复幼合剂通过PI3K/Akt信号通路对性早熟SD雌鼠的治疗作用观察

郭春彦; 柳静; 张怡; 丁倩; 王谦; 张萌; 王晓玲

doi:10.16025/j.1674-1307.2024.10.011

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复幼合剂通过PI3K/Akt信号通路对性早熟SD雌鼠的治疗作用观察

实验研究 | 更新时间：2024-11-26

- 复幼合剂通过PI3K/Akt信号通路对性早熟SD雌鼠的治疗作用观察
- Efficacy of Fuyou Mixture in treatment of precocious puberty in SD female rats based on PI3K/Akt signaling pathway
- 北京中医药 2024年43卷第10期页码：1131-1137
- 作者机构：
  
  1.国家儿童医学中心首都医科大学附属北京儿童医院药学部，北京 100045
  2.国家儿童医学中心首都医科大学附属北京儿童医院中医科，北京 100045
- 作者简介：
  
  郭春彦，女，40岁，硕士，副主任药师。研究方向：中药药理学、临床中药学。
  王晓玲，E-mail：wangxiaoling@bch.com.cn
- 基金信息：
  
  重大新药创制科技重大专项(2018ZX09721003-002-001);首都医科大学附属北京儿童医院“萌芽杯”儿童用药专项(MYQY202201)
- DOI：10.16025/j.1674-1307.2024.10.011
  中图分类号：
- 纸质出版日期：2024-10-25，
  
  收稿日期：2023-12-05，
- 稿件说明：
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郭春彦，柳静，张怡，等.复幼合剂通过PI3K/Akt信号通路对性早熟SD雌鼠的治疗作用观察［J］.北京中医药，2024，43（10）：1131-1137.

GUO Chunyan,LIU Jing,ZHANG Yi,et al.Efficacy of Fuyou Mixture in treatment of precocious puberty in SD female rats based on PI3K/Akt signaling pathway[J]. Beijing Journal of Traditional Chinese Medicine,2024,43(10):1131-1137.
郭春彦，柳静，张怡，等.复幼合剂通过PI3K/Akt信号通路对性早熟SD雌鼠的治疗作用观察［J］.北京中医药，2024，43（10）：1131-1137. DOI： 10.16025/j.1674-1307.2024.10.011.

GUO Chunyan,LIU Jing,ZHANG Yi,et al.Efficacy of Fuyou Mixture in treatment of precocious puberty in SD female rats based on PI3K/Akt signaling pathway[J]. Beijing Journal of Traditional Chinese Medicine,2024,43(10):1131-1137. DOI： 10.16025/j.1674-1307.2024.10.011.

摘要

目的

探究复幼合剂通过磷脂酰肌醇3-激酶-蛋白激酶B（PI3K/Akt）信号通路对性早熟（PP）雌鼠下丘脑促性腺激素释放激素（GnRH）分泌的影响。

方法

取75只SD雌鼠，用随机数字表法将其分为正常组、模型组、复幼合剂组、亮丙瑞林组、槲皮素组各15只。母乳喂养至23日龄。在第5日龄时，除正常组外，其余4组均一次性颈背部皮下注射25 μL达那唑溶液建立PP模型。大鼠阴道开口时间、首次发情时间及正常发情周期显著提前为造模成功。造模10 d，亮丙瑞林组于左侧下腹部皮下注射亮丙瑞林100 μg/kg；复幼合剂组每天给予6 mL/kg复幼合剂灌胃；槲皮素组每天给予4 mg/kg槲皮素灌胃；正常组、模型组给予2 mL/kg生理盐水灌胃。20日龄开始检查阴道开口情况，通过苏木精-伊红（HE）染色观察性腺发育情况；酶联免疫吸附检测血清中促黄体生成素（LH）、促卵泡刺激素（FSH）、雌二醇（E

）；免疫组织化学法检测下丘脑GnRH蛋白表达；实时荧光定量聚合酶链反应（RT-qPCR）检测下丘脑GnRH、吻素（Kiss-1）、G蛋白偶联受体54（GPR54）、胰岛素生长因子（IGF-1）、雌激素受体α（ESR1）mRNA表达，Western blotting法检测GnRH、磷脂酰肌醇3-激酶（PI3K）、磷酸化磷脂酰肌醇3-激酶（p-PI3K）、蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）蛋白表达。

结果

与正常组比较，模型组阴道开口提前。模型组子宫系数、卵巢系数较正常组大（

0.05）。复幼合剂组子宫系数较模型组小（

0.01），亮丙瑞林组子宫系数、卵巢系数较模型组小（

0.05）。模型组、复幼合剂组、亮丙瑞林组、槲皮素组子宫、卵巢细胞形态结构无明显病理变化，表明达那唑不会造成性器官病理性增长。各组卵巢发育情况大致相同，均可见初级、次级、成熟卵泡。模型组子宫壁厚度与正常组比较差异无统计学意义（

0.05），复幼合剂组子宫壁厚度较模型组小，但差异无统计学意义（

0.05），亮丙瑞林组子宫壁厚度较模型组小（

0.05），槲皮素子宫壁厚度与模型组比较差异无统计学意义（

0.05）。模型组血清E

、LH、FSH水平较正常组高（

0.05）；亮丙瑞林组E

及FSH水平较模型组低（

0.05），复幼合剂组、槲皮素组血清E

水平较模型组低（

0.01）。模型组下丘脑GnRH细胞平均光密度值较正常组高（

0.05）；复幼合剂组、亮丙瑞林组光密度值较模型组低（

0.01），槲皮素组光密度值与模型组比较差异无统计学意义（

0.05）。显微镜下可见各组深浅不等的棕黄色GnRH阳性细胞，细胞结构清晰，核仁明显，且以Broca斜角带分布最为集中。模型组下丘脑GnRH、Kiss-1、GPR54、ESR1、IGF-1 mRNA表达较正常组高（

0.01）。复幼合剂组、亮丙瑞林组下丘脑GnRH、Kiss-1、GPR54、ESR1、IGF-1 mRNA表达较模型组低（

0.05），槲皮素组下丘脑GnRH、Kiss-1、ESR1 mRNA表达较模型组低（

0.05）。模型组下丘脑GnRH蛋白相对表达量及p-Akt/Akt、p-PI3K/PI3K均较正常组高（

0.01），复幼合剂组、亮丙瑞林组、槲皮素组GnRH蛋白相对表达量及p-Akt/Akt、p-PI3K/PI3K均较模型组低（

0.05）。

结论

复幼合剂通过下调IGF-1 mRNA以及p-Akt/Akt、p-PI3K/PI3K蛋白表达，抑制IGF-1/PI3K/Akt信号通路激活，进而降低Kiss-1、GPR54 mRNA表达，减少GnRH以及血清E

的分泌，从而抑制下丘脑-垂体-性腺轴（HPGA）的启动，发挥治疗大鼠PP的作用。

Abstract

Objective

To investigate the effects of Fuyou Mixture on the secretion of gonadotropin-releasing hormone（GnRH）in the hypothalamus of female rats with precocious puberty through the phosphoinositide 3-kinase-protein kinase B（PI3K/Akt）signaling pathway.

Methods

A total of 75 female SD rats were randomly divided into a normal group，a model group，a Fuyou Mixture group，a leuprolide group，and a quercetin group according to a random number table，with 15 rats in each group.The rats were fed breast milk until 23 days of age.On day 5，except for the normal group，the other four groups received a single subcutaneous injection of 25 μL of danazol solution in the neck to establish the precocious puberty model.Successful modeling was confirmed by an earlier vaginal opening time， earlier first estrus， and abnormal estrous cycles. After 10 days， the leuprolide group received subcutaneous injections of leuprolide（100 μg/kg）on the left lower abdomen， the Fuyou Mixure group received Fuyou Mixture at 6 mL/kg by gavage daily，and the quercetin group received quercetin at 4 mg/kg by gavage daily. The normal and model groups received normal saline at 2 mL/kg by gavage.Vaginal opening was checked starting from day 20，and ovarian development was observed using hematoxylin-eosin （HE） staining.Serum levels of luteinizing hormone（LH），follicle-stimulating hormone（FSH），and estradiol（E

）were measured by enzyme-linked immunosorbent assay（ELISA）.Immunohistochemistry was used to detect GnRH protein expression in the hypothalamus.Real-time quantitative polymerase chain reaction（RT-qPCR）was used to detect mRNA expression of GnRH， Kiss-1， G protein-coupled receptor 54（GPR54），insulin-like growth factor（IGF-1），and estrogen receptor α（ESR1）in the hypothalamus. Western blotting was used to assess the protein expression of GnRH，PI3K，phosphorylated PI3K（p-PI3K），Akt，and phosphorylated Akt（p-Akt）.

Results

Compared with the nor

mal group， the model group showed an earlier vaginal opening.The model group had higher uterine and ovarian coefficients than the normal group（

0.05）.The Fuyou Mixture group had a smaller uterine coefficient than the model group（

0.01）， and the leuprolide group showed smaller uterine and ovarian coefficients than the model group（

0.05）.No significant pathological changes were observed in the morphology of ovarian and uterine cells among the model，Fuyou Mixture，leuprolide，and quercetin groups，indicating that danazol did not cause pathological growth of the sexual organs. Ovarian development in all groups showed primary，secondary，and mature follicles.There was no significant difference in the uterine wall thickness between the model group and the normal group（

0.05）.The Fuyou Mixture group showed a smaller uterine wall thickness than the model group，but the difference was not statistically significant（

0.05），while the leuprolide group had a significantly thinner uterine wall than the model group（

0.05）.No significant differences in uterine wall thickness were observed between the quercetin group and the model group（

0.05）.The serum levels of E

，LH，and FSH were significantly higher in the model group than in the normal group（

0.05）.E

and FSH levels were lower in the leuprolide group than in the model group（

0.05），and both the Fuyou Mixture and quercetin groups showed significantly lower serum E

levels than the model group（

0.01）.The average optical density of GnRH cells in the hypothalamus was higher in the model group than in the normal group（

0.05）.The Fuyou Mixture and leuprolide groups showed lower optical density values than the model group（

0.01），while the quercetin group showed no significant difference from the model group（

0.05）.Under the microscope，GnRH-positive cells with varying shades of brown-yellow were observed in all groups，with clear cell structure and prominent nucleoli，most concentrated in the Broca's diagonal band.The mRNA expression levels of GnRH，Kiss-1，GPR54，ESR1，and IGF-1 in the hypothalamus were significantly higher in the model group than in the normal group（

0.01）.The Fuyou Mixture and leuprolide groups showed lower mRNA expression of GnRH， Kiss-1， GPR54， ESR1， and IGF-1 than the model group（

0.05），while the quercetin group showed lower mRNA expression of GnRH，Kiss-1，and ESR1 than the model group（

0.05）.The relative protein expression of GnRH， p-Akt/Akt， and p-PI3K/PI3K in the hypothalamus was significantly higher in the model group than in the normal group（

0.01）.The Fuyou Mixture， leuprolide， and quercetin groups showed lower protein expression of GnRH， p-Akt/Akt， and p-PI3K/PI3K than the model group（

0.05）.

Conclusion

Fuyou Mixture exerts therapeutic effects on precocious puberty in rats by downregulating IGF-1 mRNA expression and the protein expression of p-Akt/Akt and p-PI3K/PI3K and inhibiting the IGF-1/PI3K/Akt signaling pathway to reduce the expression of Kiss-1 and GPR54 mRNA，thereby decreasing GnRH secretion and serum E

levels and inhibiting the activation of the hypothalamic-pituitary-gonadal axis.

关键词

性早熟复幼合剂磷脂酰肌醇3-激酶-蛋白激酶B信号通路下丘脑促性腺激素释放激素大鼠

Keywords

precocious pubertyFuyou MixturePI3K/Akt signaling pathwayGnRHrat

references

戴丽凤,田华,杨群燕,等.儿童性早熟流行病学特征及相关影响因素分析[J].中国公共卫生管理,2017,33(1):136-137,140.

朱铭强,傅君芬,梁黎,等.中国儿童青少年性发育现状研究[J].浙江大学学报(医学版),2013,42(4):396-402,410.

中华医学会儿科学分会内分泌遗传代谢学组,中华儿科杂志编辑委员会.中枢性性早熟诊断与治疗专家共识(2022)[J].中华儿科杂志,2023,61(1):16-22.

KANETY H,KARASIK A,PARIENTE C,et al.Insulin⁃like growth factor⁃1 and IGF binding protein⁃3 remain high after GnRH analogue therapy in girls with central precocious puberty[J].Clinical endocrinology,1996,45(1):7-12.

WEISE M,FLOR A,BARNES KM,et al.Determinants of growth during gonadotropin⁃releasing hormone analog therapy for precocious puberty[J].J Clin Endocrinol Metab,2004,89(1):103-107.

CISTERNINO M,DRAGHI M,LAURIOLA S,et al.The acid⁃labile subunit of human ternary insulin⁃like growth factor⁃binding protein complex in girls with central precocious puberty before and during gonadotropin releasing hormone analog therapy[J].J Clin Endocrinol Metab,2002,87(10):4629-4633.

胡亚美,江载芳,申昆玲.诸福棠实用儿科学[M].9版.北京:人民卫生出版社,2015:2119.

刘慧丽,柳静,刘桂琴.复幼合剂治疗女童真性性早熟60例临床研究[J].北京中医药,2009,28(8):588-589,611.

戴方伟.达那唑诱导雌性大鼠性早熟模型的建立及其评估[D].杭州:浙江大学,2009.

MORISHITA H,TAKEMOTO M,KONDO H,et al.Induction of true precocious puberty by neonatal treatment with danazol in female rats[J].Neurosci Lett,1993,157(1):33-36.

尹蔚萍.基于“肝肾同源”辨治女童性早熟经验总结及疏肝泻火方治疗CPP的机制研究[D].南京:南京中医药大学,2021.

杨立,王青,王志,等.Kisspeptin对下丘脑⁃垂体⁃卵巢轴的调控作用[J].中华生殖与避孕杂志,2021,41(2):177-183.

XU LX,XUE HL,LI SN,et al.Seasonal differential expression of KiSS⁃1/GPR54 in the striped hamsters(Cricetulus barabensis)among different tissues[J].Integrative Zoology,2017,12(3):260-268.

CASTELLANO JM,BENTSEN AH,SANCHEZ MA,et al.Early metabolic programming of puberty onset: impact of changes in postnatal feeding and rearing conditions on the timing of puberty and development of the hypothalamic kisspeptin system[J].Endocrinology, 2011,152:3396-3408.

TALI H,DAGAN H,MELANIJA T,et al.Role of PI4K and PI3K⁃AKT in ERK1/2 activation by GnRH in the pituitary gonadotropesp[J].Mol Cell Endocrinol,2015,415:12-23.

HINEY JK,VINOD KS,DANIELLE NV,et al. Regulation of kisspeptin synthesis and release in the preoptic/anterior hypothalamic region of prepubertal female rats:actions of IGF-1 and alcohol[J].Alcohol Clin Exp Res,2018,42(1):61-68.

SRIVASTAVA VK,JILL KH,WILLIAM D Manganese⁃stimulated kisspeptin is mediated by the IGF⁃1/Akt/mammalian target of rapamycin pathway in the prepubertal female rat[J].Endocrinology,2016,157(8):3233-3241.

LIU T,WANG YZ,YANG MDet al.Di⁃(2⁃ethylhexyl) phthalate induces precocious puberty in adolescent female rats[J].Iran J Basic Med Sci,2018,21(8):848-855.

ERGENOGLU M,YILDIRIM N,YILDIRIM AG,et al.Effects of resveratrol on ovarian morphology,plasma anti⁃mullerian hormone,IGF⁃1 levels and oxidative stress paramaters in a rat model of polycystic ovary syndrome[J].Reprod Sci,2015,22(8):942-947.

CASCIO S,BARTELLA V,GAROFALA C,et al.Insulin⁃like growth factor 1 differentially regulates estrogen receptor⁃dependent transcription at estrogen response element and AP⁃1 sites in breast cells[J].J Biol Chem,2007,282(6):3498-3506.

LIANG J,SHANG Y.Estrogen and cancer[J].Annu Rev Physiol,2013,75:225-240.

SHAO P,WANG Y,ZHANG M,et al.The interference of DEHP in precocious puberty of females mediated by the hypothalamic IGF⁃1/PI3K/Akt/mTOR signaling pathway[J].Ecotox Environ Safe,2019,181:362-369.

罗小娟,曹科,叶炳均,等.双酚Ａ和邻苯二甲酸二丁酯暴露对雌性幼鼠青春期发育的影响[J].检验医学与临床,2021,18(19):2803-2808.

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