L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF-κB translocation in cortical neurons/glia Cocultures.

Ya-Ni Huang,Jia-Yi Wang,Chien-Tsai Chiu,Chien-Cheng Lai,Jhen-Jhe Lin

doi:10.1371/journal.pone.0097276

Ya-Ni Huang, Jia-Yi Wang + Show 3 more

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https://doi.org/10.1371/journal.pone.0097276

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In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS) to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C) affected neuroinflammation. LPS (100 ng/ml) induced the expression of inducible NO synthase (iNOS) and the production of NO, interleukin (IL)-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2) in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM) attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation) of mitogen-activated protein kinases (MAPKs), such as p38 at 30 min and extracellular signal-regulated kinases (ERKs) at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK) as these inhibitors. Vit. C also reduced LPS-induced IκB-α degradation and NF-κB translocation. Thus, Vit. C suppressed the LPS-stimulated production of inflammatory mediators in neuron/glia cocultures by inhibiting the MAPK and NF-κB signaling pathways.

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Neuroinflammation plays a crucial role in the pathogenesis of acute brain insults, such as bacterial infections [1,2], cerebral ischemia [3], and traumatic brain injury [4], and in chronic neurodegenerative diseases such as Alzheimer’s disease [5]
We found that IL-6 and MIP-2 significantly increased in the cultured cells exposed to LPS for 9 h and remained elevated at up to 24 h (p,.001; Figs. 1C and 1D)
To examine whether the activation of p38 and extracellular signal-regulated kinases (ERKs) mitogen-activated protein kinases (MAPKs) are involved in LPS-induced neuroinflammatory responses, we examined the phosphorylation of p38 and ERK and the effects of inhibitors of p38 and ERK

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Introduction

Neuroinflammation plays a crucial role in the pathogenesis of acute brain insults, such as bacterial infections [1,2], cerebral ischemia [3], and traumatic brain injury [4], and in chronic neurodegenerative diseases such as Alzheimer’s disease [5]. Neuroinflammation involves a complex interplay of glia cells beginning in microglial cells, which activate astrocytes (reactive gliosis) resulting in the release of inflammatory molecules [4,6] that can cause neuronal damage. Glial cells, and astrocytes and microglia, have been shown to provide the early sources of proinflammatory cytokines in cerebrospinal fluid (CSF) and brain tissues when meningoencephalitis is caused by a Gram-negative bacterium (Klebsiella pneumonia) infection [1,2]. Cell culture studies have demonstrated that glial cells activated by lipopolysaccharide (LPS), a Gram-negative bacterial cell wall endotoxin, time-dependently expressed inducible nitric oxide synthase (iNOS) [7] and proinflammatory cytokines and chemokines [8]. Toll-like receptor 4 (TLR4) is the receptor of LPS and is expressed in neurons and glial cells [9]. The activation of TLR4 by LPS causes the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), and the nuclear factor (NF)-kB signaling pathway in brain cells, yielding neuroinflammation [9,10]

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