Exposure to mono (2‐ethylhexyl) phthalate facilitates apoptosis and pyroptosis of human endometrial microvascular endothelial cells through NLRP3 inflammasome

Abstract

Mono (2‐ethylhexyl) phthalate (MEHP) is a major metabolite of di (2‐ethylhexyl) phthalate (DEHP). This study aimed to observe the toxic effect of MEHP on human endometrial microvascular endothelial cells (HEMECs) and its potential molecular mechanism. HEMECs were exposed to different concentrations of MEHP (0, 50, 100, and 200 nM). Cell viability and apoptosis were assessed by cell counting kit‐8 (CCK‐8) and flow cytometry assays. Western blot was performed to examine the expression of apoptosis‐related proteins (Bcl‐2, Bax, and Caspase‐3). Moreover, the expression of pyroptosis‐related Caspase‐1 was detected by western blot and immunofluorescence assays. Lactate dehydrogenase (LDH) release levels were evaluated in HEMECs treated with MEHP and/or Caspase‐1 inhibitor Ac‐YVAD‐CHO. After exposure to MEHP, NLRP3 expression was examined by reverse transcription quantitative polymerase chain reaction (RT‐qPCR) and western blot. LDH release and apoptosis levels were tested in HEMECs induced by MEHP and/or siNLRP3. MEHP significantly induced cell viability and inhibited apoptosis for HEMECs, with a concentration‐dependent manner. Furthermore, Bcl‐2/Bax ratio was distinctly reduced and Caspase‐3 expression was increased in HEMECs after exposure to MEHP. Western blot and immunofluorescence results confirmed that MEHP markedly augmented Caspase‐1 expression in HEMECs. Furthermore, LDH release levels were fortified in HEMECs treated with MEHP, which were improved following cotreatment with Ac‐YVAD‐CHO. At the mRNA and protein levels, NLRP3 expression was prominently increased in HEMECs exposed to MEHP. NLRP3 knockdown markedly ameliorated the increase in LDH release and apoptosis induced by MEHP exposure in HEMECs. Our findings suggested that exposure to MEHP facilitates apoptosis and pyroptosis of HEMECs through NLRP3 inflammasome.

Exposure to the non-phthalate plasticizer di-heptyl succinate is less disruptive to C57bl/6N mouse recovery from a myocardial infarction than DEHP, TOTM or related di-octyl succinate

“…Increased NLRP3, AIM and P2xR7 expression and increased secretion of active p20kD Caspase-1 and p17kD IL-1β was found in THP-1 monocytes exposed to doses of DEHP expected post-surgery. Consistently, DEHP, its metabolite MEHP and di-butyl phthalate have activated NLRP3 and increased Il-1β secretion in human and rat monocyte cell lines [ 33 – 36 ] as well as other cell lines [ 71 – 74 ]. In contrast, increases in pattern recognition receptor or effector expression were not detected with TOTM, DOS or DHPS exposure in THP-1 cells suggesting that these chemicals do not induce substantial inflammasome activation in macrophages.…”

Introduction to special issue endothelial cells in toxicology: Current status and future perspectives

“…An showed that mono (2‐ethylhexyl) phthalate (MEHP), a major metabolite of di (2‐ethylhexyl) phthalate (DEHP), induced apoptosis and pyroptosis of human endometrial microvascular endothelial cells (HEMECs). The cytotoxicity of MEHP is related with NLRP3 inflammasome, and knock‐down of NLRP3 ameliorated the cytotoxicity of MEHP (An, 2020). Liang et al showed that exposure to cadmium (Cd) induced cytotoxicity to human microvascular endothelial cells (HMEC‐1).…”