The First Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510405, China.
Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China.
J Ethnopharmacol. 2024 Dec 5;335:118659. doi: 10.1016/j.jep.2024.118659. Epub 2024 Aug 2.
Endometriosis (EMS) is a common gynecological disease that causes dysmenorrhea, chronic pelvic pain and infertility. Luoshi Neiyi Prescription (LSNYP), a traditional Chinese medicine (TCM) formula, is used to relieve EMS in the clinic.
This study aimed to examine the active components of LSNYP and the possible mechanism involved in its treatment of EMS.
Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used to identify the chemical components of LSNYP. Human primary ectopic endometrial stromal cells (ecESCs) and eutopic endometrial stromal cells (euESCs) were isolated, and the expression levels of hypoxia inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and steroidogenic factor 1 (SF-1) were detected by immunofluorescence and qPCR. Cobalt chloride (CoCl) was utilized to construct an in vitro hypoxic environment, and lentiviruses were engineered to downregulate HIF1A and EZH2 and upregulate EZH2. Subsequently, the expression levels of HIF1A, EZH2, and SF-1 were measured using qPCR or western blotting. The binding of EZH2 to the SF-1 locus in ESCs was examined via ChIP. Furthermore, the effects of LSNYP on the HIF1A/EZH2/SF-1 pathway were evaluated both in vitro and in vivo.
A total of 185 components were identified in LSNYP. The protein and gene expression levels of HIF1A and SF-1 were increased, whereas those of EZH2 were decreased in ecESCs. After treating euESCs with 50 μmol L CoCl for 24 h, cell viability and estradiol (E) production were enhanced. Hypoxia decreased EZH2 protein expression, while si-HIF1A increased it. SF-1 was increased when EZH2 was downregulated in normal and hypoxic environments, whereas the overexpression of EZH2 led to a decrease in SF-1 expression. ChIP revealed that hypoxia reduced EZH2 binding to the SF-1 locus in euESCs. In vitro, LSNYP-containing serum decreased E and prostaglandin E (PGE) production, inhibited cell proliferation and invasion, and reduced the expression of HIF1A, SF-1, steroidogenic acute regulatory protein (StAR), and aromatase cytochrome P450 (P450arom). In vivo, LSNYP suppressed inflammation and adhesion and inhibited the HIF1A/EZH2/SF-1 pathway in endometriotic tissues.
LSNYP may exert pharmacological effects on EMS by inhibiting E synthesis and inflammation through regulation of the HIF1A/EZH2/SF-1 pathway. These results suggest that LSNYP may be a promising candidate for the treatment of EMS.
子宫内膜异位症（EMS）是一种常见的妇科疾病，会导致痛经、慢性盆腔疼痛和不孕。罗氏内异方（LSNYP）是一种中药方剂，临床上用于缓解 EMS。
本研究旨在探讨 LSNYP 的活性成分及其治疗 EMS 的可能机制。
采用超高效液相色谱-四极杆飞行时间质谱联用（UPLC-Q/TOF-MS）技术鉴定 LSNYP 的化学成分。分离人原代异位子宫内膜基质细胞（ecESCs）和在位子宫内膜基质细胞（euESCs），通过免疫荧光和 qPCR 检测低氧诱导因子 1A（HIF1A）、增强子的锌指蛋白 2（EZH2）和类固醇生成因子 1（SF-1）的表达水平。利用氯化钴（CoCl）构建体外低氧环境，通过慢病毒下调 HIF1A 和 EZH2 并上调 EZH2。然后，通过 qPCR 或 Western blot 检测 HIF1A、EZH2 和 SF-1 的表达水平。通过 ChIP 检测 ESCs 中 EZH2 与 SF-1 基因座的结合情况。此外，还在体外和体内评估了 LSNYP 对 HIF1A/EZH2/SF-1 通路的影响。
在 LSNYP 中鉴定出 185 种成分。ecESCs 中 HIF1A 和 SF-1 的蛋白和基因表达水平升高，而 EZH2 表达水平降低。用 50 μmol·L CoCl 处理 euESCs 24 h 后，细胞活力和雌二醇（E）的产生增加。低氧降低了 EZH2 蛋白表达，而 si-HIF1A 则增加了它。在正常和低氧环境下下调 EZH2 可增加 SF-1 的表达，而过表达 EZH2 则导致 SF-1 的表达减少。ChIP 显示低氧降低了 euESCs 中 EZH2 与 SF-1 基因座的结合。体外，含 LSNYP 的血清降低了 E 和前列腺素 E（PGE）的产生，抑制了细胞增殖和侵袭，并降低了 HIF1A、SF-1、类固醇急性调节蛋白（StAR）和细胞色素 P450 芳香化酶（P450arom）的表达。体内，LSNYP 抑制了子宫内膜异位组织中的炎症和粘连，并抑制了 HIF1A/EZH2/SF-1 通路。
LSNYP 可能通过抑制 HIF1A/EZH2/SF-1 通路来抑制 EMS 中 E 的合成和炎症，从而发挥对 EMS 的药理作用。这些结果表明，LSNYP 可能是治疗 EMS 的一种有前途的候选药物。
