The current study demonstrated that anesthesia with 3% sevoflurane for 4 hours caused significant upregulation of Cx43 and Bax, decreased the level of the Bcl-2 protein and increased the expression of cleaved caspase-3 in the hippocampus after 1 day. Cbx [14], injected i.c.v. 2 hours before sevoflurane exposure could not decrease the upregulation of Cx43, Bax and cleaved caspase-3 or increase Bcl-2 expression in the hippocampus. Cbx also could not improve rat cognitive impairment after 30 days, and the role of Cx43 HCs and GJs in sevoflurane-induced development of neurotoxicity needs further investigation.
Cx43 protein HCs exhibit a low open probability under normal conditions to maintain normal synaptic function. HCs could be deleterious by releasing excitotoxins (e.g., ATP and glutamate), disturbing the concentration of intracellular Ca2+ or altering cytoplasmic ionic and osmotic balance. When the CNS is stimulated by various inflammatory mediators (cytokines, NO, ROS, etc.), it first enhances the HCs activity of astrocytes and microglia. For example, microglia stimulated by detrimental agents will elevate the release of TNF-α and IL-1β via microglial Cx32 hemichannels, leading to an increase in astrocyte Cx43 protein HCs activity. Then, astrocyte Cx43 HCs opening mediated by microglia could increase Ca2+ influx and release glutamate, thus disrupting normal synaptic function.
Cx43 HCs opening under pathological conditions could release many glial transmitters, such as ATP and glutamate. When the extracellular Ca2+ concentration decreases or the synaptic glutamate concentration increases, Cx43 HCs open and ATP is released into the synaptic space. ATP activates the P2Y1 purine receptor of inhibitory intermediate neurons [15], thereby enhancing synaptic inhibition. At the same time, the Cx43 HCs-dependent release of ATP can also enhance the synaptic transmission of glutamatergic neurons, but this mechanism is still unclear. It has been speculated that ATP activates the AMPA receptor of the postsynaptic membrane through the P2X7 receptor, which increases excitotoxicity. D-serine is also released in a HC-dependent form intracellularly to extracellularly to enhance synaptic transmission and synaptic plasticity. D-serine is a coagonist of the NMDA receptor in the postsynaptic membrane of neurons. Studies have shown that D-serine could act on glutamatergic receptors in the postsynaptic membrane and play a synaptic role [16]. Data have also shown that Cx43 HCs of hippocampal astrocytes also control long-term potentiation (LTP) by releasing D-serine. These results indicate that Cx43 HCs play an important role in gliotransmitter release in the synaptic space [17].
Similarly, Cx43 GJs play an important role in the CNS [18-20]. GJs in astrocytes contribute to the formation of functional syncytial bodies, promote the removal of ions and neurotransmitters during neuronal activity, and allow the transmission of Ca2+ waves between astrocytes. Neurotransmitters released by neurons can bind to receptors in astrocyte membranes and produce metabolites such as inositol 1,4,5 triphosphate (IP3), ATP, glutamate and D-serine, which diffuse through GJs and affect nerve function. This intercellular communication based on Cx43 GJs enables astrocytes to perceive and integrate local and global synaptic activity and respond to glial transmitters affecting presynaptic and postsynaptic transmission. Therefore, Cx43 GJs and gliotransmitters are important participants in the regulation of the nerve activity, behavior and homeostasis of brain function.
An increase or decrease in Cx43 expression does not directly indicate whether its role is protective or damaging in the CNS, because Cx43 plays a role in the formation of HCs and GJs [21]. It is generally believed that under normal conditions, only a small number of Cx43 HCs are open in order to play some basic roles. HCs will open in large quantities enabling intracellular and extracellular communication directly when the CNS suffers various stimuli, such as ischemia, hypoxia, trauma and growth factors. Harmful toxic substances should be released from the cells to the extracellular environment, which produces adverse effects [22]. GJs have been shown to be both neuroprotective and neurodestructive in ischemic situations. When damage is limited to a small extent, GJs can buffer the toxic substances between adjacent cells and allow the cells to survive. When the damage increases, GJs can spread toxic substances a longer distance, causing more cell death. Many diseases or pathophysiological conditions seem to be associated with Cx43 HCs and GJs which have become new pharmacological targets in recent years [23].
To date, there have been many reagents and drugs that inhibit Cx43 HCs and GJs, including Cbx, quinine, mefloquine, connexin mimetic peptides GAP26, and GAP27 and so on [24,25]. Although the structures of Cx43 HCs and GJs have been studied for a long time, there is still no specific inhibitors within Cx43 HC and GJ. Cbx, a semisynthetic derivative of glycyrrhetinic acid (GA), is widely used as a Cx43 HCs and GJs blocker in various pathological processes of the brain. It has been confirmed that Cbx displays different protective effects in a model of Parkinson’s disease [26], ischemic brain injury [27], posttraumatic epilepsy [28], allergic airway inflammation [29] and fatty liver disease in obsess mice [30]. Also, Cbx did not induce significant side effects even at the highest dose tested (50 mg/Kg) [31]. Unfortunately, little is known about the clear mechanism of Cbx on connexin HCs and GJs [32]. Meanwhile, Cbx was previously shown to leave Cx43 expression unaffected [33]. Similar findings were reported in our study.
Why Cbx failed to attenuate sevoflurane-induced neurotoxicity in developing hippocampus in our study. First, Cbx, a more water-soluble derivate of GA, has different cellular targets and also acts as an inhibitor of 11β-hydroxysteroid dehydrogenase [34]. Second, in addition to blocking Cx43 HCs and GJs, Cbx can block many other channels and receptors including voltage-gated Ca2+ currents, homomeric Panx1 and heteromeric Panx1/Panx2 channels, P2X7 receptors, Cx26 and Cx38 HCs [35-37]. Third, Cbx was widely used as a potent blocker of the major astroglia-to-astroglia and astroglia-to-neuron HCs and GJs in previous studies [38]. Future study is need to determine which type of glial cell (microglia or astrocyte)is most affected by Cbx. Last but not least, because there are no specific inhibitors for HCs and GJs, it is necessary to develop Cx43 HCs and GJs specific inhibitors to clarify the role of Cx43 HCs and GJs in sevoflurane-induced CNS toxicity in the future.