**Background:** Reactive oxygen species (ROS) are implicated in numerous diseases, including cancer, neurodegeneration, and aging. Mitochondria are a major source of intracellular ROS, particularly through electron leakage from complexes I and III of the electron transport chain. While it is known that ROS can initiate signal transduction pathways, whether mitochondrial ROS (mtROS) directly activate nuclear signaling has been controversial. The Nrf2/Keap1 pathway is a key antioxidant response system; under oxidative stress, Keap1 is modified, releasing Nrf2 to translocate to the nucleus and drive expression of protective genes such as HO-1, HO-2, MnSOD, GCL, GST, and NQO1. This study aimed to test the hypothesis that mtROS initiate Nrf2/Keap1 signaling by using MnSOD transfection to reduce mtROS and observing downstream effects.
**Methods:** The study used two rat gastric mucosal cell lines: RGM1 (normal, low mtROS) and RGK1 (tumorized, high mtROS). Mitochondrial ROS levels were measured using HPF, a hydroxyl radical-sensitive fluorescent dye. MnSOD activity was assessed by native gel-based SOD activity assay and Western blotting. Protein expression of Nrf2, Keap1, HO-1, HO-2, MnSOD, GCL, GST, and NQO1 was evaluated by immunofluorescence staining and quantified by fluorescence intensity. To directly test the role of mtROS, RGK1 cells were transiently transfected with a human MnSOD (hMnSOD) expression vector or empty vector. After transfection, MnSOD mRNA was measured by qRT-PCR, and MnSOD activity was confirmed. Changes in Nrf2, Keap1, HO-1, and HO-2 protein levels were assessed by immunofluorescence. Additionally, an antioxidant response element (ARE) dual-luciferase reporter assay was performed in RGK1 cells stably expressing an ARE-luciferase construct, then transiently transfected with hMnSOD or vector, to quantify Nrf2 transcriptional activity.
**Key Results:** HPF fluorescence was significantly higher in RGK1 cells than RGM1 cells (p < 0.01), confirming higher mtROS in tumor cells. MnSOD activity and protein expression were also higher in RGK1 cells. Immunofluorescence showed significantly higher levels of Nrf2, Keap1, HO-1, HO-2, MnSOD, GCL, GST, and NQO1 in RGK1 versus RGM1 cells (all p < 0.05 or p < 0.01). After hMnSOD transfection into RGK1 cells, MnSOD mRNA and activity were significantly increased (p < 0.01), while CuZnSOD activity remained unchanged. Importantly, hMnSOD transfection significantly reduced the fluorescence intensity of Nrf2, Keap1, HO-1, and HO-2 compared to vector-only controls (p < 0.01). The ARE-luciferase reporter assay showed that hMnSOD-transfected cells had significantly lower luciferase activity than vector controls (p < 0.01), directly demonstrating that reducing mtROS decreases Nrf2-mediated transcriptional activation.
**Clinical Implications:** This study provides direct experimental evidence that mitochondrial ROS can exit the mitochondria and activate Nrf2/Keap1 signaling in the nucleus, placing mtROS at a central position in cellular signaling regulation. The findings have significant implications for understanding how cancer cells adapt to oxidative stress and develop resistance to chemotherapy and radiation, as these treatments often increase ROS. By demonstrating that MnSOD overexpression suppresses this signaling, the study suggests that targeting mitochondrial ROS or enhancing antioxidant defenses could modulate the Nrf2 pathway, potentially improving therapeutic outcomes. The results also support the concept that mitochondrial dysfunction contributes to disease pathology through ROS-mediated signaling, offering new avenues for intervention in conditions where oxidative stress plays a key role, such as neurodegeneration, ischemia-reperfusion injury, and metabolic diseases.