Effect Of GnIH On POMC Neurons In Hypothalamus Of Estrogen-supplemented Rats After Ovariectomy

Hypothalamus is the main nerve center regulating energy balance and reproduction.It has complex structure and contains many nerve nuclei.The complex regulatory network is formed between different nuclei,including dorsal medial hypothalamic nucleus(DMH),ventromedial hypothalamic nucleus(VMH),paraventricular nucleus of hypothalamus(PVH)and arcuate nucleus(ARC),all of which are involved in regulating energy balance and reproduction.And the regulation of energy balance and reproduction is interrelated.Gonadotropin inhibitory hormone(GnIH)is a new RFamide peptide found in the brain of Japanese quail.It is named gonadotropin inhibitory hormone because of its function of inhibiting the release of gonadotropin.GnIH/RFRP-3(gonadotropin inhibitory hormone/ RFamide-related peptide-3,GnIH/RFRP-3)neurons are mainly located in paraventricular nucleus(PVH)、ventromedial hypothalamic nucleus(VMH)and dorsomedial hypothalamic nucleus(DMH),and their nerve fibers can project to many areas,including the hypothalamic arcuate nucleus(ARC).At the same time,GnIH/RFRP-3 is a multipotent hormone,which plays an important role in food intake、energy balance and reproduction.Recent studies have shown that GnIH/RFRP-3 is a neuropeptide that negatively regulates reproduction and stimulates food intake.Pro-opiomelanocortin(POMC)is an important appetite regulatory peptide located in the arcuate nucleus.POMC is a precursor protein.After hydrolysis by specific protease,many small molecular polypeptides related to energy balance and food regulation are produced.Recent studies have shown that POMC has the effect of inhibiting food intake and stimulating reproductive.It was also found that GnIH/RFRP-3 has a role in inhibiting POMC neurons,and there was a synaptic relationship between them.Moreover,GnIH could inhibit POMC neurons,which was consistent with the effect of GnIH/RFRP-3 on food intake.However,whether GnIH/RFRP-3 plays a regulatory role by directly acting on POMC neurons has not been clearly established at home and abroad.This topic will carry on the validation research to this.Objective:To observe the expression of GnIH/RFRP-3 and POMC in rat hypothalamic neurons by immunofluorescence.To observe the interaction between GnIH/RFRP-3 and POMC by co-precipitation immunoassay(CO-IP),and to detect the changes of the expression of POMC protein in rat hypothalamus after different time of GnIH/RFRP-3 microinjection into lateral ventricle by Western Blotting method,so as to explore whether GnIH/RFRP-3 acts through POMC neurons.Methods:1.Grouping of experimental animals:65 adult female Sprague-Dawley rats,2 randomly selected for immunofluorescence double-labeling experiments,3 randomly selected for immuncoprecipitation experiments(CO-IP),and the remaining 60 were established ovariectomized estrogen primed(OEP)rat model.They were randomly divided into GnIH injection group and saline control group(GnIH and saline were respectively 4 μl),with thirty rats in each group.And each group was divided into six subgroups,including 20,40,60,120,240,360 minutes,with five rats in each group.2.Observation of hypothalamic nuclei in rats by Nissl staining.3.The expression of GnIH/RFRP-3 and POMC in rat hypothalamic neurons was observed by immunofluorescence double labeling method.4.The interaction between GnIH/RFRP-3 and POMC was detected by CO-IP.5.Western Blotting method was used to detect the expression of POMC protein in hypothalamus after microinjection of GnIH/RFRP-3 into lateral ventricle at different time.Result:1.Referring to the stereotaxic map of rat brain,the dorsomedial nucleus of hypothalamus,ventromedial nucleus of hypothalamus and arcuate nucleus of hypothalamus can be seen by Nissl staining.2.Immunofluorescence double labeling results showed that both GnIH/RFRP-3 neurons and POMC neurons were cytoplasmically stained,GnIH/RFRP-3 fluorescence was red,POMCfluorescence was green,nucleolus was blue,and GnIH/RFRP-3 co-expressed with POMC in the same neuron.3.CO-IP results showed that POMC antibody was used to co-precipitate the total protein of rat hypothalamic tissue lysate.After electrophoresis,immunoblotting was performed with POMC antibody and GnIH/RFRP-3 antibody respectively.The results showed that POMC protein and GnIH/RFRP-3 protein were contained in the immune complex,and there were bands in the immunoblot which were phase with rat hypothalamic tissue lysate.The bands of allergens are in the same position.4.Western Blotting results showed that the expression of POMC protein in hypothalamus of rats in 20 minutes after microinjection of GnIH/RFRP-3 into lateral ventricle was significantly higher than that in control group(P < 0.05),and there was no notable difference between groups in 40 minutes(P > 0.05),the expression of POMC protein in experimental group was significantly lower than that in control group at 60,120,240 and 360 minutes(P < 0.05);Compared with the 20-minute experimental group,the expression of POMC protein in 60,120 and 240-minute groups decreased in turn,while that in 360-minute group increased,but was still lower than that in 20-minute group,(P < 0.05)the difference was statistically significant.Conclusions:GnIH/RFRP-3 may play a role by directly acting on POMC neurons.