Bushen Huoxue Prescription Replenishes Diminished Ovarian Reserve in Rats Through PI3K/Akt/mTOR Signaling Pathway

Jing-ling ZHAO; Li XIE; Xiao-cui JIANG; Min XIAO; Cheng-yin LI; Si WU

doi:10.13422/j.cnki.syfjx.20211409

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Bushen Huoxue Prescription Replenishes Diminished Ovarian Reserve in Rats Through PI3K/Akt/mTOR Signaling Pathway

Pharmacology | 更新时间：2021-07-15

- Bushen Huoxue Prescription Replenishes Diminished Ovarian Reserve in Rats Through PI3K/Akt/mTOR Signaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 15, Pages: 29-35(2021)
- 作者机构：
  
  1.湖北中医药大学，武汉 430065
  2.湖北省中医院，武汉 430061
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20211409
  CLC： R2-0;R22;R285.5;R289;R33
- Received：10 April 2021，
  
  Published Online：08 June 2021，
  
  Published：05 August 2021
- 稿件说明：
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赵井苓,谢莉,江晓翠等.补肾活血方通过调节PI3K/Akt/mTOR信号通路改善DOR大鼠卵巢储备功能[J].中国实验方剂学杂志,2021,27(15):29-35.

ZHAO Jing-ling,XIE Li,JIANG Xiao-cui,et al.Bushen Huoxue Prescription Replenishes Diminished Ovarian Reserve in Rats Through PI3K/Akt/mTOR Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(15):29-35.
赵井苓,谢莉,江晓翠等.补肾活血方通过调节PI3K/Akt/mTOR信号通路改善DOR大鼠卵巢储备功能[J].中国实验方剂学杂志,2021,27(15):29-35. DOI： 10.13422/j.cnki.syfjx.20211409.

ZHAO Jing-ling,XIE Li,JIANG Xiao-cui,et al.Bushen Huoxue Prescription Replenishes Diminished Ovarian Reserve in Rats Through PI3K/Akt/mTOR Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(15):29-35. DOI： 10.13422/j.cnki.syfjx.20211409.

摘要

目的

观察补肾活血方调节磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/雷帕霉素靶蛋白（mTOR）通路相关因子改善卵巢储备功能下降（DOR）大鼠卵巢储备功能的作用机制。

方法

运用Ataya法腹腔注射环磷酰胺复制60只DOR模型大鼠，将其随机分为模型组、戊酸雌二醇组（0.000 9 g·kg

-1

），补肾活血方高、中、低剂量组（33，16.5，8.25 g·kg

-1

），同时另设正常组，每组12只；各组相应药物治疗，正常组和模型组给予等体积生理盐水，连续灌胃14 d，每天1次。治疗结束后，苏木素-伊红（HE）染色卵巢组织，光镜观察初级卵泡数、成熟卵泡数、总卵泡数的变化；免疫组化检测卵巢组织中血管内皮生长因子（VEGF）的表达，蛋白免疫印迹法（Western blot）检测卵巢组织中PI3K，Akt，mTOR，半胱氨酸天冬氨酸蛋白水解酶-3（Caspase-3）蛋白表达；实时荧光定量聚合酶链式反应（Real-time PCR）法检测卵巢组织中PI3K，Akt，mTOR mRNA表达。

结果

与正常组比较，模型组卵巢组织中成熟卵泡数量、总卵泡数量明显减少（

0.05，

0.01），卵巢组织中VEGF，Caspase-3表达升高（

0.05），PI3K，Akt，mTOR蛋白及mRNA表达水平降低（

0.05，

0.01）；与模型组比较，补肾活血方中、高剂量组成熟卵泡数量、总卵泡数量均有不同程度上升（

0.05，

0.01），VEGF补肾活血方中剂量组升高最为明显（

0.05），补肾活血方低、中、高剂量组及戊酸雌二醇组Caspase-3下降，补肾活血方中、高剂量组PI3K，Akt，mTOR蛋白及mRNA表达水平均升高（

0.05，

0.01），且补肾活血方中剂量组更加接近于正常组。

结论

补肾活血方可以有效地改善DOR大鼠卵巢储备功能，促进卵泡数量增加。作用机制可能与补肾活血方通过增加卵巢组织中VEGF的表达有关，进而激活PI3K/Akt/mTOR信号通路，从而调节Caspase-3的含量，最后促进卵泡数量的增加。

Abstract

Objective

To explore the mechanism of Bushen Huoxue prescription in regulating the related factors in phosphatidylinositol 3-kinase （PI3K）/ protein kinase B （Akt）/ mammalian target of rapamycin （mTOR） pathway and improving ovarian reserve function of rats with diminished ovarian reserve （DOR）.

Method

Sixty DOR model rats were duplicated by Ataya method （intraperitoneal injection of cyclophosphamide） and then randomized into the model group， estradiol valerate （0.000 9 g·kg

-1

） group， and high- （33 g·kg

-1

）， middle- （16.5 g·kg

-1

） and low-dose （8.25 g·kg

-1

） Bushen Huoxue prescription groups， with 12 rats in each group. Another 12 healthy rats were classified into the blank control group. The rats in each group were given the corresponding drugs by gavage， while those in the blank control group and model group received the same volume of normal saline， once per day， for 14 successive days. After treatment， the ovarian tissue was stained with hematoxylin-eosin （HE） for observing the changes in quantities of primary follicles， mature follicles， and total follicles under a light microscope， followed by the detection of vascular endothelial growth factor （VEGF） expression in the ovarian tissue by immunohistochemistry. The protein expression levels of PI3K， Akt， mTOR， and cysteine-dependent aspartate-directed protease-3 （Caspase-3） in the ovarian tissue were assayed by Western blot， whereas the mRNA expression levels of PI3K， Akt， and mTOR were measured by Real-time polymerase chain reaction （Real-time PCR）.

Result

As revealed by comparison with the blank control group， the quantities of mature follicles and total follicles in the ovarian tissue of model group were significantly reduced （

0.05，

0.01）. The protein expression levels of VEGF and Caspase-3 in the ovarian tissue were increased （

0.05）， while the protein and mRNA expression levels of PI3K， Akt， and mTOR were decreased （

0.05，

0.01）. Compared with the model group， Bushen Huoxue prescription at the high and middle doses elevated the quantities of mature follicles and total follicles to varying degrees （

0.05，

0.01）， and VEGF increased most significantly in the middle-dose Bushen Huoxue prescription group （

0.05）. Caspase-3 in the low-， middle-， and high-dose Bushen Huoxue prescription groups and the western medicine group declined. The protein and mRNA expression levels of PI3K， Akt， and mTOR were up-regulated in the middle- and high-dose Bushen Huoxue prescription groups （

0.05，

0.01）， and the levels in the middle-dose Bushen Huoxue prescription group were closer to those in the blank control group.

Conclusion

Bushen Huoxue prescription effectively improves the ovarian reserve function of rats with DOR and increases the number of follicles possibly by up-regulating VEGF expression in the ovarian tissue， activating the PI3K/Akt/mTOR signaling pathway， and regulating the content of Caspase-3.

关键词

Keywords

references

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