Department of Gynecology, Cangzhou Central Hospital, Cangzhou, China.
J Appl Toxicol. 2021 May;41(5):755-764. doi: 10.1002/jat.4106. Epub 2020 Nov 7.
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.
邻苯二甲酸单（2-乙基己基）酯（MEHP）是邻苯二甲酸二（2-乙基己基）酯（DEHP）的主要代谢物。本研究旨在观察 MEHP 对人子宫内膜微血管内皮细胞（HEMECs）的毒性作用及其潜在的分子机制。将 HEMECs 暴露于不同浓度的 MEHP（0、50、100 和 200 nM）中。通过细胞计数试剂盒-8（CCK-8）和流式细胞术检测细胞活力和细胞凋亡。通过 Western blot 检测凋亡相关蛋白（Bcl-2、Bax 和 Caspase-3）的表达。此外，通过 Western blot 和免疫荧光检测细胞焦亡相关 Caspase-1 的表达。用 MEHP 和/或 Caspase-1 抑制剂 Ac-YVAD-CHO 处理 HEMECs 后，评估乳酸脱氢酶（LDH）释放水平。通过逆转录定量聚合酶链反应（RT-qPCR）和 Western blot 检测 MEHP 处理后 NLRP3 的表达。在 MEHP 和/或 siNLRP3 诱导的 HEMECs 中检测 LDH 释放和细胞凋亡水平。MEHP 显著诱导 HEMECs 的细胞活力并抑制细胞凋亡，呈浓度依赖性。此外，MEHP 处理后 HEMECs 中的 Bcl-2/Bax 比值明显降低，Caspase-3 表达增加。Western blot 和免疫荧光结果证实 MEHP 明显增加了 HEMECs 中 Caspase-1 的表达。此外，用 MEHP 处理的 HEMECs 中 LDH 释放水平增强，用 Ac-YVAD-CHO 共同处理后得到改善。在 mRNA 和蛋白水平上，MEHP 暴露后 HEMECs 中 NLRP3 的表达明显增加。NLRP3 敲低显著改善了 MEHP 暴露诱导的 HEMECs 中 LDH 释放和细胞凋亡的增加。我们的研究结果表明，MEHP 通过 NLRP3 炎性小体促进 HEMECs 的细胞凋亡和细胞焦亡。
