NLRP3 Pathway Research Articles

P2X7R-NEK7-NLRP3 Inflammasome Activation: A Novel Therapeutic Pathway of Qishen Granule in the Treatment of Acute Myocardial Ischemia.

BackgroundAcute myocardial ischemia (AMI) is a common heart disease with increasing morbidity and mortality year by year. Persistent and sterile inflammatory infiltration of myocardial tissue is an important factor triggering of acute myocardial ischemia secondary to acute myocardial infarction, and NLRP3 inflammasome activation is an important part of sterile inflammatory response after acute myocardial ischemia. Previous studies have shown that Qishen granule (QSG) can significantly inhibit the inflammatory injury of myocardial tissue caused by ischemia, but its effect and specific mechanism of inhibiting the activation of NLRP3 inflammasome have not been reported. This study was to investigate the specific mechanism of QSG inhibiting inflammation after AMI, and to validate the possible targets.MethodsThe myocardial ischemia model in mice was established by ligation of the left anterior descending coronary artery. Echocardiography was used to evaluate the cardiac function of the mice. Plasma CK-MB and cTnl were detected by ELISA to evaluate the degree of myocardial injury. The extent of myocardial tissue inflammation in mice was assessed by HE staining and immunohistochemistry of IL-18, IL-1β. The expressions of NLRP3, ASC, Caspase-1, and CD86 were detected by immunofluorescence; detection of key pathway proteins P2X7R, NEK7, NLRP3, ASC, Caspase-1, and effector proteins IL-18, IL-1β by Western blot. In vitro experiments, ATP+LPS was used to construct a RAW264.7 macrophage NLRP3 inflammasome activation model. Immunofluorescence and Western blot analysis were performed to detect the expression of NLRP3 pathway activator and effector proteins. Plasmid-transfected P2X7R overexpression and immunoprecipitation assays were used to evaluate the QSG-regulated NLRP3 inflammasome activation pathway.ResultsQSG rescued cardiac function and further reduced inflammatory effects in mice by inhibiting NLRP3 inflammasome activation. In vitro, QSG inhibited LPS combined with ATP-induced NLRP3 inflammasome activation in RAW264.7 macrophages by downregulating the expression of NLRP3 inflammasome key pathway proteins. In addition, inhibition or overexpression of P2X7R in RAW264.7 macrophages and immunoprecipitated protein interactions further confirmed that QSG reduces macrophages inflammasome activation via the P2X7R-NEK7-NLRP3 pathway.ConclusionP2X7R-NEK7-NLRP3 inflammasome activation is a novel therapeutic mechanism of QSG in the treatment of acute myocardial ischemia.

Trichodimerol inhibits inflammation through suppression of the nuclear transcription factor-kappaB/NOD-like receptor thermal protein domain associated protein 3 signaling pathway.

Excessive inflammation causes chronic diseases and tissue damage. Although there has been drug treatment, its side effects are relatively large. Searching for effective anti-inflammatory drugs from natural products has become the focus of attention. First isolated from Trichoderma longibraciatum, trichodimerol is a natural product with TNF inhibition. In this study, lipopolysaccharide (LPS)-induced RAW264.7 macrophages were used as a model to investigate the anti-inflammatory activity of trichodimerol. The results of nitric oxide (NO) detection, enzyme-linked immunosorbent assay (ELISA), and reactive oxygen species (ROS) showed that trichodimerol could reduce the production of NO, ROS, and the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α. Western blotting results showed that trichodimerol could inhibit the production of inflammatory mediators such as cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) and the protein expression of nuclear transcription factor-kappaB (NF-κB), p-IKK, p-IκB, Toll-like receptor 4 (TLR4), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteinyl aspartate specific proteinase (Caspase)-1, and ASC, which indicated that trichodimerol may inhibit inflammation through the NF-κB and NLRP3 pathways. At the same time, molecular docking showed that trichodimerol can directly combine with the TLR4-MD2 complex. Hence, trichodimerol inhibits inflammation by obstructing the interaction between LPS and the TLR4-MD2 heterodimer and suppressing the downstream NF-κB and NLRP3 pathways.

Fetal and maternal NLRP3 signaling is required for preterm labor and birth.

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. One of every 4 preterm neonates is born to a mother with intra-amniotic inflammation driven by invading bacteria. However, the molecular mechanisms underlying this hostile immune response remain unclear. Here, we used a translationally relevant model of preterm birth in Nlrp3-deficient and -sufficient pregnant mice to identify what we believe is a previously unknown dual role for the NLRP3 pathway in the fetal and maternal signaling required for the premature onset of the labor cascade leading to fetal injury and neonatal death. Specifically, the NLRP3 sensor molecule and/or inflammasome is essential for triggering intra-amniotic and decidual inflammation, fetal membrane activation, uterine contractility, and cervical dilation. NLRP3 also regulates the functional status of neutrophils and macrophages in the uterus and decidua, without altering their influx, as well as maternal systemic inflammation. Finally, both embryo transfer experimentation and heterozygous mating systems provided mechanistic evidence showing that NLRP3 signaling in both the fetus and the mother is required for the premature activation of the labor cascade. These data provide insights into the mechanisms of fetal-maternal dialog in the syndrome of preterm labor and indicate that targeting the NLRP3 pathway could prevent adverse perinatal outcomes.

Characterisation of NLRP3 pathway-related neuroinflammation in temporal lobe epilepsy.

Inflammation of brain structures, in particular the hippocampal formation, can induce neuronal degeneration and be associated with increased excitability manifesting as propensity for repetitive seizures. An increase in the abundance of individual proinflammatory molecules including interleukin 1 beta has been observed in brain tissue samples of patients with pharmacoresistant temporal lobe epilepsy (TLE) and corresponding animal models. The NLRP3-inflammasome, a cytosolic protein complex, acts as a key regulator in proinflammatory innate immune signalling. Upon activation, it leads to the release of interleukin 1 beta and inflammation-mediated neurodegeneration. Transient brain insults, like status epilepticus (SE), can render hippocampi chronically hyperexcitable and induce segmental neurodegeneration. The underlying mechanisms are referred to as epileptogenesis. Here, we have tested the hypothesis that distinct NLRP3-dependent transcript and protein signalling dynamics are induced by SE and whether they differ between two classical SE models. We further correlated the association of NLRP3-related transcript abundance with convulsive activity in human TLE hippocampi of patients with and without associated neurodegenerative damage. Hippocampal mRNA- and protein-expression of NLRP3 and associated signalling molecules were analysed longitudinally in pilocarpine- and kainic acid-induced SE TLE mouse models. Complementarily, we studied NLRP3 inflammasome-associated transcript patterns in epileptogenic hippocampi with different damage patterns of pharmacoresistant TLE patients that had undergone epilepsy surgery for seizure relief. Pilocarpine- and kainic acid-induced SE elicit distinct hippocampal Nlrp3-associated molecular signalling. Transcriptional activation of NLRP3 pathway elements is associated with seizure activity but independent of the particular neuronal damage phenotype in KA-induced and in human TLE hippocampi. These data suggest highly dynamic inflammasome signalling in SE-induced TLE and highlight a vicious cycle associated with seizure activity. Our results provide promising perspectives for the inflammasome signalling pathway as a target for anti-epileptogenic and -convulsive therapeutic strategies. The latter may even applicable to a particularly broad spectrum of TLE patients with currently pharmacoresistant disease.

PCSK9 Promotes Endothelial Dysfunction During Sepsis Via the TLR4/MyD88/NF-κB and NLRP3 Pathways.

Endothelial dysfunction often accompanies sepsis. We aimed to explore the role of PCSK9 in septic endothelial dysfunction. Sepsis was induced by lipopolysaccharide (LPS) treatment of human umbilical vein endothelial cells (HUVECs) in vitro and cecal ligation and puncture (CLP) surgery in mice in vivo. Evolocumab (EVC) and Pep 2-8, PCSK9 inhibitors, were subsequently used to determine the role of PCSK9 in sepsis-induced endothelial dysfunction in vitro and in vivo, respectively. In addition, the TLR4 agonist, Kdo2-Lipid A ammonium (KLA), was used to determine the related mechanism. Protein expression of eNOS, VE-cadherin, PCSK9, TLR4, MyD88, p-p65, p65, NLRP3, ASC, and caspase-1 p20 in mice aortas and HUVECs was measured by western blotting, while mRNA expression of TNFα, IL-1β, and IL-18 was determined by qRT-PCR. The level of inflammatory cytokines of mouse aortas was visualized by immunohistochemistry. Vasodilation of the aorta was detected by vascular reactivity experiments. The 96-h survival rate after CLP was assessed. Our findings showed that the expression of eNOS and VE-cadherin decreased, and PCSK9 expression increased, in septic HUVECs or mice. Inhibition of PCSK9 increased eNOS and VE-cadherin expression. Activation of the TLR4/MyD88/NF-κB and NLRP3 pathways may be responsible for PCSK9-induced endothelial dysfunction in sepsis. Vascular reactivity tests and survival studies showed that inhibition of PCSK9 could prevent the decrease in endothelium-dependent vasodilation function and improve the survival rates of septic mice. In summary, our results suggested that increased PCSK9 expression during sepsis activated the TLR4/MyD88/NF-κB and NLRP3 pathways to induce inflammation, which resulted in vascular endothelial dysfunction and decreased survival rates. Thus, inhibition of PCSK9 may be a potential clinical therapeutic target to improve vascular endothelial function in sepsis.

The role of PP2A /NLRP3 signaling pathway in ambient particulate matter 2.5 induced lung injury

Ambient particulate matter 2.5 (PM2.5) exposure has been linked to pulmonary fibrosis. However, the key signaling pathways remained unclear. In the present study, we applied a mouse model with myeloid-specific deletion of Ppp2r1a gene (encoding protein phosphatase 2 A (PP2A) A subunit) to identify the key signaling pathways involved in PM2.5-induced pulmonary fibrosis. PP2A Aα−/− homozygote mice and matched wild-type (WT) littermates were exposed to filtered air (FA), unfiltered air (UA), and concentrated PM2.5 (CA) in a real-ambient PM exposure system for 8 weeks and 16 weeks, respectively. The mice exposed to PM2.5 displayed a progressive inflammation and pulmonary fibrosis. Moreover, the expressions of NLRP3, pro-caspase-1, caspase-1, ASC and IL-1β were increased in mice lung following PM2.5 exposure, indicating PM2.5 exposure caused pulmonary inflammation by the NLRP3 pathways activation. Furthermore, the effects of PM exposure on pulmonary inflammation, pulmonary fibrosis, oxidative stress, and pulmonary function damage were significantly enhanced in PP2A−/− mice compared to WT mice, indicating the role of PP2A in the regulation of pulmonary injury induced by PM exposure. In vitro study confirmed that PP2A was involved in the PM2.5-induced inflammation response and NLRP3 inflammasome activation. Importantly, we identified PP2A regulated the activation of NLRP3 pathways by direct dephosphorylating IRE1α in response to PM2.5 exposure. Taken together, our results demonstrated that PP2A-IRE1α-NLRP3 signaling pathway played a crucial role in regulating the inflammation response, triggering the lung fibrogenesis upon PM2.5 exposure. Our findings provide new insights into regulatory role of PP2A in human diseases upon the PM exposure.

Sacubitril/Valsartan Improves Progression of Early Diabetic Nephropathy in Rats Through Inhibition of NLRP3 Inflammasome Pathway.

PurposeDiabetic nephropathy (DN), a global disease, is the leading cause of end-stage renal disease. There is a lack of specific treatment for this disease, and early intervention in disease progression is essential. In this paper, we used a rat model of early diabetic nephropathy to explore the therapeutic mechanism of sacubitril/valsartan in rats with early diabetic nephropathy.Materials and MethodsRats were grouped into 1 normal group; 2. Model group (DN group): STZ (45 mg/kg/d) induced early diabetic nephropathy rats; 3. Sac group: DN rats + Sac group (orally, 60 mg/kg/d) for 6 weeks. After 6 weeks, the levels of serum albumin (ALB), glucose (GLU), creatinine (Cr), urea nitrogen (BUN) and 24-h urinary protein excretion were measured. In renal tissue homogenates, NLRP3 inflammasome, proinflammatory factors IL1-β and TNF-α, oxidative stress MDA and pro-fibrotic cytokine TGF-β1 were performed. Histological analysis of kidneys by hematoxylin and eosin (HE), PAS and Masson trichrome staining.Results1. Sacubitril/valsartan (Sac) significantly improved renal hypertrophy, proteinuria and serum albumin levels in rats with early diabetic nephropathy (P < 0.001), and decreased GLU, Scr (P<0.001), and BUN levels (P < 0.01).2. Light microscopy of renal tissues showed glomerular hypertrophy and interstitial inflammatory cell infiltration, and mean glomerular area (MGA) and mean glomerular volume (MGV) were crucially increased in early diabetic nephropathy (P < 0.001), and the Sac group showed reduced renal pathology and improved MGA and MGV (P < 0.001).3. Kidney tissue homogenate levels of NLRP3, Caspase-1, IL1-β, TNF-α, MDA and TGF-β1 were critically, increased in DN rats (P < 0.001), and SOD was significantly decreased. All these indicators above were improved after treatment (P < 0.0001).ConclusionNlrp3-inflammasome promote progression of diabetic nephropathy through inflammation, fibrosis and oxidative stress; sacubitril/valsartan ameliorated early diabetes-induced renal damage by inhibiting NLRP3 pathway activation.

GYY4137 ameliorates sepsis-induced cardiomyopathy via NLRP3 pathway

Sepsis-induced cardiomyopathy (SICM) has a poor prognosis, with no effective therapeutic strategy currently. This study aimed to explore the mechanism underlying SICM and investigate the protective role of the hydrogen sulfide (H2S) donor GYY4137. This study included patients with SICM and animal models of SICM with wild-type and Nlrp3−/− mice, which were treated with or without GYY4137. Echocardiography, ELISA, TUNEL staining, and immunofluorescence were used to investigate phenotypic alterations. Serum levels of H2S and cytokines were measured. Inflammatory cell infiltration in the myocardial tissue was identified using immunohistochemistry and immunofluorescence. RNA expression profiles were identified using RNA sequencing. The protective mechanism of GYY4137 was further validated in the crosstalk between macrophages and cardiomyocytes using immunoblotting, real-time polymerase chain reaction (RT-PCR), and immunofluorescence when conditional medium of macrophages boosted by LPS were co-cultured with cardiomyocytes. Patients and animal models of SICM presented with lower serum H2S levels and heart dysfunction. GYY4137 reduced macrophage infiltration in septic heart tissue. GO analysis suggested that GYY4137 was involved in the inflammatory process. GYY4137 inhibited NLRP3 inflammasome activity in macrophages, reduced the secretion of inflammatory factors, and decreased the production of reactive oxygen species (ROS) in cardiomyocytes, thus exerting protective effects against SICM. We further found that the protective effects of GYY4137 were absent in Nlrp3-knockout models. GYY4137 ameliorates myocardial injury in SICM via the NLRP3 pathway by inhibiting the inflammatory response and reducing the production of myocardial ROS.

Inhibition of the JAK/STAT Pathway With Baricitinib Reduces the Multiple Organ Dysfunction Caused by Hemorrhagic Shock in Rats.

The aim of this study was to investigate (a) the effects of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway inhibitor (baricitinib) on the multiple organ dysfunction syndrome (MODS) in a rat model of hemorrhagic shock (HS) and (b) whether treatment with baricitinib attenuates the activation of JAK/STAT, NF-κB, and NLRP3 caused by HS. Posttraumatic MODS, which is in part due to excessive systemic inflammation, is associated with high morbidity and mortality. The JAK/STAT pathway is a regulator of numerous growth factor and cytokine receptors and, hence, is considered a potential master regulator of many inflammatory signaling processes. However, its role in trauma-hemorrhage is unknown. An acute HS rat model was performed to determine the effect of baricitinib on MODS. The activation of JAK/STAT, NF-κB, and NLRP3 pathways were analyzed by western blotting in the kidney and liver. We demonstrate here for the first time that treatment with baricitinib (during resuscitation following severe hemorrhage) attenuates the organ injury and dysfunction and the activation of JAK/STAT, NF-κB, and NLRP3 pathways caused by HS in the rat. Our results point to a role of the JAK/STAT pathway in the pathophysiology of the organ injury and dysfunction caused by trauma/hemorrhage and indicate that JAK inhibitors, such as baricitinib, may be repurposed for the treatment of the MODS after trauma and/or hemorrhage.

The NLRP3 inhibitor and Nrf2 Agonist, RRx-001, Ameliorates Non-alcoholic Fatty Liver Disease in Rats

Lead Author's Financial Disclosures EpicentRx. Study Funding EpicentRx Inc. Background/Synopsis The development and progression of non-alcoholic steatohepatitis (NASH), an increasingly prevalent disease for which no pharmacological therapy has been approved, is linked to oxidative stress, inflammation, and fibrosis of the liver. Here, we investigated the novel mechanism of RRx-001, an NLRP3 inhibitor and Nrf2 agonist, which is in Phase 3 clinical trials for the treatment of cancer, as a therapeutic option for ameliorating NASH in a diet-induced mice model. Objective/Purpose To investigate whether intraperitoneal administration of RRx-001 improved high-fat diet-induced NASH in rats. Methods Sprague-Dawley rats (6-weeks-old male; about 180g weight) were randomly divided into three groups, one subjected to normal diet (ND) and two groups in high-fat, atherogenic diet (HFD, 40 kcal % fat, 1.25% cholesterol, 0.5% sodium cholate) for 12 weeks. One of HFD groups was treated IP with (5 mg/kg of RRx-001 every other week) for the last 8 weeks before characterization. Several markers of inflammation and steatosis were evaluated. Results Every other week administrations of RRx-001 reduced hepatic steatosis scores, serum ALT and AST levels, and hepatic levels of cholesterol and triglycerides in rats fed a high-fat diet. These were correlated with the suppression of NLRP3 inflammasome activation by RRx-001 as evidenced by decreased levels of NLRP3, caspase-1, and IL-1β levels on ELISA. Conclusions The findings of this study demonstrate that RRx-001 reduced inflammation with an anti-lipidemic and anti-NASH effect mediated at least in part by inhibition of the NLRP3 pathway. Thus, targeting the NLRP3 inflammasome with RRx-001 may contribute to the prevention and treatment of NASH. EpicentRx.

Colchicine alleviates inflammation and improves diastolic dysfunction in heart failure rats with preserved ejection fraction

PurposeSeveral studies have reported that colchicine attenuates cardiac inflammation and improves cardiac function in myocardial infarction and atrial fibrillation. However, no study has investigated its effect on heart failure with preserved ejection fraction (HFpEF). Hence, this study aimed to assess its efficacy in a high salt diet (HSD)-induced HFpEF rat model. MethodsA rat hypertension-induced HFpEF model was created by treating Dahl/SS salt-sensitive rats with an HSD for 6 weeks. Colchicine was given via gavage daily as treatment. Cardiac function and inflammation were assessed using echocardiography, histology, and ELISA. Furthermore, the expression levels of NLRP3 and NF-κB signaling pathways were examined. ResultsTreatment with colchicine increased survival and attenuated cardiac dysfunction, as indicated by decreased echocardiographic E/A ratio and longer exercise endurance along with reduced ventricular fibrosis and remodeling in HSD-induced Dahl rats. The treatment also reduced cardiac oxidative stress and inflammatory cell infiltration, as inferred from lower mRNA expressions of TNFα and CCL2 as well as protein expressions of NLRP3 and NF-κB pathways. ConclusionThe findings signify that colchicine plays a crucial role in alleviating systemic inflammation and NLRP3 inflammation activation as well as in attenuating cardiac dysfunction and fibrosis in HSD-induced HFpEF model. Colchicine, therefore, holds therapeutic potential for further clinical applications.

Abstract 5608: Deciphering role of NLRP3 inflammasome in acute myeloid leukemia microenvironment

Abstract Acute Myeloid Leukemia (AML), the most common form of leukemia in adults. Despite intensive chemotherapy regimen, approximately 60% of patients subsequently relapse. Inflammatory cells and mesenchymal stem cells (MSC) which are part of bone marrow (BM) microenvironment, play a critical role in cancer progression. Current strategies are focused on understanding cell-cell contact dependent/independent communication within the leukemic stem cell niche. NLRP3 is a part of innate immune mechanisms, resulting in the release of IL-1β and IL-18 immunomodulatory cytokines. Current study was aimed at analyzing NLRP3 pathway regulation at phenotypic and genotypic level in AML BM-MSC compared to healthy BM-MSC and their cross-talk with immune function in AML stem cell niche. MSC were isolated and cultured from AML BM that exhibited heterogenous fibroblastoid shape. Multi-color flow cytometry of AML BM-MSC revealed characteristic high expression of CD90 and minimal expression of CD45. They also expressed immune checkpoint markers TIM3, PDL-1, adenosine receptors A1R/A2BR, vimentin and NLRP3. Cultured AML BM MSC and fresh leukemic blasts demonstrated tunneling nanotubules, increased vesicles and increase in mitochondria which were of irregular shapes and sizes as seen by transmission electron microscopy. Microarray data revealed genes of NLRP3 damage-associated molecular patterns (DAMP) pathway viz. Nlrp3, NME8, Pycard and IL-18 were distinctly upregulated in AML BM-MSC compared to uninvolved BM-MSC. AML peripheral blood (PB) plasma showed immunosuppressive effect on healthy peripheral blood mononuclear cells. NK cell cytotoxicity was found to be significantly reduced in presence of culture supernatant of AML cell line and further reduced with supernatant of cells activated for NLRP3 pathway. Immunofluorescence demonstrated NLRP3 expression in AML BM-MSC and also in vesicles released from MSC. NLRP3 pathway activation was observed in AML cell line which was significantly abrogated upon treatment with NLRP3 inhibitor. NLRP3 pathway is stimulated using basic DAMP agent LPS and secondary activators ATP or Nigericin. Activation was visualized by colocalization of NLRP3 and caspase-1, in the presence/absence of NLRP3 inhibitor. Expression of IL-1β was observed using immunofluorescence and its release in culture supernatants was measured by ELISA. Inhibition of NLRP3 corroborated with decreased secretion of IL-1β in AML cell line. Thus, a positive correlation of IL-1β release was observed with expression of NLRP3. Paraffin sections of AML BM are being evaluated for NLRP3 pathway markers by immunohistochemistry. Understanding remodelling of BM-MSC, leukemia target and immune cell dynamics through NLRP3 pathway regulation in stroma microenvironment may provide crucial leads in therapeutic modulation of the immune response within the stem cell niche. Citation Format: Monalisa Sahoo, Manasi Nagare, Naythan Dcunha, Meena Patkar, Manju Sengar, Navin Khattry, Anant Gokarn, Sachin Punatar, Sridhar Epari, Tanuja Shet, Jyoti Kode. Deciphering role of NLRP3 inflammasome in acute myeloid leukemia microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5608.

Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4222.

The NLRP3 Inflammasome Pathway: A Review of Mechanisms and Inhibitors for the Treatment of Inflammatory Diseases.

The NLRP3 inflammasome is a multiprotein complex that plays a pivotal role in regulating the innate immune system and inflammatory signaling. Upon activation by PAMPs and DAMPs, NLRP3 oligomerizes and activates caspase-1 which initiates the processing and release of pro-inflammatory cytokines IL-1β and IL-18. NLRP3 is the most extensively studied inflammasome to date due to its array of activators and aberrant activation in several inflammatory diseases. Studies using small molecules and biologics targeting the NLRP3 inflammasome pathway have shown positive outcomes in treating various disease pathologies by blocking chronic inflammation. In this review, we discuss the recent advances in understanding the NLRP3 mechanism, its role in disease pathology, and provide a broad review of therapeutics discovered to target the NLRP3 pathway and their challenges.

Resveratrol Ameliorates Deep Vein Thrombosis-Induced Inflammatory Response Through Inhibiting HIF-1α/NLRP3 Pathway.

Deep vein thrombosis (DVT) has become a prevalent and increasingly serious problem globally and resveratrol (Res) is a natural antitoxin that inhibits arterial thrombosis. To investigate the effect of Res on DVT and further explore its mechanism, thrombosis was monitored at different time points and the pathological changes occurring in the inferior vena cava (IVC) and lung tissue were observed in Sprague-Dawley rats. The protein expression of HIF-1α and NLRP3 in the IVC and lung tissue and the concentrations of D-dimer (D2D), prothrombin fragment 1 + 2 (F1 + 2), interleukin-1β (IL-1β), caspase-1, and tissue factor (TF) in the plasma were determined. After setting different doses of Res groups and using low-molecular-weight heparin (LMWH) as a positive control to determine the effective experimental dose of Res, rats were further divided into sham, DVT, HIF-1α inhibitor, Res, and HIF-1α inhibitor + Res groups. The above indicators were tested repeatedly. The DVT was formed on the 1st day of modeling. With the extension of time, DVT was gradually institutionalized and finally recanalized. Lesions in the IVC and lung tissue were effectively ameliorated, and thrombosis was significantly decreased in the LMWH or 60mg/kg Res-treated groups. The levels of D2D, F1 + 2, IL-1β, caspase-1, TF, and the expression of HIF-1α and NLRP3 were significantly reduced in the HIF-1α inhibitor, Res, and HIF-1α inhibitor + Res groups. Res can ameliorate DVT in rats by inhibiting HIF-1α/NLRP3 pathway, which provides a novel therapeutic strategy for DVT treatment.

Low doses of advanced glycation end-products and fructosilation products promotes premature cell death of human cardiac cells and increases drug resistance of human breast cancer cells exposed to doxorubicin through NLRP3 and MyD88 pathways.

e24055 Background: Advanced glycosylation end-products (AGEs) are non-enzyme, protein glycosylation products caused by glucose-induced metabolic disorder, and play an important role in aging, cancer, metabolic syndrome and cardiovascular diseases. We aimed to assess whether AGEs and fructosilation products, such as methylglyoxal (MG) and N-carboxymethyllysine (N-CML) could increase susceptibility to cell damages induced by doxorubicin. Methods: Human cardiomyocytes were pre-exposed for 24h with low doses (50 mmol/L) of methylglyoxal (MG) or N-carboxymethyllysine (N-CML). After, cells were exposed to subclinical concentration of doxorubicin (at 100 and 200 nM) for 48 and 72h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of peroxisome proliferator-activated receptor-α; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Expression of p53 was also performed through western blot method. Results: Pre-exposure to methylglyoxal (MG) but especially to N-carboxymethyllysine (N-CML) increase cell mortality to doxorubicin of 48-53% compared to control. Pre-exposure to N-CML promotes premature death of cardiomyocytes to doxorubicin through NLRP3-driven pathways. Notably, MG and N-CML increased significantly the cardiotoxicity through NLRP3 inflammasome, Myd88 myddosome and cytochrome C-mediated apoptosis. Pre-exposure to N-CML and MG increase the secretion of interleukin-6 that increase cell apoptosis through a paracrine and autocrine mechanism. Induction of IL-6 reduces the expression of p53 resulting in the induction of the apoptotic process. Conclusions: These observations suggest that AGEs and fructosilation producs promotes premature cardiotoxicity of human cardiac cells exposed to doxorubicin by activation of NLRP3, Myd88-related pathways and downregulation of p53.

Effects of Probiotics at the Interface of Metabolism and Immunity to Prevent Colorectal Cancer-Associated Gut Inflammation: A Systematic Network and Meta-Analysis With Molecular Docking Studies.

BackgroundDysbiosis/imbalance in the gut microbial composition triggers chronic inflammation and promotes colorectal cancer (CRC). Modulation of the gut microbiome by the administration of probiotics is a promising strategy to reduce carcinogenic inflammation. However, the mechanism remains unclear.MethodsIn this study, we presented a systematic network, meta-analysis, and molecular docking studies to determine the plausible mechanism of probiotic intervention in diminishing CRC-causing inflammations.ResultsWe selected 77 clinical, preclinical, in vitro, and in vivo articles (PRISMA guidelines) and identified 36 probiotics and 135 training genes connected to patients with CRC with probiotic application. The meta-analysis rationalizes the application of probiotics in the prevention and treatment of CRC. An association network is generated with 540 nodes and 1,423 edges. MCODE cluster analysis identifies 43 densely interconnected modules from the network. Gene ontology (GO) and pathway enrichment analysis of the top scoring and functionally significant modules reveal stress-induced metabolic pathways (JNK, MAPK), immunomodulatory pathways, intrinsic apoptotic pathways, and autophagy as contributors for CRC where probiotics could offer major benefits. Based on the enrichment analyses, 23 CRC-associated proteins and 7 probiotic-derived bacteriocins were selected for molecular docking studies. Results indicate that the key CRC-associated proteins (e.g., COX-2, CASP9, PI3K, and IL18R) significantly interact with the probiotic-derived bacteriocins (e.g., plantaricin JLA-9, lactococcin A, and lactococcin mmfii). Finally, a model for probiotic intervention to reduce CRC-associated inflammation has been proposed.ConclusionProbiotics and/or probiotic-derived bacteriocins could directly interact with CRC-promoting COX2. They could modulate inflammatory NLRP3 and NFkB pathways to reduce CRC-associated inflammation. Probiotics could also activate autophagy and apoptosis by regulating PI3K/AKT and caspase pathways in CRC. In summary, the potential mechanisms of probiotic-mediated CRC prevention include multiple signaling cascades, yet pathways related to metabolism and immunity are the crucial ones.

Effect of Er Miao San on peritoneal macrophage polarisation through the miRNA-33/NLRP3 signalling pathway in a rat model of adjuvant arthritis

Context Er Miao San (EMS) is a formulation that contains Atractylodis Rhizoma and Phellodendri Cortex in 1:1 ratio, and is commonly used to treat rheumatoid arthritis (RA) and other inflammatory diseases. Objective We investigated the mechanism of action and effects of EMS on peritoneal macrophage differentiation in a rat model of adjuvant arthritis (AA). Materials and methods EMS (3, 1.5 and 0.75 g/kg; once daily) and methotrexate (0.5 mg/kg; once every 3 days) were administered orally from days 21 to 35 after immunisation. Paw swelling and arthritis index were measured; pathological changes in the ankle joint were observed using x-ray and haematoxylin eosin staining. The ratio of CD86/CD206 in macrophages was detected by flow cytometry. Examination of the miRNA-33/NLRP3 signalling pathway was examined by RT-qPCR and western blotting. The levels of cytokines in the serum and cell supernatants were tested by ELISA. Results EMS significantly reduced the AA index in rats (from 11.0 to 9.3) and pathological changes in the ankle joint (from 3.8 to 1.4). The ratio of CD86/CD206 was reduced, and polarisation to M1 improved (from 0.9 to 0.6) in macrophages of EMS-treated rats. EMS downregulated the miRNA-33/NLRP3 pathway. Furthermore, EMS treatment increased IL-10 and TGF-β levels in the serum and supernatant of macrophages of AA rats and simultaneously decreased the levels of IL-1β and TNF-α. Discussion and conclusions Our results suggest that EMS may reduce macrophage polarisation to the M1 inflammatory phenotype by downregulating the miRNA-33/NLRP3 pathway in AA rats. These findings may provide new insights into the treatment of RA.

HMGB1-NLRP3-P2X7R pathway participates in PM2.5-induced hippocampal neuron impairment by regulating microglia activation

Neuroinflammation is a key mechanism underlying the cognitive impairment induced by PM2.5, and activated microglia plays an important role in this process. However, the mechanisms by which activated microglia induced by PM2.5 impair hippocampal neurons have not been fully elucidated. In this study, we focused on the role of HMGB1-NLRP3-P2X7R pathway which mediated the microglia activation in hippocampal neurons impairment induced by PM2.5 using a co-culture model of microglia and hippocampal neurons. We found that PM2.5 resulted in activated microglia and HMGB1-NLRP3 inflammatory pathway, and elevated proinflammatory cytokines of IL-18 and IL-1β in a dose-dependent manner. Notably, we next utilized previously reported pharmacological inhibitors or siRNA for HMGB1 and found that they significantly inhibited the activation of downstream NLRP3 and MAPK pathways derived from PM2.5 exposure, and down-regulated IL-18 and IL-1β in microglia. Furthermore, we employed co-cultured hippocampal neurons and microglia and found that reducing HMGB1 significantly decreased neuron impairment, apoptosis related protein of cl-caspase3, synaptic damage, and neurotransmitter receptor of 5-HT2A, along with notably elevated presynaptic and postsynaptic proteins of SYP and PSD-95, as well as learning and memory related proteins of p-CREB and BDNF. The neuronal impairment induced by PM2.5 could not be prevented in the case of simultaneous employment of HMGB1 siRNA and NLRP3 agonist. After silencing NLRP3 alone in microglia, hippocampal neurons demonstrated decreased excessive autophagy and up-regulated synaptic protein of GAP43 as well as learning and memory related protein of NCAM1. Therefore, we further studied how hippocampal neurons affected microglia under PM2.5 exposure, Further investigation indicated that silencing HMGB1 could affect the activation of P2X7R and reduce the release of ATP from hippocampal neurons, thus protecting the interaction between microglia and hippocampal neurons. The present work suggests that regulation of HMGB1-NLRP3-P2X7R pathway can inhibit the microglia activation induced by PM2.5 to alleviate hippocampal neuron impairment and stabilize the microenvironment between microglia and neurons. This contributes to maintaining the normal function of hippocampal neurons and alleviating the cognitive impairment derived from PM2.5 exposure.

Interleukin-6 mediated inflammasome activation promotes oral squamous cell carcinoma progression via JAK2/STAT3/Sox4/NLRP3 signaling pathway.

BackgroundInterleukin-6 (IL-6) has been reported to be critical in oral squamous cell carcinoma (OSCC). However, the set of pathways that IL-6 might activate in OSCC are not fully understood.MethodsIL-6 and Sox4 expressions were first determined with RT-qPCR, ELISA, Western blot, or immunohistochemistry in OSCC tissues, and correlations between IL-6 and Sox4 expression and patient pathological characteristics were examined, and Kaplan–Meier approach was employed for evaluating the prognostic utility in OSCC patients. CCK-8, EdU stain and colony formation assays were utilized to test cell proliferation in vitro. Mechanistically, downstream regulatory proteins of IL-6 were verified through chromatin immunoprecipitation, luciferase reporter, pull-down, and the rescued experiments. Western blot was used for detecting protein expression. A nude mouse tumorigenicity assay was used to confirm the role of IL-6 and Sox4 in vivo.ResultsIL-6 was upregulated in OSCC tissues, and Sox4 expression was positively correlated with IL-6 expression. High IL-6 and Sox4 expression was closely related to tumor size, TNM stage, and a poorer overall survival. Besides, IL-6 could accelerate OSCC cell proliferation by activating inflammasome via JAK2/STAT3/Sox4/NLRP3 pathways in vitro and in vivo. Furthermore, STAT3 played as a transcription factor which positively regulated Sox4, and IL-6 promotes Sox4 expression by activating JAK2/STAT3 pathway. Moreover, through the rescue experiments, we further confirmed that IL-6 could promote proliferation and NLRP3 inflammasome activation via JAK2/STAT3/Sox4 pathway in OSCC cells. Finally, knockdown of Sox4 suppressed OSCC growth in vivo, and antagonized the acceleration of IL-6 on tumor growth.ConclusionsWe confirmed that IL-6 plays an oncogenic role in OSCC progression by activating JAK2/STAT3/Sox4/NLRP3 pathway, which might be the therapeutic targets for OSCC remedy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-022-02376-4.