Molecular hydrogen protects against oxidative stress-induced RAW 264.7 macrophage cells through the activation of Nrf2 and inhibition of MAPK signaling pathway

Background: Oxidative stress is involved in the development of many inflammatory, metabolic and aging diseases. Objective: In this study we investigated, the protective effects of H₂ on RAW 264.7 macrophage cell against LPS-and H₂O₂-induced oxidative stress by the inhibition of MAPK pathway and also activate the Nrf2 pathway. Results: Our results showed H₂ increased the macrophage cell proliferation and generated ROS and NO against LPS stimulation to exert an active immune response. Similarly, H₂ protected the macrophage cell from H₂O₂-induced oxidative stress. H₂ reduced the LPS-and H₂O₂-induced inflammatory cytokine production and intracellular calcium influxes. H₂ inhibited the LPS-and H₂O₂-induced phosphorylation of MAPK pathway and its downstream signaling molecules. Furthermore, H₂ protected the macrophage cell from mitochondrial apoptosis. H₂ increased Nrf2 protein expression indicating its strong anti-oxidative effects against oxidative stress. Conclusion: Collectively, our results indicate the strong antioxidant role of H₂ against LPS-and H₂O₂-induced oxidative stress on macrophage cells by activating the Nrf2 pathway and inhibiting the MAPK-signaling pathway. Graphic Abstract: Our results clearly showed that LPS increased the cellular ROS by recognizing the TLR4 and H₂O₂ rapidly increased the cellular (1) and mitochondrial (2) oxidative stress. Excessive ROS/NO molecules cause intracellular calcium influxes (3) As a results imbalance the cellular membrane homeostasis and activate the stress response MAPK signaling pathway with its downstream signaling protein and mitochondrial caspase protein (4) that collapse the anti-oxidant mechanisms and induced the inflammatory cytokine secretion; leads to cell apoptosis (5) Whether H₂ reduced the cellular and mitochondrial oxidative stress, intracellular calcium influxes and inhibits the stress response MAPK, caspase cell signaling pathway through the activation of Nrf2/ARE signaling pathway (6) Consequently, increased the antioxidant enzymes and reduced the inflammatory cytokine that influences the macrophage cell proliferation (7) to protect the cell from apoptosis. The different effects of ROS and H₂ used in this study are indicated in red and green, red arrow depicts ROS effects in the cell, green arrow depicts H₂ effects.[Figure not available: see fulltext.].