Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348 Krakow, Poland.
Department of Clinical Sciences, Polytechnic University of Marche, Via Ranieri 65, 60131 Ancona, Italy.
Oxid Med Cell Longev. 2020 Oct 14;2020:4678252. doi: 10.1155/2020/4678252. eCollection 2020.
Nrf2 is a master regulator of antioxidant cellular defence, and agents activating the Nrf2 pathway have been tested in various diseases. However, unexpected side effects of cardiovascular nature reported for bardoxolone methyl in patients with type 2 diabetes mellitus and stage 4 chronic kidney disease (the BEACON trial) still have not been fully explained. Here, we aimed to characterize the effects of bardoxolone methyl compared with other Nrf2 activators-dimethyl fumarate and L-sulforaphane-on human microvascular endothelium. Endothelial toxicity, bioenergetics, mitochondrial membrane potential, endothelin-1 (ET-1) release, endothelial permeability, Nrf2 expression, and ROS production were assessed in human microvascular endothelial cells (HMEC-1) incubated for 3 and 24 hours with 100 nM-5 M of either bardoxolone methyl, dimethyl fumarate, or L-sulforaphane. Three-hour incubation with bardoxolone methyl (100 nM-5 M), although not toxic to endothelial cells, significantly affected endothelial bioenergetics by decreasing mitochondrial membrane potential (concentrations ≥ 3 M), decreasing spare respiratory capacity (concentrations ≥ 1 M), and increasing proton leak (concentrations ≥ 500 nM), while dimethyl fumarate and L-sulforaphane did not exert such actions. Bardoxolone methyl at concentrations ≥ 3 M also decreased cellular viability and induced necrosis and apoptosis in the endothelium upon 24-hour incubation. In turn, endothelin-1 decreased permeability in endothelial cells in picomolar range, while bardoxolone methyl decreased ET-1 release and increased endothelial permeability even after short-term (3 hours) incubation. In conclusion, despite that all three Nrf2 activators exerted some beneficial effects on the endothelium, as evidenced by a decrease in ROS production, bardoxolone methyl, the most potent Nrf2 activator among the tested compounds, displayed a distinct endothelial profile of activity comprising detrimental effects on mitochondria and cellular viability and suppression of endothelial ET-1 release possibly interfering with ET-1-dependent local regulation of endothelial permeability.
Nrf2 是抗氧化细胞防御的主要调节剂，已在各种疾病中测试了激活 Nrf2 途径的药物。然而， bardoxolone 甲基在 2 型糖尿病和 4 期慢性肾病患者中的心血管不良反应（BEACON 试验）仍未得到充分解释。在这里，我们旨在比较 bardoxolone 甲基与其他 Nrf2 激活剂——二甲基富马酸和 L-苏氨酸对人微血管内皮的作用。在培养 3 小时和 24 小时后，用 100 nM-5 M 的 bardoxolone 甲基、二甲基富马酸或 L-苏氨酸孵育人微血管内皮细胞（HMEC-1），评估内皮毒性、生物能量学、线粒体膜电位、内皮素-1（ET-1）释放、内皮通透性、Nrf2 表达和 ROS 产生。虽然 3 小时内孵育 100 nM-5 M 的 bardoxolone 甲基对内皮细胞没有毒性，但它显著影响内皮生物能量学，降低线粒体膜电位（浓度≥3 M），降低备用呼吸能力（浓度≥1 M），并增加质子泄漏（浓度≥500 nM），而二甲基富马酸和 L-苏氨酸则没有这种作用。浓度≥3 M 的 bardoxolone 甲基还降低了内皮细胞的细胞活力，并在 24 小时孵育后诱导内皮细胞坏死和凋亡。相反，内皮素-1 在皮摩尔范围内降低内皮细胞的通透性，而 bardoxolone 甲基降低 ET-1 释放并增加内皮通透性，即使在短期（3 小时）孵育后也是如此。总之，尽管所有三种 Nrf2 激活剂都对内皮产生了一些有益的影响，表现为 ROS 产生减少，但在测试的化合物中，最有效的 Nrf2 激活剂 bardoxolone 甲基对内皮细胞表现出明显的活性特征，包括对线粒体和细胞活力的有害影响，以及抑制内皮 ET-1 释放，这可能干扰内皮通透性的内皮依赖性局部调节。
