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Abstract
Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) relative to the antioxidant defense and plays a critical role in the pathogenesis of various diseases. The body naturally produces antioxidants such as glutathione (GSH) to counteract ROS under oxidative stress. Phytochemicals from certain plants such as tef (Eragrostis tef) have been shown to have antioxidant activities. Tef, an annual grass native to Ethiopia and Eritrea, is believed to be one of the earliest plants domesticated by humans. It is grown for human consumption in East Africa, and also serves as a valuable forage grass in other countries, including the United States. Tef has higher nutrient content as compared to other grains. Although in vitro studies have shown the antioxidant properties of tef grain extracts, studies using relevant cell models are lacking. Moreover, the molecular mechanisms involved in tef-induced antioxidant activity remained unknown. In this study, we analyzed the antioxidant properties of brown and ivory tef seeds and biomass using THP-1 (human leukemia monocytic cell line) due to their capability to differentiate into macrophages and induce GSH response. Our findings showed that tef extracts do not have cytotoxic effects. Grain extracts, particularly for the brown organic fractions increased cellular GSH levels in THP-1 monocytes when treated at 50 µg/mL for 24 hours. However, tef biomass extracts showed no increase in GSH levels in THP-1 monocytes. Separation (HPLC) and purification (ISCO) of the brown organic fraction further increased GSH levels in treated THP-1 cells. To understand the mechanism of tef extract-induced glutathione levels, we studied the modulation of the Nrf2 and NF-κB luciferase activity using a Luciferase reporter cell line. Our findings showed that the tef extracts increased the Nrf2-regulated luciferase activity by 20-fold while the NF-κB-regulated luciferase activity was only slightly increased in the presence of the inflammatory cytokine TNF-α. Furthermore, tef extracts enhance the transcription of Nrf2-regulated genes including HO-1, NQO-1, GR, GCLC, and GCLM. Gene set enrichment analysis (GSEA) of the transcriptome data revealed the enrichment of genes involved in various pathways including the TNF-α signaling via NF-κB, the inflammatory response, and the ROS signaling pathways in THP-1 cells treated with tef extracts compared to the control group. Furthermore, heatmap of differentially expressed genes (DEGs) showed increased expressions of over 90 genes involved in various pathways including iron storage protein-ferritin pseudo genes FTH1P10, FTH1P7, FTH1P11, FTH1P20, SLC11A1, SLC45A3, SLC12A7, SLC7A7, and SLC43A2, ZNF, GSR and TNF in cells treated with the tef extracts compared to control. Taken together, our findings indicate that tef grains possess phytochemicals that increase antioxidant levels. The increase in antioxidant levels from tef extracts is primarily attributed to the Nrf2-signaling pathways. This is the first investigation of the cellular mechanisms of increased antioxidant levels in human leukemia monocytic cell lines by tef grain phytochemicals.