Effect of interferon-γ combined with tumor necrosis factor-related apoptosis-inducing ligand on programmed necrosis of HaCaT cells and its mechanisms / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the inductive effect of interferon-γ(IFN-γ) combined with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on programmed necrosis of the human immortalized keratinocyte cell line HaCaT,and to explore its mechanisms.Methods In vitro cultured HaCaT cells were divided into several groupsnegative control group receiving no treatment,IFN-γ group treated with 50 μg/L IFN-γ,TRAIL group treated with 4 μg/L TRAIL,and cytokine combination group treated with 50 μg/L IFN-γ and 4 μg/L TRAIL or zVAD combination group pretreated with 40 μmo/L zVAD for 1 hour followed by the treatment with 50 μg/L IFN-γand 4 μg/L TRAIL.After 48-hour treatment,the morphology of HaCaT cells were observed under a light microscope,methyl-thiazolyl-tetrazolium assay was performed to evaluate the inhibitory effect of the treatment on the proliferation of HaCaT cells,and double staining flow cytometry to detect the necrosis of HaCaT cells.Meanwhile,real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of receptor interaction protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL),Western blot analysis to determine the expression of RIP1,RIP3,MLKL proteins and their phosphorylated forms (pRIP1,pRIP3,pMLKL),immunofluorescent staining to observe the distribution of pRIP3 and pMLKL in HaCaT cells,and the level of reactive oxygen species (ROS) in HaCaT cells in the above groups was detected by the fluorescence probe DCFH-DA.Statistical analysis was carried out with SPSS 22 software by using one-way analysis of variance (ANOVA) for comparing indices among different groups,and least significant difference (LSD)-t test for multiple comparisons.Results After 48-hour treatment,HaCaT cells in the cytokine combination group and zVAD combination group showed necrosis-like morphologic features.Methyl-thiazolyl-tetrazoliumassay revealed significant differences in the survival rate of HaCaT cells among the IFN-γgroup,TRAIL group,cytokine combination group,zVAD combination group and negative control group (73.16％ ± 5.71％,81.46％ ± 4.68％,72.18％ ± 2.93％,69.67％ ± 3.24％ and 100％,respectively;F =24.34,P ＜ 0.001).The necrosis rate of HaCaT cells was notably higher in the cytokine combination group and zVAD combination group (9.86％ ± 1.31％,10.33％ ± 2.16％,respectively) than in the negative control group (5.26％ ± 0.91％,t =4.61,5.07,respectively,both P ＜ 0.05).qPCR revealed that the mRNA expression of RIP3 and MLKL significantly increased in the cytokine combination group and zVAD combination group compared with the negative control group (tRIP3 =0.99,1.84,tMLKL =1.51,2.17,respectively,all P ＜ 0.05).Western blot analysis suggested that the protein expression of RIP1,RIP3,MLKL,pRIP1,pRIP3 and pMLKL significantly increased in the cytokine combination group compared with the negative control group (all P ＜ 0.05),and the zVAD combination group showed significantly decreased caspase 8 expression and increased expression of the above proteins compared with the cytokine combination group.Fluorescence microscopy showed that enhanced green dot-like or clump-like fluorescent spots (representing pRIP3) could be observed in the cytoplasm,and red fluorescent spots (representing pMLKL) could be seen on the cell membrane in the cytokine combination group.The average fluorescence intensity of ROS was significantly higher in the cytokine combination group than in the negative control group (t =702.00,P ＜ 0.05).Conclusion IFN-γcombined with TRAIL can induce the programmed necrosis of HaCaT cells with increased level of ROS.