Role Of Brain PrRP In Electroacupuncture Normalizing The Dysfunction Of Hypothalamus-pituitary-ovarian Axis And Regulation Of Oestrous Cycle In Female Rats

Objective: To explore the neuroendocrinologic role and mechanism of brain PrRP in electroacupuncture normalizing the dysfunction of hypothalamus-pituitary-ovarian axis and regulation of oestrous cycle in adult female rats. Methods: (1) Adult female Sprague-Dawley rats with normal oestrous cycle were randomly divided into four groups, intact (INT), intact with EA (INT+EA), ovariectomized (OVX), ovariectomized with EA (OVX+EA).The animals in OVX and OVX+EA were undergone an ovariectomy , and the INT+EA and OVX+EA rats were accepted EA treatment at "Guanyuan" acupoint (RN4), "Zhongji" acupoint (RN3), "Zigong" acupoints (EXTRA22) and "Sanyinjiao" acupoint (SP6). The stimulation was generated by a G6805 EA apparatus and lasted for 30 min (8:00-10:00 AM everyday), Q.D, for 3 days altogether. The stimulation parameters were 1～2 mA of density and a low-burst frequency of 2 Hz. Radioimmunoassay, immunohistochemistry, real time RT-PCR technologies were used to observe the changes of PrRP expression in medulla oblongata and hypothalamus. (2) Adult female Sprague-Dawley rats with regular 4-day oestrous cycle were randomly divided into four groups according to examination of exfoliative epithelia in vaginal smears: diestrus group(DI), proestrus group(P), estrus group(E) and metaestrus group(M). Radioimmunoassay, immunohistochemistry, RT-PCR technologies were used to observe the PrRP mRNA and protein expression in medulla oblongata and hypothalamus, and the alteration of PrRP receptor of brain in oestrous cycle. (3) Adult female rats were adopted to observe the coexistence of PrRP receptor and GnRH by immunofluorescent double labeling histochemistry combining laser confocal scanning microscope (CLSM). The hypothalamic slices incubation of ovariectomized rats was adopted to study the effect and mechanisms of PrRP to GnRH releasing in vitro. Results: (1) EA might normalize the abnormal release of pituitary LH induced by ovariectomy in female rates. The levels of blood E₂, uterine organ coefficient were increased, the figure and structure of uterus tended to be normal after EA treatment in ovariectomized rats. (2) The number of PrRP neurons in nucleus of solitary tract (NTS) and ventrolateral reticular nucleus (VLRN), as well as the level of relative optic density of PrRP immunoreactivity fibers inhypothalamic bed nucleus of stria terminalis (BST) was decreased significantly in OVX than those in INT and INT+EA. However, there was an evident increase of the numbers of PrRP neuron and positive immunoreactive PrRP fibers in OVX+EA. (3) The mRNA expression of PrRP in medulla oblongata and hypothalamus also reduced in OVX compared with INT and INT+EA. Similarly, there was an enhancement of PrRP mRNA expression in above brain regions of OVX+EA. (4) For adult female rats, PrRP immunoreactive neurons of NTS and VLRN in proestrus were less than those in dioestrus, estrus and metaestrus. Meanwhile, the relative optical density of PrRP-ir fibers of BST in proestrus was decreased compared with that in dioestrus, estrus and metaestrus. (5) The PrRPmRNA signals of medulla oblongata and hypothalamus in proestrus and estrus were higher than that in metaestrus and dioestrus. (6) No significant difference was found in PrRP receptor-GPR10 in pituitary and brain regions in oestrous cycle. (7) The co-localization of GPR10-immunoreactivity (GPR10-ir) and GnRH-immunoreactivity (GnRH-ir) neurons in hypothalamic medial preoptic area (MPOA) was observed and GPR10 seemed to exist in the cytomembrane of GnRH neurons. (8) To add NS (Control) and 1nmol PrRP-31, 10nmol PrRP-31 and 100nmol PrRP-31 to hypothalamic slices and pituitary slices of ovariectomized rats, GnRH and LH were detected by RIA. PrRP could inhibit the abnormal GnRH releasing and show evident dose dependent effect induced by PrRP-31 in ovariectomized rats, but there was no variance in LH level. (9) Administration of different doses of PrRP- 31 could induce the change of some classic monoamine and amino acid neurotransmitters in the incubatation of hypothalamic slices. Conclusions: (1) PrRP in brain may involve in the neuroendocrinological mechanism of EA normalizing the dysfunction of HPOA. (2) Brain PrRP might be responsible for the physiological regulation of the oestrous cycle in adult female rat. (3) At the physiologic E₂ level, brain PrRP can promote the releasing of hypothalamic GnRH, while inhibit the releasing of hypothalamic GnRH at subnormal E₂ level, this effect might be mediated by PrRP receptor. (4) Some classic monoamine and amino acid neurotransmitter might participate in the mechanism of PrRP regulating GnRH release in rat hypothalamus.