and the expression of LIF mRNA decreased gradually along with the increasing doses, and It was significant between experimental groups (P<0.01).The results indicate that saffron steep can change the morph of endometrium, cause the asynchronism development between embryo and endometrium, and thus affect the microenvironment in endometrium during implantation and result in failure of nidation; saffron steep may have effect on anti-earlypregnancy through inhibiting the expression of LIF in the endometrium; there is relationship between the reduction of LIF protein and the reduction of LIF mRNA, and the dose-dependent relationship is obvious between expression of LIF and saffron steep.In conclusion, our researches can offer some referenced and theoretic proof for the clinic use which target the Chinese drug saffron.