Study on acupuncture combined with umbilical cord mesenchymal stem cells to improve ovarian reserve function in rats with premature ovarian failure

LIU Wenzhao; CHEN Dongmei; CHEN Miao; HU Na; XING Shasha; LUO Jiaqi; TIAN Ruiying; WANG Jing; YANG Li; HE Rui; YANG Hua; MA Huiming

doi:10.3969/j.issn.1006-2157.2023.03.022

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Study on acupuncture combined with umbilical cord mesenchymal stem cells to improve ovarian reserve function in rats with premature ovarian failure

Acupuncture Moxibustion and Tuina | 更新时间：2023-04-07

- Study on acupuncture combined with umbilical cord mesenchymal stem cells to improve ovarian reserve function in rats with premature ovarian failure
- Journal of Beijing University of Traditional Chinese Medicine Vol. 46, Issue 3, Pages: 435-444(2023)
- 作者机构：
  
  1.宁夏医科大学生育力保持教育部重点实验室　银川　750004
  2.宁夏医科大学总医院宁夏干细胞与再生医学重点实验室
  3.宁夏医科大学基础医学院
  4.宁夏医科大学实验动物中心
  5.宁夏医科大学中医学院
- 作者简介：
  
  A/Prof. MA Huiming, Ph. D. , Master’s Supervisor. Ningxia Medical University, No. 1160, Shengli Street, Xingqing District, Yinchuan 750004. E-mail: mhm289@nxmu.edu.cn
- 基金信息：
  
  National Natural Science Foundation of China(81901439);Ningxia Key R&D Program(2020BEG3042);Ningxia Science and Technology Leading Talent Project(2020GKLRLX11)
- DOI：10.3969/j.issn.1006-2157.2023.03.022
  CLC： R245.9
- Received：01 September 2022，
  
  Published Online：05 January 2023，
  
  Published：30 March 2023
- 稿件说明：
移动端阅览
LIU Wenzhao, CHEN Dongmei, CHEN Miao, et al. Study on acupuncture combined with umbilical cord mesenchymal stem cells to improve ovarian reserve function in rats with premature ovarian failure[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(3): 435-444.
DOI：

LIU Wenzhao, CHEN Dongmei, CHEN Miao, et al. Study on acupuncture combined with umbilical cord mesenchymal stem cells to improve ovarian reserve function in rats with premature ovarian failure[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(3): 435-444. DOI： 10.3969/j.issn.1006-2157.2023.03.022.

摘要

目的

观察针刺联合脐带间充质干细胞对卵巢早衰大鼠卵巢储备功能的影响。

方法

70只雌性SD大鼠随意分为造模组和正常组。造模组大鼠给予去氧乙烯基环己烯(80 mg/kg)腹腔注射，制备卵巢早衰大鼠模型。造模成功后，再将其随意分为模型组、针刺组、干细胞组、联合治疗组，每组12只，剩余12只正常大鼠为空白组。针刺组针刺“关元”“气海”“足三里”；干细胞组在干预的第1、8天，经大鼠尾静脉注入人类脐带间充质干细胞液0.5 mL(细胞数为2.5×10

个)；联合治疗组进行针刺和尾静脉注入人类脐带间充质干细胞液。干预后，检测大鼠卵巢湿重和卵巢指数；酶联免疫吸附测定检测血清睾酮(T)、雌二醇(E

)、促卵泡激素(FSH)、黄体生成素(LH)和抗米勒管激素(AMH)水平及抗氧化指标丙二醛(MDA)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)水平；HE染色检测各组大鼠卵巢形态结构变化；免疫组织化学法及蛋白质印迹法检测卵巢组织中蛋白激酶B(Akt)、磷酸化蛋白激酶B(p-Akt)、B淋巴细胞瘤－2(Bcl-2)、Bcl-2相关X蛋白(Bax)的蛋白表达水平。

结果

与空白组比较，模型组大鼠卵巢湿重、卵巢指数降低(

0.01)；血清LH、T、FSH、MDA水平上升(

0.05，

0.01)，E

、AMH、GSH、SOD水平下降(

0.05，

0.01)；HE染色显示卵巢内成熟卵泡减少，闭锁卵泡增多；卵巢组织中Akt、p-Akt、Bcl-2相对表达量下降(

0.05，

0.01)，Bax相对表达量上升(

0.01)。与模型组比较，针刺组、干细胞组、联合治疗组大鼠卵巢湿重、卵巢指数上升(

0.05，

0.01)；血清中的T、FSH水平下降(

0.05，

0.01)；血清中E

、AMH、GSH、SOD水平上升(

0.05，

0.01)；HE染色显示卵巢异常情况减少；卵巢组织中p-Akt、Bcl-2阳性表达与蛋白相对表达量上升(

0.05，

0.01)，Bax阳性表达与蛋白相对表达量下降(

0.05，

0.01)。与针刺组和干细胞组比较，联合治疗组卵巢湿重上升(

0.05)，血清T、FSH水平下降(

0.05，

0.01)，AMH水平上升(

0.05，

0.01)，Akt阳性表达上升(

0.05)。与针刺组比较，联合治疗组SOD、GSH水平上升(

0.01)，Bax蛋白相对表达量下降(

0.01)。与干细胞组比较，联合治疗组血清MDA水平下降(

0.05)。

结论

针刺、干细胞和联合治疗对卵巢早衰大鼠均能起到积极治疗作用，均能改善卵巢早衰大鼠卵巢储备功能，促进卵巢颗粒细胞增殖，抑制卵泡凋亡；且联合治疗效果优于单纯针刺和干细胞治疗。

Abstract

Objective

We aimed to observe the effect of acupuncture combined with umbilical cord mesenchymal stem cells on ovarian reserve function in rats with premature ovarian failure.

Methods

Seventy female Sprague-Dawley rats were divided into the modeling group and the normal group. Modeling group rats were injected intraperitoneally with vinyl cyclohexene (80 mg/kg) to establish a rat model of premature ovarian failure. After the model was successfully established

the rats were divided into the model group

the acupuncture group

the stem cell group

and the combined treatment group (12 rats per group). The remaining 12 normal rats served as the blank group. The acupuncture group was treated with acupuncture at RN4

RN6 and ST36. In the stem cell group

0.5 mL human umbilical cord mesenchymal stem cell fluid was injected into the tail vein of rats on the first and eighth days of intervention (2.5×10

cells). Rats in the combined treatment group received acupuncture and tail vein injection of human umbilical cord mesenchymal stem cell fluid. After the intervention

wet ovarian weight and the ovarian index were determined. The contents of serum testosterone (T)

estradiol (E

)

follicle stimulating hormone (FSH)

luteinizing hormone (LH)

anti-Mullerian hormone (AMH) and the contents of antioxidant indices malondialdehyde (MDA)

glutathione (GSH)

superoxide dismutase (SOD) were detected by ELISA. HE staining was used to detect the morphological changes of ovaries in each group. The relative expression levels of Akt

p-Akt

Bcl-2 and Bax in ovaries were detected by immunohistochemistry and Western blotting.

Results

Compared with the blank group

the wet ovarian weight and ovarian index of the model group were decreased significantly (

0.01). The serum levels of LH

FSH

and MDA were increased (

0.05

0.01)

while the levels of E

AMH

GSH and SOD were decreased (

0.05

0.01). HE staining showed that the number of mature follicles in the ovary was decreased and the number of atretic follicles was increased. The relative expression of Akt

p-Akt and Bcl-2 in ovarian tissue was decreased (

0.05

0.01)

and the relative expression of Bax was increased (

0.01). Compared with the model group

the wet ovarian weight and ovarian index of rats in the acupuncture group

stem cell group and combined treatment group were increased (

0.05

0.01). The serum levels of T and FSH were decreased (

0.05

0.01). The serum levels of E

AMH

GSH and SOD were increased (

0.05

0.01). HE staining showed a decrease in ovarian abnormalities. In ovarian tissue

the positive expression of p-Akt and Bcl-2 and the relative expression of these proteins were increased (

0.05

0.01)

while the positive expression of Bax and the relative expression of this protein were decreased (

0.05

0.01). Compared with the acupuncture group and the stem cell group

the wet weight of ovaries in the combined treatment group was increased significantly (

0.05)

the serum levels of T and FSH were decreased significantly (

0.05

0.01)

the level of AMH was increased significantly (

0.05

0.01)

and the positive expression of Akt was increased significantly (

0.05). Compared with the acupuncture group

the levels of SOD and GSH in the combined treatment group were increased significantly (

0.01)

and the relative expression of Bax was decreased significantly (

0.01). Compared with the stem cell group

the serum MDA level in the combined treatment group was decreased significantly (

0.05).

Conclusion

Acupuncture

stem cell treatment

and combined treatment can play an active role in the treatment of premature ovarian failure rats

improve the ovarian reserve function of premature ovarian failure rats

promote the proliferation of ovarian granulosa cells

and inhibit apoptosis in follicles. The effect of combined treatment is better than that of acupuncture or stem cell therapy only.

关键词

Keywords

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