益母草碱激活p62/Nrf2/HO-1信号通路抑制肾小管上皮细胞铁死亡的作用机制

Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway

为探讨益母草碱(leonurine,Leo)对erastin诱导的人肾小管上皮细胞(HK-2细胞)铁死亡的保护作用及其潜在机制,在体外构建erastin诱导的HK-2细胞铁死亡模型,检测Leo对铁死亡细胞活力、细胞状态、铁死亡相关指标及相关信号通路蛋白的影响。首先,体外培养HK-2细胞,CCK-8法检测10、20、40、60、80、100μmol·L^(-1)的Leo对正常HK-2细胞活性的影响,以此确定Leo的安全给药剂量范围;用铁死亡经典诱导剂erastin诱导铁死亡细胞模型并筛选出合适的造模浓度;CCK-8法检测Leo(20、40、80μmol·L^(-1))及阳性药铁死亡抑制剂ferrostatin-1(Fer-1,1、2μmol·L^(-1))对铁死亡模型细胞活性的影响,同时在相差显微镜下观察细胞形态学的变化。其次,通过Western blot法检测核转录因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)的入核激活情况,并筛选后续机制研究的最佳浓度;进一步用透射电镜观察铁死亡发生的特征性显微形态学改变,应用流式细胞术检测活性氧(reactive oxygen species,ROS)变化,使用还原型谷胱甘肽(glutathione,GSH)测定试剂盒检测GSH含量。最后,采用Western blot法定量检测各组谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、通路蛋白p62及Nrf2下游蛋白血红素加氧酶1(heme oxygenase 1,HO-1)的表达,探讨Leo对HK-2铁死亡调控作用的具体机制。结果显示,Leo在浓度为10~100μmol·L^(-1)时对正常HK-2细胞活性无影响,表明该浓度范围的药物对细胞无明显毒副作用;HK-2细胞活性随着铁死亡诱导剂erastin浓度的增高而降低,5μmol·L^(-1) erastin能够显著诱导HK-2细胞发生铁死亡;与模型组相比,20、40、80μmol·L^(-1)的Leo呈剂量依赖性提高HK-2细胞活性,并且能有效改善细胞形态;80μmol·L^(-1) Leo能显著上调HK-2细胞核中Nrf2的表达。进一步研究表明,Leo可以显著改善erastin造成的铁死亡特征性显微结构损伤,抑制细胞内ROS的释放,升高GSH含量,促进Nrf2向细胞核内移位,增强GPX4,显著上调p62、HO-1蛋白的表达。综上所述,Leo对erastin诱导的人肾小管上皮细胞铁死亡具有保护作用,其机制可能是通过激活p62/Nrf2/HO-1信号通路实现抗氧化应激从而抑制铁死亡。

To investigate the protective effect and the potential mechanism of leonurine(Leo)against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells),an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins.HK-2 cells were cultured in vitro,and the effects of Leo on the viability of HK-2 cells at 10,20,40,60,80 and 100μmol·L~(-1)were examined by CCK-8 assay to determine the safe dose range of Leo administration.A ferroptosis cell model was induced by erastin,a common ferroptosis inducer,and the appropriate concentrations were screened.CCK-8 assay was used to detect the effects of Leo(20,40,80μmol·L~(-1))and positive drug ferrostatin-1(Fer-1,1,2μmol·L~(-1))on the viability of ferroptosis model cells,and the changes of cell morphology were observed by phase contrast microscopy.Then,the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2)activation,and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis.Flow cytometry was performed to detect reactive oxygen species(ROS),and the level of glutathione(GSH)was measured using a GSH assay kit.The expressions of glutathione peroxidase 4(GPX4),p62,and heme oxygenase 1(HO-1)in each group were quantified by Western blot.Results showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100μmol·L~(-1).The viability of HK-2 cells decreased as the concentration of erastin increased,and 5μmol·L~(-1)erastin significantly induced ferroptosis in the cells.Compared with the model group,Leo dose-dependently increased cell via-bility and improved cell morphology,and 80μmol·L~(-1)Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus.Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin,inhibited the release of intracellular ROS,elevated GSH and GPX4,promoted the nuclear translocation of Nrf2,and significantly upregulated the expression of p62 and HO-1 proteins.In conclusion,Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells,which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.