益肾疏肝汤介导AMPK/PINK1/Parkin信号通路调控线粒体自噬修复DOR大鼠卵巢损伤的机制研究*

作者：肖 潇1，滕秀香1，危一飞2，佟 庆1

单位：1.首都医科大学附属北京中医医院，北京 100010； 2.中国中医科学院望京医院，北京 100102

引用：引用：肖潇,滕秀香,危一飞,佟庆.益肾疏肝汤介导AMPK/PINK1/Parkin信号通路调控线粒体自噬修复DOR大鼠卵巢损伤的机制研究[J].中医药导报,2025,31(11):20-25,50.

DOI：10.13862/j.cn43-1446/r.2025.11.004

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摘要：

目的：观察益肾疏肝汤对雷公藤所致卵巢储备功能减退（DOR）模型大鼠卵巢损伤的修复作用并探讨其机制。方法：32只SD大鼠随机分为空白组8只和造模组24只，予雷公藤制备DOR模型，成模后将造模组大鼠再随机分为模型组、中药组和西药组，每组8只。中药组予益肾疏肝汤灌胃，西药组予戊酸雌二醇药液灌胃，空白组和模型组予等体积生理盐水灌胃，连续15 d。观察大鼠动情周期，计算卵巢指数和子宫指数；酶联免疫吸附试验（ELISA）检测血清抗米勒管激素（AMH)、卵泡刺激素（FSH）、雌二醇（E2）水平；HE染色观察卵巢组织形态；比色法检测卵巢中超氧化物歧化酶（SOD）活力、丙二醛（MDA）含量；免疫组化法检测卵巢颗粒细胞中线粒体自噬相关蛋白的表达；蛋白质印迹法（Western blotting）检测卵巢组织中腺苷酸活化蛋白激酶（AMPK）/PTEN诱导假定激酶1（PINK1）/帕金森病蛋白2（Parkin）信号通路相关蛋白的表达。结果：与空白组比较，模型组大鼠动情周期延长、紊乱，性腺指数显著降低；FSH水平显著升高，AMH、E2水平显著降低；卵巢组织病理切片显示生长卵泡数量明显减少，闭锁卵泡数量增多，SOD活性显著降低，MDA含量显著升高；卵巢颗粒细胞中微管相关蛋白1轻链3B（LC3B）蛋白表达光密度（IOD）值明显上升，PINK1、Parkin明显下降；AMPK、PINK1、Parkin蛋白相对表达量均明显降低（P<0.01）。与模型组比较，中药组、西药组SD大鼠动情周期缩短、趋于正常，性腺指数升高，血清FSH水平显著下降；AMH、E2水平显著提高；卵巢组织病理切片提示卵泡数目增多，损伤减少，SOD活性显著升高，MDA含量显著下降。卵巢颗粒细胞中LC3B蛋白IOD值明显下降，PINK1、Parkin明显上升；AMPK、PINK1、Parkin蛋白相对表达量均明显上升（P<0.05）。与中药组比较，西药组大鼠血清E2含量上升，卵巢LC3B蛋白IOD值增加，卵巢组织中的AMPK、Parkin蛋白相对表达量下降（P<0.01），其余指标未见差异。结论：益肾疏肝汤能改善DOR模型大鼠卵巢受损状态，提高卵巢储备，其机制可能与激活AMPK/PINK1/Parkin信号通路、激活卵巢颗粒细胞线粒体自噬有关。

关键词：卵巢储备功能减退；益肾疏肝汤；线粒体自噬；AMPK/PINK1/Parkin；大鼠

Abstract：

Objective: To observe the repair effects of Yishen Shugan decoction on ovarian damage in a rat model of diminished ovarian reserve (DOR) induced by tripterygium glycosides and to explore its underlying mechanisms. Methods: A total of 32 SD rats were randomly divided into control group (n=8) and model establishment group (n=24). The DOR model was established using tripterygium glycosides. After successful modeling, the model establishment group was further randomized into model group, Chinese medicine (Yishen Shugan decoction) group, and Western medicine (estradiol valerate) group, with 8 rats in each. The Chinese medicine group received Yishen Shugan decoction via intragastric administration, and the Western medicine group received estradiol valerate solution. The control group and model group received an equal volume of saline for 15 consecutive days. Ovarian injury repair was assessed by observing estrous cycles, calculating ovarian and uterine indices, measuring serum anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2) levels via enzyme-linked immunosorbent assay (ELISA), analyzing ovarian histopathology via hematoxylin-eosin (HE) staining, evaluating superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in ovarian tissues using colorimetric assays, detecting mitochondrial autophagy-related protein expression in ovarian granulosa cells via immunohistochemistry, and analyzing adenosine monophosphate-activated protein kinase (AMPK)/PTEN-induced putative kinase 1 (PINK1)/Parkinson disease protein 2 (Parkin) signaling pathway via Western blotting. Results: Compared with the control group, the model group exhibited prolonged and irregular estrous cycles, significantly reduced ovarian and uterine indice, elevated FSH levels, decreased AMH and E2 levels, reduced ovarian follicular development with increased atretic follicles, decreased SOD activity, increased MDA content, upregulated LC3B protein expression, down-regulated PINK1 and Parkin protein expression, and reduced AMPK, PINK1 and Parkin protein levels (P<0.01). Compared with the model group, the Chinese medicine grou and Western medicine group showed improved estrous cycles, increased ovarian and uterine indices, reduced FSH levels, elevated AMH and E2 levels, enhanced ovarian follicular development, reduced ovarian damage, increased SOD activity, decreased MDA content, reduced LC3B expression, increased PINK1 and Parkin expression, and up-regulated AMPK, PINK1 and Parkin protein levels (P<0.05). Compared with the Chinese medicine group, the Western medicine group showed higher E2 level, increased LC3B expression, and reduced AMPK and Parkin protein levels (P<0.01), with no significant differences in other parameters. Conclusion: Yishen Shugan decoction can alleviate ovarian damage and improves ovarian reserve in DOR rats by activating the AMPK/PINK1/Parkin signaling pathway, thereby promoting mitochondrial autophagy in ovarian granulosa cells.

Key words：decreased ovarian reserve function; Yishen Shugan decoction; mitophagy; AMPK/PINK1/Parkin; rat

发布时间：2025-11-29

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