Hao Jianheng, Wang Haijun, Cao Yuxia, Ji Laixi
College of Acupuncture-Moxibustion and Tuina, Chengdu University of TCM, Chengdu 610075, Sichuan Province, China; Second Clinical College of Shanxi University of CM, Jinzhong 030619.
Second Clinical College of Shanxi University of CM, Jinzhong 030619.
Zhongguo Zhen Jiu. 2024 Jul 12;44(7):821-30. doi: 10.13703/j.0255-2930.20230610-k0001.
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.
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•d) 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.
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).
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.
基于PI3K/AKT/FOXO3a通路观察针刺“秩边”（BL 54）透“水道”（ST 28）对卵巢反应不良（POR）小鼠的保护作用，探讨针刺抑制POR小鼠卵巢颗粒细胞凋亡的可能机制。
将45只动情周期规律的小鼠随机分为空白组、模型组和针刺组，每组15只。模型组和针刺组小鼠给予雷公藤多苷混悬液（50 mg•kg•d）灌胃2周以建立POR模型。造模成功后，针刺组小鼠针刺“秩边”（BL 54）透“水道”（ST 28）2周，每日1次，每次20分钟。干预结束次日开始诱导排卵，诱导排卵后从每组取材。采用阴道涂片观察小鼠动情周期变化。测量取到的卵母细胞数量、卵巢湿重、终末体重及卵巢指数。采用酶联免疫吸附测定法（ELISA）检测血清中抗苗勒管激素（AMH）、促卵泡生成素（FSH）、雌二醇（E2）及促黄体生成素（LH）水平。采用苏木精-伊红（HE）染色观察卵巢组织形态。采用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）染色检测卵巢颗粒细胞凋亡情况。采用实时荧光定量聚合酶链反应（PCR）检测卵巢组织中PI3K、AKT及FOXO3a的mRNA表达。采用蛋白质免疫印迹法检测卵巢组织中Bcl-2相关X蛋白（BAX）、半胱天冬酶-3（caspase-3）、磷酸化磷脂酰肌醇3激酶（p-PI3K）及磷酸化蛋白激酶B（p-AKT）的蛋白表达。
与空白组比较，模型组动情周期紊乱率升高（<0.01）；与模型组比较，针刺组动情周期紊乱率降低（<0.01）。与空白组比较，模型组取到的卵母细胞数量、卵巢湿重、卵巢指数及终末体重降低（<0.01）；与模型组比较，针刺组取到的卵母细胞数量、卵巢指数及卵巢湿重升高（<0.01，<0.05），终末体重差异无统计学意义（>0.05）。与空白组比较，模型组血清FSH、LH水平升高（<0.01），血清AMH、E2水平降低（<0.01）；与模型组比较，针刺组血清FSH、LH水平降低（<0.01，<0.05），血清AMH、E2水平升高（<0.01，<0.05）。与空白组比较，模型组卵巢组织中正常发育卵泡数量减少、形态欠佳，闭锁卵泡数量增多；与模型组比较，针刺组卵泡数量、形态及颗粒细胞结构均有不同程度改善，闭锁卵泡数量减少。与空白组比较，模型组卵巢颗粒细胞凋亡率升高（<0.01）；与模型组比较，针刺组卵巢颗粒细胞凋亡率降低（<0.01）。与空白组比较，模型组卵巢组织中FOXO3a mRNA表达及caspase-3、BAX蛋白表达升高（<0.01），卵巢组织中PI3K、AKT mRNA表达及p-PI3K、p-AKT、p-FOXO3a蛋白表达降低（<0.01）；与模型组比较，针刺组卵巢组织中FOXO3a mRNA表达及caspase-3、BAX蛋白表达降低（<0.05，<0.01），卵巢组织中PI3K、AKT mRNA表达及p-PI3K、p-AKT、p-FOXO3a蛋白表达升高（<0.01，<0.05）。
针刺“秩边”（BL 54）透“水道”（ST 28）可抑制POR小鼠卵巢细胞凋亡，改善卵巢功能，其机制可能与调节PI3K/AKT/FOXO3a通路关键因子有关。
