Wu Tingting, Guo Yan
4th Department of Gynecology, NHC Key Laboratory of Birth Defect for Research and Prevention（Hunan Provincial Maternal and Child Health Care Hospital）, Changsha City, Hunan Province, China.
2nd Department of Obstetrics, NHC Key Laboratory of Birth Defect for Research and Prevention（Hunan Provincial Maternal and Child Health Care Hospital）, Changsha City, Hunan Province, China.
Am J Reprod Immunol. 2025 Aug;94(2):e70132. doi: 10.1111/aji.70132.
Endometriosis (EMS) is a chronic inflammatory disorder with ectopic endometrial tissues arising in extrauterine areas. We investigated the mechanism of adenosine triphosphate (ATP)/P2X4 regulating inflammation and oxidative stress in EMS.
Normal endometrial tissues and ectopic endometrial tissues were collected, and determined for P2X4 expression by immunohistochemical staining. Normal (nESCs) and ectopic endometrial stromal cells (eESCs) were isolated and manipulated with Apyrase (a soluble ATP-diphosphohydrolase), 5-BDBD (a P2X4 receptor antagonist), or Nigericin (a NOD-like receptor 3 [NLRP3] inflammasome activator). The ATP concentration in endometrial tissues and cells were assessed through the ATP colorimetric/fluorescence assay, and cellular P2X4 expression was determined by RT-qPCR. Fluo 3-AM calcium ion fluorescence probe was utilized for detecting calcium ion concentration. Levels of inflammation-associated proteins (interleukin [IL]-1β, tumor necrosis factor-alpha [TNF-α], IL-6, IL-18), oxidative stress indicators (malondialdehyde [MDA], superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GSH-Px]), reactive oxygen species (ROS), and the NLRP3 inflammasome pathway-related proteins were determined by ELISA, DCFH-DA fluorescent probe, and Western blot.
ATP and P2X4 were upregulated in EMS. Apyrase or 5-BDBD treatment or P2X4 knockdown reduced the concentration of Ca and levels of IL-1β, TNF-α, IL-6, MDA, and ROS, but increased the activities of SOD, GSH-Px, and CAT in eESCs. Besides, 5-BDBD treatment decreased the expression levels of the NLRP3 inflammasome pathway-related proteins in eESCs and suppressed the secretion of IL-1β and IL-18. Nigericin could reverse the inhibitory impact of 5-BDBD on NLRP3 inflammasome activation.
Altogether, ATP/P2X4 aggravates inflammation and oxidative stress in EMS by activating NLRP3.
子宫内膜异位症（EMS）是一种慢性炎症性疾病，异位子宫内膜组织出现在子宫外区域。我们研究了三磷酸腺苷（ATP）/P2X4调节EMS中炎症和氧化应激的机制。
收集正常子宫内膜组织和异位子宫内膜组织，通过免疫组织化学染色测定P2X4表达。分离正常（nESCs）和异位子宫内膜基质细胞（eESCs），并用Apyrase（一种可溶性ATP二磷酸水解酶）、5-BDBD（一种P2X4受体拮抗剂）或尼日利亚菌素（一种NOD样受体3 [NLRP3]炎性小体激活剂）进行处理。通过ATP比色/荧光测定法评估子宫内膜组织和细胞中的ATP浓度，通过RT-qPCR测定细胞P2X4表达。使用Fluo 3-AM钙离子荧光探针检测钙离子浓度。通过ELISA、DCFH-DA荧光探针和蛋白质免疫印迹法测定炎症相关蛋白（白细胞介素[IL]-1β、肿瘤坏死因子-α [TNF-α]、IL-6、IL-18）、氧化应激指标（丙二醛[MDA]、超氧化物歧化酶[SOD]、过氧化氢酶[CAT]和谷胱甘肽过氧化物酶[GSH-Px]）、活性氧（ROS）以及NLRP3炎性小体途径相关蛋白的水平。
EMS中ATP和P2X4上调。Apyrase或5-BDBD处理或P2X4基因敲低降低了eESCs中Ca的浓度以及IL-1β、TNF-α、IL-6、MDA和ROS的水平，但增加了SOD、GSH-Px和CAT的活性。此外，5-BDBD处理降低了eESCs中NLRP3炎性小体途径相关蛋白的表达水平，并抑制了IL-1β和IL-18的分泌。尼日利亚菌素可逆转5-BDBD对NLRP3炎性小体激活的抑制作用。
总之，ATP/P2X4通过激活NLRP3加重EMS中的炎症和氧化应激。
