WNK1 Antagonist Decreased Sevoflurane-induced Neurotoxicity in HT22 Hippocampal Neurons via the WNK1/NKCC1 Signalling Pathway

Abstract Background: The GABAAR plays an important role in the neurotoxicity caused by sevoflurane, but the mechanism of its regulation in this context is unknown. The present study aimed to reveal the WNK1/NKCC1 pathway as a regulator of the neurotoxicity caused by sevoflurane on the HT22 hippocampal neuron cell line. Methods: HT22 cultured hippocampal neurons were divided into three groups: control group, 4.1% sevoflurane treatment group for 6h, WNK-463 (specific antagonist of WNK1, 1µmol) + sevoflurane treatment group. Cell viability and the optimum concentration of WNK-463 were measured by MTS method. Cell apoptosis was detected by Tunel method, and the content of cleaved caspase-3 protein apoptosis factor was detected by Western blot. Pathway protein detection including the expression of WNK1, NKCC1 were detected. Calcium imaging measures intracellular calcium ion concentration and verifies downstream targets.Results: The neurotoxic effects of sevoflurane on hippocampal neurons were observed. As setting the results of control group is “1”, compared with sevoflurane group, WNK-463 downregulated the protein expression of cleaved caspase-3 (1.96±0.49 vs 1.29±0.38), increased cell viability (0.68 vs 0.96) and decreased apoptosis (13.31±4.67/vision vs 7.05±3.82/vision, ***P < 0.001). Compared with the control group, sevoflurane treatment increased the expression of WNK1 kinase and NKCC1 protein, whereas WNK-463 reversed this increase without affecting the control HT22 cells (****P < 0.0001). Compared with the control group, sevoflurane exposure in HT22 cells increased intracellular Ca2+ concentrations, while WNK-463 reversed this change (1.0 vs 2.08±1.36 vs 1.26±0.77, **P < 0.01). Conclusion: This study demonstrated a neuroprotective role of the WNK1 antagonist WNK-463 in sevoflurane-induced neurotoxicity. WNK-463 promoted hippocampal neuron viability and reduced the apoptosis and intracellular calcium overload caused by sevoflurane on HT22 hippocampal neurons, possibly via the modulation of the WNK1/NKCC1 pathway.