ATP/P2X4 Regulates Inflammation and Oxidative Stress in Endometriosis Through NLRP3 Inflammasome-Dependent Mechanisms.

PURPOSE: 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.

METHODS

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.

RESULTS

ATP and P2X4 were upregulated in EMS. Apyrase or 5-BDBD treatment or P2X4 knockdown reduced the concentration of Ca2+ 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.

CONCLUSION

Altogether, ATP/P2X4 aggravates inflammation and oxidative stress in EMS by activating NLRP3.