Modulation of the HMGB1/TLR4/NF-κB signaling pathway in the CNS by matrine in experimental autoimmune encephalomyelitis

Highlights

• Matrine suppressed inflammation, demyelination and inflammatory cytokines in the CNS of EAE.

• Matrine inhibited HMGB1, TLR4 expression in both astrocytes and microglia.

• Matrine also inhibited MyD88, TRAF6 expression and NF-κB activation, with upregulated expression of IκB-α, an inhibitory molecule for NF-κB activation in the CNS.

Abstract

The inflammatory mediator high-mobility group box 1 (HMGB1)-induced signaling pathway has been shown to play an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophorae flavescens, has the capacity to effectively suppress EAE. However, the impact of MAT treatment on HMGB1-induced signaling is not known. In the present study, we show that MAT treatment alleviated disease severity of ongoing EAE, reduced inflammatory infiltration and demyelination, and reduced the production of inflammatory factors including TNF-α, IL-6, and IL-1β in the CNS. Moreover, MAT administration significantly reduced the protein and RNA expression of HMGB1 and TLR4 in the spinal cord, particularly in astrocytes and microglia/infiltrating macrophages. The expression of MyD88 and TRAF6, and the phosphorylation of NF-κB p65, was also down-regulated after MAT treatment. In contrast, the level of IκB-α, an inhibitory molecule for NF-κB activation, was significantly increased. Furthermore, the direct inhibitory effect of MAT on HMGB1/TLR4/NF-κB signaling in macrophages was further confirmed in vitro. Taken together, these findings demonstrate that MAT treatment alleviated CNS inflammatory demyelination and activation of astrocytes and microglia/macrophages in EAE rats, and that the mechanism underlying these effects may be closely related to modulation of HMGB1/TLR4/NF-κB signaling pathway.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), which affects more than 2.3 million people worldwide, imposing a heavy burden on patients, their families, medical systems and society (Reich et al., 2018). In MS and its animal model, experimental autoimmune encephalomyelitis (EAE), myelin-reactive T cells are activated in the peripheral immune system. These cells, upon migrating into the CNS, activate resident immune-related cells such as microglia and astrocytes, and trigger a cascade of inflammatory responses, resulting in demyelination and axonal damage. Thus far, the mechanism underlying inflammation and immune modulation in the CNS tissue damage is not fully understood, and elucidating the underlying pathogenic mechanism is crucial to developing effective treatments for this disease.

Accumulating evidence has recently shown a key role of the high-mobility group box 1 (HMGB1), a nuclear nonhistone protein, in the pathogenesis of MS (Andersson et al., 2008) and EAE (Robinson et al., 2013; Sun et al., 2015). HMGB1 is released from necrotic and dying neural cells, as well as from infiltrating monocytes in MS lesions, and is secreted into the extracellular space as a pro-inflammatory cytokine during injury, infection, and inflammation responses (Kang et al., 2014; Zhu et al., 2018). HMGB1 targets the receptor for advanced glycation end products or through toll-like receptors 2 (TLR2) and TLR4 (Andersson et al., 2008), which interact with myeloid differentiation factor 88 (MyD88) to activate the downstream signaling molecules IL-1 receptor associated kinase (IRAK) and tumor necrosis factor receptor-associated factor 6 (TRAF6). This signaling pathway then activates nuclear factor-kappa B (NF-κB), which increases the production of pro-inflammatory cytokines and results in an inflammatory response (Li et al., 2018; Lin et al., 2008). Inhibiting HMGB/NF-κB signaling pathway could thus be an important approach to suppressing MS/EAE (Paudel et al., 2019).

Matrine (MAT), a quinolizidine alkaloid component extracted from the root of Sophorae flavescens, has been shown to have anti-inflammatory, anti-allergic and anti-tumor effects (Qiu et al., 2014). We have previously reported that MAT decreased clinical EAE severity, reduced CNS inflammatory infiltration and demyelination, inhibited production of pro-inflammatory cytokines/chemokines such as IFN-γ, IL-17, IL-23, ICAM-1 and VCAM-1 (Kan et al., 2013; Zhao et al., 2011), and promoted the production of anti-inflammatory molecules such as IL-4 and IL-10 (Liu et al., 2014). In addition, MAT protected neurons and astrocytes against focal cerebral ischemia by inhibiting the expression of NF-κB (Xu et al., 2012). These results suggested that MAT had significant anti-inflammatory potential for the treatment of EAE/MS. However, to the best of our knowledge, whether MAT plays a protective role via regulating the HMGB1-induced signaling pathway in the CNS of EAE has not yet been studied. The aim of this study was, therefore, to investigate this important question.