Overexpression of Thioredoxin-1 alleviates morphine-induced neuroinflammation in BV-2 cells and the ventral tegmental area of mice

The clinical utility of morphine is limited by the development of tolerance and addiction. These processes are linked to neuroinflammation involved microglial responses. Thioredoxin-1 (Trx-1) is a crucial endogenous antioxidant with neuroprotective properties, and our previous work has shown that Trx-1 overexpression blocks morphine-induced conditioned place preference (CPP). However, the role of Trx-1 in morphine-induced microglial inflammatory responses remains unclear. Here, we investigated the effects and underlying mechanisms of Trx-1 overexpression on morphine-induced microglial pro-inflammatory shifts, both in the microglial cell line BV-2 and in the ventral tegmental area (VTA) of mice. In vitro, morphine treatment increased intracellular Reactive Oxygen Species (ROS) in BV-2 cells, upregulated markers associated with microglial reactivity (IBA-1 and MHC-II), activated the NF-κB pathway (evidenced by increased p-IκBα and nuclear p65 translocation), and elevated pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) while reducing the anti-inflammatory cytokine IL-10. Overexpression of Trx-1 via adenoviral transduction reversed all these morphine-induced effects. In vivo, using a CPP paradigm, we confirmed that morphine CPP was blocked in Trx-1 transgenic (TG) mice. Biochemical analysis of the VTA from these mice revealed that Trx-1 overexpression similarly attenuated morphine-induced upregulation of IBA-1, MHC-II, and pro-inflammatory cytokines, consistent with in vitro results. Mechanistically, co-immunoprecipitation assays demonstrated an endogenous interaction between Trx-1 and the mu-opioid receptor (MOR). Overexpression of Trx-1 significantly enhanced this interaction and was associated with increased MOR expression. These results reveal that overexpression of Trx-1 suppresses morphine-induced neuroinflammation by binding MOR and reducing ROS-driven NF-κB activation in microglia. This mechanism positions Trx-1 as a potential therapeutic target for improving the clinical safety and efficacy of opioid addiction.