基于Keap1/Nrf2/ARE信号通路探讨高良姜总黄酮对铅诱导HK-2细胞损伤的保护作用

Protective effects of total flavonoids from Alpinia officinarum on lead-induced HK-2 cell injury via Keap1/Nrf2/ARE signaling pathway

目的 探讨高良姜总黄酮对铅(Pb)诱导人肾皮质近曲小管上皮细胞(HK-2)细胞损伤的保护作用。方法 体外培养HK-2细胞,MTT法检测不同质量浓度高良姜总黄酮和Pb对HK-2细胞活力的影响。设置对照组、高良姜总黄酮对照组、模型组和高良姜总黄酮组,除对照组和高良姜总黄酮对照组外各组均给予200μmol/L Pb诱导细胞损伤,同时高良姜总黄酮对照组和高良姜总黄酮组给予100μg/mL高良姜总黄酮干预24 h。通过Hoechst 33258荧光染色法联合annexin V-FITC/PI双标记流式细胞术检测细胞凋亡情况,荧光显微镜法观察Pb诱导及高良姜总黄酮干预对细胞内ROS水平的影响,试剂盒法检测细胞内氧化应激指标ROS、MDA、GSH水平和GSH-Px、CAT、SOD活性,ELISA法检测细胞培养上清液IL-1β、IL-6、TNF-α水平,Western bolt法检测细胞Keap1/Nrf2/HO-1信号通路相关蛋白及caspase-3蛋白表达。结果 与对照组比较,高良姜总黄酮(12.5~200μg/mL)对HK-2细胞活力没有显著影响。与模型组比较,高良姜总黄酮可减少Pb诱导HK-2细胞的凋亡(P<0.05),减轻细胞皱缩等细胞凋亡形态学变化,上调细胞内SOD、CAT、GSH-Px活性及GSH水平(P<0.05),降低细胞内MDA、ROS水平(P<0.05),上调Keap1/Nrf2/HO-1信号通路相关蛋白及caspase-3蛋白表达(P<0.05)。结论 高良姜总黄酮可能通过调控Keap1/Nrf2/ARE信号通路相关蛋白表达,对Pb诱导HK-2细胞损伤起保护作用。

AIM To investigate the protective effects of total flavonoids from Alpinia officinarum Hance(TFAO)on lead-induced HK-2 cell injury.METHODS HK-2 cells cultured in vitro had their viability detected by MTT assay under the influence of TFAO or Pb.The control group,the TFAO control group,the model group,and the TFAO group were set up and exposed to 200μmol/L Pb to induce cell injury in the groups except for the two control groups,meanwhile 100μg/mL TFAO was given to the two TFAO groups for 24 h.After Pb-induction,the HK-2 cells had their apoptosis observed by Hoechst 33258 fluorescence staining combined with annexin V-FITC/PI double labeling flow cytometry;their intracellular ROS levels under Pb induction and TFAO intervention observed by fluorescence microscopy;their levels of ROS,MDA and GSH and the activities of GSH-Px,CAT and SOD determined by kit method;their levels of IL-1β,IL-6 and TNF-αin the supernatant of cell culture detected by ELISA;and their expressions of Keap1/Nrf2/HO-1 signaling pathway related proteins and caspase-3 protein detected by Western blot as well.RESULTS Compared with the control group,TFAO(12.5-200μg/mL)intervention showed no significant effect on HK-2 cell viability.Compared with the model group,the TFAO group shared less apoptosis of Pb-induced HK-2 cells(P<0.05);alleviation of a series of apoptotic morphological changes including cell collapse,up-regulated activities of SOD,CAT and GSH-Px enzymes and GSH levels(P<0.05);lower levels of MDA and ROS(P<0.05);up-regulated expressions of Keap1/Nrf2/HO-1 signaling pathway related proteins and caspase-3 protein(P<0.05).CONCLUSION TFAO may play a protective role against Pb-induced HK-2 cell damage by regulating the expressions of proteins related to Keap1/Nrf2/ARE signaling pathway.