Esketamine is neuroprotective against traumatic brain injury through its modulation of autophagy and oxidative stress via AMPK/mTOR-dependent TFEB nuclear translocation.

Abstract

Recent clinical studies highlight the neuroprotective effects of esketamine, but its benefits following traumatic brain injury (TBI) have not been defined. Here, we investigated the effects of esketamine following TBI and its associated neuroprotection mechanisms. In our study, controlled cortical impact injury on mice was utilized to induce the TBI model in vivo. TBI mice were randomized to receive vehicle or esketamine at 2 h post-injury for 7 consecutive days. Neurological deficits and brain water content in mice were detected, respectively. Cortical tissues surrounding focal trauma were obtained for Nissl staining, immunofluorescence, immunohistochemistry, and ELISA assay. In vitro, esketamine were added in culture medium after cortical neuronal cells induced by H₂O₂ (100μM). After exposed for 12h, neuronal cells were obtained for western blotting, immunofluorescence, ELISA and CO-IP assay. Following administration of 2-8 mg/kg esketamine, we observed that 8 mg/kg esketamine produced no additional recovery of neurological function and ability to alleviate brain edema in TBI mice model, so 4 mg/kg esketamine was selected for subsequent experiments. Additionally, esketamine can effectively reduce TBI-induced oxidative stress, the number of damaged neurons, and the number of TUNEL-positive cells in the cortex of TBI models. Meanwhile, the levels of Beclin 1, LC3 II, and the number of LC3-positive cells in injured cortex were also increased following esketamine exposure. Western blotting and immunofluorescence assays showed that esketamine accelerated the nuclear translocation of TFEB, increased the p-AMPKα level and decreased the p-mTOR level. Similar results including nuclear translocation of TFEB, the increases of autophagy-related markers, and influences of AMPK/mTOR pathway were observed in H₂O₂-induced cortical neuronal cells; however, BML-275 (AMPK inhibitor) can reverse these effects of esketamine. Furthermore, TFEB silencing not only decreased the Nrf2 level in H₂O₂-induced cortical neuronal cells, but also alleviated the oxidative stress. Importantly, CO-IP confirmed the interaction between TFEB and Nrf2 in cortical neuronal cells. These findings suggested that esketamine exerts the neuroprotective effects of esketamine in TBI mice model via enhancing autophagy and alleviating oxidative stress; its mechanism involves AMPK/mTOR-dependent TFEB nuclear translocation-induced autophagy and TFEB/Nrf2-induced antioxidant system.

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