Wang Shuyi, Zhu Xiaoling, Xiong Lize, Ren Jun
Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY 82071 USA.
Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY 82071 USA; Department of Anesthesiology, Xijing Hospital, the Fourth Military Medical University, Xi'an, China.
Toxicol Lett. 2017 Mar 5;269:1-14. doi: 10.1016/j.toxlet.2017.01.009. Epub 2017 Jan 20.
Paraquat is a quaternary nitrogen herbicide triggering oxidative stress, mitochondrial damage and multi-organ injuries including hearts. To date, effective measure to combat paraquat toxicity is still lacking. Recent evidence has revealed a role for Akt in cardiac homeostasis. To this end, this study was designed to examine the role of Akt2 in acute paraquat exposure-induced cardiac contractile and mitochondrial injury using a unique murine model of Akt2 knockout. Cardiac contractile and intracellular Ca properties were evaluated. Mitochondrial integrity, ROS production, lipid peroxidation, ER stress and apoptosis were evaluated using aconitase assay, citrate synthase activity, DHE staining, mitochondrial permeation pore opening, 4-hydroxy-nonenal (4-HNE) and Western blot. Our results revealed compromised echocardiographic, contractile and intracellular Ca handling properties along with overt mitochondrial damage (reduced levels of PGC-1α, aconitase, citrate synthase activity and NAD) in mice challenged with paraquat (45mg/kg, single injection, i.p.), the effects of which were attenuated by Akt ablation. Paraquat triggered O production, lipid peroxidation and apoptosis as evidenced by increased DHE staining, 4-HNE, caspase-3 activity, Bax and reduced Bcl-2 levels in association with unchanged ER stress. The redox signaling molecule nuclear factor erythroid related factor 2 (Nrf2) was upregulated in response to paraquat challenge. Findings from in vitro study revealed that stimulation of Nrf2 using sulforaphane (10μM) negated Akt2 ablation-offered beneficial effect against paraquat whereas inhibition of Nrf2 using luteolin (20μM) mimicked Akt2 ablation-induced beneficial effect against paraquat challenge. Taken together, our data indicate that Akt2 ablation may protect against paraquat toxicity-induced cardiac contractile defect and apoptosis possibly via regulation of Nrf2 activation and mitochondrial homeostasis.
百草枯是一种季铵盐类除草剂，可引发氧化应激、线粒体损伤以及包括心脏在内的多器官损伤。迄今为止，仍缺乏对抗百草枯毒性的有效措施。最近的证据表明Akt在心脏稳态中发挥作用。为此，本研究旨在使用独特的Akt2基因敲除小鼠模型，研究Akt2在急性百草枯暴露诱导的心脏收缩和线粒体损伤中的作用。评估了心脏收缩功能和细胞内钙特性。使用乌头酸酶测定、柠檬酸合酶活性、DHE染色、线粒体通透性转换孔开放、4-羟基壬烯醛（4-HNE）和蛋白质印迹法评估线粒体完整性、活性氧生成、脂质过氧化、内质网应激和细胞凋亡。我们的结果显示，在用百草枯（45mg/kg，单次腹腔注射）攻击的小鼠中，超声心动图、收缩功能和细胞内钙处理特性受损，同时伴有明显的线粒体损伤（PGC-1α、乌头酸酶、柠檬酸合酶活性和NAD水平降低），而Akt基因敲除可减轻这些影响。百草枯引发了活性氧生成、脂质过氧化和细胞凋亡，这可通过DHE染色增加、4-HNE、半胱天冬酶-3活性、Bax升高以及Bcl-2水平降低得到证明，同时内质网应激未发生变化。氧化还原信号分子核因子红系相关因子2（Nrf2）在百草枯攻击后上调。体外研究结果显示，使用萝卜硫素（10μM）刺激Nrf2可消除Akt2基因敲除对百草枯的有益作用，而使用木犀草素（20μM）抑制Nrf2则可模拟Akt2基因敲除对百草枯攻击的有益作用。综上所述，我们的数据表明，Akt2基因敲除可能通过调节Nrf2激活和线粒体稳态来预防百草枯毒性诱导的心脏收缩缺陷和细胞凋亡。
