Evaluation of the true precocious puberty rats induced by neonatal administration of Danazol: therapeutic effects of nourishing "Yin"-Removing "Fire" Chinese herb mixture

Abstract

Background: Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture, a traditional herb-based formulation, has been successfully used for the management of idiopathic true precocious puberty (IPP) for more than thirty years. Precocious puberty rat model by neonatal administration of Danazol was used to investigate the effects of the herb mixture on the advanced sexual development of the rats, and the expression of hypothalamic gonadotropin-releasing hormone (GnRH), which is the important regulator for the hypothalamus-pituitary-gonadal axis, particularly at puberty.

Methods: Female Sprague-Dawley rats were divided into five groups: intact normal (N), IPP model (M), vehicle with no IPP (V), IPP model exposed to herb mixture (HM) and IPP model exposed to saline (S). Rats at 5 days of age were given a single subcutaneous injection of 300 microgram of Danazol dissolved in 25 microliter vehicle of propylene glycol-ethanol (1:1, v/v), to establish the precocious puberty model. From the day 15, rats in HM and S groups were continuously fed with either Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture 2 ml or saline 2 ml, until 3 consecutive regular estrous cycles were established. The day of vaginal opening and the day of setup regular estrous cycle of the rats were observed. Blood concentration of estrogen was determined by radioimmunoassay. Immunohistochemistry and RT-PCR analysis were used to explore the expression of GnRH.

Results: The day of vaginal opening and first estrous showed significant advancement in M compared with N and V (p < 0.05, respectively). The blood estrogen level increased significantly in M compared with those in other groups (about 28 days of age, at the time of vaginal opening in M rats) (p < 0.05, respectively). GnRH cells in rostral medial septum (MS), Broca diagonal band nucleus (DBB) and the medial preoptic area (MPOA), were calculated. The number in M was less than those in N and V (p < 0.05, respectively). The number was significantly higher in HM than that in M (p < 0.05). The GnRH mRNA expression increased significantly in M compared with that in N and V (p < 0.05).

Conclusion: The true precocious puberty model by neonatal administration of Danazol in female rats showed augmented expression of hypothalamic GnRH; the Nourishing "Yin"-Removing "Fire" Chinese Herb Mixture down-regulated the increased GnRH expression, and significantly delayed the sexual development of the precocious puberty rat.

Figure 1

Effects of herb mixture on GnRH expression by immunohistochemistry. N: intact normal, M: IPP model, V: vehicle with no IPP, HM: IPP model exposed to herb mixture, S: IPP model exposed to saline. A: The statistical analysis of (spiny GnRH cells/total cells) % (n = 6). * p < 0.05 vs M; ^ P < 0.05 vs HM. B: Total GnRH cells of the rats (n = 6). * p < 0.05 vs N and V, respectively; ^ P < 0.05 vs HM. C: Light micrography illustrating the different types of GnRH cells at the region of MS at low power (n = 6). Sm: smooth type cell; Sp: spiny type cell.

Figure 2

Effects of herb mixture on GnRH mRNA expression by RT-PCR. The upper picture shows the gel electrophoresis of the RT-PCR products for the GnRH. Densitometric analysis of the mRNA concentration using GnRH/β-actin expressed as the mean with SEM bar (n = 6) in each column indicated in the lower panel. N: intact normal, M: IPP model, V: vehicle with no IPP, HM: IPP model exposed to herb mixture, S: IPP model exposed to saline. *p < 0.05 vs N and V, respectively; ^ p < 0.05 vs M and S, respectively.