Transcriptome characteristics of hypothalamus and pituitary in rats with bilateral ovariectomy and regulatory effect of Erxian Decoction

PAN Zhiqiang; XIE Haina; CAO Linna; LIU Xiaomei; LU Wenli; JIA Dongwei; PENG Peike; FANG Zhaoqin

doi:10.16305/j.1007-1334.2024.2309133

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Transcriptome characteristics of hypothalamus and pituitary in rats with bilateral ovariectomy and regulatory effect of Erxian Decoction

更新时间：2024-07-04

- Transcriptome characteristics of hypothalamus and pituitary in rats with bilateral ovariectomy and regulatory effect of Erxian Decoction
- Shanghai Journal of Traditional Chinese Medicine Vol. 58, Issue 7, Pages: 65-76(2024)
- 作者机构：
  
  上海中医药大学中医学院（上海 201203）
- 作者简介：
- 基金信息：
- DOI：10.16305/j.1007-1334.2024.2309133
  CLC：
- Published：10 July 2024，
  
  Received：25 September 2023，
- 稿件说明：
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潘志强,谢海纳,曹琳娜,等.双侧卵巢切除大鼠下丘脑‑垂体的转录组学特征及二仙汤的调节效应[J].上海中医药杂志,2024,58(7):65-76.

PAN Zhiqiang,XIE Haina,CAO Linna,et al.Transcriptome characteristics of hypothalamus and pituitary in rats with bilateral ovariectomy and regulatory effect of Erxian Decoction[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(7):65-76.
潘志强,谢海纳,曹琳娜,等.双侧卵巢切除大鼠下丘脑‑垂体的转录组学特征及二仙汤的调节效应[J].上海中医药杂志,2024,58(7):65-76. DOI： 10.16305/j.1007-1334.2024.2309133.

PAN Zhiqiang,XIE Haina,CAO Linna,et al.Transcriptome characteristics of hypothalamus and pituitary in rats with bilateral ovariectomy and regulatory effect of Erxian Decoction[J].Shanghai Journal of Traditional Chinese Medicine,2024,58(7):65-76. DOI： 10.16305/j.1007-1334.2024.2309133.

摘要

目的

利用RNA测序技术研究围绝经期综合征模型大鼠下丘脑-垂体的转录组学特征及二仙汤的作用。

方法

以大鼠双侧卵巢切除术模拟围绝经期综合征雌激素骤然下降状态。48只雌性SD大鼠，随机取假手术组12只，其余36只大鼠双侧卵巢切除术后再随机分为双侧卵巢切除组、17β-雌二醇组、二仙汤组，每组12只。大鼠双侧卵巢切除术后第7天，17β-雌二醇组灌胃17β-雌二醇（0.1 mg·kg

-1

·d

-1

），二仙汤组灌胃二仙汤提取液（8 g·kg

-1

·d

-1

），每日灌胃1次，持续6周；假手术组与双侧卵巢切除组均灌胃等量的灭菌水。采用酶联免疫吸附测定（ELISA）法检测血清雌二醇（E

）、促性腺激素释放激素（GnRH）与β-内啡肽（β-EP）含量。采用mRNA转录组测序技术检测下丘脑与垂体，根据FPKM值与edgeR算法筛选组间差异表达基因，进行基因本体（

GO）与京都基因与基因组百科全书（KEGG）富集分析，并以实时荧光定量逆转录聚合酶链式反应（RT-qPCR）法验证下丘脑雌激素受体α（

Esr1

）与线粒体编码的NADH脱氢酶亚基4（

nd4

）、垂体钙/钙调素依赖性蛋白激酶Ⅱα（

Camk2a

）与甲状腺激素受体α（

Thra

）共4个差异表达基因。

结果

①与假手术组比较，双侧卵巢切除组大鼠血清E

与β-EP显著下降（

0.05）；与双侧卵巢切除组比较，二仙汤组E

与β-EP水平显著升高（

0.05）。②大鼠下丘脑与垂体可检测基因数量均为30 957个。③与假手术组比较，双侧卵巢切除大鼠下丘脑上调基因135个、下调基因348个，垂体上调基因409个、下调基因82个。与双侧卵巢切除组比较，17β-雌二醇治疗后大鼠下丘脑上调基因126个、下调基因225个，垂体上调基因93个、下调基因273个；二仙汤治疗后大鼠下丘脑上调基因91个、下调基因92个，垂体上调基因390个、下调基因376个。④GO分析表明，下丘脑与垂体差异表达基因富集于细胞过程、生物学调控、代谢过程等。⑤KEGG呈现了下丘脑与垂体的差异表达基因富集前30个信号通路，其中二仙汤调节尼古丁成瘾、氮代谢、突触囊泡循环等信号通路。⑥RT-qPCR所验证的二仙汤显著调节的

Esr1、Mt

nd4、Camk2a

基因表达变化与转录测序结果一致。

结论

双侧卵巢切除大鼠下丘脑与垂体的神经内分泌系统处于紊乱状态，17β-雌二醇侧重于调节下丘脑基因，二仙汤对垂体调节更广、更深。

Abstract

Objective

To study the transcriptome characteristics of hypothalamus and pituitary in perimenopausal syndrome model rats by RNA-Seq and the regulatory effect of Erxian Decoction.

Methods

The perimenopausal syndromes model rats were established by bilateral ovariectomy. Forty eight female SD rats were randomly divided into sham operation group， bilateral ovariectomy group， estradiol intervention group and Erxian Decoction intervention group， with 12 rats in each group. On the 7th day after bilateral oophorectomy， the rats were administered respectively 0.1 mg·kg

-1

·d

-1

17β-estradiol or 8 g·kg

-1

·d

-1

Erxian Decoction for 6 weeks， once daily for 6 weeks； an equal amount of sterilized water were gavaged in both sham operation group and bilateral ovariectomy group. Then， the serum estradiol （E

）， gonadotropin-releasing hormone （GnRH） and β-Endorphin （β-EP） were detected by ELISA. The hypothalamus and pituitary were detected by RNA-Seq， and the differentially expressed ge

nes between the groups were screened according to FPKM and edger methods， and GO and KEGG enrichment analysis were performed. The differentially expressed genes such as

Esr1

and

nd4

in the hypothalamus，

Camk2a

and

Thra

in the pituitary were tested by real time quantitative PCR（RT-qPCR）.

Results

①Compared with the sham operation group， the serum E

and β-EP levels in the bilateral ovariectomy group were significantly decreased （

0.05）； compared with the bilateral ovariectomy group， the E

and β‑EP levels in the Erxian Decoction group were significantly increased （

0.05）. ②The number of genes in the hypothalamus and pituitary of rats is 30，957. ③Compared with the sham operation group， 135 genes up-regulated and 348 genes down-regulated were found in the hypothalamus of ovariectomized rats； there are 409 genes up-regulated and 82 genes down-regulated in the pituitary gland. Compared with the bilateral ovariectomy group， after estradiol treatment， 126 genes were up-regulated and 225 genes were down-regulated in the hypothalamus of the rats； 93 genes up-regulated and 273 genes down-regulated in the pituitary gland； After treatment with Erxian Decoction， 91 genes were up-regulated and 92 genes were down-regulated in the hypothalamus of the rats； 390 genes were up-regulated and 376 genes were down-regulated in the pituitary gland.④The differentially expressed genes of the hypothalamus and pituitary were enriched in cellular processes， biological regulation and metabolic processes by GO analysis.⑤After KEGG analysis， the first 30 signaling pathways enriched with differentially expressed genes in the hypothalamus and pituitary gland， among which Erxian Decoction regulated signaling pathways such as nicotine addiction， nitrogen metabolism and synaptic vesicle circulation. ⑥The expression changes of

Esr1

，

nd4

and

Camk2a

by RT-qPCR were consistent with the RNA-Seq results.

Conclusion

The neuroendocrine system of hypothalamus and pituitary is in a state of disorder in bilateral ovariectomized rats. Estradiol focuses on regulating hypothalamic genes， and Erxian Decoction has a broader and deeper regulation in pituitary.

关键词

围绝经期综合征二仙汤下丘脑垂体RNA测序作用机制中药研究

Keywords

perimenopausal syndromeErxian DecoctionhypothalamuspituitaryRNA-Seqmechanism of actiontraditional Chinese medicine research

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