王不留行黄酮苷通过抑制铁死亡减轻阿米卡星导致的肾小管上皮细胞损伤  

Effects of vaccarin on amikacin-induced damage to renal tubular epithelial cells

作　　者：郑松 储超群 岳琳 黄申卓凡 温家根 Zheng Song;Chu Chaoqun;Yue Lin;Huangshen Zhuofan;Wen Jiagen(Dept of Tuberculosis,Anhui Chest Hospital,Hefei 230032;Dept of Basic and Clinical Pharmacology,School of Pharmacy,Anhui Medical University,Hefei 230032)

机构地区：[1]安徽省胸科医院结核科,合肥230032 [2]安徽医科大学药学院基础与临床药理学教研室,合肥230032

出　　处：《安徽医科大学学报》2024年第4期653-659,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年项目(编号:81800606);安徽省转化医学研究院科研基金项目(编号:2022zhyx-C08);安徽医科大学药学创新基金科研项目(编号:YXCX202203)。

摘　　要：目的探讨建立阿米卡星(AKN)体外肾损伤模型的方法,研究在AKN体外肾损伤模型中王不留行黄酮苷(VA)的保护作用及机制。方法体外培养人肾小管上皮细胞(HK-2),孵育不同浓度的AKN或VA,MTT法检测细胞活力,确定药物浓度;采用二氢乙锭(DHE)探针和谷胱甘肽(GSH)、丙二醛(MDA)检测试剂盒检测细胞内氧化应激状态的变化;提取总RNA,实时荧光定量PCR(RT-qPCR)检测肾损伤分子-1(KIM-1)和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)基因表达的变化;提取总蛋白,Western blot检测铁死亡相关的蛋白溶质载体蛋白家族47成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)的水平。结果高浓度AKN在体外可以显著引起HK-2细胞活力的下降,AKN对HK-2细胞的半数抑制浓度(IC50)为(5.74±0.47)mmol/L。25~100μmol/L的VA可以提高AKN刺激后的HK-2细胞活力(P<0.05)。AKN(4 mmol/L)处理后,KIM-1和NGAL的mRNA表达水平显著高于阴性对照(NC)组(P<0.001);VA(50μmol/L)可以显著降低KIM-1(P<0.01)和NGAL(P<0.05)的mRNA表达水平。AKN处理3 h后DHE染色强度升高但差异无统计学意义,6~24 h后DHE染色强度显著大于0 h组(P<0.01)。此外,AKN处理6~24 h后MDA水平显著上升,GSH水平显著下降,差异均有统计学意义(P<0.05)。AKN刺激6~24 h后,铁死亡相关蛋白SLC7A11和GPX4表达均明显下降(P<0.001)。VA同时孵育24 h,显著逆转DHE染色、GSH和MDA水平的变化及SLC7A11和GPX4蛋白的下降(P<0.001)。结论该研究通过高浓度AKN体外刺激HK-2细胞建立AKN体外肾损伤模型,并发现VA可能通过抑制过氧化应激相关的铁死亡途径减轻AKN导致的肾小管细胞损伤。Objective To establish an in vitro renal injury model of amikacin(AKN)and investigate the protective effect and mechanism of vaccarin(VA)in the AKN-induced in vitro renal injury model.Methods Human renal tubular epithelial cells(HK-2)were cultured in vitro and incubated with different drugs of AKN or/and VA to determine the optimal drug concentration based on cell viability tested by MTT.The changes in intracellular oxidative stress were assessed using the dihydroethidium(DHE)probe and malondialdehyde(MDA)/glutathione(GSH)assay kits at different time points.Total RNA was extracted,and RT-qPCR was performed to detect the changes in the gene expression of kidney injury molecule-1(KIM-1)and neutropil gelatinase-associated lipocalin(NGAL).Western blot analysis was performed to detect the levels of ferroptosis-related markers solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)in HK-2 cell lysis.Results High concentrations of AKN significantly decreased the viability of HK-2 cells in vitro,with a half maximal inhibitory concentration(IC50)of(5.74±0.47)mmol/L.VA at concentrations of 25-100μmol/L increased the viability of AKN-stimulated HK-2 cells(P<0.05).After treatment with AKN(4 mmol/L),the mRNA expression levels of KIM-1 and NGAL were significantly higher than those of the negative control(NC)group(P<0.001).VA(50μmol/L)significantly reduced the mRNA expression levels of KIM-1(P<0.01)and NGAL(P<0.05).The intensity of DHE staining increased after 3 hours of AKN treatment,but the difference was not statistically significant.However,the intensity of DHE staining was significantly higher in the 6-24 hours group compared to the 0-hour group(P<0.01).Furthermore,MDA levels significantly increased,while GSH levels significantly decreased after 6-24 hours of AKN treatment,with statistically significant differences(P<0.05).After 6-24 hours of AKN stimulation,the ferroptosis-related proteins SLC7A11 and GPX4 both significantly decreased(P<0.001).Co-incubation with VA for 24 hours effectively reversed

关 键 词：阿米卡星 肾小管上皮细胞 王不留行黄酮苷 氧化应激 铁死亡 药物性肾损伤 

分 类 号：R965.1[医药卫生—药理学]