Effects of <italic style="font-style: italic">Bushen Tiaojing Formula</italic> （补肾调经方） on Blastocyst Cell Apoptosis and Endoplasmic Reticulum Stress in Repeated Controlled Ovarian Stimulation Model Mice

XIONG Wenyan; SHI Beibei; SHEN Lulu; DU Huilan

doi:10.13288/j.11-2166/r.2025.15.012

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Effects of Bushen Tiaojing Formula （补肾调经方） on Blastocyst Cell Apoptosis and Endoplasmic Reticulum Stress in Repeated Controlled Ovarian Stimulation Model Mice

更新时间：2025-07-31

- Effects of Bushen Tiaojing Formula （补肾调经方） on Blastocyst Cell Apoptosis and Endoplasmic Reticulum Stress in Repeated Controlled Ovarian Stimulation Model Mice
- Journal of Traditional Chinese Medicine Vol. 66, Issue 15, Pages: 1579-1587(2025)
- 作者机构：
  
  1.河北中医药大学中西医结合学院，河北省石家庄市桥西区新石南路326号，050200
  2.河北省中西医结合生殖疾病协同创新中心
- 作者简介：
- 基金信息：
- DOI：10.13288/j.11-2166/r.2025.15.012
  CLC：
- Received：26 February 2025，
  
  Revised：2025-05-31，
  
  Published：02 August 2025
- 稿件说明：
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熊文艳,史蓓蓓,申路路等.补肾调经方对重复控制性卵巢刺激模型小鼠囊胚细胞凋亡及内质网应激的影响[J].中医杂志,2025,66(15):1579-1587.

XIONG Wenyan,SHI Beibei,SHEN Lulu,et al.Effects of Bushen Tiaojing Formula （补肾调经方） on Blastocyst Cell Apoptosis and Endoplasmic Reticulum Stress in Repeated Controlled Ovarian Stimulation Model Mice[J].Journal of Traditional Chinese Medicine,2025,66(15):1579-1587.
熊文艳,史蓓蓓,申路路等.补肾调经方对重复控制性卵巢刺激模型小鼠囊胚细胞凋亡及内质网应激的影响[J].中医杂志,2025,66(15):1579-1587. DOI： 10.13288/j.11-2166/r.2025.15.012.

XIONG Wenyan,SHI Beibei,SHEN Lulu,et al.Effects of Bushen Tiaojing Formula （补肾调经方） on Blastocyst Cell Apoptosis and Endoplasmic Reticulum Stress in Repeated Controlled Ovarian Stimulation Model Mice[J].Journal of Traditional Chinese Medicine,2025,66(15):1579-1587. DOI： 10.13288/j.11-2166/r.2025.15.012.

摘要

目的

探讨补肾调经方对重复控制性卵巢刺激（COS）早期胚胎质量的影响及其可能作用机制。

方法

150只ICR雌性小鼠随机分为正常组、模型组、抑制剂组和补肾调经方低、高剂量组，每组30只。正常组、抑制剂组和模型组小鼠每日以蒸馏水0.25 ml灌胃，补肾调经方低、高剂量组分别给予补肾调经方25.6、51.2 g/（kg·d）灌胃，均每日灌胃1次，持续13天为1个周期。正常组小鼠于灌胃第11天腹腔注射生理盐水0.1 ml，其余4组小鼠建立COS模型，抑制剂组在造模前1 h注射内质网应激抑制剂4-苯基丁酸0.2 ml。各组灌胃及腹腔注射连续3个周期，每个周期间隔4天。各组小鼠均于第3次腹腔注射后立即与同品系雄鼠以2∶1的比例合笼，次日8：00检查阴栓，见栓或阴道涂片见精子者记为妊娠。于妊娠第4天下午处死小鼠，倒置显微镜下获取囊胚，观察囊胚形态并记录总囊胚数、优质囊胚数，计算优质囊胚率；Tunel法检测囊胚细胞凋亡情况，计算细胞凋亡率；免疫荧光法及RT-qPCR法分别检测内质网分子伴侣蛋白葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）、B淋巴细胞瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）、半胱氨酸天冬氨酸蛋白酶12（Caspase-12）蛋白荧光信号强度及其mRNA表达。

结果

正常组囊胚形态规整，形态发育好，碎片率少；模型组囊胚发育较差，细胞形态不规则，碎片率高，有部分停滞；抑制剂组及补肾调经方高、低剂量组可见细胞形态发育较好，形态较规则，碎片率较少。与正常组比较，模型组优质囊胚率降低，囊胚细胞凋亡率升高，囊胚中GRP78、CHOP、Caspase-12、Caspase-3、Bax蛋白荧光信号强度及其mRNA表达升高，Bcl-2蛋白荧光信号强度及其mRNA降低（

＜0.01）。与模型组比较，抑制剂组及补肾调经方高、低剂量组优质囊胚率升高，囊胚细胞凋亡率降低，囊胚中GRP78、CHOP、Caspase-12、Caspase-3、Bax蛋白荧光信号强度及其mRNA表达降低，Bcl-2蛋白荧光信号强度及其mRNA表达升高（

＜0.01）。与抑制剂组比较，补肾调经方高剂量组GRP78、CHOP、Caspase-12、Bax蛋白荧光信号强度及其mRNA表达升高，补肾调经方低剂量组Caspase-12、Caspase-3、Bax蛋白荧光信号强度及其mRNA表达升高（

＜0.05或

＜0.01）。

结论

补肾调经方可能通过改善内质网应激，抑制重复控制性卵巢刺激导致的囊胚细胞凋亡，以发挥提高早期胚胎质量的作用。

Abstract

Objective

To explore the effects of

Bushen Tiaojing Formula

（补肾调经方） on the quality of early embryos in repeated controlled ovarian stimulation （COS） and its possible mechanism.

Methods

A total of 150 ICR female mice were divided into a normal group， a model group， an inhibitor group， a low-dose and a high-dose

Bushen Tiaojing Formula

group. Mice in the normal group， inhibitor group and model group were gavaged with distilled water 0.25 ml a day， and mice in the low- and high-dose groups were given 25.6 and 51.2 g/（kg·d） of

Bushen Tiaojing Formula

， respectively. All groups were gavaged once a day for 13 days as a cycle. Mice in the normal group were injected intraperitoneally with 0.1 ml normal saline on the 11th day of gavage， while mice in the other four groups were used to establish COS models. The inhibitor group was injected with the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid 0.2 ml one hour before modelling. All groups were injected by gavage and intraperitoneal injection for 3 consecutive cycles， with an interval of 4 days between cycles. Immediately after the third intraperitoneal injection， mice were grouped with male mice of the same strain in a ratio of 2∶1， and at 8AM of the second day， the mice were examined， and those with spermatozoa or spermatozoa in the vaginal smears were recorded as pregnant. The mice were executed in the afternoon of the 4th day of pregnancy， and the blastocysts were obtained under an inverted microscope. The morphology of the blastocysts was observed， and the total number of blastocysts and the number of high-quality blastocysts were recorded to calculate the rate of high-quality blastocysts； the apoptosis of blastocyst cells was detected by the Tunel method， and the apoptosis rate was calculated； the endoplasmic reticulum molecular chaperone protein glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， B-lymphoblastoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax），

cystathionin 3 （Caspase-3） and cystathionin 12 （Caspase-12） protein levels and their mRNA expression were detected by immunofluorescent assay and RT-qPCR assay， respectively.

Results

In the normal group， blastocysts had regular morphology， good morphological development and low fragmentation rate； in the model group， blastocysts were poorly developed， cell morphology was irregular， fragmentation rate was high， and there was some stagnation； in the inhibitor group and high-dose， low-dose

Bushen Tiaojing Formula

groups， cell morphology could be seen to be better developed， with regular morphology and less fragmentation rate. Compared with the normal group， the rate of high-quality blastocysts in the model group reduced， the rate of apoptosis of blastocyst cells increased， the levels of GRP78， CHOP， Caspase-12， Caspase-3， Bax protein and its mRNA expression in blastocysts increased， while the level of Bcl-2 protein and its mRNA reduced （

＜0.01）. Compared with the model group， the rate of high-quality blastocysts increased， the rate of apoptosis of blastocysts decreased， the levels of GRP78， CHOP， Caspase-12， Caspase-3， Bax protein and their mRNA expression decreased， and the levels of Bcl-2 protein and its mRNA expression increased in the blastocysts in the inhibitor group and the high-dose and low-dose

Bushen Tiaojing Formula

groups （

＜0.01）. Compared with the inhibitor group， GRP78， CHOP， Caspase-12， Bax protein levels and their mRNA expressions increased in the high-dose

Bushen Tiaojing Formula

group， and Caspase-12， Caspase-3， Bax protein levels and their mRNA expressions increased in the low-dose formula group （

＜0.05 or

＜0.01）.

Conclusion

Bushen Tiaojing Formula

can inhibit the apoptosis of blastocyst cells caused by repeated controlled ovarian stimulation by improving endoplasmic reticulum stress， which plays a r

ole in improving the quality of early embryos.

关键词

Keywords

references

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Effects of Bushen Tiaojing Formula （补肾调经方） on Blastocyst Cell Apoptosis and Endoplasmic Reticulum Stress in Repeated Controlled Ovarian Stimulation Model Mice

DOI：10.13288/j.11-2166/r.2025.15.012

摘要

Abstract

关键词

Keywords

references