RTA-408是一种抗氧化炎症调节剂(AIM)，可激活Nrf2并抑制一氧化氮(NO)

IC50&Target：Nrf2

体外

为了评估RTA-408的抗炎活性，用RTA-408处理RAW 264.7小鼠巨噬细胞2小时，然后加入IFNγ以刺激NO产生并释放到培养基中。RTA-408剂量依赖性地降低培养基中的NO浓度，IC50值为4.4±1.8nM。RTA-408在该测定中的效力类似于Bardoxolone甲基的效力，其具有1.9±0.8nM的IC 50值。AIM介导的NO抑制需要Nrf2激活。在bardoxolone甲基处理的RAW 264.7细胞中观察到一氧化氮合酶2（Nos2）蛋白水平的降低，当通过siRNA降低Nrf2 mRNA水平时，其减弱。为了评估RTA-408的抗癌活性，用RTA-408处理来自不同来源的肿瘤的一组八种人细胞系，并在72小时后使用磺酰罗丹明B（SRB）测定法测量细胞生长。RTA-408抑制所有肿瘤细胞系的生长，平均GI50值为260±74nM。为了确定RTA-408是否诱导细胞凋亡，将肿瘤细胞组用RTA-408和半胱天冬酶底物DEVD-AFC处理24小时。RTA-408剂量依赖性地增加DEVD-AFC切割，表明RTA-408处理触发癌细胞中的胱天蛋白酶活化。在相同浓度的RTA-408中通过蛋白质印迹也观察到Caspase-3和caspase-9裂解，这增加了DEVD-AFC裂解

体内

为了确定在骨髓致死剂量的全身照射（TBI）后RTA-408是否是造血急性放射综合征的有效缓和剂，在TBI后24小时开始每天3次注射17.5mg/kg RTA-408。用RTA-408治疗导致100％的7 Gy（LD40/35）TBI小鼠（P<0.05）和60％的7.5 Gy（LD100/13）TBI小鼠（P<0.0001）的35天存活率

Cell Assay

MEFs,PANC-1,A549,A375,A549/NF-κB-Luc ａnd HeLa/NF-κB-Luc cells are cultured in Gibco high glucose DMEM with 10%FBS.G-361 cells are cultured in McCoy’s 5A medium with 10%FBS.All other cell lines are cultured in RPMI 1640 medium with 10%FBS.For growth inhibition assays,cells are plated in duplicate 96-well culture dishes at 3×103 cells per well.The following day,one plate is treated with RTA-408(200,400,600,800 ａnd 1000 nM)and the other is immediately processed for the sulforhodamine B(SRB)assay(time 0).Cells in the RTA-408-treated plate are processed for the SRB assay 72 hours after the start of treatment.Percentage of growth relative to vehicle-treated cells is calculated.Dose-response curves are plotted in GraphPad Prism ａnd GI50 values are calculated.For cell counting experiments,MEFs are plated in 6-well culture dishes at 5×104 cells per well ａnd treated with RTA-408 the following day.Following treatment,cells are counted using a Vi-CELL XR cell analyzer.For clonogenic assays,wild-type(1×103 cells per well)and Keap1-/-(0.5×103 cells per well)MEFs are seeded in 6-well dishes.Six hours later,MEFs are treated with RTA-408.After seven days,colonies are fixed with a 1:7 solution of acetic acid:MeOH ａnd stained with 0.5%crystal violet in MeOH.Colonies consisting of≥50 cells are counted[.

MCE has not independently confirmed the accuracy of these methods.They are for reference only

更新时间：2024/1/2 10:10:55