| Objective: To investigate the potential role of Kiss-1/GPR54 in precocious puberty and the effects of nourishing "Yin" removing "Fire" Chinese herb mixture on the hypothalamic Kiss-1/GPR54 expression, female precocious model rats induced by danazol were used. Methods: (1) Female Sprague-Dawley rats at 3 days of age in company with the mothers were randomly divided into normal (N) , vehicle (V) and model (M) groups. The model litters at postnatal day 5 were given a single subcutaneous injection of 300μg of danazol. Immunohistochemistry, Western blot and RT-PCR technologies were used to observe the characteristic of hypothalamic Kiss-1/GPR54 mRNA and protein expression on the day of prepuberty, onset-puberty and post-puberty stages. (2) Precocious rats on the day of onset-puberty were adopted to observe the colocaliazation of GPR54 and GnRH by immunofluorescent double labeling histochemistry. The neutralization that intracerebraventricular administered with GPR54 antibody was used on postnatal day 20, the day of vaginal opening and the hypothalamic kisspeptin, GnRH expression were observed respectively to evaluate the role of kisspeptin/GPR54 in precocious puberty. (3) The expression of hypothalamic kisspeptin, GPR54, GnRH mRNA and protein in precocious rats after administered with nourishing "Yin" removing "Fire" Chinese herb mixture were observe by immunohistochemistry and RT-PCR technologies. In addition, precocious rats were administered with nourishing "Yin" removing "Fire" Chinese herb mixture from the postnatal day 14 to 20, then intracerebraventricular injected kisspeptin peptide 2 nmol, 4 nmol was administered on the postnatal day 20．Vaginal opening and the hypothalamic GPR54, GnRH expression were observed to probe the effect and mechanism of nourishing "Yin" removing "Fire" Chinese herb mixture on PP. Results: (1) The day of vaginal opening and establishment of two regular estrous cycles were advanced, the organ coefficient of uterus and ovary were increased in female rats that subcutaneously administered with danazol, at the same time, the hypothalamic GnRH mRNA and protein expression increased significantly than that of the normal rats. There were not significant kisspeptin/GPR54 immunoreactive cells in hypothalamus in model rats at pre-puberty stage compare with normal rats. Kisspeptin/GPR54 immunoreactive cells were increased in the arcuate nucleus (ARC), periventricular nucleus (PeN) and preoptic area (POA) in model rats on the day of onset-puberty. On the day of post-puberty, however, the number of kisspeptin immunoreactive cells in ARC and PeN decreased while the number of those cells in POA increased. However, the number of GPR54 immunoreactive cells in ARC and PeN increased on the day of post-puberty while the number of those cells in POA decreased. Furthermore, hypothalamic Kiss-1/GPR54 mRNA expression was detected on the different pubertal stages in precocious rats. The Kiss-1/GPR54 mRNA increased to maximum and the expression higher significantly than the nomal rats on the day of vaginal opening. Kiss-1/GPR54 mRNA expression decreased on the day of post-puberty day. (2) GPR54 colocalize with GnRH neuron in POA in precocious rats on the day of onset-puberty. The day of vaginal opening in the model rats that intracerebraventricular administered with GPR54 antibody were delayed compared with that of the model rats. No difference was observed in kisspeptin expression, but the GnRH immunohistochemistry positive cells in MS, DBB and POA were increased. (3) The nourishing "Yin" removing "Fire" Chinese herb mixture could down-regulate the kisspeptin, GPR54 and GnRH mRNA and protein expression in ARC, PeN and POA, regulate the GnRH expression in MS, DBB and POA. (4) The vaginal opening was observed on the postnatal day 22 in the precocious rats that intracerebraventricular injected with kisspeptin peptide 2 nmol, 4 nmol on the postnatal day 20．Conclusion: (1) The high expression of hypothalamic Kiss-1/GPR54 mRNA, the abnormal expression of kisspeptin/GPR54 in in ARC, PeN and POA on the day of onset-puberty of precocious rats might involve in the advance onset-puberty in PP. (2) Kisspeptin might participate in the advance onset-puberty in precocious rats through its receptor GPR54．Intracerebraventricular injected with GPR54 antibody might inhibit GnRH protein release in precocious rats and then delay the puberty development. (3) The nourishing "Yin" removing "Fire" Chinese herb mixture might regulate the abnormal function of HPGA in PP through down-regulate the expression of hypothalamic Kiss-1/GPR54 mRNA and protein, and then inhibit GnRH mRNA expression and protein release. |
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