TLR4 aggravates microglial pyroptosis by promoting DDX3X-mediated NLRP3 inflammasome activation via JAK2/STAT1 pathway after spinal cord injury.

Toll-like receptor 4 (TLR4) participates in the initiation of neuroinflammation in various neurological diseases, including central nervous system injuries. NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated microglial pyroptosis is crucial for the inflammatory response during secondary spinal cord injury (SCI). However, the underlying mechanism by which TLR4 regulates NLRP3 inflammasome activation and microglial pyroptosis after SCI remains uncertain. We established an in vivo mouse model of SCI using TLR4-knockout (TLR4-KO) and wild-type (WT) mice. The levels of pyroptosis, tissue damage and neurological function recovery were evaluated in the three groups (Sham, SCI, SCI-TLR4-KO). To identify differentially expressed proteins, tandem mass tag (TMT)-based proteomics was conducted using spinal cord tissue between TLR4-KO and WT mice after SCI. For our in vitro model, mouse microglial BV2 cells were exposed to lipopolysaccharides (1 µg/ml, 8 h) and adenosine triphosphate (ATP) (5 mM, 2 h) to induce pyroptosis. A series of molecular biological experiments, including Western blot (WB), real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemical (IHC), chromatin immunoprecipitation (ChIP), Dual-Luciferase Reporter assay (DLA) and co-immunoprecipitation (Co-IP), were performed to explore the specific mechanism of microglial pyroptosis in vivo and in vitro. Our results indicated that TLR4 promoted the expression of dead-box helicase 3 X-linked (DDX3X), which mediated NLRP3 inflammasome activation and microglial pyroptosis after SCI. Further analysis revealed that TLR4 upregulated the DDX3X/NLRP3 axis by activating the JAK2/STAT1 signalling pathway, and importantly, STAT1 was identified as a transcription factor promoting DDX3X expression. In addition, we found that biglycan was increased after SCI and interacted with TLR4 to jointly regulate microglial pyroptosis through the JAK2/STAT1/DDX3X/NLRP3 axis after SCI. Our study preliminarily identified a novel mechanism by which TLR4 regulates NLRP3 inflammasome-mediated microglial pyroptosis in response to SCI-providing a novel and promising therapeutic target for SCI.

Wang J ，Zhang F ，Xu H ，Yang H ，Shao M ，Xu S ，Lyu F ... -  《Clinical and Translational Medicine》

Paeonol regulates NLRP3 inflammasomes and pyroptosis to alleviate spinal cord injury of rat.

Spinal cord injury (SCI) is a life-threatening traumatic disorder. Paeonol has been confirmed to be involved in a variety of diseases. The purpose of this study is to investigate the role of paeonol on SCI progression. Sprague Dawley (SD) rat was used for the establishment of SCI model to explore the anti-inflammation, anti-oxidation, and neuroprotective effects of paeonol (60 mg/kg) on SCI in vivo. For in vitro study, mouse primary microglial cells (BV-2) were induced by lipopolysaccharide (LPS)/adenosine triphosphate (ATP) treatment. The effect of paeonol on the polarization of LPS/ATP-induced BV-2 cells was determined by detection the expression inducible nitric oxide synthase (iNOS), tumour necrosis factor alpha (TNF-α), arginase-1 (Arg-1), and interleukin (IL)-10 using qRT-PCR. ELISA was used to assess the levels of IL-1β, IL-18, TNF-α, malondialdehyde (MDA), and glutathione (GSH). Western blotting was conducted to determine the levels of NLRP3 inflammasomes and TLR4/MyD88/NF-κB (p65) pathway proteins. Paeonol promoted the recovery of locomotion function and spinal cord structure, and decreased spinal cord water content in rats following SCI. Meanwhile, paeonol reduced the levels of apoptosis-associated speck-like protein (ASC), NLRP3, active caspase 1 and N-gasdermin D (N-GSDMD), repressed the contents of IL-1β, IL-18, TNF-α and MDA, and elevated GSH level. In vitro, paeonol exerted similarly inhibiting effects on pyroptosis and inflammation. Meanwhile, paeonol promoted BV-2 cells M2 polarization. In addition, paeonol also inactivated the expression of TLR4/MyD88/NF-κB (p65) pathway. Paeonol may regulate NLRP3 inflammasomes and pyroptosis to alleviate SCI, pointing out the potential for treating SCI in clinic.

Zhao H ，Wang X ，Liu S ，Zhang Q ... -  《BMC NEUROSCIENCE》

Deficiency of the microglial Hv1 proton channel attenuates neuronal pyroptosis and inhibits inflammatory reaction after spinal cord injury.

Li X ，Yu Z ，Zong W ，Chen P ，Li J ，Wang M ，Ding F ，Xie M ，Wang W ，Luo X ... -  《Journal of Neuroinflammation》

TREM2 Deficiency Aggravates NLRP3 Inflammasome Activation and Pyroptosis in MPTP-Induced Parkinson's Disease Mice and LPS-Induced BV2 Cells.

Microglia-mediated neuroinflammation plays a crucial role in the pathogenesis of Parkinson's disease (PD). Triggering receptor expressed on myeloid cells 2 (TREM2) confers strong neuroprotective effects in PD by regulating the phenotype of microglia. Recent studies suggest that TREM2 regulates high glucose-induced microglial inflammation through the NLRP3 signaling pathway. This study aimed to investigate the effect of TREM2 on NLRP3 inflammasome activation and neuroinflammation in PD. Mice were injected with AAV-TREM2-shRNA into both sides of the substantia nigra using a stereotactic injection method, followed by intraperitoneal injection of MPTP to establish chronic PD mouse model. Behavioral assessments including the pole test and rotarod test were conducted to evaluate the effects of TREM2 deficiency on MPTP-induced motor dysfunction. Immunohistochemistry of TREM2 and tyrosine hydroxylase (TH), immunohistochemistry and immunofluorescence Iba1, Western blot of NLRP3 inflammasome and its downstream inflammatory factors IL-1β and IL-18, and the key pyroptosis factors GSDMD and GSDMD-N were performed to explore the effect of TREM2 on NLRP3 inflammasome and neuroinflammation. In an in vitro experiment, lentivirus was used to interfere with the expression of TREM2 in BV2 microglia, and then lipopolysaccharide (LPS) and adenopterin nucleoside triphosphate (ATP) were used to stimulate inflammation to construct a cellular inflammation model. The expression differences of NLRP3 inflammasome and its components were detected by qPCR and Western blot. In vivo, TREM2 knockdown aggravated the loss of dopaminergic neuron and the decline of motor function. After TREM2 knockdown, the number of activated microglia was significantly increased, and the expression of cleaved caspase-1, NLRP3 inflammasome, IL-1β, GSDMD, and GSDMD-N was increased. In vitro, TREM2 knockdown aggravated the inflammatory response of BV2 cells stimulated by LPS and promoted the activation of NLRP3 inflammasome through the NF-κB pathway. In addition, TREM2 knockdown also promoted the expression of TLR4/MyD88, an upstream factor of the NF-κB pathway. Our vivo and vitro data showed that TREM2 knockdown promoted NLRP3 inflammasome activation and downstream inflammatory response, promoted pyroptosis, and aggravated dopaminergic neuron loss. TREM2 acts as an anti-inflammatory in PD through the TLR4/MyD88/NF-κB pathway, which extends previous findings and supports the notion that TREM2 ameliorates neuroinflammation in PD.

Huang P ，Zhang Z ，Zhang P ，Feng J ，Xie J ，Zheng Y ，Liang X ，Zhu B ，Chen Z ，Feng S ，Wang L ，Lu J ，Liu Y ，Zhang Y ... -  《-》

Zinc Promotes Microglial Autophagy Through NLRP3 Inflammasome Inactivation via XIST/miR-374a-5p Axis in Spinal Cord Injury.

Zinc has reported to play a neuroprotective role in the development of spinal cord injury (SCI). The protective mechanism of zinc remains to be uncovered. The aim of the current study was to investigate the neuroprotective mechanism of zinc in the progression of SCI. The C57BL/6J mouse SCI model was established to confirm the protective role of zinc in vivo, while the cellular model was induced in mouse microglial BV2 cells by using lipopolysaccharide (LPS). The expression levels of XIST, miR-374a-5p and NLRP3 inflammasome as well as the autophagy-related proteins were detected using real-time PCR and immunoblotting. Cell viability was assessed by CCK-8 assay. Apoptosis was evaluated by TUNEL staining, flow cytometry, the determination of apoptosis-related proteins. The target relationship was confirmed by luciferase reporter assays. Zinc improved locomotor function in SCI mice and alleviated LPS-induced BV2 cell injuries by inhibiting apoptosis and initiating autophagy processes. XIST and NLRP3 inflammasome was upregulated while miR-374a-5p was downregulated in spinal cords of SCI mice and LPS-treated BV2 cells. All these effects were inhibited by Zinc treatment. XIST knockdown triggered microglial autophagy-mediated NLRP3 inactivation in LPS-induced BV2 cells by regulating miR-374a-5p. Zinc treatment protected BV2 cells from LPS-induced cell injury by the downregulation of XIST. This process might be through autophagy‑mediated NLRP3 inflammasome inactivation by targeting miR-374a-5p. Zinc downregulates XIST and induces neuroprotective effects against SCI by promoting microglial autophagy-induced NLRP3 inflammasome inactivation through regulating miR-374a-5p. Our finding provides novel opportunities for the understanding of zinc-related therapy of SCI.

Zhao X ，Sun J ，Yuan Y ，Lin S ，Lin J ，Mei X ... -  《-》