草酸钙晶体诱导人肾小管上皮细胞铁死亡的机制  

Mechanism of calcium oxalate crystal-induced ferroptosis in human renal tubular epithelial cell

作　　者：周凯 赵嘉闻 黎承杨[2] Zhou Kai;Zhao Jiawen;Li Chengyang(Deparment of Nephrology,Jinhua Peoples Hospital,Jinhua 32000,China;Department of Urology,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

机构地区：[1]金华市人民医院肾内科,金华321000 [2]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8期622-629,共8页Chinese Journal of Urology

摘　　要：目的探讨草酸钙(CaOx)晶体诱导人肾小管上皮细胞(HK-2细胞)发生铁死亡的机制。方法2021年3—9月使用CaOx晶体悬浊液干预HK-2细胞,构建HK-2-CaOx反应模型。设置CaOx晶体浓度分别为0、0.25.0.5、1.0、2.0、4.0、8.0mmol/L,干预HK-2细胞24h,干预完成后提取HK-2细胞蛋白。采用最佳干预浓度的CaOx晶体干预HK-2细胞,分别于干预后0、3、6、9、12、24、48h提取细胞蛋白。蛋白质印迹法检测细胞内铁死亡标志蛋白谷胱甘肽过氧化物酶4(GPX4)的表达情况。用铁死亡诱导剂爱拉斯汀(Erastin)和铁死亡抑制剂3-氨基4-环已基氨基苯甲酸乙酯(Fer-1)干预HK-2细胞以调控细胞内铁死亡水平。将HK-2细胞分为4组:正常对照组(NC组),无干预处理,单纯使用完全培养基培养;CaOx晶体刺激组(CaOx组),使用含4.0mmol/LCaOx晶体的完全培养基培养;CaOx晶体+Erastin处理组(CaOx+Erastin组),使用含10.0μmol/LErastin和4.0mmol/LCaOx晶体的完全培养基培养;CaOx晶体+Fer-1处理组(CaOx+Fer-1组),使用含1.0μmol/LFer-1和4.0mmol/LCaOx晶体的完全培养基培养。培养24h后,采用蛋白质印迹法和免疫荧光技术检测GPX4、长链脂酰辅酶A合成酶4(ACSL4)和溶质载体家族7成员11(SLC7A11)在HK-2细胞内的表达情况;检测HK-2细胞内谷胱甘肽含量;采用DCFH-DA荧光染色法观察HK-2细胞活性氧(ROS)表达情况。通过光学显微镜观察各组HK-2细胞内CaOx黏附情况,DAPI染色检测HK-2细胞核损伤情况。结果采用0、0.25、0.5、1.0、2.0、4.08.0mmol/L浓度CaOx晶体干预24h后,细胞中CPX4的表达量分别为5.67±1.05、5.60±0.02、4.99±0.94、4.82±0.93、4.50±0.70、4.14±0.53、0.97±0.53,4.0mmol/L组与0mmol/L组相比差异有统计学意义(P=0.026)。选用4.0mmol/L作为最佳浓度干预细胞,干预0、3、6、9、12、24、48h后细胞中CPX4的表达量分别为11.73±1.29、11.68±1.32、11.72±1.30、10.97±1.28、10.63±1.21、8.79±1.10、8.03±1.06,24 h组与Oh组相比差异有统计�Objective To investigate the role of ferroptosis in calcium oxalate(Calcium Oxalate,CaOx)crystal-induced injury of human renal tubular epithelial cells(HK-2 cells).Methods From March 2021 to September 2021,I used calcium oxalate crystal suspension to intervene HK-2 cells to build a HK-2-CaOx reaction model.Set the concentration gradient group and time gradient of calcium oxalate crystal intervention in HK-2 cells:7 groups of calcium oxalate crystals with different concentrations(0,0.25,0.5,1.0,2.0,4.0,8.0 mmol/L)were used to intervene HK-2 cells24 hours,the HK-2 cell protein was extracted after the intervention;HK-2 cells were intervened with calcium oxalate crystals at optimum concentration,and extract proteins at different time points(O,3,6,9,12,24,48 h)after intervention,the expression of intracellular ferroptosis marker protein glutathione peroxidase 4(GPX4)was detected by Western blot.Intervention of HK-2 cells with ferroptosis inducer Erastin and ferroptosis inhibitor ethyl 3-amino-4-cyclohexylaminobenzoate(Ferrostatin-1,Fer-1)to regulate intracellular ferroptosis Level.HK-2 cells were divided into 4 groups:normal control group(NC;no intervention treatment,cultured in complete medium only);calcium oxalate crystal stimulation group(CaOx;cultured in complete medium containing 4.O mmol/L CaOx crystals);calcium oxalate crystals+erastine treatment group(CaOx+Erastin;cultured in complete medium containing 10.Oμmol/L erastine and 4.0 mmol/L calcium oxalate crystals);calcium oxalate crystals+Fer-1 Treatment group(CaOx+Fer-l;cultured in complete medium containing 1.Oμmol/L Fer-1 and 4.O mmol/L calcium oxalate crystals).After 24 hours,the expression of ferroptosis-related protein GPX4,long-chain fatty acyl-CoA synthase 4(ACSL4)and solute carrier family 7 member 11(SLC7A11)in HK-2 cells was analyzed by western blot and immunofluorescence techniques;the content of glutathione in HK-2 cells was detected;DCFH-DA fluorescence staining was used to observe the expression of reactive oxygen species(ROS)in HK-2 cells.The ad

关 键 词：活性氧 铁铁死亡 细胞损伤 草酸钙晶体 人肾小管上皮细胞 

分 类 号：R692.6[医药卫生—泌尿科学]