目的 探究桂枝茯苓胶囊-散结镇痛胶囊-宫血宁胶囊联用（GSG）方案对人子宫腺肌病异位内膜衍生细胞（adenomyosis derived cells，AMDC）异常增殖的抑制作用，评价对子宫腺肌病（adenomyosis，AM）模型小鼠的改善效果，并探讨相关分子机制。方法 提取AMDC原代细胞并培养，采用CCK-8法检测GSG对AMDC活力的影响；采用克隆形成实验、EdU增殖检测实验确定GSG对AMDC克隆形成和增殖能力的影响。新生ICR小鼠滴喂他莫昔芬制备AM模型，分为对照组、模型组、复方米非司酮（3.25 mg/kg）组和GSG低、中、高剂量（0.635、1.270、2.539 g/kg）组，给药干预后，采用苏木素-伊红（hematoxylin-eosin，HE）染色观察小鼠子宫、肝、脾和肾的形态学改变；免疫组化法检测子宫组织中磷酸化磷脂酰肌醇3-激酶（phosphorylated phosphatidylinositol 3-kinase，p-PI3K）、磷酸化蛋白激酶B（phosphorylated protein kinase B，p-Akt）和缺氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）的表达；检测血清中丙氨酸氨基转移酶（alanine aminotransferase，ALT）活性和血尿素氮（blood urea nitrogen，BUN）水平。结果 GSG能有效抑制AMDC活力和克隆形成能力，并呈剂量相关性地抑制AMDC增殖能力（P＜0.001）。子宫HE染色结果显示，模型组子宫内膜与肌层边界模糊，多处腺体深度侵入子宫肌层；GSG低剂量组边界欠清晰，有腺体侵入子宫肌层，浸润深度减轻，数量减少；复方米非司酮组和GSG中剂量组边界较清晰，偶见腺体侵入肌层；GSG高剂量组边界清晰，少见明显腺体侵入肌层。免疫组化结果表明，与对照组比较，模型组子宫组织p-PI3K、p-Akt和HIF-1α表达显著增加（P＜0.01、0.001）；与模型组比较，各给药组p-PI3K、p-Akt的表达显著降低（P＜0.01、0.001），复方米非司酮组和GSG中、高剂量组HIF-1α的表达显著降低（P＜0.05、0.01）。各组肝、脾和肾形态学均未见明显改变，血清ALT活性和BUN水平无显著性差异。结论 GSG方案在体内实验中可通过抑制PI3K/Akt/HIF-1α通路阻断AM进展，所选GSG剂量在动物体内未见明显不良反应，是潜在的AM治疗方案。

Objective To investigate the inhibitory effects of Guizhi Fuling Capsules (桂枝茯苓胶囊)-Sanjie Zhentong Capsules (散结镇痛胶囊)-Gongxue Ning Capsules (宫血宁胶囊) combination (GSG) on abnormal proliferation of human adenomyosis-derived cells (AMDC), and evaluate its therapeutic efficacy and mechanism in a mouse model of adenomyosis (AM). Methods Primary AMDCs were collected and cultured, cell viability was assessed using CCK-8 assay. The effects of GSG on AMDC colony formation and proliferation capabilities were determined through colony formation and EdU proliferation assays. Neonatal ICR mice were orally administered with tamoxifen to induce AM model and divided into control group, model group, compound mifepristone (3.25 mg/kg) group and GSG low-, medium-, and high-dose (0.635, 1.270, 2.539 g/kg) groups. After drugs intervention, hematoxylin-eosin (HE) staining was used to observe morphological changes in uterus, liver, spleen and kidney tissues of mice. Immunohistochemistry (IHC) was employed to detect the expressions of phosphorylated phosphoinositide 3-kinase (p-PI3K), phosphorylated protein kinase B (p-Akt) and hypoxia-inducible factor-1α (HIF-1α) in uterine tissues of mice. Alanine aminotransferase (ALT) activity and blood urea nitrogen (BUN) level in serum were measured. Results GSG effectively inhibited the viability and colony formation capacity of AMDCs and showed a dose-dependent inhibition of AMDC proliferation (P < 0.001). HE staining results indicated that the boundary between the endometrium and myometrium was unclear in model group, with extensive glandular infiltration into the myometrium. In GSG low-dose group, the boundary was less clear, with glandular infiltration into the myometrium and reduced depth and number of glandular infiltrations. The boundary was relatively clear with occasional glandular infiltration in compound mifepristone group and GSG medium-dose group. In GSG high-dose group, the boundary was clear, and significant glandular infiltration was rare. IHC results showed a significant increase in expressions of p-PI3K, p-Akt and HIF-1α in model group compared to control group (P < 0.01, 0.001). Compared to model group, the expressions of p-PI3K and p-Akt were significantly reduced in each administration group (P < 0.01, 0.001), and HIF-1α expression was significantly reduced in compound mifepristone group and GSG medium-, high-dose groups (P < 0.05, 0.01). There were no significant changes in the morphology of liver, spleen and kidney in each group, and there was no significant difference in ALT activity and BUN level in serum. Conclusion GSG therapy can blockade of AM progression by inhibiting PI3K/Akt/HIF-1α pathway in in vivo experiments, with the selected GSG dose showing no significant adverse reactions in animals, indicating it is a potential treatment option for AM.

中国博士后科学基金面上项目（2022M722000）；泰山学者青年专家项目（tsqn201909185）；山东省自然联合基金项目（ZR2023LZY024）