高糖通过激活活性氧介导的NF-κB信号通路诱导HK-2人肾小管上皮细胞转分化

High glucose induces transdifferentiation of HK-2 human renal tubular epithelial cells by activating reactive oxygen species-mediated NF-kappa B signaling pathway

目的基于活性氧/核因子κB(ROS/NF-κB)信号通路探讨高糖诱导的人肾小管上皮细胞转分化机制。方法 HK-2正常人近端肾小管上皮细胞随机分为空白对照组、渗透压对照组、高糖组、吡咯烷二硫氨基甲酸(PDTC)组。用相差显微镜观察细胞形态、噻唑蓝(MTT)法检测细胞活性,流式细胞术检测细胞内ROS含量, ELISA检测丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;Western blot法检测细胞NF-κBp65、磷酸化的NF-κB抑制蛋白α(p-IκBα)和IκB激酶α(IKKα)、单核细胞趋化蛋白1(MCP-1)和细胞间黏附分子1(ICAM-1)的蛋白水平;免疫细胞化学法检测细胞NF-κBp65、上皮型钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)表达。结果高糖组细胞变成长梭形或不规则形,边缘可呈现放射状,细胞间隙变大,折光度下降,排列不整齐。同时细胞活性随处理时间的延长不断下降, PDTC组细胞活性较高糖组高。与空白对照组相比,高糖组ROS及MDA含量增加、SOD活性下降, PDTC组ROS及MDA含量较高糖组减少、SOD活性较高糖组增强。与空白组比较,高糖组NF-κBp65、p-IκBα、IKKα、MCP-1、ICAM-1蛋白水平增加;与高糖组比较PDTC组以上蛋白水平降低。与空白组比较,高糖组NF-κBp65及α-SMA的阳性细胞所占比率升高、E-cadherin的阳性细胞所占比率降低;与高糖组比较, PDTC组NF-κBp65及α-SMA的阳性细胞所占比率降低、E-cadherin的阳性细胞比率升高。结论高糖可以诱导HK-2细胞发生上皮间质转化, ROS介导的NF-κB信号通路的激活参与以上进程, PDTC可阻断该过程。

Objective To explore the mechanism of transdifferentiation of human renal tubular epithelial cells induced by high glucose based on reactive oxygen species(ROS)/NF-κB signaling pathway. Methods HK-2 normal human proximal tubular epithelial cells were randomly divided into blank control group, osmotic pressure control group, high glucose group and pyrrolidine dithiocarbamate(PDTC) group. Cell morphology was observed by phase contrast microscope. Cell viability was measured by MTT assay. Flow cytometry was used to detect intracellular ROS content. Malondialdehyde(MDA) content and superoxide dismutase activity were tested by ELISA. Western blot analysis was used to examine the protein levels of NF-κBp65, phosphorylated IκBα(p-IκBα) and IKKα, monocyte chemoattractant protein-1(MCP-1) and intercellular adhesion molecule-1(ICAM-1). The expressions of NF-κBp65, epithelial cadherin(E-cadherin) and alpha smooth muscle actin(α-SMA) were assessed by immunocytochemistry. Results In high glucose group, the cells became spindle-shaped or irregular, with radial edges, enlarged intercellular space, decreased refractive index and irregular arrangement. At the same time, the cell activity decreased with the prolongation of treatment time, and the cell activity in PDTC group was higher than that in high glucose group. Compared with the blank control group, the content of ROS and MDA increased and the activity of SOD decreased in the high glucose group, while the content of ROS and MDA decreased and the activity of SOD increased in the PDTC group compared with the high glucose group. Compared with the blank group, the protein levels of NF-κBp65, p-IκBα, IKKα, MCP-1 and ICAM-1 increased in the high glucose group, while the protein levels above decreased in the PDTC group compared with the high glucose group. Compared with the blank group, the proportion of positive cells of NF-κBp65 and α-SMA increased and the proportion of positive cells of E-cadherin decreased in the high glucose group. Compared with the high glucose group, the proportion of positive cells of NF-κBp65 and α-SMA decreased and the proportion of positive cells of E-cadherin increased in the PDTC group. Conclusion High glucose can induce epithelial-mesenchymal transition in HK-2 cells. Activation of NF-κB signaling pathway mediated by ROS participates in the above process, which can be blocked by PDTC.