左卡尼汀对草酸钙诱导肾小管上皮细胞铁死亡的影响  

Effect of L-carnitine on ferroptosis in renal tubular epithelial cells induced by calcium oxalate

作　　者：黄默然 赵嘉闻 黎承杨[1] Huang Moran;Zhao Jiawen;Li Chengyang(Department of Urology,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院泌尿外科,南宁530021

出　　处：《中华泌尿外科杂志》2023年第4期292-300,共9页Chinese Journal of Urology

基　　金：国家自然科学基金(81660125)。

摘　　要：目的探讨左卡尼汀对草酸钙诱导肾小管上皮细胞(HK-2)铁死亡的影响。方法采用蛋白质印迹法检测不同浓度(0、2、4、8 mmol/L)草酸钙对HK-2细胞铁死亡相关蛋白长链脂酰辅酶A合成酶4(ACSL4)、胱氨酸转运蛋白系统(XCT)和谷胱甘肽过氧化物酶4(GPX4)表达的影响, 筛选实验最适的草酸钙浓度。将HK-2细胞分为4组:①对照组, 细胞贴壁后用正常培养基培养12 h, 然后继续用正常培养基;②左卡尼汀组, 细胞贴壁后用含5 mmol/L左卡尼汀的培养基预处理12 h, 然后更换为含有5 mmol/L左卡尼汀的培养基;③草酸钙组, 细胞贴壁后用正常培养基培养12 h, 然后更换为含有最适浓度草酸钙的培养基;④草酸钙+左卡尼汀组, 细胞贴壁后用含5 mmol/L左卡尼汀的培养基预处理12 h, 然后更换为含有5 mmol/L左卡尼汀和最适浓度草酸钙的培养基。4组更换培养基后继续培养24 h, 收集细胞或上清液, 采用蛋白质印迹法检测铁死亡相关蛋白醌氧化还原酶(NQO1)、ACSL4、XCT、GPX4蛋白表达水平。采用相应试剂盒分别检测超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛水平;活性氧试剂盒检测细胞活性氧水平;二价铁离子探针检测细胞内铁离子蓄积情况。采用乳酸脱氢酶(LDH)试剂盒检测细胞损伤程度。采用透射电镜观察线粒体损伤情况。对细胞行DAPI染色观察细胞核损伤情况;行苏木素染色观察草酸钙晶体黏附情况。结果蛋白质印迹法检测结果显示, 0、2、4、8 mmol/L草酸钙组的ACSL4蛋白表达分别为0.37±0.16、0.68±0.16、0.73±0.09、0.89±0.03, XCT蛋白表达分别为1.11±0.10、0.91±0.14、0.83±0.09、0.80±0.07, GPX4蛋白表达分别为1.23±0.13、0.99±0.17、0.81±0.05、0.72±0.06, 与0 mmol/L组相比, 2、4、8 mmol/L组ACSL4蛋白表达升高, XCT、GPX4蛋白表达降低, 差异均有统计学意义(P<0.05), 其中4 mmol/L组与0 mmol/L组比较差异更显著(P<0.01), 故将4 mmol/L作为最适�Objective To investigate the effect of L-carnitine on calcium oxalate-induced ferroptosis in renal tubular epithelial cells(HK-2).Methods The effects of calcium oxalate(0,2,4 and 8 mmol/L)on the expression of ferroptosis-related protein long chain fatty acyl-CoA synthetase 4(ACSL4),cystine/glutamate transporter(XCT)and glutathione peroxidase 4(GPX4)in HK-2 cells were detected by Western blotting.The experiment was then divided into four groups:control group,cells were cultured in normal medium for 12 hours,then continued to use normal medium;②L-carnitine group,cells were pretreated with medium containing 5mmol/L L-carnitine for 12 hours,then changed to medium containing 5mmol/L L-carnitine;calcium oxalate group,cells were cultured in normal medium for 12 hours,and then replaced with medium containing 4 mmol/L calcium oxalate;calcium oxalate+L-carnitine group,the cells were pretreated with medium containing 5mmol/L L-carnitine for 12 h,and then replaced with 5mmol/L L-carnitine and 4mmol/L calcium oxalate medium.After changing the culture medium for 24 hours,the cells or supernatants were collected,and the expression levels of ferroptosis-related protcin quinone oxidoreductase(NQO1),ACSL4,XCT and CPX4 were detected by Western blotting.The levels of superoxide dismutase(SOD),glutathione(CSH)and malondialdehyde were detected by corresponding kit,and the level of reactive oxygen species in cells was detected by reactive oxygen species kit.Results The results of Western blotting showed that the expression of ACSL4 protein in 0,2,4,8 mmol/L calcium oxalate was 0.37±0.16,0.68±0.16,0.73±0.09,0.89±0.03 respectively.The expression of XCT protein was 1.11±0.10,0.91±0.14,0.83±0.09,0.80±0.07,respectively.The expression of GPX4 protein was 1.23±0.13,0.99±0.17,0.81±0.05,0.72±0.06,respectively.Compared with Ommol/L group,the expression of ACSL4 protein increased and the expression of XCT and GPX4 decreased in 2,4 and 8 mmol/L groups,and the difference was more significant between 4 mmol/L group and O mmol/L group.S

关 键 词：草酸钙 左卡尼汀 肾小管上皮细胞 氧化应激 铁死亡 

分 类 号：R692[医药卫生—泌尿科学]