槲皮素介导铁死亡途径对高渗刺激诱导的干眼细胞模型的保护作用

Protective effect of quercetin mediated ferroptosis pathway on hypertonic stimulation induced dry eye cell model

目的 探讨槲皮素对高渗透压诱导的人角膜上皮细胞HCE-2损伤的影响及其作用机制。方法 将HCE-2细胞随机分为对照组(正常渗透压)、模型组(高渗透压)、低剂量实验组(高渗透压+31.25μg·mL^(-1)槲皮素)、中剂量实验组(高渗透压+62.50μg·mL^(-1)槲皮素)、高剂量实验组(高渗透压+125.00μg·mL^(-1)槲皮素)、Erastin组(高渗透压+125.00μg·mL^(-1)槲皮素+30.00μmol·L^(-1)铁死亡诱导剂Erastin)。用细胞计数试剂盒-8检测细胞存活率,用C11-BODIPY 581/591探针染色检测活性氧(ROS)水平,用试剂盒法测定细胞谷胱甘肽(GSH)和丙二醛(MDA)水平,用实时荧光定量聚合酶链反应实验和蛋白质印迹法检测谷胱甘肽过氧化物酶4(GPX4)、二氢乳酸脱氢酶(DHODH)、铁死亡抑制蛋白1(FSP1)表达水平。结果 对照组、模型组、高剂量实验组、Erastin组细胞存活率分别为(100.00±3.97)%、(50.05±5.83)%、(86.35±7.35)%和(58.32±4.66)%,ROS水平分别为1.00±0.09、2.45±0.16、1.19±0.05和2.09±0.30,GPX4蛋白水平分别为1.09±0.11、0.34±0.03、0.91±0.12和0.30±0.04,FSP1蛋白水平分别为0.92±0.06、0.25±0.03、0.89±0.07和0.39±0.07,DHODH蛋白水平分别为0.89±0.11、0.31±0.04、0.86±0.11和0.41±0.04。对照组上述指标与模型组比较,在统计学上差异均有统计学意义(均P<0.05);高剂量实验组上述指标与模型组比较,在统计学上差异均有统计学意义(均P<0.05);Erastin组上述指标与高剂量实验组比较,在统计学上差异均有统计学意义(均P<0.05)。结论 槲皮素可通过抑制铁死亡途径改善高渗透压诱导的HCE-2细胞损伤。

Objective To investigate the effect of quercetin on HCE-2injury of human corneal epithelial cells induced by high osmotic pressure and its mechanism.Methods HCE-2 cells were randomly divided into control group(normal osmotic pressure),model group(high osmotic pressure),experimental-L group(high osmotic pressure+31.25μg·mL^(-1) quercetin),experimental-M group(high osmotic pressure+62.50 μg·mL^(-1) quercetin),experimental-H group(high osmotic pressure+125.00 μg·mL^(-1) quercetin),erastin group(high osmotic pressure+125.00 μg·mL^(-1) quercetin+30.00 μmol·L^(-1) iron death inducer erastin).Cell survival rate was detected by cell counting kit 8;reactive oxygen species(ROS) levels was detected by C11-BODIPY581/591 probe staining;glutathione(GSH) and malondialdehyde(MDA) levels were determined by kit method;the expression levels of glutathione peroxidase 4(GPX4),dihydrolactate dehydrogenase(DHODH) and ferroptosis suppressor protein 1(FSP1) were detected by real-time quantitative polymerase chain reaction and Western blot.Results The cell survival rates of control group,model group,experimental-H group and erastin group were(100.00±3.97) %,(50.05±5.83) %,(86.35±7.35) % and(58.32±4.66) %,respectively;ROS levels were1.00±0.09,2.45±0.16,1.19±0.05 and 2.09±0.30,respectively;GPX4 protein levels were 1.09±0.11,0.34±0.03,0.91±0.12 and 0.30±0.04,respectively;FSP1 protein levels were 0.92±0:06,0:25±0.03,0.89±0.07 and 0.39±0.07,respectively;DHODH protein levels were 0.89±0.11,0.31±0.04,0.86±0.11,0.41±0.04,respectively.Compared with model group,the above indexes in control group were statistically significant(all P <0.05);the differences between experimental-H group and model group were statistically significant(all P <0.05);the above indexes in erastin group were significantly different from those in experimental-H group(all P <0.05).Conclusion Quercetin can ameliorate HCE-2 cell damage induced by high osmotic pressure by inhibiting iron death pathway.