Live Cell Imaging Observing Glutathione S-transferase MU 5 Nuclear Translocation in Human Bronchus Epithelial Induced by Tumor Necrosis Factor Alpha
ZENG Yanting
ZHANG Jiake
YUAN Weifeng
HE Wenyuan
XU Hong
LI Li
Abstract：Objective To establish cell inflammation model of human bronchus epithelial(16HBE)induced by tumor necrosis factor alpha(TNF-α),observe the dynamics of glutathione S-transferase MU 5(GSTM5)nuclear translo-cation and investigate the effects of GSTM5 on inflammation and oxidative stress.Methods After 16HBE cells were transfected by GSTM5 eukaryotic expression vector and small interfering RNA(siRNA),then followed by TNF-α treat-ment.The experiments were divided into seven groups:16HBE group,16HBE+TNF-α group(TNF-α stimulation),16HBE+con083+TNF-α group(transfected with con083-GFP recombinant plasmid,TNF-α stimulation),16HBE+GSTM5 group(transfected with GV230-GSTM5-GFP),16HBE+GSTM5+TNF-α group(transfected with GV230-GSTM5-GFP,TNF-α stimulation),16HBE+siGSTM5+TNF-α group(adding silencing GSTM5 siRNA,TNF-αstimulation)and 16HBE+NCsiRNA+TNF-α group(adding negative control siRNA,TNF-α stimulation).The dynamic nuclear translocation of GSTM5 was ob-served by Western blot analysis,live cell imaging and confocal microscopy.Cell viability of 16HBE was assessed by methylthiazolyldiphenyl-tetrazolium bromide(MTT),while oxidative stress levels were evaluated by malondialdehyde(MDA)and total antioxidative capacity(T-AOC).The concentrations of interleukin 6(IL-6)and interleukin 10(IL-10)in the cell supernatant were determined by enzyme-linked immunosorbent assay(ELISA).Results After 16HBE cells were treated with TNF-α for 0.5 h,green fluorescence in the nucleus enhanced,indicating that GSTM5 translocated into the nuclear of 16HBE cells.TNF-α could significantly de-crease 16HBE cell viability and T-AOC,and GSTM5 could reduce IL-6,IL-10 and MDA levels.IL-6 and IL-10 in 16HBE+TNF-α group was significantly higher than those in the 16HBE+GSTM5+TNF-α group(P＜0.001).Conclusion The imbalance of inflammation and oxidation induced by TNF-α stimulation can induce GSTM5 nuclear translocation in 16HBE,and GSTM5 nuclear translocation induced by TNF-α is recorded by live cell imaging system dynamically.
Keywords：Glutathione S-transferase MU 5Human bronchus epithelialNuclear translocationTumor necrosis factor alphaLive cell imaging
Publication Date：2025-05-28
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 371-377 )