镉通过Nrf2/HO-1通路诱导猪肾PK-15细胞铁死亡

Cadmium induces ferroptosis by modulating Nrf2/HO-1 pathway in PK-15 cells

镉(Cd)是非必需且难降解的重金属元素之一,镉蓄积可导致肾损伤。本研究旨在揭示镉暴露诱导猪肾PK-15细胞铁死亡的作用机制。首先,CCK-8法检测梯度浓度氯化镉(CdCl_(2))对PK-15细胞活力的影响,以筛选出合适的工作浓度。其次,用10μmol/L CdCl_(2)处理PK-15细胞3、6和12 h,通过比色法检测细胞上清中乳酸脱氢酶(LDH)和细胞中丙二醛(MDA)的含量,Western blot检测铁死亡相关蛋白的表达水平,荧光探针法检测细胞中亚铁离子(Fe^(2+))含量。最后,用10μmol/L的HO-1抑制剂锌原卟啉(ZnPP)预处理PK-15细胞6 h,再用10μmol/L CdCl_(2)处理PK-15细胞12 h,通过相差显微镜观察细胞形态,Western blot检测铁死亡相关蛋白表达水平。结果显示,CdCl_(2)处理后细胞活力下降,LDH、MDA和Fe^(2+)水平极显著升高(P<0.01),Nrf2、HO-1和ALOX5蛋白表达水平极显著升高(P<0.01),FTH1蛋白表达水平极显著下降(P<0.01)。与CdCl_(2)处理组相比,ZnPP可以显著改善CdCl_(2)处理诱导的猪肾PK-15细胞铁死亡。ZnPP预处理组细胞形态明显好转,Nrf2、HO-1和ALOX5蛋白表达水平极显著下降(P<0.01),FTH1蛋白表达水平极显著升高(P<0.01)。结果表明,镉通过Nrf2/HO-1通路引发细胞铁过载和脂质过氧化,诱导猪肾PK-15细胞铁死亡。

Cadmium is a non-essential,non-degradable heavy metal element,the accumulation of cadmium can cause kidney damage.The purpose of this study was to reveal the ferroptosis mechanism induced by cadmium exposure in porcine kidney PK-15 cells.First of all,cell viability of gradient concentration of cadmium chloride(CdCl_(2))on PK-15 cells was detected by CCK-8 method to screen suitable work concentration of CdCl_(2).Secondly,PK-15 cells were treated with 10μmol/L CdCl2 for 3,6 and 12 h.The contents of lactate dehydrogenase(LDH)malondialdehyde(MDA)were detected by colorimetry,the expression of ferroptosis related protein were detected by Western blot,and the content of ferrous ion(Fe^(2+))was detected by fluorescence probe.Finally,PK-15cells were pretreated with 10μmol/L HO-1 inhibitor zinc protoporphyrin(ZnPP)for 6 h,and then treated with 10μmol/L CdCl2 for 12 h.The morphology of PK-15 cells was observed by phase contrast microscope and the expression of ferroptosis related proteins was detected by Western blot.The results showed that CdCl_(2)treatment caused a significant decrease in cell viability.The levels of LDH,MDA and Fe^(2+),the protein levels of Nrf2,HO-1 and ALOX5,and the expression level of FTH1 protein were significantly decreased in CdCl_(2)treatment cells(P<0.01).ZnPP could significantly improve the ferroptosis of PK-15 cells induced by CdCl_(2)treatment.Compared with CdCl_(2)treatment cells,the cell morphology was significantly improved,the protein levels of Nrf2,HO-1 and ALOX5 protein were significantly decreased,and the protein levels of FTH1 protein were significantly increased in ZnPP pretreatment group(P<0.01).The results showed that cadmium induced iron overload and lipid peroxidation,which result in ferroptosis in PK-15 cells through Nrf2/HO-1 pathway.