Epilepsy is one of the most prevalent and serious neurological disorders, affecting over 70 million people globally and posing a significant public health issue. Patients with epilepsy experience spontaneous and recurrent seizures, which can continuously damage neural cells and potentially lead to brain impairment in some cases. Altered consciousness, persistent neurocognitive impairments, and multiple neurological or psychiatric comorbidities are typically observed during epileptogenesis [[1], [2], [3]]. Despite the development of some anti-seizure medications (ASMs) that can temporarily control epileptic manifestations, no available therapies can completely prevent, cease, or cure epileptogenesis to date [3]. Existing therapeutic strategies are effective in only 70 % of patients with epilepsy, leaving the remaining 30 % with uncontrollable seizures [4]. The precise culprit of epileptogenesis remains unclear. Well-known hypotheses include multiple etiologies, such as genetic mutations, imbalances in excitatory/inhibitory systems, and dysfunction of ion channels. Central nervous system (CNS) injuries caused by other neurological diseases, such as traumatic brain injury (TBI), stroke, and infection, may also contribute to approximately 20 % of seizures or status epilepticus (SE), indicating common neuropathological mechanisms [5]. Particularly, neuroinflammation has been considered as one of the contributors to epileptogenesis.

A increasing number of reports indicate that neuroinflammation, a common occurrence in patients with epilepsy and in several epileptic models, may play a crucial role in epileptic activity [[6], [7], [8]]. The neuroinflammation process during epileptogenesis is characterized by the release of pro-inflammatory molecules, such as interleukin-1β (IL-1β) and IL-18. As an adaptive immune response, the neuroinflammatory reaction triggered by seizures is important for eliminating dead cells debris and harmful molecules. However, prolonged neuroinflammation creates a pro-inflammatory micro-environment, which partially facilitates disease progression by promoting programmed cell death (PCD) [9]. The dying neural cells not only adversely affect brain function, but also continue to release pro-inflammatory mediators [10,11], exacerbating the epileptic condition. Therefore, a better understanding of the neuroinflammation-associated PCDs is conducive for delineating the underlying mechanism of epilepsy.

Pannexin 1 (Panx1) is a member of the pannexin family, which consists of non-selective large pore membrane channels with a structure similar to gap junction hemichannels: known as connexins, but with unique functions. Panx1 is one of the prominent isoforms that form plasma membrane ion channels and metabolite channels distributed in multiple types of neural cells in the CNS, especially in the brain, from early development through adulthood. The opening Panx1 is highly permeable to small molecules, such as chloride ions (Cl-) and calcium ions (Ca2+), as well as larger molecules, including adenosine triphosphate (ATP) and its metabolites [12]. Since excessive extracellular ATP is considered as a crucial damage-associated molecular pattern (DAMP), a dangerous signal for living cells, Panx1 is tightly related to initiate the inflammatory responses. Prolonged channel opening can also be triggered by caspase-dependent cleavage of the C terminus in apoptotic/pyroptotic cells, suggesting that Panx1 can modulate PCDs [13,14]. For example, the activation of Panx1 in myeloid cells was a major contributor to acute brain inflammation following TBI [15]. The critical role of Panx1 in pyroptosis induced by the noncanonical inflammasome has been demonstrated in sepsis [16]. Interestingly, both extrinsic and intrinsic apoptosis could activate Panx1 to drive NLRP3 inflammasome assembly, indicating that Panx1 may participate in the crosstalk between different PCDs [17]. In the context of epilepsy, abnormal Panx1 levels have been observed during epileptogenesis, implying a pro-epileptic property. The Panx1 protein level was significantly higher in the temporal lobe cortex of patients with temporal lobe epilepsy (TLE) and drug-resistant epilepsy (DRE) compared to controls, indicating that Panx1 activation may accelerate seizure generation [18,19]. Conversely, Panx1 has also been reported to negatively regulate epileptiform activity due to the inhibitory effect of extracellular ATP metabolites on neurons, making the function of Panx1 in epileptogenesis complex to discern [20,21]. Importantly, there remains a huge knowledge gap regarding Panx1's involvement in neuronal cell loss related to neuroinflammation following seizures.

Recently, PANoptosis has been proposed as a mechanism that coordinates a unique lytic inflammatory cell death process, integrating three classical cell death patterns: pyroptosis, apoptosis, and necroptosis [[22], [23], [24]]. This novel inflammatory PCD pathway exhibits the characteristics of pyroptosis, apoptosis, and/or necroptosis, but cannot be fully explained by any of these three cascades alone [25]. The PANoptosome, a multifaceted protein complex, governs PANoptosis by integrating key proteins from other PCD pathways. Acting as a molecular scaffold or platform, it assembles DAMP / pathogen-associated molecular patterns (PAMPs) sensors (such as ZBP1 and AIM2), adaptors (to connect sensors with effectors, such as ASC), and catalytic effectors (such as Caspase-1, Caspase-8, RIPK1, and RIPK3) in response to cellular stress to mediate PANoptosis [26]. Due to the continuous release of both cytokines and DAMPs, PANoptosis in macrophages can cause robust inflammation, culminating in organ injury and even lethality in patients with various infections [27]. To date, PANoptosis has been demonstrated in a range of human diseases, including ulcerative colitis gut mucosa [28], cancers [29,30], and neurological diseases [31,32]. Nevertheless, whether and how PANoptosis is involved in epilepsy-associated neuron death remains to be fully clarified and requires further investigation. Furthermore, given the implication of Panx1 in diverse PCD forms, it is of particular interest to explore its potential role in this novel form of inflammatory PCD in the context of epilepsy.

In the current study, we aim to investigate the hypothesis that pharmacological inhibition of Panx1 mitigates epileptic damage induced by pilocarpine through decreasing PANoptosis-like neuron death. We first detected that Panx1 was up-regulated in pilocarpine-treated mice and HT22 cells. Pharmacological inhibition of the Panx1 significantly improved epileptic behaviors in animals and alleviated neuron loss both in vivo and in vitro. We observed that cell death caused by pilocarpine could not be completely ameliorated by inhibitors of pyroptosis, apoptosis, or necroptosis alone, suggesting the occurrence of PANoptosis-like cell death. The neuron loss was consistent with features of PANoptosis in mice, and blocking Panx1 with probenecid or 10PANX could mitigate PANoptosis-like cell death.