背景:Empagliflozin(EMPA)改善暴露于10%拉伸的人内皮细胞(EC)中的活性氧(ROS)生成,但潜在的机制仍不清楚。病理拉伸被认为通过增加细胞内钙(Ca2)刺激蛋白激酶C(PKC),因此激活烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX)并促进人EC中ROS的产生。我们假设EMPA通过阻止PKC活化来抑制拉伸诱导的NOX活化和ROS生成。
方法:将人冠状动脉内皮细胞(HCAECs)预孵育2小时,然后暴露于循环拉伸(5%或10%),EMPA或PKC抑制剂LY-333531或PKCsiRNA。PKC活性,24h后检测NOX活性和ROS产生。Ca2+螯合剂BAPTA-AM,NCX抑制剂ORM-10962或NCXsiRNA,应用钠/钾泵抑制剂乌巴因和钠氢交换剂(NHE)抑制剂cariporide探讨NHE/Na/NCX/Ca2参与EMPA的ROS抑制能力。
结果:与5%拉伸相比,10%显著增加PKC活性,通过EMPA和PKC抑制剂LY-333531减少。EMPA和LY-333531对10%拉伸诱导的NOX活性和ROS产生具有相似的抑制能力,两种药物联合治疗并未增强。PKC-β敲低抑制Ca2+和10%拉伸诱导的NOX活化。BAPTA,药物或遗传NCX抑制和cariporide降低了静态HCAECs中的Ca2+,并阻止了10%拉伸细胞中PKC和NOX的激活。Ouabain增加了暴露于5%拉伸的细胞中的ROS生成。
结论:EMPA通过NHE/Na+/NCX/Ca2+/PKC轴的衰减降低NOX活性,导致暴露于10%拉伸的HCAECs中ROS生成减少。
BACKGROUND: Empagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca2+), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation.
METHODS: Human coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca2+ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na+/NCX/Ca2+ in the ROS inhibitory capacity of EMPA.
RESULTS: Compared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-β knockdown inhibits the NOX activation induced by Ca2+ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca2+ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch.
CONCLUSIONS: EMPA reduced NOX activity via attenuation of the NHE/Na+/NCX/Ca2+/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.