Multiple sclerosis (MS) is a chronic, autoimmune-mediated and demyelinating disorder characterized by a neuroinflammatory process that impacts the central nervous system (CNS). It is estimated that over 2.1 million individuals globally are affected by MS, which stands as the primary cause of neurological disability among young and middle-aged adults [1]. Pathologically, MS is marked by extensive demyelinated lesions in the brain, accompanied by the infiltration of inflammatory immune cells, including microglia and macrophages. Additionally, there is notable proliferation of astrocytes and the synthesis of glial fibers [2]. Although the exact etiology of MS remains elusive, numerous studies indicate that neural network dysfunction and the activation of pyroptosis may play significant roles in the pathogenesis of the disease [3, 4, 5, 6].

Recent research has increasingly focused on the interactions between oligodendrocytes and neurons, as well as between microglia and astrocytes, due to their distinct yet complementary roles in various biological processes, including apoptosis, inflammation, neurodegeneration, and myelination [4]. Microglia, which represent approximately 10% of all cells in the CNS, serve as immune cells and facilitate tissue repair within the brain. However, in the context of MS, microglial activation occurs at an early stage, resulting in the production of significant quantities of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-$\alpha$, interleukin-1$\beta$ (IL-1$\beta$) [7, 8]. Subsequently, activated microglia can trigger the transformation of astrocytes into highly reactive and neurotoxic cells in MS [9, 10]. Furthermore, these cytokines may exacerbate mitochondrial damage in neurons [11]. Collectively, findings position microglia as pivotal players in the pathogenesis of MS, underscoring the necessity for a deeper understanding of neuroimmune interactions to inform therapeutic strategies for this condition.

Pyroptosis represents a novel form of programmed cell death, distinguished by the release of pro-inflammatory intracellular components alongside cellular demise. In the canonical pyroptotic pathway, the activation of caspase-1 is initiated by NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes, which subsequently leads to the cleavage of gasdermin-D (GSDMD). This cleavage results in the formation of an N-terminal fragment with the capacity to create membrane pores. Furthermore, IL-1$\beta$ and IL-18 are activated by caspase-1 and subsequently released into the extracellular environment through the GSDMD pore [12]. Research has indicated that patients diagnosed with MS exhibit increased levels of IL-1$\beta$ and GSDMD-N in peripheral blood mononuclear cells and the post-mortem brain tissue [13]. In the context of experimental autoimmune encephalomyelitis (EAE), an animal model for MS, the inhibitors targeting the NLRP3 inflammasome have demonstrated the ability to provide protection against neuroinflammation and demyelination [14]. Consequently, there is a pressing need to investigate the molecular mechanisms that regulate pyroptosis in the context of MS.

High Mobility Group Box 1 (HMGB1) is a highly conserved chromatin-associated protein extensively expressed in neuronal cells. The HMGB1/toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-$\kappa$B) signaling pathway has been recognized as a pivotal mediator in inflammatory responses [15, 16]. Nonetheless, the underlying mechanisms and regulatory aspects of this pathway in pyroptosis and the interactions among various cell types in MS remain inadequately elucidated. Consequently, a comprehensive understanding of HMGB1’s role could yield significant therapeutic insights for MS.

Our objective in this investigation was to investigate the role of HMGB1 in the inflammatory immune response and pyroptosis associated with MS through the use of EAE mice and conditioned medium (CM) obtained from activated BV-2 cells.

Although the molecular mechanisms underlying MS remain incompletely elucidated, intercellular communication, immune dysregulation, and inflammation are widely acknowledged as central contributors to its pathogenesis. Recent reviews have highlighted the involvement of several key signaling pathways—including TNF-$\alpha$, TGF-$\beta$, NF-$\kappa$B, and Wnt—mediated by exosomal contents such as miRNAs and HMGB1, which offer novel insights into both the underlying mechanisms and potential therapeutic strategies for MS [21]. In this study, we observed that EAE mice displayed increased concentrations of HMGB1 within the CNS. The downregulation of HMGB1 may serve to inhibit microglial activation and pyroptosis, thereby mitigating the inflammatory response and enhancing neurological function. Furthermore, we noted an increase in HMGB1 release in the MS cell model, which was associated with neuronal and astrocytic cell death and pyroptosis. Consequently, HMGB1 may act as a fundamental triggering factor in MS and represents a potential therapeutic target for intervention.

Among a variety of contributing factors, persistent activation of microglia is recognized as a significant driver of MS [22]. Research has demonstrated that microglial activation can lead to the formation of the NLRP3 inflammasome and enhance GSDMD-mediated pyroptosis [23], which is regulated by HMGB1 in cases of neonatal hypoxic-ischemic brain injury [24]. Therefore, it is imperative to investigate the roles of HMGB1 and pyroptosis within the pathophysiological mechanisms of MS. Recent studies have identified HMGB1 as a potent pro-inflammatory mediator that facilitates the M1 polarization of microglia [24, 25]. Furthermore, it has been observed that there is an elevation of HMGB1 and TLR4 in the cerebrospinal fluid and white matter regions of MS patients [26]. In the context of cardiac ischemia/reperfusion injury, TLR4 may regulate NF-$\kappa$B expression, thereby amplifying the inflammatory response and initiating pyroptosis through the activation of the NLRP3 inflammasome [27]. Our current investigation revealed that microglia were activated, and that the HMGB1/TLR4/NF-$\kappa$B signaling pathways, along with proteins associated with pyroptosis, were upregulated in EAE mice. Additionally, the downregulation of HMGB1 was found to reverse the increase in TLR4/NF-$\kappa$B signaling pathway proteins and pyroptosis-related protein levels in the EAE mice.

Conversely, HMGB1 can be released into the CNS by activated microglia and necrotic cells during MS. Recent studies indicate that serum concentrations of HMGB1 are significantly higher in MS patients [28, 29, 30]. Additionally, a notable positive correlation has been identified between HMGB1 levels and both physical and psychological well-being in these patients [29]. Furthermore, research has demonstrated that extracellular HMGB1 may induce an overexpression of NF-$\kappa$B-regulated genes and facilitate NF-$\kappa$B nuclear translocation in oligodendrocyte progenitor cells [31]. Consequently, extracellular HMGB1 may serve as a critical mediator in triggering the inflammatory cascade within the CNS by interacting with TLR4 and activating the HMGB1/TLR4/NF-$\kappa$B signaling pathways. However, to our knowledge, no studies have specifically investigated the role of HMGB1 in the interactions between microglia and neurons or astrocytes in the context of MS. In our investigation, we found that CM from BV-2 cells treated with MOG35-55 resulted in cell death, the secretion of inflammatory cytokines, and pyroptosis in HT-22 and Ma-c cells. Notably, the downregulation of HMGB1 was found to mitigate these inflammatory responses. Therefore, it is plausible that HMGB1 may function as a central hub for neuroimmune interactions and facilitate communication between microglia and astrocytes. Furthermore, recent research has underscored the significant involvement of NLRP3 inflammasome activation and pyroptosis in microglia in the pathogenesis of MS [32]. In addition, the inhibition of NLRP3 through selective small molecules [33], exosomal miR-23b-3p [34], and ketogenic diets [6] has been shown to exert inhibitory effects on neuroinflammation and pyroptosis, potentially alleviating central neuropathic pain associated with MS. Our study further demonstrated that the suppression of HMGB1 expression in microglia could inhibit pyroptosis and reduce the release of TNF-$\alpha$ and IL-1$\beta$ from neurons and astrocytes via the HMGB1/TLR4/NF-$\kappa$B signaling pathways. Thus, HMGB1 inhibitors may represent novel therapeutic strategies for MS and NLRP3-related inflammatory diseases.

To date, novel pharmacological agents targeting HMGB1 have been utilized in therapeutic research, including anti-HMGB1 antibodies, the HMGB1 A box (a recognized inhibitor of HMGB1) [35], ethyl pyruvate (EP) [36], and chloroquine [37]. Additionally, certain medications have shown promising results in treating neurological disorders primarily through the inhibition of HMGB1 in microglial cells. Zhan Zhang et al. [38] have reported that pregabalin may alleviate microglial activation and neuronal damage by modulating the HMGB1-TLR2/TLR4/RAGE signaling pathway in cases of radiation-induced brain injury. Similarly, Bo Wang et al. [39] have demonstrated that minocycline can mitigate depressive-like behaviors by inhibiting the release of HMGB1 from microglia and neurons. Consequently, based on our research findings and the conclusions drawn from other studies, the downregulation of HMGB1 expression in microglia may represent a novel therapeutic approach for MS. However, the current literature assessing HMGB1-targeting agents in the context of EAE and MS patients remains limited [40, 41, 42]. Furthermore, there is a significant lack of studies documenting the clinical outcomes associated with the administration of HMGB1 neutralizing agents in MS patients. As a result, the involvement of HMGB1 in MS necessitates further investigation, although it faces certain challenges that may impede its future clinical application. Previous studies have identified two isoforms of HMGB1, fully reduced HMGB1 (fr-HMGB1) and disulfide HMGB1 (ds-HMGB1), both of which are believed to interact with receptors and contribute to pro-inflammatory processes, but another isoform of HMGB1, fully oxidized HMGB1 (oxHMGB1), is regarded as inert [43, 44, 45, 46]. The coexistence of these distinct HMGB1 isoforms within the extracellular matrix complicates the determination of the specific functions of individual antagonists. Moreover, the pharmacological inhibition of HMGB1 at inappropriate times may hinder tissue repair rather than diminish inflammation [47], underscoring a potential risk associated with the use of anti-HMGB1 therapies within the CNS.

As indicated above, increased serum HMGB1 is associated with raised depression levels and poor sleep in MS patients [29]. Poor sleep and depression are commonly associated with decreased pineal and local melatonin production [48], with melatonin increasing sirtuin-1 to deacetylate HMGB1 and decreasing its cytoplasmic translocation, thereby decreasing HMGB1 levels and capacity to induce the TLR4/NF-$\kappa$B pathway [49]. The suppression of pineal and local melatonin production, commonly observed in MS, may therefore be an important upstream factor in the HMGB1 rise in MS. This includes MS microglia, where the induction of the microglia melatonergic pathway and the autocrine effects of melatonin shift microglia from a pro-inflammatory M1-like phenotype to a pro-phagocytic M2-like phenotype [50]. The beneficial effects of exogenous melatonin on knee muscle strength, manual dexterity, static postural balance, mood, and cognition in MS patients would support this [51]. It is also important to note that alterations in the gut microbiome have long been associated with MS pathogenesis and pathophysiology, including the suppression of the gut short-chain fatty acid, butyrate [52]. Butyrate is an epigenetic regulator and, like melatonin, suppresses HMGB1 [53], whilst the loss of pineal melatonin will contribute to the increased gut permeability and gut dysbiosis evident in MS [54]. Pineal melatonin loss at night may be intimately linked to the pathogenesis of a host of diverse medical conditions [55], partly mediated by an alteration in how biomedical processes are dampened and reset at night, including via alterations in the gut microbiome and its products. The interactions of such circadian and systemic factors in a night-time pathogenesis of MS will be important to determine in future research.

In summary, the results of the present study suggest that the downregulation of HMGB1 expression may alleviate the symptoms observed in EAE mice by reducing microglial activation and pyroptosis within the CNS. Additionally, exposure to MOG35-55 was found to trigger the active release of HMGB1 from BV-2 cells. The supernatant derived from BV-2 cells cultured with MOG35-55 was shown to promote the secretion of TNF-$\alpha$ and IL-1$\beta$, and induce the expression of pyroptosis-related proteins in HT-22 and Ma-c cells. However, our study had some limitations. First, the present study primarily relied on in vitro cellular models and the EAE mouse model, which, while widely used, may not fully recapitulate the complex immunopathological features of human MS. Validation of these findings in human tissue samples or clinical settings is necessary to assess their translational relevance. Second, we did not employ direct inhibitors targeting extracellular HMGB1, leaving the immediate effects of extracellular HMGB1 blockade to be further investigated. A comprehensive exploration of the diverse roles of HMGB1 at various stages of the disease may enhance therapeutic strategies for MS. Although we observed the activation of pyroptotic pathways and pro-inflammatory cytokine secretion following HMGB1 release, our study did not evaluate upstream signaling events such as pattern recognition receptor engagement or downstream cell-specific responses within the broader neuroimmune environment. Future studies should incorporate primary cell cultures or animal models that better mimic in vivo conditions to further investigate the mechanism of HMGB1 in MS pathogenesis.

Our findings indicate that HMGB1 serves as a facilitator of weight loss and neurological impairments in EAE mice. The down-regulation of HMGB1 expression has the potential to significantly alleviate these symptoms by modulating microglial activation and pyroptosis. Furthermore, the release of HMGB1 into the extracellular environment by microglia contributes to the inflammatory response and pyroptosis in both neurons and astrocytes. These results underscore the role of HMGB1 as a promoter of pyroptosis and a mediator in the inflammatory immune response. Consequently, the modulation of HMGB1 expression may represent a promising therapeutic target in the treatment of MS.

MS, Multiple sclerosis; CNS, Central nervous system; IL-1$\beta$, interleukin 1$\beta$; EAE, Experimental autoimmune encephalomyelitis; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; HMGB1, High mobility group box 1; SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide; TLR4, Toll-like receptor 4; NF-$\kappa$B, Nuclear factor-kappa B; CM, Conditioned medium; DMEM, Dulbecco’s Modified Eagle’s Medium; MOG35-55, Myelin Oligodendrocyte Glycoprotein 35-55; MOI, Multiplicity of infection; NLRP3, NOD-like receptor family pyrin domain containing 3; HE, Haematoxylin-Eosin; PBS, phosphate-buffered saline; ELISA, Enzyme-Linked Immunosorbent Assay; WB, Western Blot; PVDF, Polyvinylidene Difluoride; GSDMD-N, N-terminus of GSDMD; DAPI, 4^′,6^′-diamidino-2-phenylindole; EP, Ethyl Pyruvate.

The datasets involved in the present study can be provided under reasonable request from the corresponding author.

YF: Conceptualization, Formal analysis, Investigation, Methodology, Software, Writing — original draft; LW: Investigation, Methodology; ZM: Investigation, Methodology; WW: Conceptualization, Funding acquisition, Project administration, Supervision, Writing. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The animal study was approved by the Ethic Committee of The Second Hospital of Hebei Medical University (No. 2024-R004), and all the procedures were conducted under the guidelines for the use of live animals of the National Institute of Health.

The present study was supported by the Hebei Natural Science Foundation (NO.H2022206492).

The authors declare no conflict of interest.

The authors used ChatGPT only to improve the language of the Introduction and Discussion section, with an emphasis on improving clarity and correcting grammar. The scientific content was written entirely independently by the authors, without the use of AI tools. We hope that this clarification will resolve any issues with the use of AI in our submitted manuscript. The authors read the full article after using the AI tool and is responsible for the article.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL37838.