Base Sequence ; Child, Preschool ; Humans ; Male ; Point Mutation ; Polymerase Chain Reaction ; Puberty, Precocious ; genetics ; Receptors, LHRH ; genetics ; Sequence Analysis, DNA ; Testosterone ; blood

OBJECTIVETo probe the mechanism of Chinese herbal medicine (CHM) for nourishing Yin and purging Fire on the expressions of gonadotropin-release hormone (GnRH) and its mRNA expression in hypothalamus and GnRH receptor mRNA in pituitary in danazol induced precocious puberty model rats.

METHODSRats were divided into the normal group, the model group, the blank control group and the CHM group. Rats, except that in the normal group, were subcutaneously administered danazol 300 micrograms at 5 days of age individually and CHM was fed to rats in the CHM group from 15 days of age, in the meantime, normal saline was fed to rats in the blank control group. Expression of GnRH in hypothalamus was observed by immunohistochemical method and expressions of GnRH mRNA in hypothalamus and GnRH receptor mRNA in pituitary were determined by RT-PCR.

RESULTSCompared with rats in the normal groups, the vaginal opening and the onset of first estrus were ahead of time, the number of GnRH immunoreactive positive cells decreased and the expressions of GnRH mRNA in hypothalamus and GnRH receptor mRNA in pituitary up-regulated in the model rats and blank control rats. Compared with the model and the blank control groups, in CHM group, all the above-mentioned abnormally changed parameters improved significantly after treatment.

CONCLUSIONCHM for nourishing Yin and purging Fire may inhibit the abnormal hyperfunction of hypothalamus-pituitary-ovary axis in precocious puberty rat induced by danazol via reducing the synthesis and release of GnRH, and lowering the responsibility of pituitary cells to GnRH. This may be the primary mechanism of CHM in effectively treating the true precocious puberty.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gonadotropin-Releasing Hormone ; biosynthesis ; genetics ; Hypothalamo-Hypophyseal System ; metabolism ; Puberty, Precocious ; metabolism ; RNA, Messenger ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, LHRH ; biosynthesis ; genetics ; Yin Deficiency ; metabolism

The homologous regulation of pituitary Gonadotropin Releasing Hormone Receptor (GnRH-R) mRNA expression by GnRH has been well demonstrated. However, the regulation of the ovarian GnRH-R is poorly understood. The present study was performed to demonstrate the presence of GnRH transcripts in addition to GnRH-R mRNA and the regulation of GnRH-R mRNA expression in the granulosa cells isolated from small antral follicles. The GnRH and GnRH-R mRNA levels were determined by a competitive reverse transcription-polymerase chain reaction (RT-PCR). The granulosa cells were obtained from immature rats implanted with diethylstilbestrol for 3 days. When GnRH transcript expression was examined in isolated granulosa cells by RT-PCR, the PCR products showed two bands. The larger band contained intronic sequences and the smaller band was a fully processed GnRH gene transcript identical to hypothalamic GnRH. This suggests that authentic GnRH gene transcripts are expressed in ovarian granulosa cells and may act on the granulosa cells in a paracrine or autocrine manner. Since GnRH action in the granulosa cells is mediated by specific GnRH-R, it is of interest to examine whether GnRH-R is synthesized in the granulosa cells. When the granulosa cells were cultured in media only, GnRH-R mRNA levels increased abruptly within 3 h and gradually decreased thereafter during the 24 h culture period. However, GnRH itself did not alter the GnRH-R mRNA expression levels in cultured granulosa cells. Interestingly, treatment with FSH decreased the GnRH-R mRNA levels in a dose-dependent manner. A time-course analysis revealed that the GnRH-R mRNA levels were significantly lower up to 9 h after FSH treatment, and returned to the basal level between 12 h-24 h. Activation of adenylate cyclase with forskolin also decreased the GnRH-R mRNA levels. It is therefore concluded that in the granulosa cells of the small antral follicles GnRH-R mRNA expression was not homologously regulated by GnRH, while FSH may negatively regulate GnRH-R mRNA expression in the granulosa cells possibly through a cAMP-protein kinase A pathway.
Animal ; Cells, Cultured ; FSH/pharmacology ; Female ; Gene Expression Regulation* ; Gonadorelin/pharmacology ; Granulosa Cells/metabolism* ; Granulosa Cells/drug effects ; RNA, Messenger/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, LHRH/genetics* ; Reverse Transcriptase Polymerase Chain Reaction

This study is to investigate the anti-tumor effect in vitro of methotrexate modified by LH-RH peptide (LH-RH-MTX). LH-RH receptors highly expressing MCF-7 human breast carcinoma cell line and lowly expressing K562 human erythroleukemia cell line were served as the tested cells. The cell proliferation inhibition rates of LH-RH-MTX were detected by MTT colorimetric assay. The effects of LH-RH-MTX on the cell cycle and apoptosis rates were detected by flow cytometry. The inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that on K562 cells, and the inhibition rate of LH-RH-MTX on MCF-7 cells was much higher than that of free MTX at the same concentration. The inhibition rate of LH-RH-MTX on rat bone marrow mononuclear cells was less than that of free MTX. The number of MCF-7 cells in S phase increased after administration of LH-RH-MTX. The apoptosis rate of LH-RH-MTX group significantly increased compared with that of the control group and MTX group. The relative expression of LHRHR mRNA of LH-RH-MTX group markedly decreased compared with that of the control group and MTX group. LH-RH-MTX is realizable to reduce drug side effects, increase the therapeutic index and achieve tumor-targeted therapy.
Animals ; Antimetabolites, Antineoplastic ; chemical synthesis ; pharmacology ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drug Delivery Systems ; Gonadotropin-Releasing Hormone ; chemistry ; pharmacology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; MCF-7 Cells ; Methotrexate ; chemical synthesis ; pharmacology ; RNA, Messenger ; metabolism ; Rats ; Receptors, LHRH ; biosynthesis ; genetics