This initial phase is characterized by oxygen and nutrient deprivation, leading to reduced ATP production and metabolic dysfunction. Mitochondrial dysfunction triggers excessive reactive oxygen species (ROS) generation (Chen et al., 2021; Grass et al., 2022). The accumulation of ROS leads to oxidative stress, damaging cellular structures and initiating cell death pathways. The mitochondrial membrane potential becomes hyperpolarized, further enhancing ROS generation and leading to mitochondrial dysfunction (Grass et al., 2022).

The reperfusion phase is a critical period of ischemia-reperfusion injury, and its negative effects (and also the main characteristics of ischemia-reperfusion injury) are mainly reflected in the following aspects: oxidative stress, inflammatory response, and cell death. Oxygen and nutrients accumulated during ischemia are rapidly depleted during reperfusion, leading to mitochondrial electron transport chain dysfunction and ROS burst (Wang et al., 2011), activating apoptotic and necrotic programs (Zhou et al., 2018). Meanwhile, ROS oxidizes polyunsaturated fatty acids in the cell membrane to produce lipid peroxides, leading to cell membrane damage and loss of function (George et al., 2024). Inflammatory response during the reperfusion phase is also an important cause of increased damage. Prolonged ischemia is associated with increased production of damage-associated molecular patterns (DAMP) released into the surrounding tissues upon cell death. These molecular patterns can increase pro-inflammatory signaling cascades in peripheral cells and activate the complement system, which in turn causes recruitment and activation of immune cells (Kaltenmeier et al., 2022), and activated inflammatory cells release a large number of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), which further contribute to inflammatory responses (Zhou et al., 2018). In addition, increased vascular permeability, excessive platelet aggregation and release of derived mediators during ischemia-reperfusion injury are alterations that lead to abnormalities in the coagulation process and exacerbate the inflammatory response (Eltzschig and Eckle, 2011). The inflammatory response also leads to a cytokine cascade, the consequences of which include further generation of reactive oxygen species (ROS), tissue hypoxia, and cell death (George et al., 2024). In conclusion, ischemia-reperfusion injury causes cell death through multiple mechanisms involving apoptosis, necrosis, and autophagy (Hotchkiss et al., 2009), resulting in severe damage to the organism, and an understanding of these mechanisms is essential for the development of therapeutic strategies to mitigate ischemia-reperfusion injury-induced tissue damage.

The volume-regulated anion channel (VRAC) is a key membrane protein complex that plays an important role in cell volume regulation and various physiological processes. Its molecular identity remained an enigma for nearly 3 decades until its essential subunit leucine-rich repeat-containing protein 8A (LRRC8A, also known as SWELL1) was discovered (Qiu et al., 2014). VRAC channels are formed by the assembly of LRRC8 family, specifically LRRC8A and its four homologous proteins LRRC8B, LRRC8C, LRRC8D and LRRC8E (Voss et al., 2014). These proteins form heterotrimeric multimeric channels (Syeda et al., 2016), and the composition of the subunits determines the properties and substrate selectivity of the channels, giving the constituent VRACs different characteristics, including their ionic selectivity and permeability to organic osmotic pressure regulators (Osei-Owusu et al., 2018).

The LRRC8 protein consists of two parts: four transmembrane helices (TM) at the N-terminal end and seventeen leucine-rich repeat sequences (LRR) at the C-terminal end (Abascal and Zardoya, 2012). The LRR structural domain is intracytoplasmic and is thought to be involved in protein-protein interactions. The TM region displays weak, but significant, sequence similarity to gap junction proteins pannexins, suggesting a possible evolutionary relationship (Osei-Owusu et al., 2018). Functional studies and mutational analyses have established that the N-terminal TM region plays an important role in the structure and function of VRAC, such as the threonine (T44) at position 44 in SWELL1, where mutation of this residue significantly alters the anion permeability ratio (Qiu et al., 2014; Syeda et al., 2016).

VRAC can be activated by a variety of stimuli, and its main activation mechanism is cell swelling, extracellular hypo-osmotic state or intracellular hyperosmotic state leading to an osmotic pressure gradient that triggers the opening of VRAC channels, mediating the transmembrane transport of a variety of anions and the formation of a kind of anionic currents to restore cell volume (Hoffmann et al., 2009; Jentsch, 2016). VRAC is therefore thought to play an important role in the cellular regulated volume reduction (RVD) role (Formaggio et al., 2019), and it has been shown that when LRRC8 gene expression is inhibited, the intensity of the VRAC current decreases and the ability to regulate cell volume is significantly reduced (Formaggio et al., 2019). VRAC can also be activated by a decrease in intracellular ionic strength, and it has been suggested that this is the initial trigger for VRAC activation (Osei-Owusu et al., 2018), rather than an increase in cell volume per se. In addition, the mechanism of VRAC activation may also involve a variety of intra- and extracellular signaling molecules. For example, the inflammatory mediator sphingosine-1-phosphate (S1P) can activate VRAC in microglia, and S1P-induced VRAC activation can lead to the release of ATP, which may further affect microglial and neuronal activity and cause neuropathic pain (Chu et al., 2023). Other activation mechanisms such as apoptosis inducers like staurosporine and cisplatin (Maeno et al., 2000; Shimizu et al., 2004), and G protein-coupled receptor (GPCR) (Liu et al., 2009; Takano et al., 2005) activation have also been reported.

The physiological mechanisms of VRAC are mainly involved in cell volume regulation and regulation of multiple physiological processes. Its most prominent role is mediating anion release and regulating cell volume as mentioned above, in addition to several other aspects, such as involvement in cell proliferation (Shen et al., 2000), apoptosis (Konishi et al., 2019), necrosis (Liu and Stauber, 2019), and migration (Liu and Stauber, 2019); In terms of metabolism, VRAC has been shown to be critical for glucose-stimulated insulin secretion in pancreatic islet β-cells. The channel contributes to membrane depolarization, causing calcium inward flow and subsequent insulin release (Best et al., 2010); Immunologically, it plays a role in the activation of the NLRP3 inflammasome, which is a key component of the innate immune response (Compan et al., 2012), in addition, VRAC contributes to T-cell activation and B-cell development, as evidenced by the phenotype of Swell1 knockout mice, which show deficits in T-cell development and B-cell function (Kumar et al., 2014); VRAC also exerts an effect on the nervous system, it is involved in the release of excitatory neurotransmitters such as glutamate (Mongin, 2016). This process may have a greater impact in conditions such as ischemic stroke, where glutamate release mediated by VRAC activated by cerebral ischemia causes damage to neurons through excitotoxicity (Zhou et al., 2020).

As the understanding of the physiological role of VRAC deepens, some emerging areas are gradually gaining attention from researchers. Since VRAC is permeable to certain apoptosis inducers such as cisplatin, it contributes to the cellular uptake of anticancer drugs (Osei-Owusu et al., 2018). Studies have shown that downregulation of the SWELL1 subunit correlates with cancer cell resistance to cisplatin (Sørensen et al., 2016), suggesting that VRAC may be a promising target for overcoming cancer cell resistance in cancer therapy. Improving cancer therapeutic efficacy by modulating VRAC activity may become a promising study in the future.

Another area that is still unclear but holds great promise is the modulation of neuropathic pain by VRAC. Given that VRAC involves the release of ATP and glutamate (Chu et al., 2023), a key neurotransmitter involved in pain signaling (Fundytus, 2001), the role played by VRAC in pain perception and transmission warrants further study to identify potential therapeutic opportunities.

In conclusion, the physiological role of VRAC encompasses many aspects of the organism’s vital activities such as cell volume regulation, immune responses, metabolic processes, and neurological functions, and an understanding of the biophysical properties and regulatory mechanisms of VRAC is essential for the development of effective therapeutic approaches.

Suppression of VRAC by genetic methods is mainly through conditional knockout of the gene encoding LRRC8A, an essential subunit of VRAC. Yang et al.'s experiments resulted in the deletion of the LRRC8A subunit of VRAC by conditional knockout of the GFAP gene in mouse astrocytes and the GFAP - cKO mice exhibited reduced infarct size and significant improvement in neurological prognosis after middle cerebral artery occlusion (MCAO) (Yang et al., 2019). Subsequently, Zhou et al. showed that ischemia increases LRRC8A-dependent VRAC activity in hippocampal neurons, which enhances glutamate release and contributes to ischemic brain injury (Zhou et al., 2020), and that mice with conditional knockout of the LRRC8A gene had significantly lower infarct volume and neurological severity scores than wild-type mice after middle cerebral artery occlusion cerebral infarction (Zhou et al., 2020). Furthermore, these findings emphasize the importance of astrocyte VRAC in the pathogenesis of ischemic stroke and suggest that targeting this pathway may provide neuroprotection.

A variety of drugs have been shown to inhibit VRAC. 4-(2-butyl-6,7-dichloro-2-cyclopentyl-1-on-5-yl) oxalylbutyric acid (DCPIB) is a potent and specific blocker of VRAC, acting on the swell1 subunit (Decher et al., 2001), and in a reversible middle cerebral artery occlusion (rMCAO) model, in-chamber administration of DCPIB resulted in a reduction in mean infarct volume by approximately 75% compared to controls (Zhou et al., 2020); in Qiu et al. DCPIB attenuated excitotoxicity during ischemic events by decreasing glutamate release, thereby reducing neuronal damage (Qiu et al., 2014); Han et al. demonstrated that DCPIB significantly inhibited hypoxia-glucose deprivation (OGD)-induced activation of microglia, with possible mechanisms including direct inhibition of VRAC in microglia and inhibition of the MAPK pathway, a key pathway mediating microglial activation (Han et al., 2014). Activation of microglia plays an important role in the inflammatory response of ischemia-reperfusion injury (Wang et al., 2023). In a recent study, Cao et al. found that DCPIB could promote the conversion of pro-inflammatory M1 microglia into anti-inflammatory M2 microglia through the MAPK signaling pathway, thereby reducing inflammatory responses and oxidative stress at the ischemic site (Cao et al., 2024). All these studies suggest that DCPIB protects neuronal cells from ischemic injury. In a model of ischemia-reperfusion injury in cardiomyocytes, DCPIB was shown to inhibit chloride efflux mediated by VRAC to attenuate autophagy and oxidative stress in cardiomyocytes (Shen et al., 2014; Xia et al., 2016). These findings suggest that inhibition of VRAC activity using DCPIB may be a promising therapeutic strategy to attenuate ischemia-reperfusion injury.

Dicumarol, another VRAC inhibitor, administered intracerebrally effectively blocked VRAC-mediated anionic currents activated by ischemic stroke in mice (Formaggio et al., 2019), and also inhibited cellular release of ATP (Formaggio et al., 2019) as well as pathologic release of glutamate (Chen et al., 2024), attenuated neuronal swelling and cell death, and provided neuroprotection after ischemic stroke, which is a promising therapeutic strategy for attenuating potential therapeutic agent for ischemic stroke-induced brain damage. In addition, Dicumarol is an FDA-approved drug (Chu et al., 2023). It has been used as an effective anticoagulant in clinical practice for the treatment of deep vein thrombosis and atrial fibrillation and is also recommended by current guidelines for the prevention of ischemic stroke in patients with high-risk atrial fibrillation (Kleindorfer et al., 2021).

In addition to DCPIB and dicumarol, several other VRAC inhibitors have shown potential in the treatment of ischemia-reperfusion injury.

Tamoxifen, a non-selective VRAC inhibitor, has been shown to inhibit injury after cerebral ischemia (Kimelberg et al., 2000), as well as inhibit nNOS (Osuka et al., 2001), scavenge oxygen free radicals (Zhang et al., 2007), and have some neuroprotective ability.

IAA-94 is also a VRAC blocker, and whole-cell membrane slice technology has demonstrated that it can inhibit VRAC currents and reduce glutamate-induced neuronal necrosis (Inoue et al., 2005).

4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS), a stilbene derivative chloride channel blocker, effectively eliminates chloride inward flow and neuronal swelling induced by intracellular sodium accumulation during ischemia-reperfusion injury (Chen et al., 2024).

Other drugs such as the carboxylate analog (NPPB), and phloretin have also been shown to inhibit VRAC (Inoue et al., 2005), but their roles in ischemia-reperfusion injury need to be further investigated.

When exploring the clinical potential of VRAC inhibitors, we must consider their efficacy, safety, and pharmacokinetic properties in the treatment of ischemia-reperfusion injury. VRAC inhibitors, such as DCPIB and Dicumarol, have been shown to have a certain ability to attenuate ischemia-reperfusion injury in animal models. However, there are certain limitations in the clinical application of these drugs, which should be addressed in order to fully utilize the therapeutic potential of VRAC inhibitors.

Currently, VRAC inhibitors are not yet used in the clinical treatment of ischemia-reperfusion injury. Because of the limitations of each of these approaches, it is important to optimize the pharmacokinetic and pharmacodynamic properties of these inhibitors and to explore their efficacy in preclinical and clinical settings.