Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway

TANG Li; MU Zhenni; SHEN Sinan; LEI Lei

doi:10.13422/j.cnki.syfjx.20222444

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Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway

更新时间：2023-01-17

- Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 4, Pages: 52-59(2023)
- 作者机构：
  
  湖南中医药大学中西医结合学院，长沙 410208
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20222444
  CLC： R2-0;R33;R289;R274.1
- Published：20 February 2023，
  
  Published Online：14 December 2022，
  
  Received：02 September 2022，
- 稿件说明：
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唐丽,牟珍妮,申思楠等.基于PI3K/Akt/mTOR通路探讨助卵汤对大鼠早发性卵巢功能不全的保护作用机制[J].中国实验方剂学杂志,2023,29(04):52-59.

TANG Li,MU Zhenni,SHEN Sinan,et al.Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(04):52-59.
唐丽,牟珍妮,申思楠等.基于PI3K/Akt/mTOR通路探讨助卵汤对大鼠早发性卵巢功能不全的保护作用机制[J].中国实验方剂学杂志,2023,29(04):52-59. DOI： 10.13422/j.cnki.syfjx.20222444.

TANG Li,MU Zhenni,SHEN Sinan,et al.Protective Mechanism of Zhuluan Decoction in Rats with Premature Ovarian Insufficiency Based on PI3K/Akt/mTOR Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(04):52-59. DOI： 10.13422/j.cnki.syfjx.20222444.

摘要

目的

观察助卵汤对环磷酰胺诱导的早发性卵巢功能不全模型大鼠卵巢储备功能的影响，并基于磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路探讨助卵汤对大鼠早发性卵巢功能不全的保护机制。

方法

60只雌性SD大鼠随机分为正常组10只，模型组50只，模型组采用环磷酰胺（第1天50 mg·kg

-1

负荷剂量+第2~15天8 mg·kg

-1

小剂量维持）腹腔注射进行造模，造模成功后随机分为模型组、阳性药物补佳乐组（0.1 mg·kg

-1

·d

-1

）、助卵汤低、中、高剂量组（14、28、56 g·kg

-1

·d

-1

），每组10只，模型组与正常组予以等体积蒸馏水灌胃，每日1次，连续给药21 d。检测各组大鼠动情周期、体质量，计算卵巢脏器指数与子宫脏器指数，苏木素-伊红（HE）染色检测卵巢组织病理情况及卵巢切面各级卵泡计数，酶联免疫吸附测定法（ELISA）检测大鼠血清抗苗勒激素（AMH）、促卵泡激素（FSH）、黄体生成素（LH）、抑制素-B（INH-B）激素含量，蛋白免疫印迹法（Western blot）检测大鼠卵巢组织PI3K、Akt、mTOR蛋白表达，免疫组化检测大鼠卵巢组织微管相关蛋白1轻链3B（LC3B）蛋白的表达。

结果

与正常组比较，模型组大鼠动情周期紊乱，体质量及卵巢脏器指数与子宫脏器指数减少，原始卵泡数量减少，次级卵泡和闭锁卵泡数量增多，FSH升高（

0.01），LH升高（

0.05），AMH水平下降（

0.05），卵巢组织PI3K、Akt、mTOR蛋白表达量均降低（

0.01），LC3B蛋白表达量显著增加（

0.01）；与模型组比较，补佳乐和助卵汤各剂量组上述指标均改善，AMH含量升高（

0.05），FSH降低（

0.05），LC3B蛋白表达量显著降低（

0.01），PI3K、Akt、mTOR蛋白表达量均有升高趋势，且补佳乐组和助卵汤低、中剂量组升高差异具有统计学意义（

0.05）。

结论

助卵汤可能通过激活PI3K/Akt/mTOR通路抑制卵巢颗粒细胞过度自噬的发生，从而逆转造模对大鼠卵巢储备功能的影响。

Abstract

Objective

To observe the effect of Zhuluan decoction on the ovarian reserve function of rats with cyclophosphamide-induced premature ovarian insufficiency， and explore the protective mechanism of Zhuluan decoction in the rat model of premature ovarian insufficiency based on the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway.

Method

Sixty female SD rats were randomly divided into normal group （

=10） and model group （

=50）. The model group was given intraperitoneal injection of cyclophosphamide （50 mg·kg

-1

loading dose on the 1

day+8 mg·kg

-1

low-dose maintenance on the 2

–15

days）. After successfully modeling， the rats were randomly divided into model group， positive drug （progynova） group （0.1 mg·kg

-1

·d

-1

）， and low-， medium-， and high-dose Zhuluan decoction groups （14， 28， 56 g·kg

-1

·d

-1

）， with 10 rats in each group. The model group and the normal group were given equal volume of distilled water by gavage， once a day， continuous administration for 21 d. The estrous cycle and body weight of rats in each group were detected， and the ovarian organ index and uterine organ index were calculated. The ovarian tissue pathology and ovarian follicle counts at all levels were determined by hematoxylin-eosin （HE） staining. The content of the serum antimullerian hormone （AMH）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and inhibin-B （INH-B） of rats was determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of PI3K， Akt， mTOR in the rat ovarian tissue were determined by Western blot. The microtubule-associated protein 1 light chain 3B （LC3B） protein expression in the rat ovarian tissue was determined by immunohistochemistry.

Result

As compared with the blank group， the estrous cycle of rats in the model group was disordered， the body weight， ovarian organ index， and uterine organ index decreased， the number of primordial follicles decreased， and the number of secondary follicles and atretic follicles increased. In the model group， FSH increased （

0.01）， LH increased （

0.05）， AMH level decreased （

0.05）， the protein expression levels of PI3K， Akt， and mTOR in the ovarian tissue decreased （

0.01）， and the protein expression level of LC3B increased significantly （

0.01）. As compared with the model group， the above indexes were improved in the progynova group and different doses of Zhuluan decoction groups， the content of AMH increased （

0.05）， and FSH decreased （

0.05）. In the progynova group and different doses of Zhuluan decoction groups， the protein expression level of LC3B decreased obviously （

0.01）， and the protein expression levels of PI3K， Akt， and mTOR all showed an increasing trend. Moreover， there was a statistically significant difference in the progynova group and low- and medium-dose Zhuluan decoction groups （

0.05）.

Conclusion

Zhuluan decoction may inhibit the occurrence of excessive autophagy in ovarian granulosa cells by activating the PI3K/Akt/mTOR pathway， thereby reversing the effect of modeling on ovarian reserve in rats.

关键词

早发性卵巢功能不全（POI）助卵汤冰山论磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路自噬

Keywords

premature ovarian insufficiencyZhuluan decoctionBingshan theoryphosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathwayautophagy

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