Novel Isoalantolactone-Based Derivatives as Potent NLRP3 Inflammasome Inhibitors: Design, Synthesis, and Biological Characterization.

The NLRP3 inflammasome has been recognized as a promising therapeutic target in drug discovery for inflammatory diseases. Our initial research identified a natural sesquiterpene isoalantolactone (IAL) as the active scaffold targeting NLRP3 inflammasome. To improve its activity and metabolic stability, a total of 64 IAL derivatives were designed and synthesized. Among them, compound 49 emerged as the optimal lead, displaying the most potent inhibitory efficacy on nigericin-induced IL-1ß release in THP-1 cells, with an IC50 value of 0.29 µM, approximately 27-fold more potent than that of IAL (IC50: 7.86 µM), and exhibiting higher metabolic stability. Importantly, 49 remarkably improved DSS-induced ulcerative colitis in vivo. Mechanistically, we demonstrated that 49 covalently bound to cysteine 279 in the NACHT domain of NLRP3, thereby inhibiting the assembly and activation of NLRP3 inflammasome. These results provided compelling evidence to further advance the development of more potent NLRP3 inhibitors based on this scaffold.

The Angiotensin II Receptor Neprilysin Inhibitor LCZ696 Inhibits the NLRP3 Inflammasome By Reducing Mitochondrial Dysfunction in Macrophages and Alleviates Dextran Sulfate Sodium-induced Colitis in a Mouse Model.

The intracellular sensor protein complex known as the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome plays a crucial role in regulating inflammatory diseases by overseeing the production of interleukin (IL)-1ß and IL-18. Targeting its abnormal activation with drugs holds significant promise for inflammation treatment. This study highlights LCZ696, an angiotensin receptor-neprilysin inhibitor, as an effective suppressor of NLRP3 inflammasome activation in macrophages stimulated by ATP, nigericin, and monosodium urate. LCZ696 also reduces caspase-11 and GSDMD activation, lactate dehydrogenase release, propidium iodide uptake, and the extracellular release of NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in ATP-activated macrophages, suggesting a potential mitigation of pyroptosis. Mechanistically, LCZ696 lowers mitochondrial reactive oxygen species and preserves mitochondrial integrity. Importantly, it does not significantly impact NLRP3, proIL-1ß, inducible nitric oxide synthase, cyclooxygenase-2 expression, or NF-κB activation in lipopolysaccharide-activated macrophages. LCZ696 partially inhibits the NLRP3 inflammasome through the induction of autophagy. In an in vivo context, LCZ696 alleviates NLRP3-associated colitis in a mouse model by reducing colonic expression of IL-1ß and tumor necrosis factor-α. Collectively, these findings suggest that LCZ696 holds significant promise as a therapeutic agent for ameliorating NLRP3 inflammasome activation in various inflammatory diseases, extending beyond its established use in hypertension and heart failure treatment.

Shikonin, an inhibitor of inflammasomes, inhibits Epstein-Barr virus reactivation.

Epstein-Barr virus (EBV) is a highly prevalent human herpesvirus that persists for life in more than 95% of the adult population. EBV usually establishes an asymptomatic life-long infection, but it is also associated with malignancies affecting B lymphocytes and epithelial cells mainly. The virus alternates between a latent phase and a lytic phase, both of which contribute to the initiation of the tumor process. So far, there is only a limited number of antiviral molecules against the lytic phase, most of them targeting viral replication. Recent studies provided evidence that EBV uses components of the NLRP3 inflammasome to enter the productive phase of its cycle following activation in response to various stimuli. In the present work, we demonstrate that shikonin, a natural molecule with low toxicity which is known to inhibit inflammasome, can efficiently repress EBV reactivation. Similar results were obtained with apigenin and OLT 1177, two other NLRP3 inflammasome inhibitors. It is shown herein that shikonin repressed the transcription of reactivation-induced NLRP3 thereby inhibiting inflammasome activation and EBV lytic phase induction.

Discovery of pterostilbene analogs as novel NLRP3 inflammasome inhibitors for potential treatment of DSS-induced colitis in mice.

The pterostilbene skeleton is a promising chemical scaffold that exerts anti-inflammatory, anti-depressant, and anti-tumor effects. In this study, we aim to reduce in vivo and in vitro toxicity of compound 32 (preliminary work) and maintain its biological activity. A series of novel pterostilbene derivatives (D1-D43) were designed and synthesized, and their anti-inflammatory activities were screened. All compounds were screened to evaluate their inhibitory effect on LPS/Nigericin-induced IL-1ß production and pyroptosis. The structure-activity relationships was deduced, and finally 1-((E)-4-(2-ethoxyethoxy)styryl)-3,5-dimethoxy-2-((E)-2-nitrovinyl)benzene (D22) was found to be a low-toxic compound with most potent inhibitory efficacy (against IL-1ß: IC50 = 2.41 µM). Preliminary mechanism studies showed that compound D22 may affect the assembly of NLRP3 inflammasome by targeting NLRP3 protein, thereby inhibiting the activation of NLRP3 inflammasome. The in vivo anti-inflammatory activity indicated that compound D22 had significant therapeutic effects on DSS-induced mouse acute colitis models.

Amniotic fluid stem cell-derived extracellular vesicles are independent metabolic units capable of modulating inflammasome activation in THP-1 cells.

An immunoregulatory role of stem cells, often mediated by their secretome, has been claimed by several studies. Stem cell-derived extracellular vesicles (EVs) are crucial components of the secretome. EVs, a heterogeneous group of membranous vesicles released by many cell types into the extracellular space, are now considered as an additional mechanism for intercellular communication. In this study, we aimed at investigating whether human amniotic stem cell-derived extracellular vesicles (HASC-EVs) were able to interfere with inflammasome activation in the THP-1 cell line. Two subsets of HASC-EVs were collected by sequential centrifugation, namely HASC-P10 and HASC-P100. We demonstrated that HASC-EVs were neither internalized into nor undertake a direct interaction with THP-1 cells. We showed that HASC-P10 and P100 were able to intrinsically produce ATP, which was further converted to adenosine by 5'-nucleotidase (CD73) and ectonucleoside triphosphate diphosphohydrolase-1 (CD39). We found that THP-1 cells conditioned with both types of HASC-EVs failed to activate the NLRP3/caspase-1/inflammasome platform in response to LPS and ATP treatment by a mechanism involving A2a adenosine receptor activation. These results support a role for HASC-EVs as independent metabolic units capable of modifying the cellular functions, leading to anti-inflammatory effects in monocytic cells.

Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.

BACKGROUND AND AIMS: HEV infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, crosstalk with antiviral interferon response, and the potential of therapeutic targeting. APPROACH AND RESULTS: We measured IL-1ß secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1ß production. A profound increase of IL-1ß secretion was further observed in HEV-infected patients (1,733 ± 1,234 pg/mL; n = 70) compared to healthy persons (731 ± 701 pg/mL; n = 70). Given that macrophages are the drivers of inflammatory response, we found that inoculation with infectious HEV particles robustly triggered NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibits HEV and inflammasome response without cross-interference. CONCLUSIONS: HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting of NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

Pectic polysaccharide from Smilax china L. ameliorated ulcerative colitis by inhibiting the galectin-3/NLRP3 inflammasome pathway.

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the colon and rectum. Although galectin-3 (Gal-3) has been reported to play a proinflammatory role in UC, it is unknown whether pectic polysaccharide, a Gal-3 inhibitor in tumor metastasis, can alleviate UC by inhibiting Gal-3. The aim of this study was to investigate the anti-inflammatory effects and underlying mechanisms of SCLP, a pectic polysaccharide purified from Smilax china L. in our previous work, on dextran sulfate sodium-induced UC in BALB/c mice. The results showed that SCLP could significantly improve symptoms, alleviate histopathological damage and reduce the secretion of inflammatory mediators in mice with UC. Analysis of the anti-colitis mechanisms indicated that SCLP could inhibit the Gal-3/NLRP3 inflammasome/IL-1ß pathway by suppressing the expression of Gal-3 and the interaction of Gal-3 and NLRP3. Our results suggested that SCLP could be a promising candidate for prevention and treatment of UC.

Mangiferin Ameliorates HFD-Induced NAFLD through Regulation of the AMPK and NLRP3 Inflammasome Signal Pathways.

Nonalcoholic fatty liver disease (NAFLD) is closely related to glycolipid metabolism and liver inflammation. And there is no effective drug approved for its clinical therapy. In this study, we focused on mangiferin (Man) and explored its effects and mechanisms on NAFLD treatment based on the regulation of glycolipid metabolism and anti-inflammatory in vivo and in vitro. The results exhibited that Man can significantly attenuate liver injury, insulin resistance, and glucose tolerance in high-fat diet- (HFD-) induced NAFLD mice and significantly reduce fat accumulation and inflammation in hepatic tissue of NAFLD mice. The transcriptome level RNA-seq analysis showed that the significantly different expression genes between the Man treatment group and the HFD-induced NAFLD model group were mainly related to regulation of energy, metabolism, and inflammation in liver tissue. Furthermore, western blots, real-time PCR, and immunohistochemistry experiments confirmed that Man significantly activated the AMPK signal pathway and inhibited NLRP3 inflammasome activation and pyroptosis in NAFLD mice. In in vitro cell experiments, we further confirmed that Man can promote glucose consumption and reduce intracellular triglyceride (TG) accumulation induced by free fatty acids in HepG2 cells and further that it can be blocked by AMPK-specific inhibitors. Western blot results showed that Man upregulated p-AMPKα levels and exhibited a significant AMPK activation effect, which was blocked by compound C. At the same time, Man downregulated the expression of NLRP3 inflammasome-related proteins and inhibited the activation of NLRP3 inflammasome, alleviating cell pyroptosis and inflammation effects. These results indicate that Man anti-NAFLD activity is mediated through its regulation of glucolipid metabolism by AMPK activation and its anti-inflammatory effects by NLRP3 inflammasome inhibition. Our study indicates that Man is a promising prodrug for the therapy of NAFLD patients.

NLRP3 Inflammasome Inhibitor BAY-117082 Reduces Oral Squamous Cell Carcinoma Progression.

Oral cancer is one of the most common human malignancies, and its incidence is increasing worldwide. In particular, oral squamous cell carcinoma (OSCC) is characterized by high rates of proliferation, invasiveness, and metastasis. Currently, standard treatment for OSCC includes surgical removal, chemotherapy, and radiotherapy; however, the survival rate of patients with OSCC remains low, thus new therapies are needed. It has been proven that excessive NLRP3 inflammasome activation and apoptosis alteration may contribute to oral cancer progression. This study aimed to investigate the effect of BAY-117082, an NLRP3 inflammasome inhibitor, in an in vitro and in vivo xenograft model of oral cancer. In vitro results revealed that BAY-117082 at concentrations of 5, 10, and 30 µM was able to reduce OSCC cell viability. BAY-117082 at higher concentrations significantly reduced NLRP3, ASC, caspase-1, IL-1ß, and IL-18 expression. Moreover, Bax, Bad, and p53 expression were increased, whereas Bcl-2 expression was reduced. Furthermore, the in vivo study demonstrated that BAY-117082 at doses of 2.5 and 5 mg/kg significantly decreased subcutaneous tumor mass, and also reduced NLRP3 inflammasome pathway activation. Therefore, based on these results, the use of BAY-117082 could be considered a promising strategy to counteract oral cancer progression, thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways.

NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis.

Endometriosis is an estrogen-dependent gynecological disease. The pathogenesis of endometriosis remains controversial, although it is generally accepted that the inflammatory immune response plays a crucial role in this process. Mast cells (MCs) are multifunctional innate immune cells that accumulate in endometriotic lesions. However, the molecular mechanism by which estrogen modulates MCs in the development of endometriosis is not well understood. Here we report that estrogen can induce the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) through estrogen receptor (ER)-α via the estrogen responsive element (ERE) in MCs. Such transcriptional regulation is necessary for the activation of NLRP3 inflammasome and the production of mature interleukin (IL)-1ß in MCs. Targeted inhibition of NLRP3 significantly restrained lesion progression and fibrogenesis in a mouse model of endometriosis. Collectively, these findings suggest that MCs contribute to the development of endometriosis through NLRP3 inflammasome activation mediated by nuclear-initiated estrogen signaling pathway.

Characterization, Antioxidant and Anti-Inflammation Capacities of Fermented Flammulina velutipes Polyphenols.

This work investigated the preparation, characterization, antioxidant, and anti-inflammation capacities of Flammulina velutipes polyphenols (FVP) and fermented FVP (FFVP). The results revealed that the new syringic acid, accounting for 22.22%, was obtained after fermentation (FFVP). FFVP exhibits higher antioxidant and anti-inflammation activities than FVP, enhancing cell viability and phagocytosis, inhibiting the secretion of NO and ROS, and reducing the inflammatory response of RAW264.7 cells. This study revealed that FFVP provides a theoretical reference for in-depth study of its regulatory mechanisms and further development of functional antioxidants that are applicable in the food and health industry.

Inhibitory Role of Berberine, an Isoquinoline Alkaloid, on NLRP3 Inflammasome Activation for the Treatment of Inflammatory Diseases.

The pyrin domain-containing multiprotein complex NLRP3 inflammasome, consisting of the NLRP3 protein, ASC adaptor, and procaspase-1, plays a vital role in the pathophysiology of several inflammatory disorders, including neurological and metabolic disorders, chronic inflammatory diseases, and cancer. Several phytochemicals act as promising anti-inflammatory agents and are usually regarded to have potential applications as complementary or alternative therapeutic agents against chronic inflammatory disorders. Various in vitro and in vivo studies have reported the anti-inflammatory role of berberine (BRB), an organic heteropentacyclic phytochemical and natural isoquinoline, in inhibiting NLRP3 inflammasome-dependent inflammation against many disorders. This review summarizes the mechanism and regulation of NLRP3 inflammasome activation and its involvement in inflammatory diseases, and discusses the current scientific evidence on the repressive role of BRB on NLRP3 inflammasome pathways along with the possible mechanism(s) and their potential in counteracting various inflammatory diseases.

Discovery of Novel Pterostilbene-Based Derivatives as Potent and Orally Active NLRP3 Inflammasome Inhibitors with Inflammatory Activity for Colitis.

Studies have shown that the abnormal activation of the NLRP3 inflammasome is involved in a variety of inflammatory-based diseases. In this study, a high content screening model targeting the activation of inflammasome was first established and pterostilbene was discovered as the active scaffold. Based on this finding, total of 50 pterostilbene derivatives were then designed and synthesized. Among them, compound 47 was found to be the best one for inhibiting cell pyroptosis [inhibitory rate (IR) = 73.09% at 10 µM], showing low toxicity and high efficiency [against interleukin-1ß (IL-1ß): half-maximal inhibitory concentration (IC50) = 0.56 µM]. Further studies showed that compound 47 affected the assembly of the NLRP3 inflammasomes by targeting NLRP3. The in vivo biological activity showed that this compound significantly alleviated dextran sodium sulfate (DSS)-induced colitis in mice. In general, our study provided a novel lead compound directly targeting the NLRP3 protein, which is worthy of further research and structural optimization.

Therapeutic targeting of the inflammasome in myeloid malignancies.

Even though genetic perturbations and mutations are important for the development of myeloid malignancies, the effects of an inflammatory microenvironment are a critical modulator of carcinogenesis. Activation of the innate immune system through various ligands and signaling pathways is an important driver of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The DAMPs, or alarmins, which activate the inflammasome pathway via the TLR4/NLR signaling cascade causes the lytic cell death of hematopoietic stem and progenitor cells (HSPCs), ineffective hematopoiesis, and ß-catenin-induced proliferation of cancer cells, leading to the development of MDS/AML phenotype. It is also associated with other myeloid malignancies and involved in the pathogenesis of associated cytopenias. Ongoing research suggests the interplay of inflammasome mediators with immune modulators and transcription factors to have a significant role in the development of myeloid diseases, and possibly therapy resistance. This review discusses the role and importance of inflammasomes and immune pathways in myeloid malignancies, particularly MDS/AML, to better understand the disease pathophysiology and decipher the scope of therapeutic interventions.

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

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

Selective Inhibition of NLRP3 Inflammasome Reverses Pressure Overload-Induced Pathological Cardiac Remodeling by Attenuating Hypertrophy, Fibrosis, and Inflammation.

Activation of the NLRP3 inflammasome promotes pathological cardiac remodeling induced by pressure overload. However, the therapeutic effects of NLRP3 inhibition after cardiac remodeling remain unknown. The present study aimed to investigate whether the selective NLRP3 inhibitor, MCC950, could reverse transverse aortic constriction (TAC)-induced cardiac remodeling. Mice were divided into four groups based on the treatment given: sham, sham + MCC950, TAC, and TAC + MCC950. MCC950 (10 mg/kg, intraperitoneal injection, once per day) was administered from two weeks after TAC or sham surgery for four weeks. Echocardiography, histological analysis, RT-PCR, and Western blotting were performed to explore the function of MCC950 after TAC. We found that MCC950 reversed cardiac dysfunction after TAC. MCC950 attenuated cardiac hypertrophy by down-regulating calcineurin expression and inhibiting MAPK activation. Further, it also alleviated cardiac fibrosis post-TAC by inhibiting the TGF-ß/Smad4 pathway, and reduced cardiac inflammation and macrophage infiltration post-TAC, including both M1 and M2 macrophages. Taken together, MCC950 can attenuate cardiac remodeling due to pressure overload by inhibiting hypertrophy, fibrosis, and inflammation. Our study provides a basis for the clinical application of NLRP3 inhibitors in the treatment of non-ischemic heart failure.

Promise of the NLRP3 Inflammasome Inhibitors in In Vivo Disease Models.

Nucleotide-binding oligomerization domain NOD-like receptors (NLRs) are conserved cytosolic pattern recognition receptors (PRRs) that track the intracellular milieu for the existence of infection, disease-causing microbes, as well as metabolic distresses. The NLRP3 inflammasome agglomerates are consequent to sensing a wide spectrum of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Certain members of the NLR family have been documented to lump into multimolecular conglomerates called inflammasomes, which are inherently linked to stimulation of the cysteine protease caspase-1. Following activation, caspase-1 severs the proinflammatory cytokines interleukin (IL)-1ß and IL-18 to their biologically active forms, with consequent commencement of caspase-1-associated pyroptosis. This type of cell death by pyroptosis epitomizes a leading pathway of inflammation. Accumulating scientific documentation has recorded overstimulation of NLRP3 (NOD-like receptor protein 3) inflammasome involvement in a wide array of inflammatory conditions. IL-1ß is an archetypic inflammatory cytokine implicated in multiple types of inflammatory maladies. Approaches to impede IL-1ß's actions are possible, and their therapeutic effects have been clinically demonstrated; nevertheless, such strategies are associated with certain constraints. For instance, treatments that focus on systemically negating IL-1ß (i.e., anakinra, rilonacept, and canakinumab) have been reported to result in an escalated peril of infections. Therefore, given the therapeutic promise of an NLRP3 inhibitor, the concerted escalated venture of the scientific sorority in the advancement of small molecules focusing on direct NLRP3 inflammasome inhibition is quite predictable.

Licochalcone E improves insulin sensitivity in palmitic acid-treated HepG2 cells through inhibition of the NLRP3 signaling pathway.

Our previous research demonstrated that compound licochalcone E can reduce glucose tolerance and lipid metabolism in diabetic rats, although its mechanism remains unknown. Here, we used palmitic acid (PA) to establish a PA-treated HepG2 model, and then examined glucose uptake, glucose consumption, and blood lipids to evaluate the effects of licochalcone E within the safe dose range in the model. Polymerase chain reaction (PCR) was used to detect the expression levels of key genes associated with liver gluconeogenesis; enzyme-linked immunosorbent assay (ELISA) was deployed to evaluate the concentration of inflammatory factors; and laser confocal microscopy and western blot were used to determine the levels of reactive oxygen species (ROS) and NLRP3 inflammasome signaling pathway-related proteins, respectively. Finally, molecular simulations were exploited to validate the interaction between licochalcone E and the NLRP3 inflammasome. The results demonstrated that licochalcone E showed no toxicity in the dose range of 2.5-40 µM. In this dose range, licochalcone E substantially increased the uptake and consumption of glucose in the insulin resistance model and dose-dependently reduced the concentration of total cholesterol. The PCR results indicated that licochalcone E dose-dependently reduced the expression of Glucose-6-phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (PEPCK) genes and increased the expression of Glucose Transporter 4 (Glut4) in PA-treated HepG2. Moreover, the ELISA results revealed that licochalcone E significantly reduced the expression of TNF-α, IL-1ß, and IL-18. Confocal microscopy results showed that licochalcone E dramatically reduced the generation of ROS and the expressions of NLRP3 and its downstream caspase-1 in PA-treated HepG2 model. Western blot results further indicated that licochalcone E significantly reduced the expression of NLRP3, caspase-1 and IL-1ß in the model. Additionally, molecular simulations demonstrated that licochalcone E has good binding affinity for the NLPR3 inflammasome. We concluded that licochalcone E has the potential to be used as an insulin sensitizer by reducing the release of ROS and inflammatory factors following inhibition of the NLPR3 signaling pathway.

Preservation of Contractile Reserve and Diastolic Function by Inhibiting the NLRP3 Inflammasome with OLT1177® (Dapansutrile) in a Mouse Model of Severe Ischemic Cardiomyopathy Due to Non-Reperfused Anterior Wall Myocardial Infarction.

Interleukin-1ß (IL-1ß), a product of the NLRP3 inflammasome, modulates cardiac contractility and diastolic function. We proposed that OLT1177® (dapansutrile), a novel NLRP3 inhibitor, could preserve contractile reserve and diastolic function after myocardial infarction (MI). We used an experimental murine model of severe ischemic cardiomyopathy through the ligation of the left coronary artery without reperfusion, and after 7 days randomly assigned mice showing large anterior MI (>4 akinetic segments), increased left ventricular (LV) dimensions ([LVEDD] > 4.4 mm), and reduced function (LV ejection fraction < 40%) to a diet that was enriched with OLT1177® admixed with the chow in the diet at 3.75 g/kg (Group 1 [n = 10]) or 7.5 g/kg (Group 2 [n = 9]), or a standard diet as the no-treatment control group (Group 3 [n = 10]) for 9 weeks. We measured the cardiac function and contractile reserve with an isoproterenol challenge, and the diastolic function with cardiac catheterization at 10 weeks following the MI surgery. When compared with the control (Group 3), the mice treated with OLT1177 (Group 1 and 2) showed significantly greater preservation of their contractile reserve (the percent increase in the left ventricular ejection fraction [LVEF] after the isoproterenol challenge was +33 ± 11% and +40 ± 6% vs. +9 ± 7% in the standard diet; p < 0.05 and p < 0.005 for Group 1 and 2, respectively) and of diastolic function measured as the lower left ventricular end-diastolic pressure (3.2 ± 0.5 mmHg or 4.5 ± 0.5 mmHg vs. 10.0 ± 1.6 mmHg; p < 0.005 and p < 0.009 respectively). No differences were noted between the resting LVEF of the MI groups. These effects were independent of the effects on the ventricular remodeling after MI. NLRP3 inflammasome inhibition with OLT1177® can preserve ß-adrenergic responsiveness and prevent left ventricular diastolic dysfunction in a large non-reperfused anterior MI mouse model. OLT1177® could therefore be used to prevent the development of heart failure in patients with ischemic cardiomyopathy.

Tanshinones inhibit NLRP3 inflammasome activation by alleviating mitochondrial damage to protect against septic and gouty inflammation.

Tanshinones, the active ingredients derived from the roots of Salvia miltiorrhiza, have been widely used as traditional medicinal herbs for treating human diseases. Although tanshinones showed anti-inflammatory effects in many studies, large knowledge gaps remain regarding their underlying mechanisms. Here, we identified 15 tanshinones that suppressed the activation of NLRP3 inflammasome and studied their structure-activity relationships. Three tanshinones (tanshinone IIA, isocryptotanshinone, and dihydrotanshinone I) reduced mitochondrial reactive-oxygen species production in lipopolysaccharide (LPS)/nigericin-stimulated macrophages and correlated with altered mitochondrial membrane potentials, mitochondria complexes activities, and adenosine triphosphate and protonated-nicotinamide adenine dinucleotide production. The tanshinones may confer mitochondrial protection by promoting autophagy and the AMP-activated protein kinase pathway. Importantly, our findings demonstrate that dihydrotanshinone I improved the survival of mice with LPS shock and ameliorated inflammatory responses in septic and gouty animals. Our results suggest a potential pharmacological mechanism whereby tanshinones can effectively treat inflammatory diseases, such as septic and gouty inflammation.