Hypothalamic Orphanin FQ Participates In Neuroendocrinologic Mechanism Of Electroacupuncture Normalizing Abnormal Release Of Pituitary LH In Ovariectomized Rats

Objective To explore the neuroendocrinologic mechanism underlying the regulation effect of eletroacupuncture (EA), the mRNA and protein expression of OFQ ( Nociceptin/Orphanin FQ) and ORLl receptor ( opioid receptor-like receptor 1) in medial basal hypothalamus (MBH) was investigated following EA normalizing the abnormal release of pituitary LH (luteinizing hormone) in ovariectomized rats. Furthermore, the effect of OFQ on pituitary LH release of ovariectomized rats and the expression of OFQ and ORLl receptor of MBH in estrous cycle was also studied for probing its pharmacological and physiological effects on the release of pituitary LH. Method (1) Female Sprague-Dawley rats were randomly divide into four groups, Sham, Sham+EA; OVX and OVX+EA. Radioimmunoassay (RIA) was used to measure serum levels of LH and estradiol (E₂) in each group. (2) The immunohistochemistry was taken to detect the change of number of OFQ immunoreactive (OFQ-IR) neurons and relative density of OFQ-IR fibers in Broca diagonal band nucleus (DBB), rostral medial septum (MS) , medial preoptic area (POA), ventromedial hypothalamus nucleus (VMH), median eminence (ME) and arcuate nucleus (ARC) of hypothalamus. In addition, the change of mRNA and protein expression of OFQ and ORLl receptor in MBH were observed by RT-PCR and western blot. (3) RIA combined with intracerebroventricular injection (i.c.v.) were adopted to study the effects of OFQ and ORLl receptor specific antagonist [Nphe']Nociceptin(1-13)NH2 , (NC13 ) on the serum LH in ovariectomized rats. The dose of OFQ was 2 , 20, 200nmol and NC13 was 20, 50, 100, 200nmol, respectively. (4) Ovariectomized rats were divided into Control, OFQ, NC13+OFQ ( or MK801+OFQ) and NC13 (or MK801, NMDA receptor blocker) group to search the influence of NC13 and MK801 on the decrease of pituitary LH release induced by OFQ. Meanwhile, the change of GnRH-IR neurons and fibers were also observed in the hypothalamus nuclei. (5) RIA and pituitary slices incubation in vitro was applied to examine the effect of OFQ on LH secretion from pituitary slices. (6) RT-PCR and western blot was used to observe the location of ORLl receptor and its mRNA in rat pituitary. (7 ) By immunohistochemistry and RT-PCR analysis, the change of OFQ and ORL1 receptor expression in MBH were determined in different stages of the ratsestrous cycle. (8) RIA and icv injection of OFQ is adopted to study its effect on LH surge in EBP-primed ovariectomized rats. Results (1) EA might normalize the abnormal release of pituitary LH induced by ovariectomy in female rats. The serum E2 level in ovariectomized rats was raised significantly after EA treatment. (2) The number of OFQ-IR neurons in medial preoptic area (POA) , ventral medial hypothalamus (VMH) as well as arcuate nucleus (ARC) and the level of relative density of OFQ-IR fibers in medial eminance(ME)were decreased in OVX compared with Sham. The mRNA expression of OFQ and ORL1 receptor in MBH also reduced in OVX. There was evident enhance in the number of OFQ-IR neurons and mRNA expression of OFQ in MBH in OVX+EA, but ORL1 receptor mRNA level did not show the similar changes. Meanwhile, No significant influence on the ORL1 receptor protein was observed in OVX following EA treatment. (3) The results showed that at 2h after icv administration of 20 and 200nmol OFQ, a dose-related inhibitory effect on serum LH level appeared. The level of serum LH was increased significantly at 2h after icv injection of lOOnmol NCI 3. (4) The significant decrease of serum LH level induced by 20 nmol OFQ might be abolished by pretreatment with 20 nmol NCI3. The relative density of GnRH-IR fibers in NC13+OFQ group was higher than that in OFQ group. The pretreatment of MK801 (0.2mg/kg,sc) might antagonize partly the decrease effect on serum LH level evoked by 200 nmol OFQ. (5) The LH release from pituitary slices showed no statistical difference between OFQ and control group during 2h incubation. (6) The results of RT-PCR and western blot analysis showed that there was no expression of ORL1 receptor and its mRNA in rat pituitary. (7) The number of OFQ-IR neurons in POA and VMH and OFQ mRNA expression in MBH in proestrus was significantly decreased than those in MBH in diestrus and estrus, while the ORL1 receptor mRNA in MBH showed no obvious change during the three stages of estrous cycle in female rats. (8) The serum LH surge of EBP-primed ovariectomized rats might be reduced by icv injection of 20 nmol OFQ. Conclusion (1) Hypothalamic OFQ may be involved in the neuroendocrinolgical mechanism underlying EA normalizing the subnormal function of hypothalamic-pituitary-ovarian axis (HPOA), resulting in the normalization of the pituitary LH release in ovariectomized rats. (2) The intracerebroventricular administration of OFQ could inhibit the hypernormal release of hypothalamic GnRH in medial eminance, which might reduce the LH release from pituitary and decrease the level of serum LH in ovariectomized rats. (3) The inhibitory effect of OFQ on GnRH release might...