| Objective: To investigate the role of mitochondrial fission mediated by dynamin related protein1(Drp1) and oxidative stress mediated by reactive oxygen species involving in apoptosis of human renal tubular epithelial cells(HKC) induced by contrast media(CM) under the serum-free and hypoxia(HSF) condition, as well as the possible protective mechanisms of atorvastatin(ATO) in the process.Methods: Human renal tubular epithelial cells(HKC) were cultured and exposed to different concentrations 10, 20, 40, 80, 100, 120, 150, 200 mg I/ml of iohexol for 2 h, and exposed to iohexol(120 mg I/ml) for 1, 2, 4, 6, 8, 12 h, respectively, under the serum-free and hypoxia condition. Cells were incubated with iohexol(80 mg I/ml) for 2, 4, 6, 8, 12 h, respectively; and cells were incubated with different concentrations 10, 20, 40, 80, 120 mg I/ml of iohexol for 2 h, respectively. In the separate experiments, cells were incubated with different concentrations of ATO(10-10,10-9,10-8,10-7 mol/L) for 24 h, and then exposed to iohexol(120 mg I/ml) for 2 h under serum-free and hypoxia. Cells were incubated with ATO(10-7mol/L) for 6, 12, 24, 36 h, respectively; and then exposed to iohexol(120 mg I/ml) for 2 h under serum-free and hypoxia. Cells were incubated with ATO(10-7mol/L), and positive contrast group diphenylene iodonium(DPI) 10-5mol/L and N-acetylcysteine(NAC) 10-5 mol/L for 24 h, respectively; and then exposed to iohexol(120 mg I/ml) for 2 h under serum-free and hypoxia. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by TUNEL. The production of malondialdehyde(MDA) and reactive oxygen species(ROS) were detected by MDA and ROS kit. The relative m RNA expression of gp91 phox, p22 phox, Bax, Bcl-2 and caspase-3 were analyzed by reverse-transcription polymerase chain reaction(RT-PCR). The relative protein expression of Bax, Bcl-2, Drp1, Cyt c and caspase-3 were analyzed by Western-blotting.Results: MTT assay showed that, compared with normal group, the proliferation ofcells were not apparently inhibited in the group of 10, 20, 40 mg I/ml; but significantly inhibited in the group of 120, 150, 200 mg I/ml and 1, 2, 4, 6, 8, 12 h, and the group of HSF+CM; while pretreated with ATO, DPI, NAC, the proliferation of cells significantly increased(P<0.05). TUNEL test displayed that, the apoptosis of the group of HSF+CM was the seriousest one compared with the normal group(P<0.05), while ATO, DPI, NAC could improve the situation of cell apoptosis(P<0.05). The detection of MDA and ROS kit indicated that, CM could promote the production of MDA and ROS(P<0.05), while ATO, DPI, NAC could produce the opposite effects(P<0.05). RT-PCR test showed that, CM made the relative m RNA expression of gp91 phox, p22 phox, Bax and caspase-3 up-regulated(P<0.05), while Bcl-2 down-regulated. However, ATO could not only down-regulate the relative m RNA expression of gp91 phox, p22 phox, Bax and caspase-3, but also up-regulate the relative m RNA expression of Bcl-2(P<0.05). Western-blot test indicated that, CM could promote the protein expression of Drp1, which was related with concentration and time(P<0.05); ATO could suppress the protein expression of Drp1, which was related with concentration and time(P<0.05). CM could significantly up-regulate the protein expression of Bax, Drp1, Cyt c and caspase-3(P<0.05), while down-regulate Bcl-2 expression. However, ATO, DPI and NAC could significantly down-regulate the protein expression of Bax, Drp1, Cyt c and caspase-3(P<0.05), while up-regulate Bcl-2 expression(P<0.05).Conclusions: Under the condition of serum-free and hypoxia, iohexol induced the apoptosis of HKC might via Drp1 mediated mitochondrial fission, while ATO can ameliorate the apoptosis of HKC through inhibitting the expression of Drp1. Meanwhile, ROS derived from NADPH oxidase was involved in apoptosis of HKC cells induced by contrast media, while ATO could protect HKC through attenuating the expression of NADPH oxidase and it derived ROS related pathway. |
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