STING棕榈酰化抑制剂C176在保护HEI-OC1细胞氧化损伤中的作用及机制研究

Study on the role and mechanism of STING palmitoylation inhibitor C176 in protecting HEI-OC1 cells against oxidative damage

陈筱颖;王洁;张园园;华清泉

1:武汉大学人民医院耳鼻咽喉头颈外科武汉大学耳鼻咽喉头颈外科研究所

摘要
目的 探索氧化应激诱导HEI-OC1细胞损伤的潜在机制,并观察干扰素基因刺激蛋白(stimulator of interferon genes, STING)棕榈酰化抑制剂C176对该损伤的保护作用。方法 用不同浓度的H_2O_2和C176处理HEI-OC1细胞,通过CCK8(cell counting kit 8)检测细胞活力,选取合适的H_2O_2(0.75 mM)和C176(2μM)浓度进行后续实验。将细胞分为空白对照组(常规培养)、H_2O_2组(0.75 mM H_2O_2处理6 h)和C176+H_2O_2组(2μM C176预处理6 h后使用0.75 mM H_2O_2处理6 h)。利用流式细胞术检测各组活性氧(reactive oxygen species, ROS)水平,利用电镜观察各组细胞线粒体损伤、实时荧光定量PCR(real-time quantitative polymerase chain reaction, qRT-PCR)检测线粒体DNA(mitochondrial DNA,mtDNA)的释放,利用酰基生物素交换法(acyl-biotin exchange, ABE)检测各组细胞中STING棕榈酰化程度,比较各组结果。最后利用蛋白组学测序检测C176+H_2O_2组相对于H_2O_2组的蛋白表达差异。结果 与空白对照组相比,H_2O_2组细胞活力降低,ROS水平明显升高,线粒体显著肿胀且线粒体嵴损伤,胞质中16SrRNA及环氧合酶-1(cyclooxygenase-1,COX1)表达升高,STING棕榈酰化程度明显升高(均为P<0.05)。与H_2O_2组相比,C176+H_2O_2组细胞活力有一定程度恢复(P<0.01),ROS水平及线粒体损伤未见显著改善(P>0.05),但STING棕榈酰化程度显著降低(P<0.05)。C176+H_2O_2组相对于H_2O_2组共鉴定出173种差异表达蛋白,主要分布于过氧化物酶、内质网中相关蛋白、马达蛋白、物质代谢及铁死亡调控等通路。在差异最显著的这五种通路中,共鉴定出16个差异表达蛋白,其中11个表达上调,5个表达下调。结论 C176对HEI-OC1细胞的保护作用并非通过直接抑制氧化应激、调控线粒体实现,而是抑制STING棕榈酰化,可能存在作用于下游特定蛋白的保护通路。
关键词
HEI-OC1;C176;氧化应激;STING棕榈酰化
基金项目(Foundation):
国家专科疾病诊疗中心建设培育资金(ZX0000000037)
作者
陈筱颖;王洁;张园园;华清泉
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陈筱颖王洁张园园华清泉