Inhibiting NLRP3 Inflammasome Activation Research Articles

Deletion of African Swine Fever Virus (ASFV) H240R Gene Attenuates the Virulence of ASFV by Enhancing NLRP3-Mediated Inflammatory Responses.

African swine fever (ASF) is a highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV), with a mortality rate of up to 100%. In order to replicate efficiently in macrophages and monocytes, ASFV has evolved multiple strategies to evade host antiviral responses. However, the underlying molecular mechanisms by which ASFV-encoded proteins execute immune evasion are not fully understood. In this study, we found that ASFV pH240R strongly inhibits transcription, maturation, and secretion of interleukin-1β (IL-1β). Importantly, pH240R not only targeted NF-κB signaling but also impaired NLRP3 inflammasome activation. In this mechanism, pH240R interacted with NF-kappa-B essential modulator (NEMO), a component of inhibitor of kappa B kinase (IKK) complex and subsequently reduced phosphorylation of IκBα and p65. In addition, pH240R bonded to NLRP3 to inhibit NLRP3 inflammasome activation, resulting in reduced IL-1β production. As expected, infection with H240R-deficient ASFV (ASFV-ΔH240R) induced more inflammatory cytokine expression both in vitro and in vivo than its parental ASFV HLJ/18 strain. Consistently, H240R deficiency reduced the viral pathogenicity in pigs compared with its parental strain. These findings reveal that the H240R gene is an essential virulence factor, and deletion of the H240R gene affects the pathogenicity of ASFV HLJ/18 by enhancing antiviral inflammatory responses, which provides insights for ASFV immune evasion mechanisms and development of attenuated live vaccines and drugs for prevention and control of ASF. IMPORTANCE African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease of domestic pigs, with a high mortality approaching 100%. ASFV has spread rapidly worldwide and caused huge economic losses and ecological consequences. However, the pathogenesis and immune evasion mechanisms of ASFV are not fully understood, which limits the development of safe and effective ASF attenuated live vaccines. Therefore, investigations are urgently needed to identify virulence factors that are responsible for escaping the host antiviral innate immune responses and provide a new target for development of ASFV live-attenuated vaccine. In this study, we determined that the H240R gene is an essential virulence factor, and its depletion affects the pathogenicity of ASFV by enhancing NLRP3-mediated inflammatory responses, which provides theoretical support for the development of an ASFV attenuated live vaccine.

Eugenol Alleviates TGEV-Induced Intestinal Injury via Suppressing ROS/NLRP3/GSDMD-Dependent Pyroptosis.

Transmissible gastroenteritis virus (TGEV), a coronavirus, is one of the main causative agents of diarrhea in piglets and significantly impacts the global swine industry. Pyroptosis is involved in the pathogenesis of coronavirus, but its role in TGEV-induced intestinal injury has yet to be fully elucidated. Eugenol, an essential plant oil, plays a vital role in antiviral innate immune responses. We demonstrate the preventive effect of eugenol on TGEV infection. Eugenol alleviates TGEV-induced intestinal epithelial cell pyroptosis and reduces intestinal injury in TGEV-infected piglets. Mechanistically, eugenol reduces the activation of NLRP3 inflammasome, thereby inhibiting TGEV-induced intestinal epithelial cell pyroptosis. In addition, eugenol scavenges TGEV-induced reactive oxygen species (ROS) increase, which in turn prevents TGEV-induced NLRP3 inflammasome activation and pyroptosis. Overall, eugenol protects the intestine by reducing TGEV-induced pyroptosis through inhibition of NLRP3 inflammasome activation, which may be mediated through intracellular ROS levels. These findings propose that eugenol may be an effective strategy to prevent TGEV infection.

Naringenin protects against acute pancreatitis-associated intestinal injury by inhibiting NLRP3 inflammasome activation via AhR signaling.

Background: In this study, we examined the functions and mechanisms by which naringenin protects against SAP (severe acute pancreatitis)-related intestinal injury by modulating the AhR/NLRP3 signaling pathway. Material and methods: Fifteen healthy male C57BL/6 mice were randomly divided into SAP (n = 12) and normal (n = 3) groups. Mice in the SAP group received caerulein and lipopolysaccharide intraperitoneal injections and were then randomly assigned to the SAP, NAR, CH223191, and Dexamethasone (DEX) groups. Pathological changes in the pancreatic and intestinal mucosa were observed by Hematoxylin & Eosin (H&E) staining. In vitro, RAW264.7 cells were exposed to lipopolysaccharide and treated with naringenin. The levels of NLRP3, AhR, IL-1β, TNF, and IL-6 in the SAP model and RAW264.7 cells were evaluated by enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qRT-PCR), western blot, and immunohistochemistry. The nuclear translocation of AhR was shown by immunofluorescence. AutoDockTools was used to predict the conformations of naringenin-AhR binding, and PyMol 2.4 was used to visualize the conformations. Results: Mouse pancreatic and intestinal injury was alleviated by treatment with naringenin. Naringenin inhibited the activation of the NLRP3 inflammasome and inhibited damage to intestinal tight junctions. Moreover, naringenin increased AhR nuclear translocation and activated the AhR pathway. Conclusion: Naringenin can reduce SAP-associated intestinal injury by inhibiting the activation of the NLRP3 inflammasome via the AhR signaling pathway.

Malvidin alleviates mitochondrial dysfunction and ROS accumulation through activating AMPK-α/UCP2 axis, thereby resisting inflammation and apoptosis in SAE mice.

This study aimed to explore the protective roles of malvidin in life-threatened sepsis-associated encephalopathy (SAE) and illustrate the underlying mechanism. SAE mice models were developed and treated with malvidin for subsequently protective effects evaluation. Malvidin restored neurobehavioral retardation, declined serum S100β and NSE levels, sustained cerebrum morphological structure, improved blood-brain barrier integrity with elevated tight junction proteins, and decreased evans blue leakage, and finally protect SAE mice from brain injury. Mechanistically, malvidin prevented cerebrum from mitochondrial dysfunction with enhanced JC-1 aggregates and ATP levels, and ROS accumulation with decreased lipid peroxidation and increased antioxidant enzymes. UCP2 protein levels were found to be decreased after LPS stimulation in the cerebrum and BV-2 cells, and malvidin recovered its levels in a ROS dependent manner. In vivo inhibition of UCP2 with genipin or in vitro interference with siRNA UCP2 both disrupted the mitochondrial membrane potential, decreased ATP levels and intensified DCF signals, being a key target for malvidin. Moreover, dorsomorphin block assays verified that malvidin upregulated UCP2 expression through phosphorylating AMPK in SAE models. Also, malvidin alleviated SAE progression through inhibition of ROS-dependent NLRP3 inflammasome activation mediated serum pro-inflammatory cytokines secretion and mitochondrial pathway mediated apoptosis with weakened apoptosis body formation and tunel positive signals, and decreased Bax, cytochrome C, caspase-3 and increased Bcl-2 protein levels. Overall, this study illustrated that malvidin targeted AMPK-α/UCP2 axis to restore LPS-induced mitochondrial dysfunction and alleviate ROS accumulation, which further inhibits NLRP3 inflammasome activation and mitochondrial apoptosis in a ROS dependent way, and ultimately protected SAE mice, providing a reference for the targeted development of SAE prophylactic approach.

Sacubitril/valsartan alleviates sepsis-induced acute lung injury via inhibiting GSDMD-dependent macrophage pyroptosis in mice.

Sepsis-induced acute lung injury (ALI) is a life-threatening disorder with intricate pathogenesis. Macrophage pyroptosis reportedly plays a vital role in ALI. Although it has been established that angiotensin receptor blockers (ARBs) can reduce sepsis-induced organ injury, the efficacy of sacubitril/valsartan (SV) for sepsis has been largely understudied. Here, we aimed to investigate the role of SV in sepsis-induced ALI. Caecal ligation and puncture (CLP) were used to induce polymicrobial sepsis and related ALI. The therapeutic effects of SV in CLP mice were subsequently assessed. Gasdermin D (GSDMD)-/- mice were used to validate the signalling pathways affected by SV. In vitro, mouse bone marrow-derived macrophages (BMDMs) and Raw264.7 cells were treated with SV following exposure to lipopolysaccharide and adenosine triphosphate. Finally, the serum obtained from 42 septic patients was used for biochemical analysis. Compared to the other ARBs, SV yielded more pronounced anti-inflammatory effects on macrophages. In vivo, SV decreased mortality rates, significantly reduced lung damage and prevented the inflammatory response in CLP mice. In addition, SV suppressed GSDMD-mediated macrophage pyroptosis in mice. In BMDMs and Raw264.7 cells, the anti-inflammatory and anti-pyroptosis properties of SV were verified. SV treatment effectively inhibited NLRP3 inflammasome activation and prevented macrophage pyroptosis in a GSDMD-dependent manner. Furthermore, we found that septic individuals had considerably higher serum angiotensin II levels. Overall, we found that SV might prevent ALI in CLP mice by inhibiting GSDMD-mediated pyroptosis of macrophages. Thus, SV might be a viable drug for sepsis-induced ALI.

Ablation of NLRP3 inflammasome attenuates muscle atrophy via inhibiting pyroptosis, proteolysis and apoptosis following denervation.

Rationale: The inflammasome has been widely reported to be involved in various myopathies, but little is known about its role in denervated muscle. Here, we explored the role of NLRP3 inflammasome activation in experimental models of denervation in vitro and in vivo. Methods: Employing muscular NLRP3 specific knock-out (NLRP3 cKO) mice, we evaluated the effects of the NLRP3 inflammasome on muscle atrophy in vivo in muscle-specific NLRP3 conditional knockout (cKO) mice subjected to sciatic nerve transection and in vitro in cells incubated with NLRP3 inflammasome activator (NIA). To evaluate the underlying mechanisms, samples were collected at different time points for RNA-sequencing (RNA-seq), and the interacting molecules were comprehensively analysed. Results : In the experimental model, NLRP3 inflammasome activation after denervation led to pyroptosis and upregulation of MuRF1 and Atrogin-1 expression, facilitating ubiquitin-proteasome system (UPS) activation, which was responsible for muscle proteolysis. Conversely, genetic knockout of NLRP3 in muscle inhibited pyroptosis-associated protein expression and significantly ameliorated muscle atrophy. Furthermore, cotreatment with shRNA-NLRP3 markedly attenuated NIA-induced C2C12 myotube pyroptosis and atrophy. Intriguingly, inhibition of NLRP3 inflammasome activation significantly suppressed apoptosis. Conclusions: These in vivo and in vitro findings demonstrate that during denervation, the NLRP3 inflammasome is activated and stimulates muscle atrophy via pyroptosis, proteolysis and apoptosis, suggesting that it may contribute to the pathogenesis of neuromuscular diseases.

Osthole: A potential AMPK agonist that inhibits NLRP3 inflammasome activation by regulating mitochondrial homeostasis for combating rheumatoid arthritis

BackgroundOsthole (OST), a characteristic coumarin compound in Angelicae pubescentis radix (APR), has shown potent efficacy in the treatment of rheumatoid arthritis (RA), but its specific targets and potential mechanism are limited. PurposeThis study aimed to explore the potential targets and molecular mechanisms of OST against RA using computer-assisted techniques in combination with RA fibroblast-like synoviocytes (FLS) inflammation model and CIA rat model. MethodsNetwork pharmacology and molecular docking were applied to initially predict the potential targets of OST for the treatment of RA. Thereafter, TNFα was used to stimulate FLS to build an in vitro model of inflammation, combined with RNA-seq technology and molecular biology such as qPCR to investigate the anti-inflammatory effects and related mechanisms of OST. Finally, the anti-RA effect of OST was demonstrated by establishing a CIA rat model. ResultsThe network model results showed that the anti-RA effect of OST was mainly related to its anti-inflammatory effect, and AMPK was identified as a potential target for the potency of OST. In the TNFα-induced FLS cells, OST inhibited the secretion of FLS inflammatory factors, which was attributed to the ability of OST to activate AMPK to inhibit the activation of the NLRP3 inflammasome. Further, it was observed that the activation of AMPK by OST facilitated mitochondrial biogenesis, and corrected abnormal mitochondrial dynamics in FLS, which was favoured to the restoration of mitochondrial homeostasis, and further promoted the occurrence of apoptosis and the decrease of ROS in FLS. Consistent with in vivo studies, administration of OST significantly improved joint deformity and toe erythema, reduced arthritis index scores and inhibited synovial inflammation in CIA rats. ConclusionOur study proposed for the first time that AMPK, served as a potential target of OST, positively participated in the anti-RA therapeutic effect of OST. By regulating mitochondrial homeostasis and function, OST can effectively inhibit the activation of inflammasome and the secretion of inflammatory factors in vitro and in vivo, and finally achieve beneficial effects in the treatment of RA, which provides support and greater possibility to make further efforts on pharmacological research and clinical application of OST.

MARCH9 Mediates NOX2 Ubiquitination to Alleviate NLRP3 Inflammasome-Dependent Pancreatic Cell Pyroptosis in Acute Pancreatitis.

The pathogenesis of acute pancreatitis mainly involves NLRP3 inflammasome-mediated pancreatic cell injury, although regulators of this inflammasome machinery are still not fully identified. Membrane-associated RING-CH 9 (MARCH9) is a member of MARCH-type finger proteins, which regulates innate immunity through catalyzing polyubiquitination of critical immune factors. The aim of present research is to examine the function of MARCH9 in acute pancreatitis. Cerulein-induced acute pancreatitis was established on pancreatic cell line AR42J and rat model. Reactive oxygen species (ROS) accumulation and NLRP3 inflammasome-dependent cell pyroptosis in pancreas were examined by flow cytometry. MARCH9 was downregulated by cerulein, but overexpressing MARCH9 could inhibit NLRP3 inflammasome activation and ROS accumulation, thus suppressing pancreatic cell pyroptosis and mitigating pancreatic injury. We further uncovered that the mechanism underlying such an effect of MARCH9 is through mediating the ubiquitination of NADPH oxidase-2, whose deficiency reduces cellular ROS accumulation and inflammasome formation. Our results suggested that MARCH9 suppresses NLRP3 inflammasome-mediated pancreatic cell injury through mediating the ubiquitination and degradation of NADPH oxidase-2, which compromises ROS generation and NLRP3 inflammasomal activation.

Artesunate Alleviates Hyperoxia-Induced Lung Injury in Neonatal Mice by Inhibiting NLRP3 Inflammasome Activation.

Bronchopulmonary dysplasia (BPD) is a chronic respiratory disease in preterm infants that may cause persistent lung injury. Artesunate exhibits excellent anti-inflammatory in lung injury caused by various factors. This study aimed to investigate the effect of the artesunate on hyperoxia-induced lung injury in neonatal mice and its mechanism. A BPD model of hyperoxic lung injury in neonatal mice was established after hyperoxia (75% oxygen) exposure for 14 days, and part of the mice received intraperitoneal injections of the artesunate. H&E staining was used to observe the pathology of lung tissue, and the degree of oxidative stress in the lung tissue was determined by commercial kits. The levels of inflammatory cytokines in the serum and lung tissues of neonatal mice were detected by an enzyme-linked immunosorbent assay. Immunohistochemical experiments were performed to further evaluate the expression of IL-1β. The real-time quantitative polymerase chain reaction was used to determine the mRNA level of the NLRP3 inflammasome. The western blot assay was used to measure the levels of NLRP3 inflammasome and NF-κB pathway-related proteins. Artesunate ameliorated weight loss and lung tissue injury in neonatal mice induced by hyperoxia. The level of malondialdehyde was decreased, while the activity of superoxide dismutase and the level of glutathione increased after artesunate treatment. Artesunate reduced the level of inflammation cytokines TNF-α, IL-6, and IL-1β in the serum and lung. Moreover, artesunate inhibited the mRNA expression and protein levels of NLRP3, ASC, and caspase-1, as well as the phosphorylation of the NF-κB and IκBα. Our findings suggest that artesunate treatment can attenuate hyperoxia-induced lung injury in BPD neonatal mice by inhibiting the activation of NLRP3 inflammasome and the phosphorylation of the NF-κB pathway.

TMEM16F may be a new therapeutic target for Alzheimer's disease.

TMEM16F is involved in many physiological processes such as blood coagulation, cell membrane fusion and bone mineralization. Activation of TMEM16F has been studied in various central nervous system diseases. High TMEM16F level has been also found to participate in microglial phagocytosis and transformation. Microglia-mediated neuroinflammation is a key factor in promoting the progression of Alzheimer's disease. However, few studies have examined the effects of TMEM16F on neuroinflammation in Alzheimer's disease. In this study, we established TMEM16F-knockdown AD model in vitro and in vivo to investigate the underlying regulatory mechanism about TMEM16F-mediated neuroinflammation in AD. We performed a Morris water maze test to evaluate the spatial memory ability of animals and detected markers for the microglia M1/M2 phenotype and NLRP3 inflammasome. Our results showed that TMEM16F was elevated in 9-month-old APP/PS1 mice. After TMEM16F knockdown in mice, spatial memory ability was improved, microglia polarization to the M2 phenotype was promoted, NLRP3 inflammasome activation was inhibited, cell apoptosis and Aβ plaque deposition in brain tissue were reduced, and brain injury was alleviated. We used amyloid-beta (Aβ25-35) to stimulate human microglia to construct microglia models of Alzheimer's disease. The levels of TMEM16F, inducible nitric oxide synthase (iNOS), proinflammatory cytokines and NLRP3 inflammasome-associated biomarkers were higher in Aβ25-35 treated group compared with that in the control group. TMEM16F knockdown enhanced the expression of the M2 phenotype biomarkers Arg1 and Socs3, reduced the release of proinflammatory factors interleukin-1, interleukin-6 and tumor necrosis factor-α, and inhibited NLRP3 inflammasome activation through reducing downstream proinflammatory factors interleukin-1β and interleukin-18. This inhibitory effect of TMEM16F knockdown on M1 microglia was partially reversed by the NLRP3 agonist Nigericin. Our findings suggest that TMEM16F participates in neuroinflammation in Alzheimer's disease through participating in polarization of microglia and activation of the NLRP3 inflammasome. These results indicate that TMEM16F inhibition may be a potential therapeutic approach for Alzheimer's disease treatment.

Dimethyl fumarate attenuates MSU-induced gouty arthritis by inhibiting NLRP3 inflammasome activation and oxidative stress.

Dimethyl fumarate (DMF) has shown anti-inflammatory and antioxidant activities. However, the effects of DMF on gouty arthritis remain elusive, and the underlying mechanism is not understood. In this study, we aim to investigate the role of DMF in gouty arthritis. Mice were gavage with DMF for consecutive 7 days at two different doses (10 mg/kg/day or 30 mg/kg/day, once daily) in advance and then monosodium sodium urate (MSU) was injected into their joint to establish an acute gout mice model. The pain and swelling of the hind paw in mice were determined. The production of pro-inflammatory cytokine in the paw tissues was assessed by Elisa and the inflammatory infiltration of the joint was determined by hematoxylin and eosin (H&E) staining. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the tissues were measured by commercial kits. In addition, the expression of nuclear factor kappa B (NF-κB) and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome and downstream genes were detected by PCR and Western blot. Furthermore, LPS-primed murine macrophages Raw 264.7 cells were treated with different concentrations of DMF (2 μM, 5 μM, 10 μM) for 2 h, and then challenged with MSU (200 μg/mL) for other 12 h to observe the effect of DMF on cell viability via cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) levels in the supernatant of culture medium. Immunofluorescent staining was used to detect the NLRP3 inflammasome activation and reactive oxygen species (ROS) production in vitro. Caspase-1 activity was measured by corresponding assay kits both in vivo and in vitro. DMF attenuated pain and swelling in MSU-induced gout mice by decreasing pro-inflammatory cytokine production and inflammatory cell infiltration, as well as improved oxidative stress. Moreover, DMF inhibited the activation of NF-κB and NLRP3 inflammasome and subsequent expression of caspase-1, interleukin-1β (IL-1β), and IL-18 at both mRNA and protein levels. Meanwhile, DMF suppressed NLRP3 inflammasome expression and ROS production in LPS and MSU-stimulated Raw 264.7 cells, thereby protecting the cells from inflammatory injury. DMF serves as a new approach for the treatment of MSU-induced gouty arthritis by suppressing NLRP3 inflammasome activation and oxidative stress.

Retracted: CY-09 Inhibits NLRP3 Inflammasome Activation to Relieve Pain via TRPA1.

[This retracts the article DOI: 10.1155/2021/9806690.].

Diterpenoids with a novel 6/5-5 spiro tricyclic skeleton from Orthosiphon wulfenioides and their NLRP3 inflammasome inhibitory activity

Wulfenioidins A–C (1–3) with an unusual 6/5-5 spiro tricyclic skeleton were isolated from O. wulfenioides. Compound 3 strikingly inhibited NLRP3 inflammasome activation by blocking the expression of caspase-1, GSDMD-NT, and IL-1β.

PS-B09-9: ENOS-NO PATHWAY REGULATES INFLAMMASOME ACTIVATION IN DIABETIC MICE VIA MODULATION OF CEBP TRANSLOCATION

Background: Diabetic kidney disease (DKD) is one of the most common vascular complications of diabetes mellitus (DM). Endothelial dysfunction is also a common pathophysiologic mechanism of diabetic vascular complications. Nitric oxide (NO) produced in the endothelial cells by endothelial nitric oxide synthase (eNOS) is an important mediator for the maintenance of vascular homeostasis. In persons with diabetes, eNOS produces superoxide anion rather than nitric oxide, referred to as eNOS uncoupling which may contribute to endothelial dysfunction, albuminuria, and diabetic nephropathy. eNOS uncoupling can then lead to dysfunction of the eNOS-NO-pathway in diabetic glomeruli. To determine the mechanism of regulation of inflammasome activation by eNOS-NO pathway in DKD, we used eNOS deficient mice and eNOS-ASC double knockout mice. Methods: We used wild-type (WT) and eNOS-deficient mice (eNOSKO) to determine the role of the eNOS-NO pathway. Diabetes was induced by a single intraperitoneal injection of streptozocin. We then divided the mice into four groups: WT, WT/STZ, eNOSKO, and eNOS/STZ. Four weeks after the induction of DM, the mice were sacrificed, and their kidney tissues were harvested. Urinary albumin excretion was checked before the sacrifice, and glomerular damage was assessed by periodic acid-Schiff (PAS) staining. To determine the role of inflammasome on the development of glomerulopathy, we generated eNOS-ASC double knockout mice (eNOS-ASC-DKO). Also, to investigate inflammasome activation in glomeruli, the glomeruli were isolated from the kidney tissue by using Dynabeads. Results: Urinary albumin excretion was increased in WT/STZ compared with WT. These urinary albumin excretions were increased much more in eNOS/STZ than in WT/STZ, while the glomeruli were more damaged in eNOSKO/STZ than in WT/STZ. Urinary albumin excretion was lower in eNOS-ASC-DKO/STZ compared with eNOSKO/STZ, and the glomerular damage also was ameliorated in eNOS-ASC-DKO after treatment with STZ. S-Nitroso glutathione (GSNO) attenuated NLRP3 inflammasome activation after treatment with LPS-ATP. These results indicate that NO directly inhibits NLRP3-inflammasome activation. In addition, GSNO decreased the expression of inflammasome-related genes. To investigate the detailed the mechanism (epigenetic regulation), we performed Assay for Transposase-Accessible Chromatin-Sequencing (ATAC-seq) analysis. The binding region of the transcription factor CCAAT/ enhancer binding protein (C/EBP) β; was closed in the LPS+GSNO group significantly, compared with the LPS group. Conclusion: In eNOS-deficient mice, inflammasomes were activated, and glomerular injuries were exacerbated. However in eNOS-ASC-STZ mice, the glomerular damage were attenuated. Our data suggested that eNOS/NO signaling attenuates inflammasome activation via regulation of CEBP β;, leading to the suppressed development of DKD.

Rutin ameliorates gout via reducing XOD activity, inhibiting ROS production and NLRP3 inflammasome activation in quail

Gout is a metabolic disease affected by monosodium urate (MSU) deposition, which is directly related to hyperuricemia. Recent reports on the prevalence and incidence of gout have been widely circulated worldwide. Currently, the anti-gout drugs in clinical practice are mainly small-molecule synthetic drugs, and the effectiveness and safety are limited. Reducing uric acid and inhibiting inflammation are the focused areas of drug research and development on gout. Rutin, a natural flavonoid, has been reported to alleviate inflammation in various diseases. However, whether rutin exerts protective effects on gout remains to be elucidated. This study used quails without urate oxidase as experimental animals to induce endogenous gout models through a high purine diet. We confirmed that quail in the model group developed gout symptoms at 30 days of the experiment. And the targets of uric acid metabolism, oxidative stress level, and NLRP3 inflammasome were dysregulated in quails. Rutin treatment improves gout and reduces inflammatory expression in quail. We further confirmed that rutin treatment reduced XOD activity and uric acid levels in quail. And rutin inhibited ROS production, restored oxidative stress balance, inhibited NLRP3 inflammasome activation, and exerted anti-inflammatory effects. We extracted and identified the fibroblast-like synoviocytes (FLS) for the first time. The results showed that rutin could reduce ROS production and NLRP3 inflammasome activation of FLS after uric acid stimulation. In conclusion, our findings underscore that rutin may be a gout protective agent by reducing XOD activity, inhibiting ROS production and NLRP3 inflammasome activation. Meanwhile, this study also provides an available animal model for the research drugs of gout.

Knockdown of PHLDA1 Alleviates Necrotizing Enterocolitis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis Through Enhancing Nrf2 Signaling

ABSTRACT Objective Pleckstrin homology-like domain family A member 1 (PHLDA1) is involved in the progression of intestine-related diseases, but its role and related mechanisms in Necrotizing enterocolitis (NEC) are unclear. The aim of this study was to better understand the function of PHLDA1 in NEC and the underlying mechanisms. Methods A neonatal mouse model of NEC was established by hypoxic hypothermia, and sh-PHLDA1 was transfected into mice to observe the mortality of each group within 4 days. The levels of IL-1β, IL-6, IL-18 and TNF-α were measured by PCR and ELISA. ROS, MDA, SOD, and GSH-Px levels were detected by Dihydroethidium (DHE) method and kit; expression of pyroptosis-related factors including NLRP3, ASC, cleaved-caspase1, GSDMD-N, IL-1β, IL-18, and Nrf2 were detected by western-blot; mechanistically, the effects of transfection of sh-PHLDA1 and ML385 (Nrf2 inhibitor) were investigated, and the expression of pyroptosis-related factors was detected again. Results PHLDA1 was highly expressed in the intestinal tissues of NEC mice, and transfection of sh-PHLDA1 improved the survival rate, alleviated intestinal lesions, improved intestinal inflammation, oxidative stress and cellular scorching in NEC. In addition, sh-PHLDA1 was able to inhibit NLRP3 activation and pyroptosis by activating Nrf2. Conclusion Knockdown of PHLDA1 attenuated necrotizing small intestinal colitis by enhancing Nrf2 expression to inhibit NLRP3 inflammasome activation and pyroptosis.

Modulation of NLRP3 inflammasomes activation contributes to improved survival and function of mesenchymal stromal cell spheroids

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells that exhibit significant therapeutic potentials in a variety of disorders. Nevertheless, their clinical efficacy is limited owing to poor survival, low rate of engraftment, and impaired potency upon transplantation. Spheroidal three-dimensional (3D) culture of MSCs (MSC3D) has been proven to better preserve their invivo functional properties. However, the molecular mechanisms underlying the improvement in MSC function by spheroid formation are not clearly understood. NLRP3 inflammasomes, a key component of the innate immune system, have recently been shown to play a role in cell fate decision of MSCs. The present study examined the role of NLRP3 inflammasomes in the survival and potency of MSC spheroids. We found that MSC3D led to decreased activation of NLRP3 inflammasomes through alleviation of ER stress in an autophagy-dependent manner. Importantly, downregulation of NLRP3 inflammasomes signaling critically contributes to the enhanced survival rate in MSC3D through modulation of pyroptosis and apoptosis. The critical role of NLRP3 inflammasome suppression in the enhanced therapeutic efficacy of MSC spheroids was further confirmed in an invivo mouse model of DSS-induced colitis. These findings suggest that 3D culture confers survival and functional advantages to MSCs by suppressing NLRP3 inflammasome activation.

Hydroxysafflower Yellow A Inhibits Vascular Adventitial Fibroblast Migration via NLRP3 Inflammasome Inhibition through Autophagy Activation

Inflammation is closely associated with progression of vascular remodeling. The NLRP3 inflammasome is the key molecule that promotes vascular remodeling via activation of vascular adventitia fibroblast (VAF) proliferation and differentiation. VAFs have a vital effect on vascular remodeling that could be improved using hydroxysafflower yellow A (HSYA). However, whether HSYA ameliorates vascular remodeling through inhibition of NLRP3 inflammasome activation has not been explored in detail. Here, we cultured primary VAFs and analyzed the migration of VAFs induced by angiotensin II (ANG II) to determine the potential effects and mechanism of HSYA on VAF migration. The results thereof showed that HSYA remarkably inhibited ANG II-induced VAF migration, NLRP3 inflammasome activation, and the TLR4/NF-κB signaling pathway in a dose-dependent manner. In addition, it is worth noting that LPS promoted ANG II-induced VAF migration and NLRP3 inflammasome assembly, which could be significantly reversed using HSYA. Moreover, HSYA could be used to inhibit NLRP3 inflammasome activation by promoting autophagy. In conclusion, HSYA could inhibit ANG II-induced VAF migration through autophagy activation and inhibition of NLRP3 inflammasome activation through the TLR4/NF-κB signaling pathway.

20(S)-Protopanaxatriol ameliorates MAFLD by inhibiting NLRP3 inflammasome

Metabolic associated fatty liver disease (MAFLD) is one of the most common chronic liver diseases and may develop into non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma, which has threatened human health. Although NLRP3 inflammasome is widely recognized in the pathogenesis of MAFLD, there are currently no drugs targeting NLRP3 inflammasome approved by regulatory agencies. Panax ginseng and its main saponin components have been used to regulate inflammatory and metabolic disorders. Notably, 20(S)-protopanaxatriol (PPT) is an active metabolite of protopanaxatriol saponins with prominent anti-inflammatory activity. However, the mechanism by which PPT ameliorates MAFLD has not been fully elucidated. Therefore, this study explored the efficacy and mechanism of PPT in treating MAFLD based on the inhibition of NLRP3 inflammasome activation. First, we screened potential NLRP3 inflammasome blockers from protopanaxadiol saponins in mouse primary bone marrow-derived macrophages (BMDMs) stimulated by LPS and different inflammasome inducers. Second, LPS-primed mouse BMDMs, mouse primary hepatocytes, mouse primary Kupffer cells and human peripheral blood mononuclear cells (PBMCs) stimulated by cholesterol and ATP were used to evaluate the effect of PPT in inhibiting NLRP3 inflammasome. Finally, MCD-induced mouse MAFLD were established to verify the therapeutic effect of PPT by inhibiting NLRP3 inflammasome. Our results showed that PPT of ginseng saponins significantly inhibited NLRP3 inflammasome activation in multiple primary cells, suppressed systemic inflammation, restored liver function, and attenuated liver inflammation as well as fibrosis in MCD--induced mouse MAFLD. Collectively, protopanaxatriol saponins metabolite PPT, may serve as a potent therapeutic agent for MAFLD by inhibiting NLRP3 inflammasome activation.

WP1130 relieves septic shock in mice by inhibiting NLRP3 inflammasome activation

To investigate the mechanism by which the small molecule compound WP1130 inhibits NLRP3 inflammasome activation and alleviates septic shock. Mouse bone marrow-derived macrophages (BMDM) and human THP-1 cells were pre-treated with WP1130 before stimulation with different NLRP3 inflammasome agonists (Nigericin, ATP, MSU and intracellular LPS transfection), and AIM2 inflammasomes were activated with poly A: T. The levels of caspase-1 and IL-1β in the cell culture supernatant were determined using Western blotting and ELISA, and mitochondrial damage in the cells was observed using confocal microscopy. In the animal experiment, male C57BL/6 mice were randomized into blank control group, septic shock group (LPS group) and WP1130 treatment group (WP1130+LPS group), and the levels of IL-1β and TNF-α in the serum and peritoneal cavity were detected using ELISA. In murine BMDM and human THP-1 cells, WP1130 significantly inhibited NLRP3 agonists-induced caspase-1 and IL-1β secretion in a dose-dependent manner (P < 0.05) but did not obviously affect the secretion of such inflammatory factors as IL-6 and TNF-α that were not associated with inflammasomes (P>0.05). Treatment with WP1130 did not significantly affect poly A: T-induced activation of AIM2 inflammasomes (P>0.05) or induce obvious changes in mitochondrial damage, an upstream signal of NLRP3 inflammasome activation. In the mouse model of LPS-induced septic shock, WP1130 treatment significantly reduced the level of IL-1β (P < 0.05) without obviously affecting TNF-α level either in the serum or in the peritoneal cavity (P>0.05). WP1130 specifically inhibits NLRP3 inflammasome activation to alleviate LPS-induced septic shock in mice.