Britannin as a novel NLRP3 inhibitor, suppresses inflammasome activation in macrophages and alleviates NLRP3-related diseases in mice.

Overactivation of the NLRP3 inflammasomes induces production of pro-inflammatory cytokines and drives pathological processes. Pharmacological inhibition of NLRP3 is an explicit strategy for the treatment of inflammatory diseases. Thus far no drug specifically targeting NLRP3 has been approved by the FDA for clinical use. This study was aimed to discover novel NLRP3 inhibitors that could suppress NLRP3-mediated pyroptosis. We screened 95 natural products from our in-house library for their inhibitory activity on IL-1ß secretion in LPS + ATP-challenged BMDMs, found that Britannin exerted the most potent inhibitory effect with an IC50 value of 3.630 µM. We showed that Britannin (1, 5, 10 µM) dose-dependently inhibited secretion of the cleaved Caspase-1 (p20) and the mature IL-1ß, and suppressed NLRP3-mediated pyroptosis in both murine and human macrophages. We demonstrated that Britannin specifically inhibited the activation step of NLRP3 inflammasome in BMDMs via interrupting the assembly step, especially the interaction between NLRP3 and NEK7. We revealed that Britannin directly bound to NLRP3 NACHT domain at Arg335 and Gly271. Moreover, Britannin suppressed NLRP3 activation in an ATPase-independent way, suggesting it as a lead compound for design and development of novel NLRP3 inhibitors. In mouse models of MSU-induced gouty arthritis and LPS-induced acute lung injury (ALI), administration of Britannin (20 mg/kg, i.p.) significantly alleviated NLRP3-mediated inflammation; the therapeutic effects of Britannin were dismissed by NLRP3 knockout. In conclusion, Britannin is an effective natural NLRP3 inhibitor and a potential lead compound for the development of drugs targeting NLRP3.

Colchicine ameliorates short-term abdominal aortic aneurysms by inhibiting the expression of NLRP3 inflammasome components in mice.

BACKGROUND: Abdominal aortic aneurysms (AAA) are often associated with chronic inflammation and pose a significant risk to affected individuals. Colchicine, known for its anti-inflammatory properties, has shown promise in managing cardiovascular diseases. However, its specific role in the development of AAA remains poorly understood. METHODS AND RESULTS: In this study, we employed a short-term AAA model induced by angiotensin II (Ang II, 1000 ng/kg/min) and calcium chloride (CaCl2, 0.5 mol/l) in male ApoE-/- and C57BL/6 mice (8-12 weeks old) to investigate the effects of colchicine on AAA progression. Colchicine (0.4 mg/kg) was administered orally once daily, starting on the same day as AAA induction. After a 4-week duration, we observed a significant reduction in AAA diameter, degradation of elastic fibers, and expression of components related to the Nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome in the vessel wall of colchicine-treated mice compared to the saline group. Mechanistically, colchicine (5 µm/l, for 24h) inhibited the expression of NLRP3 inflammasome components through the P38-ERK/MicroRNA145-toll-like receptor 4 (TLR4) pathway in RAW264.7 cells. CONCLUSIONS: Our study demonstrates the effectiveness of colchicine in suppressing NLRP3 inflammasome components, thereby delaying AAA progression in the Ang II and CaCl2-induced short-term model. These findings suggest the potential of colchicine as a pharmacological treatment option for AAA.

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.

Delayed NLRP3 inflammasome inhibition ameliorates subacute stroke progression in mice.

BACKGROUND: Ischemic stroke immediately evokes a strong neuro-inflammatory response within the vascular compartment, which contributes to primary infarct development under vessel occlusion as well as further infarct growth despite recanalization, referred to as ischemia/reperfusion injury. Later, in the subacute phase of stroke (beyond day 1 after recanalization), further inflammatory processes within the brain parenchyma follow. Whether this second wave of parenchymal inflammation contributes to an additional/secondary increase in infarct volumes and bears the potential to be pharmacologically targeted remains elusive. We addressed the role of the NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome in the subacute phase of ischemic stroke. METHODS: Focal cerebral ischemia was induced in C57Bl/6 mice by a 30-min transient middle cerebral artery occlusion (tMCAO). Animals were treated with the NLRP3 inhibitor MCC950 therapeutically 24 h after or prophylactically before tMCAO. Stroke outcome, including infarct size and functional deficits as well as the local inflammatory response, was assessed on day 7 after tMCAO. RESULTS: Infarct sizes on day 7 after tMCAO decreased about 35% after delayed and about 60% after prophylactic NLRP3 inhibition compared to vehicle. Functionally, pharmacological inhibition of NLRP3 mitigated the local inflammatory response in the ischemic brain as indicated by reduction of infiltrating immune cells and reactive astrogliosis. CONCLUSIONS: Our results demonstrate that the NLRP3 inflammasome continues to drive neuroinflammation within the subacute stroke phase. NLRP3 inflammasome inhibition leads to a better long-term outcome-even when administered with a delay of 1 day after stroke induction, indicating ongoing inflammation-driven infarct progression. These findings may pave the way for eagerly awaited delayed treatment options in ischemic stroke.

CY-09 Alleviates the Depression-like Behaviors via Inhibiting NLRP3 Inflammasome-Mediated Neuroinflammation in Lipopolysaccharide-Induced Mice.

Depression is a serious mental illness, mainly characterized as large mood swings and sleep, diet, and cognitive function disorders. NLPR3, one of the inflammasomes that can be activated by a variety of stimuli to promote the maturation and secretion of pro-inflammatory cytokines, has been considered to be involved in the pathophysiology of depression. In this study, the putative role of CY-09, a selective and direct inhibitor of NLRP3, was evaluated in the lipopolysaccharide (LPS)-induced mice. The results of the study indicated that CY-09 significantly decreased the levels of NLRP3 in the hippocampus of LPS-induced mice. In addition, CY-09 increased the sucrose preference and shortened the immobility time in LPS-induced mice, suggesting the antidepressant-like effects of inhibiting NLRP3 inflammasome. Biochemical analysis showed that LPS significantly activated the NLRP3/ASC/cytokine signaling pathway and caused microglial activation, while CY-09 prevented the changes. Moreover, CY-09 increased the brain-derived neurotrophic factor (BDNF) only in microglia but not in the whole hippocampus. Meanwhile, CY-09 did not promote neurogenesis in the hippocampus of LPS mice. In conclusion, the results of the study showed that the antidepressant-like effects of NLRP3 inhibitor CY-09 were mediated by alleviating neuroinflammation in microglia and independent of the neurotrophic function in the hippocampus.

Favorable effect of rivaroxaban against vascular dysfunction in diabetic mice by inhibiting NLRP3 inflammasome activation.

Cardiovascular disease (CVD) is the leading cause of death in various complications of type 2 diabetes mellitus (T2DM). Rivaroxaban (Xarelto; Bayer), an oral direct factor Xa (FXa) inhibitor, prevents the activation of the coagulation cascade in CVD. Considering its anticoagulant and anti-inflammatory effects, we assessed the hypothesis that rivaroxaban treatment may attenuate the vascular lesion and dysfunction in T2DM mice. C57BL/6, BKS-db/db, BKS-db/+, wild-type (WT), and NLRP3-/- mice were fed with standard chow or high-fat diet (HFD). Biochemical indexes, vascular lesions, and protein expression were evaluated using Western blot analysis, immunofluorescent staining, and RNA interference. Rivaroxaban presented favorable protection of vascular dysfunction in T2DM mice with significantly relieved vascular tension, intima-media thickness, and collagen deposition. Similar improvements in NLR family pyrin domain containing 3 (NLRP3) knockout groups and rivaroxaban pointed to the positive role of rivaroxaban against vascular dysfunction in diabetic mice by ameliorating NLRP3 inflammasome activation. Furthermore, the augmentation of inflammation and cell dysfunction in mice aortic endothelial cells (MAECs) and smooth muscle cells (MOVASs) induced by soluble FXa may be blocked by rivaroxaban via protease-activated receptors (PAR-1, PAR-2), mitogen-activated protein kinase (MAPK), and nuclear factor κ-B (NF-κB) pathway. The data indicate that the development of vascular dysfunction and inflammation in T2DM mice may be blocked by rivaroxaban in vivo and in vitro. Rivaroxaban treatment may also attenuate NLRP3 inflammasome activation via PARs, MAPK, and NF-κB pathway. This study provides mechanistic evidence of rivaroxaban therapies for vascular complications of T2DM.

Ketogenic diet ameliorates inflammation by inhibiting the NLRP3 inflammasome in osteoarthritis.

BACKGROUND: The nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been reported to be involved in the pathological process of osteoarthritis (OA) inflammation. Here, we investigated the ketogenic diet (KD), which has been previously demonstrated to inhibit NLRP3 inflammasome activation, to elucidate its protective mechanism against OA in rats. METHODS: Anterior cruciate ligament transaction (ACLT) together with partial medial meniscectomy was used to create a rat knee joint OA model. After treatment with KD or standard diet (SD) for 8 weeks, the knee specimens were obtained for testing. RESULTS: The KD significantly increased the content of ß-hydroxybutyrate (ßOHB) in rats. Compared to the SD group, the KD significantly reduced the damage caused by OA in the articular cartilage and subchondral bone. The NLRP3 inflammasome and inflammatory cytokines interleukin-1 ß (IL-1ß) and IL-18 were significantly increased in the SD group compared with the sham group, while their expression was significantly decreased in rats treated with the KD. In addition, MMP13 was significantly decreased in the KD group compared to that in the SD group, while COL2 was significantly increased. CONCLUSIONS: KD can protect the articular cartilage and subchondral bone in a rat OA model by inhibiting NLRP3 inflammasome activation and reducing the OA inflammatory response.

Fraxinol attenuates LPS-induced acute lung injury by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas and inhibiting NLRP3.

CONTEXT: Acute lung injury (ALI) is a serious heterogenous pulmonary disorder. Fraxinol was selected for this study since it is a simple coumarin compound, not previously investigated in ALI. OBJECTIVES: This study investigates the ALI therapeutic effect and mechanisms of fraxinol. MATERIALS AND METHODS: Male BALB/c mice were treated with fraxinol (20, 40, and 80 mg/kg) following intranasal injection of lipopolysaccharide (LPS; 10 µg in 50 µL). The mice in control group were intratracheally injected with 50 µL phosphate buffered saline (PBS). Raw264.7 cells were treated with fraxinol by 100 ng/mL LPS for 6 h, then treated by different concentrations of fraxinol (5, 10, and 25 µM) for 48 h. Cells in control group were treated with PBS. RESULTS: Fraxinol with doses of 20, 40, and 80 mg/kg significantly attenuated LPS-induced lung injury in mice (lung injury score, 10.4, 31.2, 50.3%). Fraxinol attenuated the apoptosis and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) activation induced by LPS (apoptosis, 18.3, 30.2, 55.6%; NLRP3, 30.0, 47.7, 63.6%). The anti-apoptosis and anti-inflammation effects of fraxinol were also confirmed in Raw264.7 cells (apoptosis, 38.8, 55.3, 68.9%; NLRP3, 20.6, 55.7, 73.9%). DISCUSSION AND CONCLUSION: The anti-ALI effects of fraxinol maybe by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas axis and inhibiting NLRP3 inflammasome. Our research provides a candidate drug in the treatment of ALI.

Nano-chemically Modified Tetracycline-3 (nCMT-3) Attenuates Acute Lung Injury via Blocking sTREM-1 Release and NLRP3 Inflammasome Activation.

BACKGROUND: Intratracheal (IT) lipopolysaccharide (LPS) causes severe acute lung injury (ALI) and systemic inflammation. CMT-3 has pleiotropic anti-inflammatory effects including matrix metalloproteinase (MMP) inhibition, attenuation of neutrophil (PMN) activation, and elastase release. CMT-3's poor water solubility limits its bioavailability when administered orally for treating ALI. We developed a nano-formulation of CMT-3 (nCMT-3) to test the hypothesis that the pleiotropic anti-inflammatory activities of IT nCMT-3 can attenuate LPS-induced ALI. METHODS: C57BL/6 mice were treated with aerosolized IT nCMT-3 or saline, then had IT LPS or saline administered 2 h later. Tissues were harvested at 24 h. The effects of LPS and nCMT-3 on ALI were assessed by lung histology, MMP level/activity (zymography), NLRP3 protein, and activated caspase-1 levels. Blood and bronchoalveolar lavage fluid (BALF) cell counts, PMN elastase, and soluble triggering receptor expressed on myelocytes-1 (sTREM-1) levels, TNF-α, IL-1ß, IL-6, IL-18, and BALF protein levels were also measured. RESULTS: LPS-induced ALI was characterized by histologic lung injury (PMN infiltration, alveolar thickening, edema, and consolidation) elevated proMMP-2, -9 levels and activity, increased NLRP-3 protein and activated caspase-1 levels in lung tissue. LPS-induced increases in plasma and BALF levels of sTREM-1, TNF-α, IL-1ß, IL-6, IL-18, PMN elastase and BALF protein levels demonstrate significant lung/systemic inflammation and capillary leak. nCMT-3 significantly ameliorated all of these LPS-induced inflammatory markers to control levels, and decreased the incidence of ALI. CONCLUSIONS: Pre-treatment with nCMT3 significantly attenuates LPS-induced lung injury/inflammation by multiple mechanisms including: MMP activation, PMN elastase, sTREM-1 release, and NLRP3 inflammasome/caspase-1 activation.

Blocking the NLRP3 inflammasome reduces osteogenic calcification and M1 macrophage polarization in a mouse model of calcified aortic valve stenosis.

BACKGROUND AND AIMS: Activated innate immune cells infiltrating the valve and their secreted cytokines drive the differentiation of valve interstitial cells into myofibroblastic and osteoblastic phenotypes in calcified aortic valve stenosis (CAVS). In this study, we investigated how NLRP3 inhibition with CY-09 reduces aortic valve stenosis and calcification. METHODS: ApoE-/- mice were fed a high-fat diet for 24 weeks with or without intraperitoneal injection of 2.5 mg/kg/day NLRP3 inhibitor CY-09 for 42 consecutive days, while the control group mice were fed a normal diet. The valve function was monitored by echocardiography; calcified nodules were assessed by Von Kossa staining; and calcification-related molecules, inflammatory factors, and white leucocyte influx into the valve were assessed by immunohistochemistry, TUNEL assay, and PCR. RESULTS: Mice treated with CY-09 exhibited improved aortic valve function and reduced valve calcification deposition. CY-09 intervention significantly downregulated the elevated expression of the NLRP3 inflammasome pathway molecules NLRP3, caspase-1, and IL-1ß and the osteogenic calcification markers RUNX2, SPARC, and BMP2 in stenotic valves, while the number of apoptotic cells and dystrophic calcification markers CDH11 and α- SMA did not change significantly. Inhibition of NLRP3 activity also reduced the ratio of M1/M2 macrophages, prevented the shift of macrophages towards the M1 phenotype, and downregulated the levels of the proinflammatory factors IL-6 and TNF-α. CONCLUSIONS: This study provides a proof-of-concept that pharmacological inhibition of the NLRP3 inflammasome is a feasible strategy for alleviating aortic valve calcification and stenosis.

Effectiveness of NLRP3 Inhibitor as a Non-Hormonal Treatment for ovarian endometriosis.

BACKGROUND: Endometriosis is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process that affects 10% of women of reproductive age. Ovarian endometriosis (OE) is the most common lesion in endometriosis and may cause infertility, in addition to dysmenorrhea. Hormonal treatments, which are the conventional treatment methods for endometriosis, suppress ovulation and hence are not compatible with fertility. The inflammasome is a complex that includes Nod-like receptor (NLR) family proteins, which sense pathogen-associated molecular patterns and homeostasis-altering molecular processes. It has been reported that the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing (NLRP) 3 inflammasome, which contributes to the activation of interleukin-1 beta (IL-1ß), might be related to the progression of endometriosis. Therefore, the aim of the present study was to evaluate non-hormonal therapies for OE, such as inhibitors of the NLRP3 inflammasome. METHODS: The expression of NLRP3 was measured in the eutopic endometrium (EM) of patients with and without endometriosis and OE samples, as well as stromal cells derived from the endometrium of patients with and without endometriosis and OE samples (endometrial stromal cells with endometriosis [ESCs] and cyst-derived stromal cells [CSCs]). The effects of an NLRP3 inhibitor (MCC950) on ESCs and CSCs survival and IL-1ß production were evaluated. We then administered MCC950 to a murine model of OE to evaluate its effects on OE lesions and ovarian function. RESULTS: NLRP3 gene and protein expression levels were higher in OE and CSCs than in EM and ESCs, respectively. MCC950 treatment significantly reduced the survival of CSCs, but not that of ESCs. Moreover, MCC950 treatment reduced the co-localization of NLRP3 and IL-1ß in CSCs, as well as IL-1ß concentrations in CSCs supernatants. In the murine model, MCC950 treatment reduced OE lesion size compared to phosphate-buffered saline treatment (89 ± 15 vs. 49 ± 9.3 mm3 per ovary; P < 0.05). In the MCC950-treated group, IL-1ß and Ki67 levels in the OE-associated epithelia were reduced along with the oxidative stress markers of granulosa cells. CONCLUSIONS: These results indicated that NLRP3/IL-1ß is involved in the pathogenesis of endometriosis and that NLRP3 inhibitors may be useful for suppressing OE and improving the function of ovaries with endometriosis.

Effect of NLRP3 repression on NLRP3 inflammasome activation in human corneal epithelial cells with black carbon exposure.

PURPOSE: To investigate the inhibitory effects of NLRP3 siRNA on NLRP3 inflammasome activation in human corneal epithelial cells (HCECs) with fresh black carbon (FBC) particles and ozone-oxidized BC (OBC) particles treatment. METHODS: HCECs were transfected with NLRP3 siRNA or control siRNA for 48 h, followed by 200 µg/ml FBC or OBC suspension for an additional 72 h. Untreated controls were cells with no siRNA transfection or BC treatment. RT-qPCR and Western blot were used to measure mRNA and protein levels of components of the NLRP3 inflammasome (NLRP3, ASC, and Caspase-1) and downstream cytokine (IL-1ß), respectively. RESULTS: Compared with untreated control cells, mRNA levels of NLRP3, ASC, Caspase-1, and IL-1ß were significantly higher (p < 0.05) in control siRNA transfected cells with BC treatments. Compared with the control siRNA transfected cells, NLRP3 siRNA transfection reduced the expression of NLRP3 and ASC, whereas it had a limited effect on the expression of Caspase-1 and IL-1ß with FBC or OBC exposures. Under FBC treatment, the reductions of NLRP3 and Caspase-1 mRNA levels were 53.5% (p < 0.001) and 34.2% (p < 0. 01), respectively, and NLRP3 and ASC protein levels were lowered by 58.2% (p < 0.001) and 45.4% (p < 0.001), respectively. Under OBC treatment, the reductions of NLRP3 and Caspase-1 mRNA levels were 39.8% (p < 0.001) and 25.6% (p < 0.05), respectively, and NLRP3 and ASC protein levels were lowered by 44.8% (p < 0.001) and 41.7% (p < 0.001), respectively. Moreover, mRNA levels of ASC and IL-1ß, the protein levels of Caspase-1 and IL-1ß showed a tendency to decrease in NLRP3 siRNA transfected cells, it was statistically insignificant (p > 0.05). CONCLUSIONS: NLRP3 siRNA transfection could partially reverse the increased mRNA levels of NLRP3 and Caspase-1, the protein levels of NLRP3 and ASC in HCECs with BC treatment, whereas the reductions of protein levels of Caspase-1 and IL-1ß were not significant, indicating that NLRP3 siRNA has a limited inhibitory effect on the activation of NLRP3 inflammasome triggered by BC.

Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation.

SignificanceThe nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that forms an inflammasome. The cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950 has established the structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation and the mechanism of action of the NLRP3 specific inhibitor. The inactive NLRP3 oligomer represents the NLRP3 resting state, capable of binding to membranes and is likely disrupted for its activation. Visualization of the inhibitor binding mode will enable optimization of the activity of NLRP3 inflammasome inhibitor drugs.

NLRP3 priming due to skin damage precedes LTP allergic sensitization in a mouse model.

Allergic sensitization is initiated by protein and epithelia interaction, although the molecular mechanisms leading this encounter toward an allergic phenotype remain unknown. Here, we apply the two-hit hypothesis of inflammatory diseases to the study of food allergy sensitization. First, we studied the effects of long-term depilation in mice by analyzing samples at different time points. Several weeks of depilation were needed until clear immunological changes were evidenced, starting with upregulation of NLRP3 protein levels, which was followed by overexpression of Il1b and Il18 transcripts. Secondly, we assessed the effects of allergen addition (in this case, Pru p 3 in complex with its natural lipid ligand) over depilated skin. Systemic sensitization was evaluated by intraperitoneal provocation with Pru p 3 and measure of body temperature. Anaphylaxis was achieved, but only in mice sensitized with Prup3_complex and not treated with the NLRP3 inhibitor MCC950, thus demonstrating the importance of both hits (depilation + allergen addition) in the consecution of the allergic phenotype. In addition, allergen encounter (but not depilation) promoted skin remodeling, as well as CD45+ infiltration not only in the sensitized area (the skin), but across several mucosal tissues (skin, lungs, and gut), furtherly validating the systemization of the response. Finally, a low-scale study with human ILC2s is reported, where we demonstrate that Prup3_complex can induce their phenotype switch (↑CD86, ↑S1P1) when cultured in vitro, although more data is needed to understand the implications of these changes in food allergy development.

Effect of parthenolide, an NLRP3 inflammasome inhibitor, on insulin resistance in high-fat diet-obese mice.

The activation of Nod-like receptor proteins (NLRP3) containing the pyrin domain inflammasome is a hallmark of the pathogenesis of metabolic disorders. Inhibition of the NLRP3 inflammasome by phytoconstituents has been attempted as a strategy to mitigate these disorders. Therefore, the present study aimed to evaluate the efficacy of an NLRP3 inflammasome inhibitor, parthenolide (PN; 5 mg/kg i.p.) against inflammation and insulin resistance in high-fat diet (HFD) - obese mice. Treatment with PN and pioglitazone (PIO; 30 mg/kg p.o.) attenuated lipopolysaccharide (LPS; 1 ng/ml) - induced elevation of tumor necrosis factor-α and interleukin-1ß in mouse peritoneal macrophages in a dose-dependent manner. Sixty days of PN and PIO treatment marginally reduced obesity-induced insulin resistance in HFD-obese mice. PN treatment also decreased blood glucose from 14th to 60th day, supporting the hypothesis of simultaneous attenuation of inflammation and insulin resistance in obese mice. Thus, PN treatment was also evident with significant improvement in glucose tolerance and peripheral insulin resistance validated through the respective tolerance tests. Therefore, the present study suggests that PN, an NLRP3 inflammasome inhibitor, could be a possible therapeutic agent for attenuating obesity-induced insulin resistance.

NLRP3 Inhibitor Tranilast Attenuates Gestational Diabetes Mellitus in a Genetic Mouse Model.

BACKGROUND AND OBJECTIVE: This study was designed to explore the protective effects of a clinically available NLR family Pyrin domain-containing receptor 3 (NLRP3) inhibitor, tranilast, in gestational diabetes mellitus (GDM) mice. METHODS: We used pregnant C57BL/KsJdb/+ (db/+) female mice as GDM mice, then orally administered 20 mg/kg of tranilast or metformin daily for 2 weeks. A glucose tolerance test and an insulin resistance test were used to evaluate the severity of diabetes in tranilast/metformin-treated GDM mice. After delivery, newborn mice were counted and weighed to measure their protective role on the reproductive outcome of GDM mice. Next, we determined the expression of NLRP3 and proinflammatory cytokines in the visceral adipose tissue and placenta of GDM mice using western blot and quantitative real-time-polymerase chain reaction. Furthermore, we determined the proinflammatory cytokines in the serum using an enzyme-linked immunosorbent assay. RESULTS: Tranilast significantly ameliorated GDM symptoms, including maternal body weight, hyperglycemia, insulin insufficiency, glucose intolerance and insulin resistance, enlarged litter size, and reduced litter body weight. Additionally, tranilast remarkably reduced the elevated expression of NLRP3 and proinflammatory cytokines. CONCLUSIONS: Our data clarified the protective role of the NLRP3 inhibitor, tranilast, on GDM by inhibiting the activation of the NLRP3 inflammasome as well as inflammatory responses. The findings mean tranilast might serve as a therapeutic drug to treat GDM.

Identification of NLRP3PYD Homo-Oligomerization Inhibitors with Anti-Inflammatory Activity.

Inflammasomes are multiprotein complexes that represent critical elements of the inflammatory response. The dysregulation of the best-characterized complex, the NLRP3 inflammasome, has been linked to the pathogenesis of diseases such as multiple sclerosis, type 2 diabetes mellitus, Alzheimer's disease, and cancer. While there exist molecular inhibitors specific for the various components of inflammasome complexes, no currently reported inhibitors specifically target NLRP3PYD homo-oligomerization. In the present study, we describe the identification of QM380 and QM381 as NLRP3PYD homo-oligomerization inhibitors after screening small molecules from the MyriaScreen library using a split-luciferase complementation assay. Our results demonstrate that these NLRP3PYD inhibitors interfere with ASC speck formation, inhibit pro-inflammatory cytokine IL1-ß release, and decrease pyroptotic cell death. We employed spectroscopic techniques and computational docking analyses with QM380 and QM381 and the PYD domain to confirm the experimental results and predict possible mechanisms underlying the inhibition of NLRP3PYD homo-interactions.

Euphzycopias A-I, macrocyclic diterpenes with NLRP3 inflammasome inhibitory activity from Euphorbia helioscopia L.

A phytochemical investigation was conducted on Euphorbia helioscopia, resulting in the isolation of thirteen compounds, including nine undescribed diterpenoids, Euphzycopias A - I (1-9), of which the skeletons of compounds 1-4 were found in E. helioscopia L. Compounds 1-3 had 5/7/6 cyclic systems, while compound 4 had a 4/11 polycyclic system with a 4,7-cyclic ether between C-4 and C-7. The anti-inflammasome test using the isolated compounds (1-6, 8-13) showed that the diterpenes from E. helioscopia L. had a strong inhibitory effect on NLRP3 inflammasomes with IC50 values of 3.34-14.92 µM.

Targeting NLRP3 signaling by a novel-designed sulfonylurea compound for inhibition of microglial inflammation.

The nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome plays an important role in microglia-mediated inflammation. Dysregulation of NLRP3 signaling results in microglial activation and triggers inflammatory responses contributing to the development of neurological disorders including ischemic stroke, schizophrenia, Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Inhibition of the NLRP3-linked inflammatory pathways reduces microglia-induced inflammation and is considered as a promising therapeutic approach for neuro-inflammatory diseases. In the present study, we report the development of AMS-17, a rationally-designed tertiary sulfonylurea compound for inhibition of inflammation in microglia. AMS-17 inhibited expression of the NLRP3, and its downstream components and cytokines such as caspase-1, tumor necrosis factor-α (TNF-α), IL-1ß and inducible nitric oxide synthase (iNOS). It also suppressed lipopolysaccharide (LPS)-induced N9 microglial cell phagocytosis in vitro and activation of the microglia in mouse brain in vivo. Together, these results provide promising evidences for the inhibitory effects of AMS-17 in inflammation. This proof-of-concept study provides a new chemical scaffold, designed with the aid of pharmacophore modeling, with NLRP3 inhibitory activity which can be further developed for the treatment of inflammation-associated neurological disorders.

Protective Effects of Remimazolam on Cerebral Ischemia/Reperfusion Injury in Rats by Inhibiting of NLRP3 Inflammasome-Dependent Pyroptosis.

INTRODUCTION: Remimazolam is a novel benzodiazepine γ-aminobutyric acid A (GABAa) receptor agonist used for sedation and the induction as well as maintenance of general anesthesia. Previous research proved that anesthetic agents acting on GABAa receptor, such as thiopentone, propofol and midazolam, have protective actions for cerebral ischemia/reperfusion (I/R) injury. We here probed into remimazolam for its protective effect and potential mechanism of action against cerebral I/R injury. MATERIAL AND METHODS: A rat model of middle cerebral artery occlusion (MCAO) with focal transient cerebral I/R injury was established and was given tail vein injection of gradient remimazolam (5, 10, 20 mg/kg) after 2 h of ischemia. Following 24 h of reperfusion, neurological function, brain infarct volume, morphology of cerebral cortical neurons, and expressions of corticocerebral NLRP3, ASC, caspase-1, GSDMD, IL-1ß and IL-18 were evaluated. RESULTS: The results showed that remimazolam could effectively improve the neurological dysfunction, reduce the infarct volume and alleviate the damage of cortical neurons after I/R injury. Notably, the expression of NLRP3 inflammasome pathway was down-regulated, suggesting that remimazolam exerted protective actions on I/R injury by suppressing pyroptosis with decreased expression and release of inflammatory factors, and the involvement of the NLRP3 inflammasome pathway might be the core during that process. Overall, our results indicate that NLRP3 inflammation is a promising target. CONCLUSION: Based on this mechanism, remimazolam may be one of the ideal anesthetic drugs for patients with ischemic stroke.