Disruption of C/EBPß-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain.

BACKGROUND: Growing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear. METHODS: A chronic constriction injury (CCI) rat model was constructed, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPß-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro. RESULTS: Significant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPß, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPß knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector. CONCLUSIONS: This pre-clinical investigation reveals that C/EBPß-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.

NLRP3-dependent pyroptosis exacerbates coxsackievirus A16 and coxsackievirus A10-induced inflammatory response and viral replication in SH-SY5Y cells.

Coxsackievirus A16 (CV-A16) and coxsackievirus A10 (CV-A10), more commonly etiological agents of hand, foot and mouth disease (HFMD), are capable of causing severe neurological syndromes with high fatalities, but their neuropathogenesis has rarely been studied. Mounting evidence indicated that pyroptosis is an inflammatory form of cell death that might be widely involved in the pathogenic mechanisms of neurotropic viruses. Our study was designed to examine the effects of NLRP3-mediated pyroptosis in CV-A16- and CV-A10-induced inflammatory neuropathologic formation. In this work, it was showed that SH-SY5Y cells were susceptible to CV-A16 and CV-A10, and meanwhile their infections could result in a decreasing cell viability and an increasing LDH release as well as Caspase1 activation. Moreover, CV-A16 and CV-A10 infections triggered NLRP3-mediated pyroptosis and promoted the release of inflammatory cytokines. Additionally, activated NLRP3 accelerated the pyroptosis formation and aggravated the inflammatory response, but inhibited NLRP3 had a dampening effect on the above situation. Finally, it was further revealed that NLRP3 agonist enhanced the viral replication, but NLRP3 inhibitor suppressed the viral replication, suggesting that NLRP3-driven pyroptosis might support CV-A16 and CV-A10 production in SH-SY5Y cells. Together, our findings demonstrated a mechanism by which CV-A16 and CV-A10 induce inflammatory responses by evoking NLRP3 inflammasome-regulated pyroptosis, which in turn further stimulated the viral replication, providing novel insights into the pathogenesis of CV-A16 and CV-A10 infections.

Oxymatrine attenuates oxidized low­density lipoprotein­induced HUVEC injury by inhibiting NLRP3 inflammasome­mediated pyroptosis via the activation of the SIRT1/Nrf2 signaling pathway.

Oxymatrine, a quinolizidine alkaloid isolated from the traditional Chinese herb Sophora flavescens Aiton, has been demonstrated to exert anti­inflammatory and atherosclerotic effects, but the molecular mechanism has yet to be elucidated. Accumulating evidence indicates an important role of NLR family pyrin domain containing 3 (NLRP3) inflammasome­mediated pyroptosis in the pathogenesis of atherosclerosis. The present study was undertaken to investigate whether oxymatrine attenuates oxidized low­density lipoprotein (ox­LDL)­induced human umbilical vein endothelial cell (HUVEC) injury, an in vitro cell model of atherosclerosis, by inhibiting NLRP3 inflammasome­mediated pyroptosis, and elucidate the role of the sirtuin (SIRT)1/nuclear factor­erythroid 2­related factor 2 (Nrf2) signaling pathway in this process. Cell viability and cytotoxicity were detected by CCK­8 assay and a lactate dehydrogenase (LDH) assay kit. Cell apoptosis was detected by flow cytometry. Reactive oxygen species (ROS) generation was detected using a ROS assay kit. The malondialdehyde (MDA) content, mitochondrial membrane potential (MMP) level, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH­Px) activities were determined using commercial kits. The inflammatory cytokines levels were measured by ELISA and protein expression was monitored by western blot analysis. The results revealed that oxymatrine alleviated ox­LDL­induced cytotoxicity and apoptosis. Concurrently, oxymatrine inhibited ox­LDL­induced NLRP3 inflammasome­mediated pyroptosis in HUVECs, as evidenced by the significant decreases in the expression of NLRP3, apoptosis­associated speck­like protein containing a C­terminal caspase recruitment domain (ASC), cleaved caspase­1, interleukin (IL)­1ß and IL­18 in HUVECs. In addition, NLRP3 siRNA transfection efficiently suppressed ox­LDL­induced pyroptosis and HUVEC injury. Furthermore, oxymatrine promoted SIRT1/Nrf2 signaling pathway activation in HUVECs subjected to ox­LDL treatment, and SIRT1 deficiency induced by SIRT1 siRNA transfection abolished the protective effect of oxymatrine against ox­LDL­induced injury. SIRT1 siRNA also mitigated the oxymatrine­induced decreases in ROS generation and MDA content, and the increases in MMP as well as the activities of SOD, CAT and GSH­Px in HUVECs. Moreover, SIRT1 siRNA transfection blocked the inhibitory effect of oxymatrine on NLRP3 inflammasome­mediated pyroptosis in ox­LDL­treated HUVECs. Collectively, these results indicated that oxymatrine may attenuate ox­LDL­induced HUVEC injury by inhibiting NLRP3 inflammasome­mediated pyroptosis via activating the SIRT1/Nrf2 signaling pathway.