The Role Of Green Autofluorescence In Non-invasive Assessment And Prediction Of Oxidative Stress Injury In HACAT Cells

Oxidative stress plays critical roles in aging and multiple diseases including cerebra ischemia myocardial ischemia,Alzheimer’s disease and diabetes.Prevention of oxidative damage has been an important strategy for both decreasing the pathological changes of multiple diseases and slowing the aging process.It is of both critical theoretical and clinical significance to search for non-invasive approaches for evaluating oxidative damage of cells and tissues.Cellular models are highly valuable for studying both the mechanisms underlying oxidative damage and the antioxidant capacity of molecules.Currently,there are two major approaches for monitoring intracellular oxidative stress:The non-invasive approaches require applications of such fluorescent probes as dichlorofluorescein（DCF）and dihydroethidium（DHE）;while the other approaches used for determining antioxidant capacity of cells and assessing oxidative damage require cell lysis.There has been no non-invasive approaches that can evaluate oxidative damage of energy metabolism.In our current study,we tested our hypothesis that the green autofluorescence（AF）intensity of cells may be used for non-invasive evaluation of oxidative stress-induced impairments of cellular energy metabolism and predicting oxidative cellular damage.Our study found that H₂O₂ dose-dependently increased the green AF of Ha Ca T cells–a human keratinocyte cell line-at non-nuclear regions assessed at 1 hr after the H₂O₂exposures,while H₂O₂ did not affect the green AF of other cell types tested in our study,including PC12 cells and BV2 microglia.We further found that the increases in the AF of Ha Ca T cells are negatively correlated with the ATP levels,the mitochondrial membrane potential and the intracellular levels of NAD⁺in the H₂O₂-treated cells 1 hr after the H₂O₂ exposures.The AF intensity was also highly correlated with the H₂O₂-induced increases in early-stage apoptosis,late-stage apoptosis and necrosis assessed at 18 hrs after the H₂O₂ exposures.Collectively,our study has suggested that green AF intensity may become the first endogenous marker for non-invasive evaluation and prediction of oxidative damage selectively for such cell types as Ha Ca T cells.Compared with traditional approaches,our approach has distinct merits including non-invasiveness,high efficiency and minimal cost.