Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response

HAO Jianheng; WANG Haijun; CAO Yuxia; JI Laixi

doi:10.13703/j.0255-2930.20230610-k0001

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Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response

Study on Mechanism | 更新时间：2024-07-11

- Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response
- Chinese Acupuncture & Moxibustion Vol. 44, Issue 7, Pages: 821-830(2024)
- 作者机构：
  
  1.成都中医药大学针灸推拿学院，四川成都　610075
  2.山西中医药大学第二临床学院，晋中　030619
- 作者简介：
- 基金信息：
- DOI：10.13703/j.0255-2930.20230610-k0001
  CLC：
- Received：10 June 2023，
  
  Online First：26 April 2024，
  
  Published：12 July 2024
- 稿件说明：
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HAO Jianheng, WANG Haijun, CAO Yuxia, et al. Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response[J]. Chinese Acupuncture & Moxibustion, 2024, 44(7): 821-830.
DOI：

HAO Jianheng, WANG Haijun, CAO Yuxia, et al. Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response[J]. Chinese Acupuncture & Moxibustion, 2024, 44(7): 821-830. DOI： 10.13703/j.0255-2930.20230610-k0001.

摘要

目的：

基于磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）/叉头蛋白O3a（FOXO3a）通路观察“秩边透水道”针刺对卵巢低反应（POR）小鼠卵巢功能的保护作用，探析针刺抑制POR卵巢颗粒细胞凋亡的可能机制。

方法：

将动情周期规律的45只小鼠随机分成空白组、模型组和针刺组，每组15只。模型组和针刺组小鼠予雷公藤多苷混悬液（50 mg·kg

-1

·d

-1

）灌胃2周制备POR模型。造模成功后针刺组小鼠予“秩边透水道”针刺干预连续2周，每日1次，每次20 min。干预结束次日开始促排卵，各组促排卵后完成取材。采用阴道脱落细胞涂片观察小鼠动情周期变化，记录小鼠取卵数、卵巢湿重、末次体质量与卵巢指数；ELISA法检测小鼠血清抗缪勒管激素（AMH）、卵泡刺激素（FSH）、雌二醇（E2）、黄体生成素（LH）含量；HE染色法观察小鼠卵巢组织形态；TUNEL染色法观察小鼠卵巢颗粒细胞凋亡情况；实时荧光定量PCR法检测小鼠卵巢组织PI3K、AKT、FOXO3a mRNA表达；Western blot法检测卵巢组织B淋巴细胞瘤-2（Bcl-2）关联X的蛋白（BAX），半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、磷酸化磷脂酰肌醇3-激酶（p-PI3K）和磷酸化蛋白激酶B（p-AKT）蛋白表达。

结果：

与空白组比较，模型组小鼠动情周期紊乱率升高（

0.01）；与模型组比较，针刺组小鼠动情周期紊乱率降低（

0.01）。与空白组比较，模型组小鼠取卵数、卵巢湿重、卵巢指数和末次体质量降低（

0.01）；与模型组比较，针刺组小鼠取卵数、卵巢指数和卵巢湿重升高（

0.01，

0.05），末次体质量比较差异无统计学意义（

0.05）。与空白组比较，模型组小鼠血清FSH、LH含量升高（

0.01），血清AMH、E2含量降低（

0.01）；与模型组比较，针刺组小鼠血清FSH、LH含量降低（

0.01，

0.05），血清AMH、E2含量升高（

0.01，

0.05）。与空白组比较，模型组小鼠卵巢组织各级正常发育卵泡数量减少且形态较差，各级闭锁卵泡增多；与模型组比较，针刺组小鼠卵泡数量、形态和颗粒细

胞结构有不同程度改善，各级闭锁卵泡减少。与空白组比较，模型组小鼠卵巢颗粒细胞凋亡率升高（

0.01）；与模型组比较，针刺组小鼠卵巢颗粒细胞凋亡率降低（

0.01）。与空白组比较，模型组小鼠卵巢组织FOXO3a mRNA及Caspase-3、BAX蛋白表达升高（

0.01），卵巢组织PI3K、AKT mRNA及p-PI3K、p-AKT和p-FOXO3a蛋白表达降低（

0.01）；与模型组比较，针刺组小鼠卵巢组织FOXO3a mRNA及Caspase-3、BAX蛋白表达降低（

0.05，

0.01），卵巢组织PI3K、AKT mRNA及p-PI3K、p-AKT、p-FOXO3a蛋白表达升高（

0.01，

0.05）。

结论：

“秩边透水道”针刺可以抑制POR小鼠卵巢颗粒细胞凋亡，改善卵巢功能，其机制可能与调节PI3K/AKT/FOXO3a通路中的关键因子有关。

Abstract

Objective

To observe the protective effect of acupuncture at "Zhibian" (BL 54) through "Shuidao (ST 28)" based on the PI3K/AKT/FOXO3a pathway in mice with poor ovarian response (POR)

and to explore the possible mechanism of acupuncture in inhibiting ovarian granulosa cells apoptosis in POR.

Methods

A total of 45 mice with regular estrous cycles were randomly divided into a blank group

a model group and an acupuncture group

with 15 mice in each group. Mice in the model group and the acupuncture group were given triptolide suspension (50 mg·kg

-1

·d

-1

) by gavage for 2 weeks to establish POR model. After successful modeling

mice in the acupuncture group were given acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) for 2 weeks

once a day

20 min each time. Ovulation induction was started the day after the intervention ended

and samples were taken from each group after ovulation induction. Vaginal smears were used to observe changes in the estrous cycle of mice. The number of oocytes retrieved

ovarian wet weight

final body weight

and ovarian index were measured. The levels of anti-Mullerian hormone (AMH)

follicle-stimulating hormone (FSH)

estradiol (E2)

and luteinizing hormone (LH) in serum were detected by ELISA. The morphology of ovarian tissue was observed by HE staining. The

apoptosis of ovarian granulosa cells was detected by TUNEL staining. The mRNA expression of PI3K

AKT

and FOXO3a in ovarian tissue was detected by real-time fluorescence quantitative PCR. The protein expression of Bcl-2 associated X protein (BAX)

caspase-3

phosphorylated phosphatidylinositol 3-kinase (p-PI3K)

and phosphorylated protein kinase B (p-AKT) in ovarian tissue was detected by Western blot.

Results

Compared with the blank group

the rate of estrous cycle disorder in the model group was increased (

0.01); compared with the model group

the rate of estrous cycle disorder in the acupuncture group was decreased (

0.01). Compared with the blank group

the number of oocytes retrieved

ovarian wet weight

ovarian index

and final body weight in the model group were decreased (

0.01); compared with the model group

the number of oocytes retrieved

ovarian index

and ovarian wet weight were increased (

0.01

0.05)

and there was no significant difference in final body weight (

0.05) in the acupuncture group. Compared with the blank group

the serum levels of FSH and LH were increased (

0.01)

and the serum levels of AMH and E2 were decreased (

0.01) in the model group; compared with the model group

the serum levels of FSH and LH were decreased (

0.01

0.05)

and the serum levels of AMH and E2 were increased (

0.01

0.05) in the acupuncture group. Compared with the blank group

the number of normal developing follicles in ovarian tissue in the model group was decreased and the morphology was poor

while the number of atretic follicles increased; compared with the model group

the number

morphology

and granulosa cell structure of follicles in the acupuncture group improved to varying degrees

and the number of atretic follicles

decreased. Compared with the blank group

the apoptosis rate of ovarian granulosa cells in the model group was increased (

0.01); compared with the model group

the apoptosis rate of ovarian granulosa cells in the acupuncture group was decreased (

0.01). Compared with the blank group

the FOXO3a mRNA expression and caspase-3 and BAX protein expression in ovarian tissue in the model group were increased (

0.01)

and the mRNA expression of PI3K and AKT and the protein expression of p-PI3K

p-AKT

and p-FOXO3a in ovarian tissue were decreased (

0.01); compared with the model group

the mRNA expression of FOXO3a and protein expression of caspase-3 and BAX in ovarian tissue in the acupuncture group were decreased (

0.05

0.01)

and the mRNA expression of PI3K and AKT and the protein expression of p-PI3K

p-AKT

and p-FOXO3a in ovarian tissue were increased (

0.01

0.05).

Conclusion

Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could inhibit ovarian cell apoptosis

and improve ovarian function in POR mice

and its mechanism may be related to the regulation of key factors in the PI3K/AKT/FOXO3a pathway.

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