背景:子宫内膜异位症(EMS)是引起痛经的常见妇科疾病,慢性盆腔疼痛和不孕症。罗氏内衣处方(LSNYP),中药(TCM)配方,用于缓解临床中的EMS。
目的:本研究旨在探讨LSNYP的活性成分及其治疗EMS的可能机制。
方法:超高效液相色谱四极杆飞行时间质谱(UPLC-Q/TOF-MS)用于鉴定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),缺氧诱导因子1A(HIF1A)的表达水平,通过免疫荧光和qPCR检测zeste同源物2(EZH2)和类固醇生成因子1(SF-1)的增强子。利用氯化钴(CoCl2)构建体外低氧环境,和慢病毒被工程化以下调HIF1A和EZH2并上调EZH2。随后,HIF1A的表达水平,使用qPCR或蛋白质印迹法测量EZH2和SF-1。通过ChIP检查EZH2与ESC中SF-1基因座的结合。此外,在体外和体内评估了LSNYP对HIF1A/EZH2/SF-1途径的影响。
结果:在LSNYP中鉴定出总共185个组分。HIF1A和SF-1的蛋白和基因表达水平升高,而EZH2在ecESCs中降低。用50μmol·L-1CoCl2处理euESCs24h后,细胞活力和雌二醇(E2)的产生增强。缺氧降低EZH2蛋白表达,而si-HIF1A增加了它。当EZH2在正常和低氧环境中下调时,SF-1增加,而EZH2的过表达导致SF-1表达降低。ChIP显示缺氧降低了EZH2与euESC中SF-1基因座的结合。体外,含LSNYP的血清降低了E2和前列腺素E2(PGE2)的产生,抑制细胞增殖和侵袭,并降低HIF1A的表达,SF-1,类固醇生成急性调节蛋白(StAR),和芳香化酶细胞色素P450(P450arom)。在体内,LSNYP抑制子宫内膜异位组织中的炎症和粘附并抑制HIF1A/EZH2/SF-1通路。
结论:LSNYP可能通过调节HIF1A/EZH2/SF-1通路抑制E2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的有希望的候选者。
BACKGROUND: 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.
OBJECTIVE: This study aimed to examine the active components of LSNYP and the possible mechanism involved in its treatment of EMS.
METHODS: 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 (CoCl2) 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.
RESULTS: 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-1 CoCl2 for 24 h, cell viability and estradiol (E2) 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 E2 and prostaglandin E2 (PGE2) 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.
CONCLUSIONS: LSNYP may exert pharmacological effects on EMS by inhibiting E2 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.