Determining the Translocation of Antioxidant Activating Transcription Factors

Translocation is a subcellular process in which activated proteins are transported into the cell nucleus as part of a signal pathway to modify cell function in response to a signaling event or condition. Transcription factors are proteins involved in the process of converting, or transcribing, DNA into RNA. NRF2 is one type of transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. In this study, scientists attempted to determine if varying concentrations of ASEA Redox Supplement placed in physical contact with living cells activates translocation of transcription factors (NRF2) associated with increased expression of antioxidants in living human endothelial cells.

Additional analysis was performed to verify the appearance of the transcription factors by western blot analysis.

Study Protocol

Human lung microvascular endothelial cell (HMVEC-L) cultures were exposed to a high concentration (5 – 20%) and a low concentration (1%) of ASEA Redox Supplement and analyzed in conjunction with cultures exposed to a phosphate buffered saline solution (PBS) as a negative control. At time points of 30, 60, 90, and 120 minutes, cell samples from each of the cultures were placed under a fluorescent microscope. The cultures were stained with a fluorescent dye designed to tag the NRF2 transcription factor along with the fluorescent nuclear stain that aids the computer software to find the nuclei. Automated imaging was used to determine the degree of nuclear accumulation of NRF2 via fluorescent analysis over several cells. NRF2 regulates the transcription of some phase II antioxidant defense enzymes and raises the possibility that additional antioxidant defense enzymes, such as glutathione transferase, may be expressed through exposure to ASEA Redox Supplement. The accumulation of NRF2 into the nucleus, as seen visually in the microscope images, is an indicator of increased antioxidant expression in the cells.

Results Summary

Results of the examination suggest that ASEA Redox Supplement at a lower concentration induces a 20 – 30% increase in the nuclear translocation of the NRF2 transcription factor in the cells over a short-lived period of 30 – 60 minutes. Researchers also observed that ASEA Redox Supplement induced a parallel decrease in the phosphorylation of an extra-nuclear protein whose phosphorylation status increases in response to hydrogen peroxide treatment, consistent with an antioxidant mode of action. Serum-starving the HMVEC-L cells significantly increased the nuclear NRF2 signal induced by ASEA Redox Supplement. NRF2 directly and dramatically amplifies the innate ability to produce antioxidant protection by signaling DNA. This study suggests that molecules from the NRF2 activating ASEA Redox Supplement may trigger the production of antioxidant molecules, providing protection against the effects of free radicals compared to standard antioxidant supplements.