A Preliminary Study On Mechanism Of RKBL Terminating The Mid-term Pregnancy In Mice

Lectin is one kind of non immune response proteins compositing of oligosaccharide, which distributed widely in animals, plants and microorganisms.Lectin has the capable to aggregate some kinds of cells,and also take an important role in biochemical and physiological process in organism.Lectin has been widely used in agriculture, animal husbandry and pharmaceutical industries. Lectin receptors are glycoproteins or glycolipids which presenting in the cell membrane.Many experiments have been shown that the lectin receptors have an important influence on fertilization,pregnancy and other reproductive process.Red kidney bean lectin(RKBL)are glycoproteins which extracted from the kidney bean plant.It’s specific binding sugar are β-galactose,α-mannose and α-acetyl glucosamine.Researches carried out in our laboratory found that KBL had the role of inhibiting fertilization and terminating different periods of pregnancy in mice,but there has not yet a systematic and comprehensive study on its mechanisms for termination pregnancy. In this dissertation,mechanisms dealing with sexual hormones changes in entire animal,morphological and relavent molecular changes in uterine and placenta tissues after administration RKBL have been tested.For purpose to understanding some mechanisms for termination pregnancy in mice after administration of RKBLObjective: To explore the influence of RKBL on serum sex hormones, placenta and the womb in mice during termination pregnancy by RKBL, At the same time,The tolerance or acute toxicity test of RKBL on female and male Kunming strain mice have been carried out. These researches will provides experimental data and establish the basement for clinic application.Methods: 1. Changes of serum progesterone(P), estradiol(E2), testosterone(T), prolactin(PRL), luteinizing hormone(LH) and follicle-stimulating hormone(FSH) from 14 days pregnant mice after administration RKBL in different time were analyzed by electrochemical luminescence, to explore the relationship between sexual hormones and abortion induced by RKBL.2.Tissues and cell morphology of endometrial and placenta have been observed; the contents of prostaglandin has beenanalyzed by 8-iso-PGF2α kit; DNA from these tissues were extracted and analyzed by electrophoresis. 3. The contents of RKBL and their agglutinin activity in the media have been analysed before or after culture of lactobacillus and dictyostelium discoideum for a period of time; and the molecular changes of RKBL in the culture media also be tested by electrophoresis. To explore whether RKBL was took in by cells or played its role outside the cells and can it be degraded or be used as energy supply substances by lactobacillus and dictyostelium discoideum. 4.To test the acute toxicity(LD50) of RKBL, administration by oral(limited test) and intraperitoneal injection have been carried out.Results : 1.During the process of termination 14 d pregnancy in mice by administration RKBL, FSH, LH, E2 and T in serum have no obviously changes.However, P and PRL reduced significantly followed a short time rose. 2. RKBL could induce the pregnancy mice appeared congestion and necrosis in placenta, The placenta edges can be deeply dyed by Typan blue staining; In addition, Methyl green-Pyronin Y staining and DNA electrophoresis bands showed that the placenta occured necrosis and apoptosis; The leve1 of 8- iso- PGF2 a which from placenta homogenate has no obvious changes but declined obviously in uterus homogenate. 3.RKBL could promote lactic acid bacteria and dictyostelium discoideum propagation by intercellular signal pathway its agglutination activity and content did not reduced or couldn’t be used as energy resources. 4. As for the acute toxicity test, after single intraperitoneal injection of KBL, the LD50 for female mice was 743.242 mg/kg, the95% confidence interval was 658.053~848.953 mg/kg; the LD50 of male mice was535.452 mg/kg, the 95% confidence interval was 480.961~598.818 mg/kg; Dose of limited test results was more than 5000 mg/kg.Conclusion : 1. The levels of E2, FSH, LH and T had not changed during termination middle-late pregnancy mice by administration RKBL, however, the levels of P and PRL changed significantly, it means that termination of pregnancy by RKBL does not interfere the neuro-endocrine axis(the hypothalamus- pituitary-sexual gland axis). 2. The phenomenon of necrosis, apoptosis and parapoptosis on mice placenta appeared after injected RKBL showed that RKBL played its roles for termination of pregnancy by followed ways such as necrosis, apoptosis andparapoptosis in placenta tissues especially in syntrophoblast; 3. RKBL can promote the lactic acid bacteria and dictyostelium discoideum proliferate by signal transduction outside the cell, its agglutination activity and content did not reduced and it couldn’t be used as energy sources or degradation by lactic acid bacteria and dictyostelium discoideum. 4. The toxicity of RKBL belonged to low toxicity on Kunming strain mice, but different sexual has different tolerance on RKBL. The tolerance of female was higher than that of males when single intraperitoneal injected RKBL.