Effects of Zigui yichong formula on premature ovarian insufficiency in mice through glycolytic metabolic pathway

ZHANG Xinmiao; LIU Xueping; XI Hongyan; TANG Siling; LI Rongxia; WU Zhongyu; DUAN Yancang

doi:10.6039/j.issn.1001-0408.2024.20.03

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Effects of Zigui yichong formula on premature ovarian insufficiency in mice through glycolytic metabolic pathway

更新时间：2024-10-25

- Effects of Zigui yichong formula on premature ovarian insufficiency in mice through glycolytic metabolic pathway
- ZHONGGUO YAOFANG Vol. 35, Issue 20, Pages: 2460-2465(2024)
- 作者机构：
  
  1.河北中医药大学研究生学院，石家庄　050091
  2.河北省中西医结合生殖疾病协同创新中心，石家庄　050091
  3.河北中西医结合肝肾病证研究重点实验室，石家庄　050091
- 作者简介：
- 基金信息：
- DOI：10.6039/j.issn.1001-0408.2024.20.03
  CLC： R965
- Published：30 October 2024，
  
  Received：15 April 2024，
  
  Revised：23 September 2024，
- 稿件说明：
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张新苗,刘雪平,郗红燕等.资癸益冲方通过糖酵解代谢途径对小鼠早发性卵巢功能不全的影响 ^Δ[J].中国药房,2024,35(20):2460-2465.

ZHANG Xinmiao,LIU Xueping,XI Hongyan,et al.Effects of Zigui yichong formula on premature ovarian insufficiency in mice through glycolytic metabolic pathway[J].ZHONGGUO YAOFANG,2024,35(20):2460-2465.
张新苗,刘雪平,郗红燕等.资癸益冲方通过糖酵解代谢途径对小鼠早发性卵巢功能不全的影响 ^Δ[J].中国药房,2024,35(20):2460-2465. DOI： 10.6039/j.issn.1001-0408.2024.20.03.

ZHANG Xinmiao,LIU Xueping,XI Hongyan,et al.Effects of Zigui yichong formula on premature ovarian insufficiency in mice through glycolytic metabolic pathway[J].ZHONGGUO YAOFANG,2024,35(20):2460-2465. DOI： 10.6039/j.issn.1001-0408.2024.20.03.

摘要

目的

研究资癸益冲方调控糖酵解代谢途径对小鼠早发性卵巢功能不全（POI）的影响。

方法

将80只SPF级C57BL/6N雌性小鼠分为正常组、模型组、资癸益冲方组（14.175 g/kg）、资癸益冲方+2-脱氧-D-葡萄糖（2-DG）组（资癸益冲方14.175 g/kg+糖酵解抑制剂2-DG 100 mg/kg），每组20只。除正常组外，其余各组小鼠腹腔注射环磷酰胺建立POI模型，造模成功后，各组小鼠给予相应药物。通过苏木精-伊红染色观察小鼠卵巢组织病理形态学变化及各级卵泡数量；采用放射免疫法检测小鼠血清中雌二醇（E

）、血清抗米勒管激素（AMH）、卵泡刺激素（FSH）水平；TUNEL法检测小鼠卵巢颗粒细胞凋亡情况；比色法检测小鼠卵巢组织中己糖激酶（HK）、丙酮酸激酶（PK）、乳酸脱氢酶（LDH）活性；Western blot法、实时荧光定量聚合酶链式反应检测小鼠卵巢组织中B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、胱天蛋白酶-3（caspase-3）、HK2、M2型PK（PKM2）、乳酸脱氢酶A（LDHA）蛋白及mRNA的表达。

结果

与模型组比较，资癸益冲方组小鼠卵巢组织中原始卵泡、生长卵泡、窦卵泡数和卵巢颗粒细胞层数均显著增加（

＜0.05），卵巢颗粒细胞排列整齐，闭锁卵泡数量、卵巢颗粒细胞凋亡数量均显著减少（

＜0.05）；血清中E

、AMH水平，卵巢组织中HK、PK、LDH活性，Bcl-2、HK2、PKM2、LDHA蛋白及mRNA表达量均显著升高（

＜0.05）；血清中FSH水平，卵巢组织中Bax、caspase-3蛋白及mRNA表达量和Bax/Bcl-2比值均显著降低（

＜0.05）。2-DG可逆转资癸益冲方对POI模型小鼠上述指标的改善作用。

结论

资癸益冲方可通过促进糖酵解来减少POI模型小鼠卵巢颗粒细胞凋亡和卵泡闭锁，改善卵巢储备功能。

Abstract

OBJECTIVE

To study the effects of Zigui yichong formula on premature ovarian insufficiency （POI） in mice through glycolysis metabolic pathway.

METHODS

Eighty SPF C57BL/6N female mice were divided into normal group， model group， Zigui yichong formula group （14.175 g/kg）， Zigui yichong formula+2-deoxy-D-arabino-hexose （2-DG） group （Zigui yichong formula 14.175 g/kg + glycolysis inhibitor 2-DG 100 mg/kg）， w

ith 20 mice in each group. Except for the normal group， POI model mice were induced by intraperitoneal administration of cyclophosphamide in the other groups. After the model was successfully established， each group was given corresponding drugs. HE staining was employed to observe the pathomorphological changes in ovarian tissue and to count follicles at all developmental stages； radioimmunoassay was conducted to measure the serum levels of estradiol （E

）， anti-Müllerian hormone （AMH）， and follicle-stimulating hormone （FSH）； TUNEL assay was employed to detect apoptosis in ovarian granulosa cells of mice； the activities of hexokinase （HK）， pyruvate kinase （PK） and lactate dehydrogenase （LDH） were detected by colorimetry； Western blot and real-time fluorescence quantitative PCR were employed to analyze the protein and mRNA expressions of B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， caspase-3， HK2， pyruvate kinase M2 （PKM2）， and lactate dehydrogenase A （LDHA）.

RESULTS

Compared with model group， the number of primordial follicles， growing follicles， antral follicles and granulosa cells were increased significantly （

＜0.05）， and granulosa cells arranged neatly， but the number of atretic follicles and granulosa cells apoptosis were decreased significantly in Zigui yichong formula group （

＜0.05）； the serum levels of E

and AMH， the activities of HK， PK and LDH， protein and mRNA expressions of Bcl-2， HK2， PKM2 and LDHA were increased significantly （

＜0.05）； the serum levels of FSH， the protein and mRNA expressions of Bax and caspase-3， Bax/Bcl-2 ratio were decreased significantly （

＜0.05）. 2-DG could reverse the improvement effects of Zigui yichong formula on the above indexes of POI model mice.

CONCLUSIONS

Zigui yichong formula may inhibit the apoptosis of ovarian granulosa cells， reduce follicle atresia and improve ovarian reserve function by promoting glycolysis levels in POI model mice.

关键词

早发性卵巢功能不全资癸益冲方糖酵解卵巢颗粒细胞细胞凋亡

Keywords

Zigui yichong formulaglycolysisovarian granulosa cellscell apoptosis

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