Dingkun Pill replenishes diminished ovarian reserve through the PI3K/AKT/mTOR signaling pathway in TWP-induced mice
Dingkun Pill replenishes diminished ovarian reserve through the PI3K/AKT/mTOR signaling pathway in TWP-induced mice
复制标题
定坤丸通过 PI3K/AKT/mTOR 信号通路补充 TWP 诱导小鼠卵巢储备功能下降
DOI：
10.1016/j.jep.2020.112993
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发表时间：
2020-11-15
影响因子：
5.4
通讯作者：
Li, Min
中科院分区：
文献类型：
--
作者：
Ma, Kun;Chen, Yanxia;Li, Min
民族药理学相关性：卵巢储备功能下降（DOR）可导致生育能力差并缩短女性的生殖寿命。定坤丸（DKP）是一种传统中成药，几个世纪以来一直是中医治疗妇科疾病的重要组成部分。相关临床研究表明DKP具有预防DOR的作用，但其作用机制尚未完全阐明。研究目的：本研究旨在从DOR的病理生理学角度探讨雷公藤多糖苷（TWP）对PI3K/AKT/mTOR通路的影响以及DKP的作用机制。材料与方法：80名女性将具有规律动情周期的 balb/c 小鼠随机分配至空白组、模型组、DKP 组和激素替代治疗 (HRT) 组。除空白组外，其他组小鼠均暴露于40 mg/kg/d TWP悬浮液30天以诱导DOR。此后，口服 DKP 或激素以确定它们对疾病进展的影响。实验过程中观察小鼠体重变化和动情周期。治疗后，使用酶联免疫吸附测定（ELISA）对血清样本中抗苗勒氏管激素（AMH）、雌二醇（E-2）、黄体生成素（LH）和卵泡刺激素（FSH）水平进行定量。然后处死小鼠以收获其卵巢进行苏木精和伊红（HE）染色。该过程可以评估卵巢形态和卵泡定量。使用TUNEL技术评估卵巢凋亡细胞，同时通过免疫组织化学（IHC）检查卵巢的Caspase-3和细胞色素C（Cyt C）表达。采用Western blotting分析定量Bax、Bcl-2、Caspase-3、Cyt C、mTOR、P-mTOR、AKT、P-AKT、P-PI3K和PI3K蛋白水平，并采用RT-PCR测定卵巢组织中Bax、Bcl-2、PI3K、AKT和mTOR mRNA水平。结果：结果显示，DKP能够改善血清激素水平，促进卵巢组织的恢复。发情周期。 DKP 增加了原始卵泡的总量，同时减少了闭锁卵泡的数量。生长卵泡凋亡指数及Bax、Cyt C、Caspase-3表达降低，Bcl-2：Bax比值升高。 DKP 抑制磷酸化水平以及 mTOR、AKT 和 PI3K mRNA 表达。结论：DKP 能够通过抑制 PI3K/AKT/mTOR 信号通路来增加卵巢储备，从而抑制原始卵泡活性并降低早期生长卵泡的凋亡水平。这凸显了它对治疗 DOR 的潜在有益作用。
Ethnopharmacological relevance: Diminished ovarian reserve (DOR) can lead to poor fertility and shorten the reproductive lifespan of females. The Dingkun Pill (DKP), a traditional Chinese-patented medication, has been an integral part of traditional Chinese medicinal treatment for the management of gynecological diseases for centuries. Relevant clinical studies have shown that DKP is able to protect against DOR, however, its mechanism of action is not yet fully elucidated.Study goals: This study was conducted with the aim of exploring the impact of tripterygium wilfordii polyglycosidium (TWP) on the PI3K/AKT/mTOR pathway in the context of the pathophysiology of DOR and the mechanism of action of DKP.Materials and methods: Eighty female balb/c mice with regular estrous cycles were assigned to Blank, Model, DKP and hormone replacement therapy (HRT) groups in a random manner. With the exception of the Blank group, mice in the other groups were exposed to 40 mg/kg/d TWP suspension for 30 days to DOR induction. Following this, either DKP or hormones were orally administrated to determine their effect on disease progression. During the experiment, changes in body weight and the estrous cycles of the mice were observed. Post treatment, serum sample anti-mullerian hormone (AMH), estradiol (E-2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were quantified using enzyme-linked immunosorbent assay (ELISA). The mice were then sacrificed in order to harvest their ovaries for hematoxylin and eosin (HE) staining. This process allowed for the assessment of ovarian morphology and follicular quantification. Apoptotic ovarian cells of the ovary were assessed using TUNEL technique, while Caspase-3 and Cytochrome C (Cyt C) expressions of the ovary were examined through immunohistochemistry (IHC). Western blotting analysis was used to quantify levels of Bax, Bcl-2, Caspase-3, Cyt C, mTOR, P-mTOR, AKT, P-AKT, P-PI3K and PI3K proteins, while mRNA levels of Bax, Bcl-2, PI3K, AKT and mTOR were measured in ovarian tissue using RT-PCR.Results: The findings revealed that DKP was able to improve levels of serum hormones and promote the recovery of the estrous cycle. DKP augmented the total amount of primordial follicles while reducing the number of follicles that were atretic follicles. The apoptosis index of growing follicles and Bax, Cyt C and Caspase-3 expressions decreased, while the Bcl-2: Bax ratio increased. DKP suppressed levels of phosphorylation and the mRNA expressions of mTOR, AKT and PI3K.Conclusions: It was demonstrated that DKP was able to increase ovarian reserves through inhibition of the PI3K/AKT/mTOR signaling pathway, which lead to the suppression of primordial follicle activity and a reduction in levels of apoptosis of early growing follicles. This highlights its potentially beneficial role for the treatment of DOR.