目的 通过网络药理学和动物实验验证探讨柴胡-当归挥发油（Bupleuri Radix-Angelicae Sinensis Radix essential oil，BAO）对乳腺增生（hyperplasia of mammary gland，HMG）大鼠的影响及其作用机制。方法 采用水蒸气蒸馏法提取BAO，通过气相色谱-质谱联用技术（gas chromatography-mass spectrometry，GC-MS）进行成分分析；基于TCMSP、PubChem及SwissTargetPrediction数据库筛选BAO活性成分作用靶点，结合GeneCards、OMIM数据库获取HMG相关靶点，构建蛋白互作网络筛选核心靶点，进行基因本体（gene ontology，GO）功能及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析。建立HMG大鼠模型，给予药物干预30 d后，采用ELISA法检测血清中雌二醇、孕酮和催乳素水平，采用生化法测定血清中超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）及谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）水平，采用试剂盒检测血清中血管内皮生长因子（vascular endothelial growth factor，VEGF）及碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）水平；采用免疫组化法测定乳腺组织中雌激素受体α（estrogen receptor α，ERα）、孕激素受体（progesterone receptor，PR）及细胞增殖标志物Ki-67的表达；采用qRT-PCR检测乳腺组织中磷酸酶与张力蛋白同源物（phosphatase and tensin homolog，PTEN）、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）、蛋白激酶B（protein kinase B，Akt）、增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、B细胞淋巴瘤超大蛋白（B-cell lymphoma-2-extra-large，Bcl-xl）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）和Bcl-2关联死亡促进因子（Bcl-2 associated agonist of cell death，Bad）mRNA表达；采用Western blotting检测乳腺组织中ERα、PR、PTEN、PI3K、Akt和p-Akt蛋白表达。结果 GC-MS鉴定出20个挥发性成分，其中Z-藁本内酯含量最高（39.24%）。网络药理学筛选出95个交集靶点，AKT1为核心靶点，主要富集于PI3K/Akt信号通路及雌激素信号通路。动物实验结果显示，与模型组比较，BAO组大鼠血清中雌二醇、催乳素、MDA、VEGF、bFGF水平显著降低（P＜0.05），血清中孕酮水平及SOD、GSH-Px活性显著升高（P＜0.05），乳腺组织中PCNA、Ki-67、ERα、PR、Bcl-2和Bcl-xl表达水平显著降低（P＜0.05），PTEN、Bax和Bad表达水平升高（P＜0.05），PI3K/Akt通路活化水平显著降低（P＜0.05），且呈剂量相关性。结论 BAO通过抑制ERα/PR及PI3K/Akt信号通路，降低雌二醇和催乳素水平，抑制血管异常增生，提高抗氧化能力，从而抑制大鼠HMG的发生发展。

Objective To explore the effect and mechanism of essential oil from Chaihu (Bupleuri Radix)-Danggui (Angelicae Sinensis Radix) (BAO) on hyperplasia of mammary gland (HMG) in rats through network pharmacology and animal experimental validation. Methods BAO was extracted via steam distillation and its constituents were analyzed using gas chromatography-mass spectrometry (GC-MS). The targets of active ingredients in BAO were screened based on TCMSP, PubChem, and SwissTarget Prediction databases, HMG-related targets were obtained from GeneCards and OMIM databases, and a protein interaction network was constructed to identify core targets, gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed. An HMG rat model was established, after 30 d of drug intervention, levels of estradiol, progesterone and prolactin in serum were measured by ELISA, levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in serum were measured by biochemical methods, levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in serum were determined by kits. Immunohistochemistry was used to measure the expressions of estrogen receptor α (ERα), progesterone receptor (PR) and cell proliferation marker Ki-67 in mammary gland tissue; qRT-PCR was used to detect mRNA expressions of phosphatase and tension homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2-extra-large (Bcl-xl), Bcl-2 associated X protein (Bax) and Bcl-2 associated agonist of cell death (Bad) in mammary gland tissue; Western blotting was used to detect the protein expressions of ERα, PR, PTEN, PI3K, Akt, and p-Akt in mammary gland tissue. Results A total of 20 volatile components were identified by GC-MS, among which Z-ligustilide had the highest content (39.24%). Network pharmacology screened 95 intersecting targets, with AKT1 as the core target, mainly enriched in PI3K/Akt signaling pathway and estrogen signaling pathway. The animal experiment results showed that compared with model group, the levels of estradiol, prolactin, MDA, VEGF and bFGF in serum of rats in BAO group were significantly reduced (P < 0.05), while the levels of progesterone and activities of SOD, GSH-Px in serum were significantly increased (P < 0.05). The expression levels of PCNA, Ki-67, ERα, PR, Bcl-2 and Bcl-xl in mammary gland tissue were significantly reduced (P < 0.05), while the expression levels of PTEN, Bax and Bad were increased (P < 0.05), the activation level of PI3K/Akt pathway was significantly reduced (P < 0.05), in a dose-dependent manner. Conclusion BAO inhibits the occurrence and development of HMG in rats by suppressing ERα/PR and PI3K/Akt signaling pathways, reducing levels of estradiol and prolactin, inhibiting abnormal angiogenesis and enhancing antioxidant capacity.

R285.5

江西省重大科技研发专项（20194ABC28009）；江西中医药大学重点学科建设（2024jzzdxk001）；校级研究生创新专项资金（XJ-S202414）；中西医结合江西省一流学科（zxyylxk20220103）