| ObjectivesTo investigate the effect of Orexin-A on central precocious puberty(CPP)model and to exam the expression of MEG3(Maternally Expressed Gene 3)and Kisspeptin in hypothalamus of central precocious puberty rat treated with Orexin-A,and analysis the potential relationships among Orexin A,MEG3 and Kisspeptin,further to explore weather Orexin-A affects CPP by changing the expression of MEG3.Method and Subjects1.Precocious puberty rat model and animal groups48 Five-days-old specific pathogen free grade female SD rats were randomly divided into four groups:Group 1(normal control,n=12),Group 2(precocious puberty rats,n=12),Group 3(precocious puberty rats treated with normal saline,n=12),Group 4(precocious puberty rats treated with Orexin-A,n= 12).After two days of adaptive feeding,each rat in experimental groups 2,3 and 4 was subcutaneously injected with 300 ?g of danazol to establish the precocious puberty model,rats in group 1 were injected with same volume normal saline as normal control.Rats in groups 4 were injected with Orexin-A(0.5 nmol/L)for 1 time/day via lateral ventricle at 15-days-old until the end of experiment.Rats in groups 3 were injected with equal volumes of normal saline.When rats were 20-days-old,vaginal openings were checked and vaginal opening time(VO)was recorded.These were recorded on the 20th day if the vaginal was open at the first inspection.For detecting the uterus index and ovary index(organ wet weight/body mass),rats in each group were sub-divided into pre puberty,onset puberty and post puberty according to the sacrificed time.Uterus and ovary specimens were taken from all rats;and uterus index and ovary index were calculated.Hypothalamic tissues were carefully cut,removed and stored in liquid nitrogen.2.Western blot analysisTissue samples were grinded from liquid nitrogen and lysed using RIPA.A Bio-Rad protein assay kit was used to determine the protein concentration.The total cellular and tissue protein extracts were separated on 10%SDS-polyacrylamide gels(SDS-PAGE)and transferred onto nitrocellulose membranes(Millipore,Bedford,MA,USA).This analysis was used to quantify the relative levels of MEG3 and Kisspeptin protein expression.3.Quantitative real-time PCR(QTR-PCR)Total RNA was isolated from tissues using the RNeasy kit according to the manufacturer's instructions.Reverse transcription reactions were carried out.Real-time PCR was performed to calculate the expression of MEG3 and Kisspeptin in tissue samples.4.Chromatin immunoprecipitation(ChIP)assayChIP assays were performed using a ChIP assay kit to assess the histone acetylation status of the Kisspeptin promoter region according to the manufacturer's instructions.The pre-cleared chromatin was incubated with anti-acetyl-H3,anti-acetyl-H4(Upstate)or normal mouse/rabbit IgG(Southern Biotechnology Associates,Inc)as negative control.A small amount of input DNA(?2.5%)was used as positive control.5.Cell culture and transfectionHT22 cells were cultured in DMEM.Cell density was maintained 80%or less confluency to attenuate excessive growth.PcDNA-MEG3 was employed to overexpress MEG3 in HT22 cells.Transfection of pcDNA-MEG3 or pcDNA was conducted using the Lipofectamine 2000 reagent(Invitrogen)according to the manufacturer's instruction.qRT-PCR and Western blot was used to detect the mRNA and protein level of Kisspeptin,and ChIP assay was used to evaluate histone H3 and H4 acetylation status of the Kisspeptin promoter region.Results1.Orexin-A delayed the symptom of central precocious pubertyWe monitored the day of vaginal opening to determine the maturation of the external genitalia and detected two regular estrus cycle days of study rats.Comparing to the female rats of the normal control group,the time of vaginal opening was found to be highly decreased in rats of Group 2 and Group 3.While,Orexin-A delayed the day of vaginal opening in rats of Group 4 comparing to that in Group 3.In the respect of regular estrus cycle days,it was also postponed in precocious puberty rats injected with Orexin-A.On the onset of puberty,ovarian indexes and uterus indexes were higher in group 2 and group 3 comparing with group 1,but ovarian and uterus indexes were lower in group 4 than in group 3.The data shown Orexin-A ameliorated the symptoms of central precocious puberty.2.Orexin-A inhibited the expression of MEG3 in hypothalamus of rats with central precocious pubertyThe expression of MEG3 in hypothalamus of rats were examined by real-time RCR.It has been shown that the mRNA level of MEG3 was significantly enhanced in precocious puberty rats comparing to that in normal controls.Moreover,lower mRNA level of MEG3 was observed in precocious puberty rats injected with orexin-A than that in precocious puberty rats injected with normal saline.3.Orexin-A decreased the expression of Kisspeptin in hypothalamus of rats with central precocious pubertyThe expression of Kisspeptin in hypothalamus of rats were detected,and the results indicated that the mRNA and protein level of Kisspeptin in hypothalamus of central precocious puberty rats were higher than that in normal controls.In addition,orexin-A reversed this dysregulation of Kisspeptin in hypothalamus of central precocious puberty rats.4.Orexin-A reduced the binding of acetyl-H4 histone to the promoter of Kisspeptin in rats with central precocious pubertyWe investigated the binding of acetyl-H3 histone(or acetyl-H4 histone)to the promoter of Kisspeptin in study rats with ChIP assay.There was no change in the binding of acetyl-H3 histone to the promoter of Kisspeptin in these four groups.However,the binding of acetyl-H4 histone to Kisspeptin was highly enhanced in precocious puberty rats comparing to normal controls.Orexin-A also reversed this binding and decreased the binding of acetyl-H4 histone to Kisspeptin in precocious puberty rats.5.Effect of MEG3 overexpression on the expression of Kisspeptin in HT22 cellsTo investigate the role of MEG3 in regulating the expression of Kisspeptin,HT22 cells were transfected with pcDNA-MEG3 or pcDNA to overexpress MEG3.Through detecting the binding of acetyl-histone to the promoter of Kisspeptin,we found that overexpressed MEG3 enhanced the binding of acetyl-H4 histone to the promoter of Kisspeptin.In addition,the mRNA and protein level of Kisspeptin were enhanced in HT22 cells transfected with pcDNA-MEG3 using qRT-PCR and Western blot.ConclusionOur results suggest Orexin A has important interactions in rats with central precocious puberty.These in vitro studies show that orexin-A ameliorates central precocious puberty in rat and MEG3 might be involved in this effect,suggesting that MEG3 might be a novel target in treating central precocious puberty.Future work will examine the effect of MEG3 on GnRH neurons in situ to prove this hypothesis. |
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