Epigenetic mechanism of Ziyin Xiehuo Formula in inhibiting bisphenol A‑induced <italic style="font-style: italic">Kiss1</italic> activation

DONG Wenke; WANG Junqi; HAN Xinghui; LU Chen; WANG Wenqin; YU Jian

doi:10.16305/j.1007-1334.2025.z20240531003

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Epigenetic mechanism of Ziyin Xiehuo Formula in inhibiting bisphenol A‑induced Kiss1 activation

更新时间：2025-03-12

- Epigenetic mechanism of Ziyin Xiehuo Formula in inhibiting bisphenol A‑induced Kiss1 activation
- Shanghai Journal of Traditional Chinese Medicine Vol. 59, Issue 3, Pages: 69-76(2025)
- 作者机构：
  
  1.复旦大学附属儿科医院中医科（上海 201102）
  2.上海交通大学医学院附属瑞金医院儿科（上海 200025）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2025.z20240531003
  CLC：
- Received：31 May 2024，
  
  Published：10 March 2025
- 稿件说明：
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董文科,王俊祺,韩兴绘,等.滋阴泻火方通过表观调控抑制双酚A诱导Kiss1基因活化的机制[J].上海中医药杂志,2025,59(3):69-76.

DONG Wenke,WANG Junqi,HAN Xinghui,et al.Epigenetic mechanism of Ziyin Xiehuo Formula in inhibiting bisphenol A‑induced Kiss1 activation[J].Shanghai Journal of Traditional Chinese Medicine,2025,59(3):69-76.
董文科,王俊祺,韩兴绘,等.滋阴泻火方通过表观调控抑制双酚A诱导Kiss1基因活化的机制[J].上海中医药杂志,2025,59(3):69-76. DOI： 10.16305/j.1007-1334.2025.z20240531003.

DONG Wenke,WANG Junqi,HAN Xinghui,et al.Epigenetic mechanism of Ziyin Xiehuo Formula in inhibiting bisphenol A‑induced Kiss1 activation[J].Shanghai Journal of Traditional Chinese Medicine,2025,59(3):69-76. DOI： 10.16305/j.1007-1334.2025.z20240531003.

摘要

目的

通过分析滋阴泻火方及内分泌干扰物双酚A（BPA）对性发育调控基因吻素1（

Kiss1

）基因的表观调控效应，初步探讨滋阴泻火方抑制BPA生殖毒性的调控机制。

方法

使用小鼠下丘脑神经元GT1-7细胞系。①采用不同浓度（2 nmol/L、2 μmol/L、40 μmol/L）的BPA干预24 h，通过实时荧光定量逆转录聚合酶链式反应（RT-qPCR）法检测

Kiss1

及其下游促性腺激素释放激素（

GnRH1

）基因的表达水平；②制备滋阴泻火方的含药血清及氯化钠溶液（9 g/L，NS）对照血清，分别刺激经BPA（40 μmol/L）干预后的GT1-7细胞，共孵育24 h，分析药物血清对

Kiss1、GnRH1

基因表达的调控；③GT1-7细胞分别加入BPA（40 μmol/L）组、磷酸盐缓冲液（溶剂对照）组、BPA（40 μmol/L）+10%中药血清组、BPA（40 μmol/L）+10% NS血清组，共孵育24 h，采用亚硫酸盐测序（BSP）方法检测

Kiss1

基因转录上游CpG位点的甲基化修饰；采用免疫共沉淀（ChIP）-qPCR法检测

Kiss1

基因启动子区域激活性组蛋白H3组蛋白4位赖氨酸三甲基化蛋白（H3K4me3）及相关修饰酶混合谱系白血病1蛋白（MLL1）的结合量。

结果

①与溶剂对照组比较，40 μmol/L BPA可促进

Kiss1

、

GnRH1

转录（

0.01，

0.05）。②滋阴泻火方的含药血清可抑制Kiss1、GnRH1的蛋白表达和mRNA表达（

0.01，

0.05）。③滋阴泻火方的含药血清可抑制

Kiss1

基因调控域的活化：BPA组的

Kiss1

起始密码子（STC）上游-500~0 bp区域内DNA总甲基化率下调，STC上游-500~-600 bp区域激活性组蛋白H3K4me3及相关修饰酶MLL1的结合量增加；滋阴泻火方血清干预后DNA甲基化率上调，伴随H3K4me3和MLL1在

Kiss1

基因启动子区域的结合量下降。

结论

BPA抑制

Kiss1

基因调控区域DNA甲基化，促进激活性组蛋白H3K4me3富集，从而激活

Kiss1

基因表达，而滋阴泻火方可通过上调DNA甲基化、抑制激活性组蛋白修饰，从而抑制BPA对

Kiss1

基因的激活效应，拮抗环境内分泌干扰物BPA的生殖发育毒性，这可能是其抑制性早熟的作用机制之一。

Abstract

Objective

To analyze the epigenetic regulatory effects of Ziyin Xiehuo Formula （ZYXH） and the endocrine disruptor bisphenol A （BPA） on the sex development-regulating gene

Kiss1

， and preliminarily explore the regulatory mechanism by which the formula inhibits the reproductive toxicity of BPA.

Methods

The GT1-7 hypothalamic neurons from mice was used. ①Different concentrations of BPA （2 nmol/L， 2 μmol/L， 40 μmol/L） were applied for 24 hours. The gene expression levels of

Kiss1

and downstream gonadotropin-releasing hormone （

GnRH1

） were detected by real-time quantitative reverse transcription PCR （RT-qPCR）.②Serum containing ZYXH was prepared， with sodium chloride solution （9 g/L， NS）-containing serum used as the control. These serums were used to stimulate GT1-7 cells that had been treated with BPA （40 μmol/L） for 24 hours， and the expression of

Kiss1

and

GnRH1

genes was analyzed to assess the regulatory effects of the serum. ③The GT1-7 cells were separately treated with BPA （40 μmol/L）， phosphate-buffered saline （solvent control）， BPA （40 μmol/L） + 10% ZYXH serum， and BPA （40 μmol/L） + 10% NS serum for 24 hours. Bisulfite sequencing （BSP） was used to detect methylation modifications at the CpG sites in the transcriptional ups

tream region of the

Kiss1

gene. Chromatin immunoprecipitation （ChIP）-qPCR was used to measure the binding of histone H3 lysine 4 trimethylation （H3K4me3） and the mixed lineage leukemia 1 （MLL1） enzyme to the promoter region of the

Kiss1

gene.

Results

①Compared with the solvent control group， BPA exposure at 40 μmol/L significantly increased the transcription of

Kiss1

and

GnRH1

（

0.01，

0.05）. ②The serum containing ZYXH significantly inhibited protein and the mRNA expression of Kiss1 and GnRH1

（

0.01，

0.05）. ③The serum containing ZYXH inhibited the activation of the

Kiss1

gene. The administration of BPA resulted in a reduction in the overall DNA methylation rate within the fragments situated between -500 and 0 bp upstream of the

Kiss1

start codon （STC）， while concurrently facilitating the binding of H3K4me3 and MLL1 within the fragment spanning -600 to -500 bp. Conversely， the serum containing ZYXH upregulated DNA methylation rate， with a reduction in the binding of H3K4me3 and MLL1 to the

Kiss1

gene promoter region.

Conclusions

BPA inhibited DNA methylation in the regulatory region of

Kiss1

gene and promoted the enrichment of activating histone H3K4me3， thereby resulting in the activation of

Kiss1

gene expression. In contrast， ZYXH inhibited BPA-induced activation of

Kiss1

gene

by upregulating DNA methylation and inhibiting the activating histone modifications， thereby antagonizing the reproductive developmental toxicity of the environmental endocrine disruptor BPA. This may be one of the underlying mechanisms through which ZYXH inhibits precocious puberty.

关键词

Keywords

references

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Epigenetic mechanism of Ziyin Xiehuo Formula in inhibiting bisphenol A‑induced Kiss1 activation

DOI：10.16305/j.1007-1334.2025.z20240531003

摘要

Abstract

关键词

Keywords

references