Preeclamptic plasma disrupts endothelial function and promotes inflammation in endothelial cells: beneficial effects of glibenclamide and MCC950 in this scenario

Preeclampsia (PE) is characterized by systemic endothelial dysfunction and remains a significant clinical challenge. Activation of NLRP3 inflammasome, reactive oxygen species (ROS) production, and pyroptosis and autophagy are important mechanisms in this condition. To evaluate the NLRP3 inhibitors effects: glibenclamide (GB) and MCC950, on markers of inflammation, endothelial dysfunction, cell death, and oxidative stress in an <i>in vitro</i> model of PE. Plasma from pregnant women with PE and normotensive pregnant women (NT) was used to investigate its impact on NLRP3 inflammasome activation (NLRP3, TLR4, MyD88, and caspase-1) in endothelial cells (ECs), analyzed by Western Blotting; effects of pharmacological inhibition on the function of ECs was assessed by the evaluation of permeability (VE-cadherin) and markers of endothelial dysfunction by flow cytometry (Flt-1, VEGFR2, E-selectin, VCAM-1, and ICAM-1), as well as cytotoxicity measured by lactate dehydrogenase (LDH), oxidative stress (ROS, nitric oxide - NO and antioxidant capacity), autophagy, and pyroptosis (interleukin IL-1β and high-mobility group box one - HMGB1). Both GB and MCC950 reduced NLRP3 inflammasome activation and its related effects in ECs exposed to PE plasma, including lowered IL-1β, caspase-1, modulated adhesion molecules expression, as well as decreased ROS and cytotoxicity. GB increased NO and restored VE-cadherin expression, while MCC950 enhanced antioxidant capacity. GB also induced autophagy, unlike MCC950. The NLRP3 inhibitors showed the potential to mitigate endothelial dysfunction, oxidative stress, and inflammation, suggesting both compounds hold potential therapeutic value for PE through distinct mechanisms. A. HUVECs exposed to PE plasma and treated with glibenclamide demonstrate: (1) activation by plasma-derived factors of (2) the NLRP3 pathway <i>via</i> TLR4/NF-κB; (3) however, (4) glibenclamide blocks NLRP3 activation by inhibiting potassium efflux. This results in (5) reduced IL-1β levels, (6) increased VE-cadherin expression and consequent reduction in endothelial permeability, (7) normalization of NO and ROS levels, among (8) other consequences that together ultimately lead to (9) a controlled inflammatory response. B. HUVECs exposed to PE plasma and treated with MCC950 demonstrate: (1) activation by plasma-derived factors of (2) the NLRP3 pathway <i>via</i> TLR4/NF-κB; (3) however, (4) MCC950 blocks NLRP3-ASC complex oligomerization. This results in (5) reduced caspase-1 levels, (6) decreased IL-1β levels, (7) normalization of ROS levels, among (8) other consequences that together ultimately lead to (9) a controlled inflammatory response.

子痫前期（Preeclampsia, PE）以全身性内皮功能障碍为核心病理特征，仍是临床亟待解决的重大难题。NLRP3炎性小体（NLRP3 inflammasome）活化、活性氧（reactive oxygen species, ROS）生成、细胞焦亡与自噬均为该病的关键发病机制。本研究旨在评估两类NLRP3抑制剂——格列本脲（glibenclamide, GB）与MCC950，在体外（in vitro）子痫前期模型中对炎症、内皮功能障碍、细胞死亡及氧化应激相关标志物的调控作用。

实验采用子痫前期孕妇与血压正常妊娠妇女（normotensive pregnant women, NT）的血浆处理内皮细胞（endothelial cells, ECs），通过蛋白质免疫印迹（Western Blotting）检测内皮细胞中NLRP3炎性小体活化相关标志物（NLRP3、Toll样受体4（TLR4）、髓系分化因子88（MyD88）及半胱天冬酶-1（caspase-1））的表达水平；通过检测细胞通透性指标（血管内皮钙黏蛋白（VE-cadherin））与流式细胞术（flow cytometry）分析内皮功能障碍标志物（Flt-1、血管内皮生长因子受体2（VEGFR2）、E选择素（E-selectin）、血管细胞黏附分子1（VCAM-1）及细胞间黏附分子1（ICAM-1））的变化，以此评估药物抑制对内皮细胞功能的影响；同时通过乳酸脱氢酶（lactate dehydrogenase, LDH）检测细胞毒性，检测氧化应激相关指标（活性氧、一氧化氮（nitric oxide, NO）及抗氧化能力）、自噬与细胞焦亡相关标志物（白细胞介素1β（interleukin IL-1β）及高迁移率族蛋白B1（high-mobility group box one, HMGB1））的表达与活性变化。

实验结果显示，格列本脲与MCC950均能显著降低暴露于子痫前期血浆的内皮细胞中NLRP3炎性小体的活化水平及其相关效应，包括下调IL-1β、半胱天冬酶-1的表达，调控黏附分子的表达模式，同时减少活性氧生成与细胞毒性。其中，格列本脲可升高NO水平并恢复VE-cadherin的正常表达，而MCC950可增强内皮细胞的抗氧化能力。此外，格列本脲可诱导自噬进程，而MCC950无此效应。两类NLRP3抑制剂均具有缓解内皮功能障碍、氧化应激与炎症反应的潜力，提示两种化合物可通过不同的作用机制对子痫前期发挥潜在的治疗价值。

A. 暴露于子痫前期血浆并经格列本脲处理的人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells, HUVECs）表现出以下特征：(1) 血浆源性因子通过(2) TLR4/NF-κB（核因子κB）通路激活(3) NLRP3信号通路；(4) 格列本脲通过抑制钾外流阻断NLRP3的活化过程。这一调控最终带来(5) IL-1β水平的下调，(6) VE-cadherin表达上调并由此降低内皮细胞通透性，(7) NO与ROS水平恢复至正常范围，以及(8) 其他一系列相关效应，最终共同促成(9) 炎症反应得到有效控制。

B. 暴露于子痫前期血浆并经MCC950处理的人脐静脉内皮细胞（HUVECs）表现出以下特征：(1) 血浆源性因子通过(2) TLR4/NF-κB通路激活(3) NLRP3信号通路；(4) MCC950通过阻断NLRP3-ASC复合物寡聚化发挥抑制作用。这一调控最终带来(5) 半胱天冬酶-1水平的下调，(6) IL-1β水平的降低，(7) ROS水平恢复至正常范围，以及(8) 其他一系列相关效应，最终共同促成(9) 炎症反应得到有效控制。