Abstract
Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation. Here, we aimed to determine the role of autophagy in α7nAChR-mediated inhibition of neuroinflammation and its underlying mechanism. Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively. The severity of EAE was evaluated with neurological scoring. Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot. Autophagosomes were observed using transmission electron microscopy and tandem fluorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux. The mRNA levels of interleukin-6 (IL-6), IL-1β, IL-18, and tumor necrosis factor-α (TNF-α) were detected by real-time PCR. We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively. Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice. PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45- or CD68-positive cells with LC3) both in spinal cord and spleen from EAE mice. The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor. In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux. Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1β, IL-18, and TNF-α mRNA. Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE.
Highlights
It is commonly believed that microglia contribute to the triggering of inflammatory responses in central nervous system (CNS), mainly through the induction of inflammatory cytokine production as well as the immune responses [1, 2]
We further investigated the association between autophagy and α7nAChR-mediated anti-inflammatory effects in vivo by examining the production of IL-6, IL-1β, IL-18, and tumor necrosis factor-α (TNF-α) in mRNA level in spinal cord and spleen obtained from mice
We found that Atg5 siRNA or the application of bafilomycin A1 significantly attenuated the inhibitory effect of PNU282987 on the mRNA levels of IL-6, IL-1β, IL-18, and TNF-α in BV2 microglia, indicating that autophagy at least partly mediated the anti-inflammatory effects of PNU282987 in LPS-stimulated BV2 microglia
Summary
It is commonly believed that microglia contribute to the triggering of inflammatory responses in central nervous system (CNS), mainly through the induction of inflammatory cytokine production as well as the immune responses [1, 2]. The overactivation of microglia may lead to the pathogenesis and aggravation of CNS damage in inflammatory diseases including multiple α7nAChR Limits Neuroinflammation via Autophagy sclerosis (MS) [3,4,5]. MS is recognized as a chronic inflammatory autoimmune disorder featuring CNS demyelination and neurodegeneration [6, 7]. Accumulation and overactivation of microglia greatly aggravates the severity of symptoms and demyelination in experimental autoimmune encephalomyelitis (EAE) mice [8, 9]. There is evidence that activation of α7nAChR contributes to the alleviation of neuroinflammation in EAE model [14,15,16], but the underlying mechanism has not been fully clarified
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