基于HIF-1α/VEGFA信号通路探讨育肾养卵方对小鼠卵巢储备功能减退改善  

Exploration of the effects of Yushen Yangluan decoction on improving ovarian reserve function in mice based on the HIF-1α/VEGFA signalingpathway

作　　者：黄家宓 夏翎晋 张兆俸 钟煜舜 毛燕燕 杜晶 张婷婷[1] Huang Jiami;Xia Linjin;Zhang Zhaofeng;Zhong Yushun;Mao Yanyan;Du Jing;Zhang Tingting(Department of Gynecology,Yueyang Hospital of Integrated Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200083,China;Shanghai-MOST Key Laboratory of Health and Disease Genomics,NHC Key Lab of Reproduction Regulation,Shanghai Institute for Biomedical and Pharmaceutical Technologies,Shanghai 200237,China)

机构地区：[1]上海中医药大学附属岳阳中西医结合医院妇科,上海200083 [2]上海市生物医药技术研究院、上海市疾病与健康基因组学重点实验室、国家卫生健康委生育调节药械重点实验室,上海200237

出　　处：《中华生殖与避孕杂志》2024年第11期1156-1163,共8页Chinese Journal of Reproduction and Contraception

基　　金：上海市进一步加快中医药传承创新发展三年行动计划(2021—2023年)(ZY(2021-2023)0302);上海市卫健委海派中医流派传承创新团队建设项目(2021LPTD-002);张婷婷教授上海市名老中医学术经验研究工作室(SHGZS-20222)。

摘　　要：目的观察育肾养卵方对卵巢储备功能减退(diminished ovarian reserve,DOR)小鼠卵巢储备功能的影响,并基于低氧诱导因子(hypoxia-inducible factor,HIF)-1α/血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)信号通路探讨其作用机制。方法基于人工智能的网络药理学进行机制分析,将50只6周龄C57BL/6雌鼠按体质量随机区组法分为对照组、模型组、中药低剂量组、中药中剂量组、中药高剂量组,每组10只。通过苏木精-伊红染色法观察卵巢窦卵泡计数,酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清激素水平,缺口末端标记法(TdT-mediated dUTP nick end labeling,TUNEL)检测卵巢颗粒细胞凋亡;免疫组织化学法检测卵巢HIF-1α的表达;蛋白印迹法检测卵巢HIF-1α、VEGFA、B淋巴细胞瘤-2(B-cell lymphoma-2,BCL2)的表达。结果采用育肾养卵方低、中、高剂量治疗DOR模型小鼠后,中药低、中、高剂量组小鼠卵巢窦卵泡计数均高于模型组(P=0.024、P<0.001、P<0.001),卵泡刺激素水平均低于模型组(P=0.003、P<0.001、P<0.001),中药中、高剂量组血清抗苗勒管激素水平均高于模型组(P=0.001、P<0.001)。网络药理学与分子结合提示育肾养卵方可能通过核心靶点HIF-1α、BCL2、VEGFA影响HIF信号通路。TUNEL提示中药低、中、高剂量治疗后小鼠卵巢颗粒细胞凋亡率均显著低于模型组(P=0.017、P=0.003、P<0.001),免疫组织化学结果显示中药低、中、高剂量治疗后小鼠卵巢颗粒细胞HIF-1α表达较模型组升高(P=0.008、P<0.001、P<0.001),蛋白印迹法显示中药中、高剂量组HIF-1α的蛋白表达量均显著高于模型组(P=0.004、P<0.001),低、中、高剂量组的VEGFA、BCL2的蛋白表达量均显著高于模型组(P=0.003、P=0.002、P=0.001;P=0.029、P=0.007、P=0.001)。结论育肾养卵方可能是通过HIF-1α/VEGFA信号通路抑制卵巢颗粒细胞凋亡达到治疗效果。Objective:To observe the effect of the Yushen Yangluan decoction on ovarian reserve function in mice with diminished ovarian reserve(DOR)and to explore its mechanism of action based on the hypoxia-inducible factor(HIF)-1α/vascular endothelial growth factor A(VEGFA)signaling pathway.Methods:Mechanism analysis was conducted using artificial intelligence-based network pharmacology.Fifty 6-week-old female C57BL/6 mice were randomly divided into five groups according to body weight,with 10 mice in each group.The groups were as follows:control group,model group,low-dose Traditional Chinese Medicine(TCM)group,medium-dose TCM group,and high-dose TCM group.Hematoxylin-eosin staining was used to observe the number of antral follicles in the ovaries,enzyme-linked immunosorbent assay was employed to detect serum hormone levels,and TdT-mediated dUTP nick end labeling(TUNEL)was used to assess apoptosis in ovarian granulosa cells.Immunohistochemistry(IHC)was performed to detect ovarian expression of HIF-1α,and Western blotting was used to measure the protein expressional levels of HIF-1α,VEGFA,and B-cell lymphoma-2(BCL2)in the ovaries.Results:After treating DOR model mice with the Yushen Yangluan decoction at low,medium,and high dosages,the ovarian antral follicle count of the mice in the TCM groups was all higher than that in the model group(P=0.024,P<0.001,P<0.001),the follicle-simulating hormone level was lower than that in the model group(P=0.003,P<0.001,P<0.001),and the serum anti-Müllerian hormone level in the medium-and high-dose TCM groups was higher than that in the model group(P=0.001,P<0.001).Network pharmacology and molecular docking suggested that the formula may influence the HIF signaling pathway through core targets such as HIF-1α,BCL2 and VEGFA.TUNEL assays indicated that the apoptosis rate of ovarian granulosa cells in the low-,medium-,and high-dose TCM groups was significantly lower than that in the model group(P=0.017,P=0.003,P<0.001).Immunohistochemistry results showed that the expression level of HIF-

关 键 词：卵巢储备功能减退 育肾养卵方 人工智能 网络药理学 HIF-1α/VEGFA信号通路 

分 类 号：R285.5[医药卫生—中药学]