黄柏酮调控Notch1/Hes1通路对雌性大鼠真性性早熟的改善作用

Improvement effect of obacunone on true precocious puberty in female rats by regulating the Notch1/Hes1 pathway

关键词:  黄柏酮  Notch1/Hes1通路  真性性早熟  下丘脑-垂体-性腺轴

Key words:  obacunone  Notch1/Hes1 pathway  true precocious puberty  hypothalamic-pituitary-gonadal axis

摘要:

[目的] 探讨黄柏酮（Oba）调控Notch1/Hes1通路对雌性大鼠真性性早熟（TPP）的改善作用。[方法] 雌性大鼠随机分为TPP组、正常组、Oba低剂量组、Oba高剂量组、亮丙瑞林组、Oba高剂量+Notch1阻断剂（DAPT）组，每组12只。除正常组外，其他组大鼠均通过皮下注射N-甲基-DL-天冬氨酸法构建TPP模型。模型构建成功后，开始给药处理，每日1次，持续2周。统计大鼠阴道开口时间；酶联免疫吸附实验（ELISA）检测大鼠血清中促黄体生成素（LH）、雌二醇（E₂）、促卵泡激素（FSH）水平；检测卵巢系数、子宫系数的变化；苏木精-伊红（HE）染色检测卵巢、子宫病理变化；免疫组化染色检测下丘脑中促性腺激素释放激素（GnRH）表达和垂体中促性腺激素释放激素受体（GnRHR）表达；蛋白质免疫印迹法（Western blot）检测下丘脑中Notch1、Hes1蛋白表达。[结果] 与正常组相比，TPP组大鼠卵巢出现多个二级卵泡和三级卵泡，子宫壁增厚，阴道开口时间提前（P<0.05），血清中LH、E₂、FSH水平、卵巢系数、子宫系数、下丘脑中GnRH表达的平均光密度值、垂体中GnRHR表达的平均光密度值升高（P<0.05），下丘脑中Notch1、Hes1蛋白表达降低（P<0.05）；与TPP组相比，Oba低剂量组、Oba高剂量组、亮丙瑞林组大鼠卵巢中二级卵泡和三级卵泡数量减少，子宫壁变薄，阴道开口时间延迟（P<0.05），血清中LH、E₂、FSH水平、卵巢系数、子宫系数、下丘脑中GnRH表达的平均光密度值、垂体中GnRHR表达的平均光密度值降低（P<0.05），下丘脑中Notch1、Hes1蛋白表达升高（P<0.05）；与Oba高剂量组相比，Oba高剂量+DAPT组大鼠卵巢中二级卵泡和三级卵泡数量增多，子宫壁增厚，阴道开口时间提前（P<0.05），血清中LH、E₂、FSH水平、卵巢系数、子宫系数、下丘脑中GnRH表达的平均光密度值、垂体中GnRHR表达的平均光密度值升高（P<0.05），下丘脑中Notch1、Hes1蛋白表达降低（P<0.05）。[结论] Oba可能通过上调Notch1/Hes1通路改善雌性大鼠TPP。

Abstract:

[Objective] To investigate the modulation of the Notch1/Hes1 pathway by obacunone（Oba） on the improvement of true precocious puberty（TPP） in female rats. [Methods] Female rats were randomly assigned to seven groups：the TPP group，normal group，Oba low-dose group，Oba high-dose group，leuprolide group，and Oba high-dose+Notch1 blocker（DAPT） group，with 12 rats in each group. Except for the normal group，the TPP model was established in the remaining groups by subcutaneous injection of N-methyl-DL-aspartic acid. After successful model induction，drug treatment was initiated and administered once daily for 2 weeks. The time of vaginal opening was recorded. Enzyme-linked immunosorbent assay（ELISA） was used to measure the serum levels of luteinizing hormone（LH），estradiol（E₂），and follicle-stimulating hormone（FSH）. Changes in ovarian and uterine coefficients were assessed. Hematoxylin and eosin（HE） staining was performed to examine pathological changes in the ovaries and uterus. Immunohistochemical staining was used to detect the expression of gonadotropin-releasing hormone（GnRH） in the hypothalamus and gonadotropin-releasing hormone receptor（GnRHR） in the pituitary gland. Additionally，the protein expression of Notch1 and Hes1 was evaluated by Western blot analysis. [Results] Compared with the normal group，rats in the TPP group showed multiple secondary follicles and tertiary follicles in the ovaries，thickening of the uterine wall，advancement of the vaginal opening time（P<0.05），elevation of serum levels of LH，E₂，FSH，ovarian coefficients，uterine coefficients，the average optical density value of GnRH expression in the hypothalamus，and GnRHR expression in the pituitary gland（P<0.05），and elevation of hypothalamic expression of Notch1 and Hes1 protein. Notch1 and Hes1 protein expression in the hypothalamus decreased（P<0.05）；compared with the TPP group，the number of secondary and tertiary follicles in the ovaries of rats in the Oba low-dose group，Oba high-dose group，and leuprolide group decreased，the uterine wall thinned，and the time of vaginal opening was delayed（P<0.05），and the levels of LH，E₂，and FSH in the serum，the coefficients of the ovary，the coefficients of the uterus，the coefficients of GnRH expression in the hypothalamus GnRH expression，the mean optical density value of GnRHR expression in the pituitary gland decreased（P<0.05），and the expression of Notch1 and Hes1 proteins in the hypothalamus was elevated（P<0.05）；compared with the Oba high-dose group，the number of secondary follicles and tertiary follicles increased in the ovaries of the rats in the Oba high-dose + DAPT group，the uterine wall thickened，and the time of vaginal opening was advanced（P<0.05），increased serum levels of LH，E₂，FSH，ovarian coefficient，uterine coefficient，mean optical density value of GnRH expression in the hypothalamus，and mean optical density value of GnRHR expression in the pituitary gland（P<0.05），and decreased protein expression of Notch1 and Hes1 in the hypothalamus（P<0.05）. [Conclusion] Oba may improve TPP in female rats by up-regulating the Notch1/Hes1 pathway.