褪黑素调控下丘脑PI3K/Akt/mTOR信号延缓雌性小鼠青春期启动的机制

Mechanism of melatonin regulation of PI3K/Akt/mTOR signaling in hypothalamus delaying the initiation of puberty in female mice

目的探讨褪黑素(MLT)对雌性小鼠青春期启动的影响以及对下丘脑磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路表达水平的影响。方法78只20日龄雌性KM小鼠,随机分为褪黑素(MLT)组和生理盐水(NS)组,每组39只。22日龄开始,给予MLT组皮下注射1 mg/kg褪黑素,给予NS组注射等体积的生理盐水。青春期启动前选取32日龄为取材点,两组分别处死13只小鼠,青春期启动后选取37、42日龄为取材点,两组分别处死13只小鼠。观察小鼠阴门开启时间;取卵巢、子宫称重计算器官指数;HE染色观察卵巢黄体数量;ELISA法检测血清黄体生成素(LH)水平;Real-time PCR和Western blotting检测下丘脑PI3K/Akt/mTOR通路mRNA和蛋白表达水平。结果与生理盐水组相比,褪黑素组小鼠阴门开启时间显著推迟(P<0.05);卵巢、子宫的大小和指数明显下降(P<0.05);血清LH水平明显下降(P<0.05);下丘脑PI3K/Akt/mTOR通路mRNA和蛋白的表达水平显著下降(P<0.05)。结论褪黑素可以延缓小鼠青春期启动,机制可能与抑制下丘脑PI3K/Akt/mTOR信号通路相关。

Objective To explore the effect of melatonin(MLT) on the initiation of puberty in female mice and on the expression level of phosphatidylinositol-3-kinases(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway in the hypothalamus. Methods Seventy-eight 20-day-old female KM mice were randomly divided into melatonin(MLT) group and normal saline(NS) group, with 39 mice in each group. Starting at 22 days of age, the MLT group was given a subcutaneous injection of 1 mg/kg melatonin and the NS group was given an equal volume of saline. Thirty-two days of age were selected as the sampling point before puberty initiation and 13 mice were executed in each of the two groups, while 37 and 42 days of age were selected as the sampling point after puberty initiation and 13 mice were executed in each of the two groups. Observation of vaginal opening time in mice, weighing of ovaries and uterus to calculate organ indices. HE staining to observe the number of ovarian corpora lutea. The levels of serum luteinizing hormone(LH)were determined by ELISA. The mRNA and protein expression levels of PI3K/Akt/mTOR pathway in hypothalamus were detected by Real-time PCR and Western blotting. Results Compared with the normal saline group, mice in the melatonin group had significantly delayed vaginal opening time(P<0.05), decreased significantly ovarian and uterine volume and index(P<0.05), decreased significantly serum LH levels(P<0.05), and decreased significantly mRNA and protein expression levels of the hypothalamic PI3K/Akt/mTOR pathway(P<0.05). Conclusion Melatonin delays puberty initiation in mice by a mechanism that may be related to inhibition of the hypothalamic PI3K/Akt/mTOR signalling pathway.