The combination of Panax ginseng and Angelica sinensis alleviates ischemia brain injury by suppressing NLRP3 inflammasome activation and microglial pyroptosis

BackgroundThe combination of Panax ginseng and Angelica sinensis (CPA) has been used to treat stroke for one thousand years and demonstrated clinically to have satisfied effects. However, the underlying mechanism remains unknown.PurposeWe investigate whether CPA has neuroprotective effects via suppressing Nod-like receptor protein 3 (NLRP3) inflammasome and microglial pyroptosis against ischemic injury in transient middle cerebral artery occlusion (MCAO) rats.MethodsMale rats were divided randomly into sham operated, MCAO, MCC950 (NLRP3-specific inhibitor) and CPA groups. Neurological deficits, glucose uptake, infarct size, activation of NLRP3 inflammasomes, microglial pyroptosis and related signaling pathways were detected. BV-2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used in in vitro experiments.ResultsCompared with sham rats, elevated level of proinflammatory interleukin-1β (IL-1β) in plasma, neurological function deficit, reduced glucose uptake in ipsilateral hemisphere, obvious infarct size, the activation of NLRP3 inflammasomes and enhanced microglial pyroptosis were presented in MCAO rats. The administrations of MCC950 and CPA respectively reversed the results. In vitro OGD/R induced the release of lactate dehydrogenase, promoted NLRP3 inflammasomes activation and pyroptosis in BV-2 cells, which was significantly suppressed by treatment with ginsenoside Rd (Rd) and Z-ligustilide (LIG). Mechanistically, OGD/R induced high expression of dynamin-related protein 1 (Drp1) and mitochondrial fission, as well as NLRP3 inflammasomes activation and pyroptosis in BV-2 cells, which was attenuated by treatment with Rd and LIG. Moreover, the increased expression of Drp1 was validated in MCAO rats, and also abolished by MCC950 or CPA treatments.ConclusionCPA treatment attenuates cerebral injury via inhibition of NLRP3 inflammasomes activation and microglial pyroptosis after stroke, which at least partially involved in the amelioration of Drp1-mediated mitochondrial fission.     

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.     

Wolf–Hirschhorn syndrome candidate 1 facilitates alveolar macrophage pyroptosis in sepsis-induced acute lung injury through NEK7-mediated NLRP3 inflammasome activation

Sepsis is a complex clinical syndrome with high incidence and mortality. Acute lung injury (ALI) is a common complication of sepsis. At present, there is no effective therapeutic strategy to treat ALI. The SET domain–containing histone methyltransferase Wolf–Hirschhorn syndrome candidate 1 (WHSC1) regulates cancer progression, while its role in sepsis-induced ALI remains unclear. Thus, this study aimed to study the effect of WHSC1 on sepsis-induced ALI and to explore the potential mechanism of action. In the study, LPS treatment induced lung injury. WHSC1 was highly expressed in LPS-induced ALI. Knockdown of WHSC1 attenuated LPS-induced ALI and pyroptosis in vivo. Besides, knockdown of WHSC1 attenuated LPS-induced alveolar macrophage pyroptosis in vitro. Furthermore, NIMA-related kinase-7 (NEK7) expression could be regulated by WHSC1, and NEK7 bound to NLRP3 in alveolar macrophages. Moreover, WHSC1 regulated alveolar macrophage pyroptosis through modulating NEK7-mediated NLRP3 inflammasome activation. In conclusion, WHSC1 was highly expressed in LPS-induced ALI. WHSC1 facilitated alveolar macrophage pyroptosis in sepsis-induced ALI through NEK7-mediated NLRP3 inflammasome activation. WHSC1 may be a valuable target for the therapy of sepsis-induced ALI.     

Genetic deletion or pharmacologic inhibition of the Nlrp3 inflammasome did not ameliorate experimental NASH

It has been postulated that inflammasomes, in particular the NLRP3 (NLR family pyrin domain containing 3) inflammasome, mediate the necroinflammation and fibrosis that characterize nonalcoholic steatohepatitis (NASH) by engaging innate immune responses. We aimed to investigate the impact of genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome on experimental steatohepatitis. Global Nlrp3 KO (expected to inhibit the NLRP3 inflammasome) or Casp1 KO (expected to inhibit all inflammasomes) mice were compared to wild type controls after 6 months on a high-fat, high-cholesterol (HFHC, 1% cholesterol) diet known to induce fibrosing steatohepatitis. Additionally, wildtype mice on a HFHC diet (0.75% or 0.5% cholesterol) for 6 months were either treated or not treated with an oral, pharmacologic inhibitor of Nlrp3 (MCC950) that was delivered in the drinking water (0.3 mg/ml). We found that genetic deletion or pharmacologic inhibition of the NLRP3 inflammasome did not ameliorate any of the histological components of fibrosing NASH in HFHC-fed mice. Collectively, these results do not support NLRP3 inhibition as a potential target for human NASH.     

炎性小体在诱导脊髓损伤神经炎症中的作用

脊髓损伤的治疗与康复一直是医学领域的重大难题,尤其是在改善损伤的神经功能方面进展甚微。继发性损伤是造成脊髓损伤后神经功能障碍的主要原因,炎症反应是继发性损伤阶段最重要的病理过程。急性期通过抑制神经炎症来减轻继发性损伤被认为可减轻神经功能损害而达到神经保护作用。炎性小体是一类蛋白质复合体,由模式识别受体中的NLRs家族和PHYIN家族的受体蛋白质作为主要框架组装并命名,常见的炎性小体包括NLRP1、NLRP3、NLRC4(IPAF)、AIM2等。在感染或受到损伤刺激时,炎性小体在细胞质内组装,并激活促炎症蛋白酶胱天蛋白酶1(caspase-1),活化的胱天蛋白酶1一方面促进促炎症细胞因子IL-1β和IL-18的前体成熟和分泌,另一方面介导细胞焦亡。细胞焦亡以细胞肿胀破裂并释放细胞内容物为特征,是在炎症和应激的病理条件下诱导的程序性细胞死亡方式。促炎症细胞因子和焦亡释放的胞内物质都可作为促炎信号引发炎症反应。近期发现,炎性小体通过诱导促炎因子释放以及介导细胞焦亡等途径, 参与激活脊髓损伤后的炎症级联反应,加重继发性神经炎症。靶向抑制炎性小体的激活可减轻炎症反应,促进神经细胞存活,达到神经保护作用。因此,炎性小体有望成为脊髓损伤治疗的新靶点。本文拟从炎性小体的结构及其在脊髓损伤中的作用、激活机制和治疗前景进行综述,以期为后续研究提供思路。     

Celastrol ameliorates Propionibacterium acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3

BackgroundCelastrol, a pentacyclic triterpenoid quinonemethide isolated from several spp. of Celastraceae family, exhibits anti-inflammatory activities in a variety of diseases including arthritis.PurposeThis study aims to investigate whether the inhibition of NLRP3 inflammasome is engaged in the anti-inflammatory activities of celastrol and delineate the underlying mechanism.MethodsThe influence of celastrol on NLRP3 inflammasome activation was firstly studied in lipopolysaccharide (LPS)-primed mouse bone marrow-derived macrophages (BMDMs) and phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells treated with nigericin. Reconstituted inflammasome was also established by co-transfecting NLRP3, ASC, pro-caspase-1 and pro-IL-1β in HEK293T cells. The changes of inflammasome components including NLRP3, ASC, pro-caspase-1/caspase-1 and pro-IL-1β/IL-1β were examined by enzyme-linked immunosorbent assay (ELISA), western blotting and immunofluorescence. Furthermore, Propionibacterium acnes (P. acnes)/LPS-induced liver injury and monosodium urate (MSU)-induced gouty arthritis in mice were employed in vivo to validate the inhibitory effect of celastrol on NLRP3 inflammasome.ResultsCelastrol significantly suppressed the cleavage of pro-caspase-1 and pro-IL-1β, while not affecting the protein expressions of NLRP3, ASC, pro-caspase-1 and pro-IL-1β in THP-1 cells, BMDMs and HEK293T cells. Celastrol suppressed NLRP3 inflammasome activation and alleviated P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis. Mechanism study revealed that celastrol could interdict K63 deubiquitination of NLRP3, which may concern interaction of celastrol and BRCA1/BRCA2-containing complex subunit 3 (BRCC3), and thereby prohibited the formation of NLRP3, ASC and pro-caspase-1 complex to block the generation of mature IL-1β.ConclusionCelastrol suppresses NLRP3 inflammasome activation in P. acnes/LPS-induced liver damage and MSU-induced gouty arthritis via inhibiting K63 deubiquitination of NLRP3, which presents a novel insight into inhibition of celastrol on NLRP3 inflammasome and provides more evidences for its application in the therapy of inflammation-related diseases.     

Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1β production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP_L is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1β. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1β production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1β expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP_L interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1β generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP_L in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.     

Adipose-derived mesenchymal stem cells overexpressing prion improve outcomes via the NLRP3 inflammasome/DAMP signalling after spinal cord injury in rat

Traumatic spinal cord injury (SCI) is a highly destructive disease in human neurological functions. Adipose-derived mesenchymal stem cells (ADMSCs) have tissue regenerations and anti-inflammations, especially with prion protein overexpression (PrPc^OE). Therefore, this study tested whether PrPc^OE-ADMSCs therapy offered benefits in improving outcomes via regulating nod-like-receptor-protein-3 (NLRP3) inflammasome/DAMP signalling after acute SCI in rats. Compared with ADMSCs only, the capabilities of PrPc^OE-ADMSCs were significantly enhanced in cellular viability, anti-oxidative stress and migration against H₂O₂ and lipopolysaccharide damages. Similarly, PrPc^OE-ADMSCs significantly inhibited the inflammatory patterns of Raw264.7 cells. The SD rats (n = 32) were categorized into group 1 (Sham-operated-control), group 2 (SCI), group 3 (SCI + ADMSCs) and group 4 (SCI + PrPc^OE-ADMSCs). Compared with SCI group 2, both ADMSCs and PrPc^OE-ADMSCs significantly improved neurological functions. Additionally, the circulatory inflammatory cytokines levels (TNF-α/IL-6) and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/TRIF/c-caspase8/FADD/p-NF-κB/NEK7/NRLP3/ASC/c-caspase1/IL-ß) and by Day 42 the protein expressions of DAMP-inflammatory signalling (HGMB1/TLR-4/MyD88/TRIF/TRAF6/p-NF-κB/TNF-α/IL-1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPc^OE-ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.     

Endothelial acid ceramidase in exosome-mediated release of NLRP3 inflammasome products during hyperglycemia: Evidence from endothelium-specific deletion of Asah1 gene

Exosomes have been demonstrated to be one of the mechanisms mediating the release of intracellular signaling molecules to conduct cell-to-cell communication. However, it remains unknown whether and how exosomes mediate the release of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome products such as interleukin-1 beta (IL-1β) from endothelial cells. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of multivesicular bodies (MVBs) to control the exosome-mediated release of NLRP3 inflammasome products during hyperglycemia. Using a streptozotocin (STZ)-induced diabetes mouse model, we found that endothelium-specific AC gene knockout mice (Asah1^fl/fl/EC^cre) significantly enhanced the formation and activation of NLRP3 inflammasomes in coronary arterial ECs (CECs). These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein compared to wild-type (WT/WT) littermates. We also observed the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was augmented in STZ-treated Asah1^fl/fl/EC^cre mice compared to WT/WT mice, which was accompanied by an increased IL-1β release of exosomes. In the primary cultures of CECs, we demonstrated that AC deficiency markedly enhanced the formation and activation of NLRP3 inflammasomes, but significantly down-regulated tight junction proteins when these cells were exposed to high levels of glucose. The CECs from Asah1^fl/fl/EC^cre mice had decreased MVB-lysosome interaction and increased IL-1β–containing exosome release in response to high glucose stimulation. Together, these results suggest that AC importantly controls exosome-mediated release of NLRP3 inflammasome products in CECs, which is enhanced by AC deficiency leading to aggravated arterial inflammatory response during hyperglycemia.     

TRPV1 channel mediates NLRP3 inflammasome-dependent neuroinflammation in microglia

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease in the central nervous system (CNS). The NLRP3 inflammasome is considered an important regulator of immunity and inflammation, both of which play a critical role in MS. However, the underlying mechanism of NLRP3 inflammasome activation is not fully understood. Here we identified that the TRPV1 (transient receptor potential vanilloid type 1) channel in microglia, as a Ca²⁺ influx-regulating channel, played an important role in NLRP3 inflammasome activation. Deletion or pharmacological blockade of TRPV1 inhibited NLRP3 inflammasome activation in microglia in vitro. Further research revealed that TRPV1 channel regulated ATP-induced NLRP3 inflammasome activation through mediating Ca²⁺ influx and phosphorylation of phosphatase PP2A in microglia. In addition, TRPV1 deletion could alleviate mice experimental autoimmune encephalomyelitis (EAE) and reduce neuroinflammation by inhibiting NLRP3 inflammasome activation. These data suggested that the TRPV1 channel in microglia can regulate NLRP3 inflammasome activation and consequently mediate neuroinflammation. Meanwhile, our study indicated that TRPV1–Ca²⁺–PP2A pathway may be a novel regulator of NLRP3 inflammasome activation, pointing to TRPV1 as a potential target for CNS inflammatory diseases.Subject terms: Neuroimmunology, Neuroimmunology     

Activated human CD4+CD45RO+ memory T-cells indirectly inhibit NLRP3 inflammasome activation through downregulation of P2X7R signalling

Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1β. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ suppresses NLRP3 inflammasome activation through an indirect mechanism involving decreased P2X7R signaling. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS.     

Inflammasome Modulation by Chemotherapeutics in Malignant Mesothelioma

Malignant mesothelioma (MM) is a fatal disease in dire need of therapy. The role of inflammasomes in cancer is not very well studied, however, literature supports both pro-and anti-tumorigenic effects of inflammasomes on cancer depending upon the type of cancer. Asbestos is a causative agent for MM and we have shown before that it causes inflammasome priming and activation in mesothelial cells. MM tumor cells/tissues showed decreased levels of inflammasome components like NLRP3 and caspase-1 as compared to human mesothelial cells or normal tissue counterpart of tumor. Based on our preliminary findings we hypothesized that treatment of MMs with chemotherapeutic drugs may elevate the levels of NLRP3 and caspase-1 resulting in increased cell death by pyroptosis while increasing the levels of IL-1β and other pro-inflammatory molecules. Therefore, a combined strategy of chemotherapeutic drug and IL-1R antagonist may play a beneficial role in MM therapy. To test our hypothesis we used two human MM tumor cell lines (Hmeso, H2373) and two chemotherapeutic drugs (doxorubicin, cisplatin). Through a series of experiments we showed that both chemotherapeutic drugs caused increases in NLRP3 levels, caspase-1 activation, pyroptosis and pro-inflammatory molecules released from MM cells. In vivo studies using SCID mice and Hmeso cells showed that tumors were smaller in combined treatment group of cisplatin and IL-1R antagonist (Anakinra) as compared to cisplatin alone or untreated control groups. Taken together our study suggests that chemotherapeutic drugs in combination with IL-1R antagonist may have a beneficial role in MM treatment.     

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd^−/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd^−/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.     

Inhibition of NLRP3 inflammasome attenuates spinal cord injury-induced lung injury in mice

Spinal cord injury (SCI) is one kind of severe traumatic injury, resulting in systemic inflammatory response syndrome and secondary lung injury, which is an important pathological basis of respiratory complications. The nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome is an important cytosolic protein complex in many inflammatory diseases. Hence, it is inescapable to explore the effect of inhibition of NLRP3 inflammasome by inhibitors in a mouse SCI model, which was conducted by using the method of 30-G closing force aneurysm clipping at T6–T7 spinal segment for 1 min, followed by assessment of edema, histology, alveolar type II cell apoptosis, mitochondrial dysfunction, and neutrophil infiltration. In brief, our results showed that, NLRP3 inflammasome inhibitor BAY 11-7082 or A438079 inhibited activation of NLRP3 inflammasome, alleviated mitochondrial dysfunction, the number of macrophage and neutrophil, thereby attenuating alveolar type II cell apoptosis, lung edema, and histological injury. Taken together, our data reveal that NLRP3 inflammasome inhibitor BAY 11-7082 or A438079 attenuates the inflammatory response, reverses mitochondrial dysfunction, and subsequently alleviates secondary lung injury following SCI.     

Loganin prevents CXCL12/CXCR4-regulated neuropathic pain via the NLRP3 inflammasome axis in nerve-injured rats

BackgroundNeuropathic pain has been shown to be modulated by the activation of the chemokine C-X-C motif ligand 12 (CXCL12)/chemokine CXC receptor 4 (CXCR4) dependent nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. Loganin, an iridoid glycoside, was proven to prevent neuropathic pain, but its underlying mechanisms related to NLRP3 activation are still unknown.PurposeThis study investigated the underlying mechanisms of loganin's effect on chronic constriction injury (CCI)-induced NLRP3 inflammasome activation in the spinal cord.MethodsSprague-Dawley rats were randomly divided into four groups: sham, CCI, sham + loganin, and CCI + loganin. Loganin (5 mg/kg/day) was administered intraperitoneally starting the day after surgery. Paw withdrawal threshold (PWT) and latency (PWL) were assessed before CCI and on days 1, 3, 7 and 14 after CCI. Spinal cords were collected for western blots and immunofluorescence studies.ResultsLoganin prevented CCI-attenuated PWT and PWL, suggesting improved mechanical allodynia and thermal hyperalgesia. The expression of CXCL12, CXCR4, thioredoxin-interacting protein (TXNIP), NLRP3 inflammasome (NLRP3, ASC, and caspase-1), IL-1β, and IL-18 were enhanced on day 7 after CCI, and all were reduced after loganin treatment. Dual immunofluorescence also showed that increased CXCL12, CXCR4, and NLRP3 were colocalized with NeuN (neuronal marker), GFAP (astrocyte marker), and Iba1 (microglial marker) on day 7 in the ipsilateral spinal dorsal horn (SDH). These immunoreactivities were attenuated in loganin-treated rats. Moreover, loganin decreased the assembly of NLRP3/ASC inflammasome after CCI in the ipsilateral SDH. Loganin appears to attenuate CCI-induced neuropathic pain by suppressing CXCL12/CXCR4-mediated NLRP3 inflammasome.ConclusionOur findings suggest that loganin might be a suitable candidate for managing CCI-provoked neuropathic pain.     

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1^trg/EC^cre) and their control littermates (Smpd1^trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1^trg/EC^cre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1^trg/EC^cre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1^trg/EC^cre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1^trg/EC^cre mice than in their control littermates. Using ECs from Smpd1^trg/EC^cre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1β levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.     

Angiostrongylus cantonensis activates inflammasomes in meningoencephalitic BALB/c mice

Angiostrongylus cantonensis is a metastrongyloid nematode that causes eosinophilic meningoencephalitis in humans. A high infestation of A. cantonensis can cause permanent brain damage or even death. The inflammasome is an oligomeric molecular platform that can detect microbial pathogens and activate inflammatory cytokines. The recognition of larval surface antigens by pattern recognition receptors (PRRs) can cause oligomerization of the NOD-like receptor (NLR) or absent in melanoma 2 (AIM2) with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) to form a caspase-1-activating scaffold. Activated caspase-1 converts pro-inflammatory cytokines into their mature, active forms. Helminths infection has been shown to activate NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasomes. In this study, we aimed to investigate the mechanism of inflammasome activation upon A. cantonensis infection in a mouse model. This study provides evidence that A. cantonensis infection can activate NLRP1B and NLRC4 inflammasomes and promote pyroptosis to cause meningoencephalitis.