Our study showed that the hippocampal neuronal HT22 cell had lower cell activity and higher apoptosis rate after exposure to sevoflurane. These changes are related to the overload of intracellular calcium and the high expression of WNK1 kinase. However, the WNK1 inhibitor WNK-463 could improve cell activity, decrease the apoptosis caused by sevoflurane, downregulate the expression of WNK1 and alleviate calcium overload. Therefore, we propose that the WNK1/NKCC1 signalling pathway may play a major role through intracellular calcium overload in sevoflurane-induced neurotoxicity, which may be a potential protective mechanism against sevoflurane-related neurotoxicity.
Most general anaesthetics are excitants of GABAAR and inhibitors of the N-methyl-D-aspartate (NMDA) receptor. Currently, relevant mechanisms have been shown, such as the hypothesis of neurotrophic apoptosis[2, 3], mitochondrial damage and accumulation of ROS[23], the effect of inflammatory factors[16, 24], the excitatory neurotoxicity hypothesis[3], the effects on the regeneration of neurons, changes in receptor subtypes[18], and changes in ion concentration[19–25]. In our previous study, we found that sevoflurane can induce neuroapoptosis in neonatal rats and cause behavioural changes during development, and that GABAAR played a role in sevoflurane’s neurotoxicity in the developing brain[18]. The toxicity of sevoflurane was demonstrated more intuitively at the cellular level. These results were similar to the findings of many studies[19].
Among all the neurotoxicity mechanisms of sevoflurane on the developing brain, GABAAR excitatory toxicity is a very important aspect. GABAAR is a ligand-gated chloride ion channel that mainly mediates inhibitory synaptic transmission. Recently, GABAAR has been found to play an excitatory role in the immature brain and an inhibitory role in the adult brain[15]. The conversion of GABAAR depends on the concentration of chloride ions in cells at different stages of development ([Cl−]i). In the early stage of brain development in mammals and humans, chloride ions in immature neuron cells are in a high concentration state (15–20 mm). After the activation of GABAAR, the channel opens and the Cl− outflow causes cell membrane depolarization and induces the action potential of neuron cells, showing the excitation effect[15, 16]. This feature plays an important role in the process of neuron growth and synapse formation. Therefore, in the newborn brain, the depolarization of the GABAAR current is a very important primary physiological and developmental neuroelectric loop that is activated. With the growth and maturation of neurons, the concentration of Cl− in neurons gradually decreases (~ 4 mM), and GABAA is transformed into an inhibitory neurotransmitter, which is used for the activation of GABAAR. As a result, the internal flow of Cl− occurs, leading to the hyperpolarization of the cell membranes of neurons and inhibition of neurons.
In the present study, we found that WNK-463, an inhibitor of WNK1 kinase, may play a protective role in developing neurons. WNK1 kinase is a Cl−-sensitive serine threonine protein kinase expressed in the central nervous system. Affected by intracellular chloride ions, this kinase can directly inhibit KCC2 activity and enhance NKCC1 activity[17]. Or postulated that SPS1-related proline/alanine-rich kinase (SPAK) or oxidative stress response protein (ORS-1) acts with KCC2 and NKCC1 and regulates nerve cell [Cl−]i[25–27]. Friedel[28] found that in the process of neuron development, GABAAR is open, which transforms from the excitatory effect of immature neurons to the inhibitory effect of mature neurons, and an important guaranteed factor is that as KCC2 transports Cl− synergistically out of cells, [Cl-]i decreases. Our previous study demonstrated that the neurotoxic effect of sevoflurane may be related to the transformation of the GABAA receptor α from subtype 1 to subtype 2[18]. As an inhibitor of NKCC1, bumetanide can attenuate the increase in apoptosis induced by sevoflurane[18, 29]. Stevens[19] also found that the NKCC1 inhibitor alleviated the decline in the learning and memory function of newborn rats caused by ketamine. Therefore, we propose that as a cotransporter of NKCC1 and KCC2, WNK1 can affect the transformation of NKCC1-mediated Cl− flow and affect the excitability of GABAAR in the immature brain. In the present study, we found that sevoflurane caused a significant decrease in cell activity and an increase in apoptosis and cleaved caspase-3 expression in the hippocampal neuronal HT22 cells of neonatal rats. After the administration of the WNK1 inhibitor WNK-463, cell activity was improved, and apoptosis and the expression of cleaved caspase-3 were decreased. These results demonstrated that the WNK1 inhibitor was a protective factor against the neuroexcitatory toxicity of sevoflurane in developing neurons, which is consistent with our hypothesis.
We also found that sevoflurane exposure caused calcium overload in HT22 cells. In the early stage of neuronal development, the membrane receptors are stage-specific; the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor is not yet fully functioning, and the activation of the NMDA receptor depends on the GABAAR. The NMDA receptor has two characteristics: ligand-gate and voltage-gate. In the posterior synaptic membrane, the activation of GABAAR leads to Cl− outflow and excitatory post-synaptic potential (EGABA). In addition, the calcium current generated by the NMDA receptor directly activates voltage-dependent Ca2+ channels (VDCCs), leading to increased intracellular Ca2+ and calcium overload. Calcium is an important regulator, and intracellular Ca2+ overload can lead to apoptosis through the activation of apoptosis-related enzymes, such as phospholipase, protease, and nucleic acid enzymes, as well as mitochondrial and cell membrane damage, and the generation of a large number of free radicals[30]. In the present study, the results of the Fluo-4 imaging of intracellular calcium showed a significant accumulation of Ca2+ in HT22 cells after treatment with sevoflurane. WNK1 inhibitor administration significantly decreased the calcium density, which demonstrated that the WNK1 inhibitor may influence NKCC1 and then GABAAR excitability and intracellular Ca2+ overload in HT22 cells after sevoflurane exposure.
Therefore, exposure to sevoflurane on the hippocampal neuronal HT22 cell line induced a decrease in cell activity and an increase in apoptosis, increasing the expression of cleaved caspase-3 and intracellular Ca2+ overload. WNK1 inhibitor (WNK-463) administration may improve HT22 cell activity and decrease apoptosis and intracellular Ca2+ overload. Therefore, WNK1 plays an important role in neurotoxicity of the sevoflurane in the developing brain. This discovery demonstrates that the WNK1/NKCC1 pathway may be a signalling pathway in sevoflurane’s neurotoxicity, and WNK1 is a promising new target for reducing the toxicity of sevoflurane. There is a lack of research in this field, which needs further study.