Americanin B inhibits pyroptosis in lipopolysaccharide-induced septic encephalopathy mice through targeting NLRP3 protein.

Sepsis is considered as a severe illness due to its high mortality. Sepsis can cause septic encephalopathy, thus leading to brain injury, behavioral and cognitive dysfunction. Pyroptosis is a type of regulated cell death (RCD) and takes a crucial part in occurrence and development of sepsis. Americanin B (AMEB) is a lignan compounds, which is extracted from Vernicia fordii. In our previous study, AMEB could inhibit microglial activation in inflammatory cell model. However, the function of AMEB in septic encephalopathy mice is uncertain. It would be worthwhile to ascertain the role and mechanism of AMEB in sepsis. Current study designs to certify the relationship between pyroptosis and septic encephalopathy, and investigate whether AMEB can restrain NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation and restrict pyroptosis by targeting NLRP3 in septic mice model. C57BL/6 mice were utilized to perform sepsis model in vivo experiments. BV-2 cell lines were used for in vitro experiments. In vivo sepsis model was established by lipopolysaccharide (LPS) intraperitoneal injection in male C57BL/6 J mice and in vitro model was exposed by LPS plus ATP in BV-2 cells. The survival rate was monitored on the corresponding days. NLRP3, apoptosis associated Speck-like protein (ASC), caspase-1, GasderminD (GSDMD), interleukin-1β (IL-1β) and interleukin-18 (IL-18) level were detected by western blotting and immunofluorescence analysis. Molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) experiments, RNAi transfection and quantitative real-time PCR were applied to confirm the potential target of AMEB. The results suggested that AMEB could rise survival percentage and lighten brain injury in LPS-induced sepsis mice. In addition, AMEB could inhibit pyroptosis and the activiation of NLRP3 inflammasome. The inhibiting function of AMEB on the activiation of NLRP3 inflammasome is weakened following si-NLRP3 transfection. Moreover, AMEB exerted anti-pyroptosis effect via targeting NLRP3 protein. Our findings first indicate NLRP3 is an effective druggable target for septic encephalopathy related brain injury, and also provide a candidate-AMEB for the treatment of septic encephalopathy. These emerging findings on AMEB in models of sepsis suggest an innovative approach that may be beneficial in the prevention of septic encephalopathy.

Liu Y ，Yao X ，Yang Y ，Mi Y ，Wang Y ，Tan S ，Fang M ，Meng Q ，Chen G ，Li N ，Hou Y ... -  《-》

Pachymaran Alleviates Acute Gouty Arthritis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis.

Yan H ，Zhou Y ，Xie Z ，Zhang X ，Yin C ，Liu J ，Yang B ... -  《ALTERNATIVE THERAPIES IN HEALTH AND MEDICINE》

Geranylgeranyl diphosphate synthase 1 knockdown suppresses NLRP3 inflammasome activity via promoting autophagy in sepsis-induced acute lung injury.

NOD-like receptor protein 3 (NLRP3) inflammasome activation has emerged as a crucial contributor to sepsis-induced lung injury. Geranylgeranyl diphosphate synthase 1 (GGPPS1) reportedly exerts the pro-inflammatory capability via activation of NLRP3 inflammasome. However, little is known about the role and mechanism of GGPPS1 in sepsis-induced lung injury. Mice underwent cecal ligation and puncture (CLP) surgery to establish the in vivo model of sepsis. The lung injury of mice was assessed by analyzing the histological changes, the lung wet/dry ratio, PaO2/FiO2 ratio, myeloperoxidase (MPO) activity, total protein content, total cell, and polymorphonuclear leukocyte counts. Mouse alveolar macrophages MH-S were exposed to LPS for developing in vitro model of sepsis. The mRNA and protein expression levels of GGPPS1, beclin-1, and autophagy and inflammasome-related genes were detected using quantitative reverse transcription-polymerase chain reaction and western blot assays. Enzyme-linked immunosorbent assay was conducted to determine the levels of interleukin (IL)-1β and IL-18. We successfully established sepsis-induced acute lung injury in vivo by CLP surgery. GGPPS1 was upregulated in the lung tissues of CLP-induced septic mice. The activation of autophagy and NLRP3 inflammasome were found in the lung tissues of CLP-induced septic mice. The addition of exogenous GGPP (synthesis products catalyzed by GGPPS1) and autophagic inhibitor 3-MA aggravated sepsis-induced hypoxemia, alveolar inflammatory response, intrapulmonary hemorrhage, and pulmonary edema, as evidenced by increased lung injury score, lung wet/dry weight ratio, MPO activity, total protein content, total cell, and PMNs counts, and decreased PaO2/FiO2 ratio. While NLRP3 inhibitor MCC950 exerted the opposite effects. Additionally, administration of exogenous GGPP could inhibit the activation of autophagy, enhance the activity of NLRP3 inflammasome, and the production of IL-1β and IL-18. Inhibition of autophagy by 3-MA treatment also promoted the activity of NLRP3 inflammasome and the production of IL-1β and IL-18. While MCC950 restrained the activity of NLRP3 inflammasome, but did not affect the activation of autophagy. Notably, the expression of GGPPS1 was unaltered in CLP-induced mice following GGPP, 3-MA, or MCC950 treatment. Moreover, GGPPS1 was upregulated in MH-S cells stimulated with LPS, and GGPPS1 knockdown enhanced the activation of autophagy and inhibited the activity of NLRP3 inflammasome in vitro. Importantly, depletion of GGPPS1 could alleviate LPS-induced inflammatory response by inducing autophagy-dependent NLRP3 inflammasome inhibition. GGPPS1 knockdown suppressed NLRP3 inflammasome activity via promoting autophagy and then attenuated sepsis-induced acute lung injury, revealing a novel target for treating sepsis-induced lung injury.

Li D ，Li C ，Wang T ，Zhang C ，Zhu Z ，Zhang G ，Fang B ... -  《-》

Prim-O-glucosycimifugin attenuates liver injury in septic mice by inhibiting NLRP3 inflammasome/caspase-1 signaling cascades in macrophages.

Liver dysfunction and liver failure are serious complications of sepsis, directly leading to septic progression and death. Now, there is no specific therapeutics available for sepsis-related liver dysfunction. Prim-O-glucosylcimifugin (POG), a chromone richest in the roots of Saposhnikovia divaricata (Turcz.) Schischk, is usually used to treat headache, rheumatoid arthritis and tetanus. While, the underlying mechanisms of POG against sepsis-induced liver damage and dysfunction are still not clear. To study the anti-sepsis effect of POG, and its pharmacological mechanism to protect liver injury by weakening the function of macrophages in septic livers through inhibiting NOD-like receptor protein 3 (NLRP3) inflammasome pathway. In vivo experiments, septic mouse model was induced by cecal ligation and puncture (CLP), and then the mortality was detected, liver inflammatory damages and plasma biomarkers of liver injury were evaluated by histopathological staining and biochemical assays, respectively. In vitro experiments, mouse primary peritoneal macrophages were treated with lipopolysaccharide (LPS) and ATP, and then the activated-inflammasomes, macrophage migration and polarization were detected by ASC immunofluorescence staining, transwell and flow cytometry assays, respectively. NLRP3 inflammasome components NLRP3, caspase-1, IL-1β and IL-18 protein expressions were detected using western blot assays, and the contents of IL-1β and IL-18 were measured by ELISA assays. POG treatment significantly decreased the mortality, liver inflammatory damages, hepatocyte apoptosis and plasma biomarkers of liver injury in CLP-challenged male WT mice, which were comparable to those in ibuprofen (a putative anti-inflammatory drug)-supplemented septic male WT mice and septic NLRP3 deficient-male mice. POG supplementation significantly suppressed NLRP3 inflammasome activation in septic liver tissues and cultured macrophages, by significantly reducing NLRP3, cleaved-caspase-1, IL-1β and IL-18 levels, the activated-inflammasome ASC specks, and macrophage infiltration and migration, as well as M1-like polarization, but significantly increasing M2-like polarization. These findings were similar to the pharmacological effects of ibuprofen, NLRP3 deficiency, and a special NLRP3 inhibitor, MCC950. POG protected against sepsis by inhibiting NLRP3 inflammasome-mediated macrophage activation in septic liver and attenuating liver inflammatory injury, indicating that it may be a potential anti-sepsis drug candidate.

Liu LL ，Yan X ，Xue KY ，Wang XM ，Li LY ，Chen HY ，Li RL ，Li H ，Lan J ，Xin JJ ，Li X ，Zhuo CL ，Wu Z ，Zhang D ，Huang WJ ，Wang YL ，Li XY ，Jiang W ，Zhang HY ... -  《-》

Microglial NLRP3 Inflammasome Induces Excitatory Synaptic Loss Through IL-1β-Enriched Microvesicle Release: Implications for Sepsis-Associated Encephalopathy.

Acute cerebral dysfunction is a pathological state common in severe infections and a pivotal determinant of long-term cognitive outcomes. Current evidence indicates that a loss of synaptic contacts orchestrated by microglial activation is central in sepsis-associated encephalopathy. However, the upstream signals that lead to microglial activation and the mechanism involved in microglial-mediated synapse dysfunction in sepsis are poorly understood. This study investigated the involvement of the NLRP3 inflammasome in microglial activation and synaptic loss related to sepsis. We demonstrated that septic insult using the cecal ligation and puncture (CLP) model induced the expression of NLRP3 inflammasome components in the brain, such as NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and IL-1β. Immunostaining techniques revealed increased expression of the NLRP3 inflammasome in microglial cells in the hippocampus of septic mice. Meanwhile, an in vitro model of primary microglia stimulated with LPS exhibited an increase in mitochondrial reactive oxygen species (ROS) production, NLRP3 complex recruitment, and IL-1β release. Pharmacological inhibition of NLRP3, caspase-1, and mitochondrial ROS all decreased IL-1β secretion by microglial cells. Furthermore, we found that microglial NLRP3 activation is the main pathway for IL-1β-enriched microvesicle (MV) release, which is caspase-1-dependent. MV released from LPS-activated microglia induced neurite suppression and excitatory synaptic loss in neuronal cultures. Moreover, microglial caspase-1 inhibition prevented neurite damage and attenuated synaptic deficits induced by the activated microglial MV. These results suggest that microglial NLRP3 inflammasome activation is the mechanism of IL-1β-enriched MV release and potentially synaptic impairment in sepsis.

Moraes CA ，Hottz ED ，Dos Santos Ornellas D ，Adesse D ，de Azevedo CT ，d'Avila JC ，Zaverucha-do-Valle C ，Maron-Gutierrez T ，Barbosa HS ，Bozza PT ，Bozza FA ... -  《-》