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补肾解郁调冲方通过 PERK-ATF4-CHOP 信号通路减少慢性应激多囊卵巢综合征大鼠模型颗粒细胞的凋亡。

Bushen Jieyu Tiaochong Formula reduces apoptosis of granulosa cells via the PERK-ATF4-CHOP signaling pathway in a rat model of polycystic ovary syndrome with chronic stress.

机构信息

Post-doctoral Mobile Station, Guang'anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China.

Department of Gynecology, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China.

出版信息

J Ethnopharmacol. 2022 Jun 28;292:114923. doi: 10.1016/j.jep.2021.114923. Epub 2021 Dec 17.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE

Polycystic ovary syndrome (PCOS) is a common and complex endocrine disorder that is also an important cause of infertility. Adverse psychological stress can aggravate the occurrence and development of PCOS. Bushen Jieyu Tiaochong Formula (BJTF), a prescription of Traditional Chinese Medicine (TCM), has been used in the treatment of PCOS and shown to be effective in reducing negative emotion. However, the therapeutic mechanism has yet to be clearly elucidated. In the current study, we investigated the potential mechanism of action of BJTF.

AIM OF THE STUDY

To investigate the role of PERK-ATF4-CHOP signaling in the molecular mechanisms that mediate the effects of BJTF in a rat model of PCOS, with chronic stress induced by letrozole and a chronic unpredictable mild stress (CUMS) paradigm.

MATERIALS AND METHODS

In addition to the normal control group, the PCOS combined with CUMS model rats were randomly assigned to a model group, a Diane-35 (ethinylestradiol 35 μg/cyproterone acetate 2 mg)-treated positive control group, or one of three BJTF-treated groups receiving a low, medium, or high dose. Behavioral testing, including the sucrose preference test and open field test, was conducted, and hematoxylin and eosin (H&E) staining was used to observe changes in the pathological morphology of ovarian tissue. Free testosterone (FT), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels in serum were quantified by enzyme-linked immunosorbent assays (ELISA). The hippocampal levels of norepinephrine (NE), 5-hydroxytryptamine/serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were measured using high-performance liquid chromatography-electrochemical detection (HPLC-ECD). Apoptotic granulosa cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to detect the expression of glucose-regulated protein 78 (GRP78) and CHOP in the ovarian tissues. The expression levels of GRP78, CHOP, PERK, and ATF4 in ovarian tissues were also measured by western blotting.

RESULTS

Treatment with either BJTF or Diane-35 ameliorated the abnormal cystic dilatation of follicles in the model rats and reduced the serum levels of FT and LH, and the LH/FSH ratio. BJTF treatment also attenuated chronic psychological stress-like behavior and regulated the expression and metabolism of cerebral monoamine neurotransmitters. The efficacy of BJTF was greater than that of Diane-35, with the optimal effects observed at the medium dose. BJTF also lowered the apoptotic index of ovarian granulosa cells and downregulated the expression of GRP78, CHOP, and ATF4. Although the expression level of PERK was not significantly altered by BJTF, the mean PERK expression level was the lowest in the medium-dose BJTF group.

CONCLUSIONS

Administration of BJTF has the therapeutic potential to promote the homeostasis of the reproductive endocrine environment and to restore follicular development and ovulation, possibly through the inhibition of the PERK-ATF4-CHOP signaling pathway, leading to downregulation of GRP78 expression to further delay ovarian granule cell apoptosis mediated by endoplasmic reticulum stress (ERS). Moreover, BJTF could improve behavioral performance by regulating cerebral monoamine neurotransmitters in this rat model. These findings provide a new perspective for treating PCOS related to psychological stress using TCM.

摘要

民族药理学相关性

多囊卵巢综合征(PCOS)是一种常见且复杂的内分泌紊乱疾病,也是导致不孕的重要原因。不良的心理应激可加重 PCOS 的发生和发展。补肾解郁调冲方(BJTF)是一种中药方剂,已被用于治疗 PCOS,并已被证明可有效减轻负面情绪。然而,其治疗机制尚未明确。在目前的研究中,我们研究了 BJTF 的潜在作用机制。

研究目的

探讨 PERK-ATF4-CHOP 信号通路在补肾解郁调冲方调节 PCOS 大鼠模型中作用的分子机制,该模型由来曲唑诱导的慢性应激和慢性不可预测轻度应激(CUMS)范式引起。

材料和方法

除正常对照组外,将 PCOS 合并 CUMS 模型大鼠随机分为模型组、Diane-35(乙炔雌二醇 35μg/环丙孕酮 2mg)阳性对照组和低、中、高剂量 BJTF 治疗组。进行行为测试,包括蔗糖偏好测试和旷场测试,并用苏木精和伊红(H&E)染色观察卵巢组织病理形态的变化。采用酶联免疫吸附试验(ELISA)定量检测血清中游离睾酮(FT)、促黄体生成素(LH)和促卵泡生成素(FSH)水平。采用高效液相色谱-电化学检测法(HPLC-ECD)测定海马去甲肾上腺素(NE)、5-羟色胺/血清素(5-HT)和 5-羟吲哚乙酸(5-HIAA)的水平。用末端脱氧核苷酸转移酶 dUTP 缺口末端标记法(TUNEL)染色检测颗粒细胞凋亡。此外,采用定量逆转录聚合酶链反应(qRT-PCR)和免疫组织化学法检测卵巢组织中葡萄糖调节蛋白 78(GRP78)和 CHOP 的表达。采用 Western blot 法检测卵巢组织中 GRP78、CHOP、PERK 和 ATF4 的表达水平。

结果

BJTF 或 Diane-35 治疗可改善模型大鼠卵泡囊性扩张的异常,并降低血清 FT 和 LH 水平,以及 LH/FSH 比值。BJTF 治疗还可减轻慢性心理应激样行为,并调节脑单胺神经递质的表达和代谢。BJTF 的疗效优于 Diane-35,中剂量组的效果最佳。BJTF 还降低了卵巢颗粒细胞的凋亡指数,并下调了 GRP78、CHOP 和 ATF4 的表达。尽管 BJTF 对 PERK 的表达水平没有显著影响,但中剂量 BJTF 组的 PERK 平均表达水平最低。

结论

BJTF 的给药具有促进生殖内分泌环境平衡和恢复卵泡发育和排卵的治疗潜力,可能通过抑制 PERK-ATF4-CHOP 信号通路,下调 GRP78 表达,进一步延缓内质网应激(ERS)介导的卵巢颗粒细胞凋亡。此外,BJTF 可通过调节大脑单胺神经递质改善该大鼠模型的行为表现。这些发现为使用中药治疗与心理应激相关的 PCOS 提供了新视角。

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