A selective inhibitor of the NLRP3 inflammasome as a potential therapeutic approach for neuroprotection in a transgenic mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of the CAG repeat in the huntingtin (HTT) gene. When the number of CAG repeats exceeds 36, the translated expanded polyglutamine-containing HTT protein (mutant HTT [mHTT]) interferes with the normal functions of many cellular proteins and subsequently jeopardizes important cellular machineries in major types of brain cells, including neurons, astrocytes, and microglia. The NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome, which comprises NLRP3, ASC, and caspase-1, is involved in the activation of IL-1ß and IL-18 and has been implicated in various biological functions. Although the existence of the NLRP3 inflammasome in the brain has been documented, the roles of the NLRP3 inflammasome in HD remain largely uncharacterized. MCC950 is a highly selective and potent small-molecule inhibitor of NLRP3 that has been used for the treatment of several diseases such as Alzheimer's disease. However, whether MCC950 is also beneficial in HD remains unknown. Therefore, we hypothesized that MCC950 exerts beneficial effects in a transgenic mouse model of HD. METHODS: To evaluate the effects of MCC950 in HD, we used the R6/2 (B6CBA-Tg[HDexon1]62Gpb/1J) transgenic mouse model of HD, which expresses exon 1 of the human HTT gene carrying 120 ± 5 CAG repeats. Male transgenic R6/2 mice were treated daily with MCC950 (10 mg/kg of body weight; oral administration) or water for 5 weeks from the age of 7 weeks. We examined neuronal density, neuroinflammation, and mHTT aggregation in the striatum of R6/2 mice vs. their wild-type littermates. We also evaluated the motor function, body weight, and lifespan of R6/2 mice. RESULTS: Systematic administration of MCC950 to R6/2 mice suppressed the NLRP3 inflammasome, decreased IL-1ß and reactive oxygen species production, and reduced neuronal toxicity, as assessed based on increased neuronal density and upregulation of the NeuN and PSD-95 proteins. Most importantly, oral administration of MCC950 increased neuronal survival, reduced neuroinflammation, extended lifespan, and improved motor dysfunction in R6/2 mice. CONCLUSIONS: Collectively, our findings indicate that MCC950 exerts beneficial effects in a transgenic mouse model of HD and has therapeutic potential for treatment of this devastating neurodegenerative disease.

Reorienting of pramipexole as a promising therapy for acute pancreatitis in a rat model by suppressing TLR4\NF-κB p65\NLRP3 inflammasome signaling.

Acute pancreatitis (AP), a disorder of global importance, has a growing incidence and prevalence, particularly in the Western world. Its complications include pseudocysts and chronic pancreatitis. Pramipexole (PMX), a D2/3 receptor selecting agonist used in Parkinsonism, was reported to have anti-inflammatory effects. This study explored the potential curative role of PMX in an l-arginine-induced acute pancreatitis rat model in addition to a possible mechanistic pathway. Rats were divided randomly into three groups: control, l-arginine, and l-arginine + PMX. Seven days after AP induction, rats were decapitated and estimated for serum amylase, lipase, glucose, pancreatic inflammatory mediators toll-like receptor-4, nuclear factor κ B p65, serum tumor necrosis factor-α, NOD-, LRR and pyrin domain- containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin 1ß, oxidative biomarkers malondialdehyde, myeloperoxidase, nitrite/nitrate, reduced glutathione, and the apoptotic marker caspase-3, with pancreatic histopathological changes. l-arginine-mediated AP was proved by elevated serum lipase and amylase and pancreatic inflammatory, oxidative, and apoptotic markers with infiltration of inflammatory cells using hematoxylin and eosin stain. PMX improved all these adverse signs of AP greatly. PMX might be considered an innovative therapy for AP due to its remarkable antioxidant, antiapoptotic, and anti-inflammatory effects, which are attributed to the suppression of the NLRP3 inflammasome and its downstream inflammatory cytokines.

Crosstalk among apoptosis, inflammation, and autophagy in relation to melatonin protective effect against contrast-induced nephropathy in rats.

Contrast medium (CM) is a chemical substance that is used for imaging anatomical boundaries and to explore normal and abnormal physiological findings; the use of CM was associated with kidney injury and acute renal failure. Melatonin (M) possesses antioxidant, anti-inflammatory, and antiapoptotic effects in addition to autophagy modulation. This study aimed to investigate the protective effect of M against contrast-induced nephropathy (CIN) and its impact on the crosstalk between inflammasome, apoptosis, and autophagy in CIN. Male albino rats received M (10, 20, and 40 mg/kg/day, intraperitoneally) for 3 days. One hour after the last administration, rats were subjected to CIN induction (10 mg/kg indomethacin, double doses of l-NAME 10 mg/kg, i.v., and meglumine diatrizoate 60% 6 mL/kg, i.v.). CIN-induced kidney damage was evidenced through elevated kidney function biomarkers and induced renal histopathological changes. Pretreatment with M caused a significant decrease in nephrotoxicity biomarkers and histopathological alterations. Moreover, CIN-induced oxidative stress, NLRP3 inflammasome, and apoptosis were attenuated by M. Furthermore, M modulates autophagy in CIN rats. M inhibits CIN-induced NLRP3-inflammasome activation and apoptosis as well as enhances autophagy.

MiR-494-3p alleviates acute lung injury through regulating NLRP3 activation by targeting CMPK2.

Acute lung injury (ALI) is a severe respiratory disorder with a high rate of mortality, and is characterized by excessive cell apoptosis and inflammation. MicroRNAs (miRNAs) play pivotal roles in ALI. This study examined the biological function of miR-494-3p in cell apoptosis and inflammatory response in ALI. For this, mice were injected with lipopolysaccharide (LPS) to generate an in-vivo model of ALI (ALI mice), and WI-38 cells were stimulated with lipopolysaccharide (LPS) to generate an in-vitro model of ALI. We found that miR-494-3p was significantly downregulated in the ALI mice and in the in-vitro model. Overexpression of miR-494-3p inhibited inflammation and cell apoptosis in the LPS-induced WI-38 cells, and improved the symptoms of lung injury in the ALI mice. We then identified cytidine/uridine monophosphate kinase 2 (CMPK2) as a novel target of miR-494-3p in the WI-38 cells. Furthermore, miR-494-3p suppressed cell apoptosis and the inflammatory response in LPS-treated WI-38 cells through targeting CMPK2. The NLRP3 inflammasome is reportedly responsible for the activation of inflammatory processes. In our study, CMPK2 was confirmed to activate the NLRP3 inflammasome in LPS-treated WI-38 cells. In conclusion, miR-494-3p attenuates ALI through inhibiting cell apoptosis and the inflammatory response by targeting CMPK2, which suggests the value of miR-494-3p as a target for the treatment for ALI.

CLEC5A knockdown protects against cardiac dysfunction after myocardial infarction by suppressing macrophage polarization, NLRP3 inflammasome activation, and pyroptosis.

Increasing evidence has shown that the NOD-like receptor protein 3 (NLRP3) inflammasome and pyroptotic cell death play vital roles in the pathophysiology of myocardial infarction (MI), a common cardiovascular disease characterized by cardiac dysfunction. C-type lectin member 5A (CLEC5A) has been reported to be strongly associated with activation of the NLRP3 inflammasome and pyroptosis. In this study, an in vivo MI model was established by ligation of the left anterior descending coronary artery in male C57BL/6 mice, and CLEC5A knockdown was further achieved by intra-myocardial injection of adenovirus delivering shRNA-CLEC5A. CLEC5A was found to be highly expressed in the left ventricle of MI mice, while CLEC5A knockdown alleviated cardiac dysfunction in MI mice. In addition, MI-induced classical activation of macrophages was significantly inhibited after CLEC5A silencing. Additionally, CLEC5A knockdown dramatically inhibited MI-triggered activation of NLRP3 inflammasome, pyroptosis, and NF-κB signaling in the left ventricle of mice. In vitro experiments further validated that CLEC5A knockdown suppressed M1 polarization in LPS/IFNγ-stimulated RAW264.7 cells and inhibited the polarized RAW264.7-induced activation of NLRP3 inflammasome/pyroptosis signaling in co-cultured cardiomyocytes. In conclusion, CLEC5A knockdown protects against MI-induced cardiac dysfunction by regulating macrophage polarization, NLRP3 inflammasome, and cell pyroptosis.

Saikosaponin-d protects against liver fibrosis by regulating the estrogen receptor-ß/NLRP3 inflammasome pathway.

Liver fibrosis is the most common pathway in most types of chronic liver damage, characterized by an imbalance of ECM degradation and synthesis. Saikosaponin-d (SSd) possesses anti-inflammatory and anti-fibrotic effects. However, the underlying mechanism by which SSd represses hepatic stellate cell (HSC) activation remains unclear. Here, we found that SSd remarkably alleviated carbon tetrachloride (CCl4)-induced liver fibrosis, as evidenced by decreased collagen levels and profibrotic marker (COl1a1 and α-smooth muscle actin (SMA)) expression. SSd repressed CCl4-induced NOD-like receptor family pyrin-domain-containing-3 (NLRP3) activation in fibrotic livers, as suggested by decreased levels of NLRP3, IL-18, and IL-ß. The primary HSCs of CCl4 mice exhibited a significant increase in profibrotic marker expression and NLRP3 activation, but SSd treatment reversed this effect. SSd also repressed TGF-ß-induced profibrotic marker expression and NLRP3 activation in vitro. Mechanistically, TGF-ß decreased the expression of estrogen receptor-ß (ERß) in HSCs, whereas SSd treatment reversed this effect. ERß inhibition enhances NLRP3 activation in HSCs. More importantly, ERß or NLRP3 inhibition partially destroyed the function of SSd in liver fibrosis. In summary, the current data suggest that SSd prevents hepatic fibrosis by regulating the ERß/NLRP3 inflammasome pathway and suggests SSd as a potential agent for treating liver fibrosis.

Age-Dependent Chronic Lung Injury and Pulmonary Fibrosis following Single Exposure to Hydrochloric Acid.

Exposure to hydrochloric acid (HCl) represents a threat to public health. Children may inhale higher doses and develop greater injury because of their smaller airways and faster respiratory rate. We have developed a mouse model of pediatric exposure to HCl by intratracheally instilling p24 mice (mice 24 days old; 8-10 g) with 2 µL/g 0.1 N HCl, and compared the profile of lung injury to that in HCl-instilled adults (10 weeks old; 25-30 g) and their age-matched saline controls. After 30 days, alveolar inflammation was observed with increased proteinosis and mononuclear cells in the bronchoalveolar lavage fluid (BALF) in both HCl-instilled groups. Young p24 animals-but not adults-exhibited higher NLR family pyrin domain containing 3 (NLRP3) inflammasome levels. Increased amounts of Transforming Growth Factor-ß (TGF-ß) mRNA and its intracellular canonical and non-canonical pathways (p-Smad2 and p-ERK) were found in the lungs of both young and adult HCl-instilled mice. Constitutive age-related differences were observed in the levels of heat shock protein family (HSP70 and HSP90). HCl equally provoked the deposition of collagen and fibronectin; however, significant age-dependent differences were observed in the increase in elastin and tenascin C mRNA. HCl induced pulmonary fibrosis with an increased Ashcroft score, which was higher in adults, and a reduction in alveolar Mean Alveolar Linear Intercept (MALI). Young mice developed increased Newtonian resistance (Rn) and lower PV loops, while adults showed a higher respiratory system resistance and elastance. This data indicate that young p24 mice can suffer long-term complications from a single exposure to HCl, and can develop chronic lung injury characterized by a stronger persistent inflammation and lesser fibrotic pattern, mostly in the airways, differently from adults. Further data are required to characterize HCl time- and dose-dependent injury in young animals and to identify new key-molecular targets.

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.

Abnormal uterine inflammation in obstetric syndromes: molecular insights into the role of chemokine decoy receptor D6 and inflammasome NLRP3.

The adaptation of the uterine environment into a favorable immunological and inflammatory milieu is a physiological process needed in normal pregnancy. A uterine hyperinflammatory state, whether idiopathic or secondary to hormonal or organic uterine disorders (polycystic ovary syndromes, endometriosis/adenomyosis and fibroids), negatively influences the interactions between decidua and trophoblast, early in gestation, and between chorion and decidua later in pregnancy. Abnormal activation of uterine inflammatory pathways not only contributes to the pathogenesis of the obstetric syndromes, i.e. recurrent pregnancy loss (RPL), pre-term delivery (PTD) and pre-eclampsia (PE), but also to correlates with severity. In this review, we summarize recent advances in the knowledge of uterine molecular mechanisms of inflammatory modulation in normal pregnancy and obstetric syndromes (RPL, PTD and PE). In particular, we focus on two regulators of uterine/placental inflammation: the NLRP3 inflammasome and the chemokines decoy receptor D6. We performed comprehensive review of the literature in PubMed and Google Scholar databases from 1994 to 2018. The available evidence suggests that: (i) the expression of inflammasome NLRP3 is increased in the endometrium of women with unexplained RPL, in the chorioamniotic membranes of women with PTL and in the placenta of women with PE; (ii) there is a role for abnormal expression and function of D6 decoy receptor at the feto-maternal interface in cases of RPL and PTD and (iii) the function of placental D6 decoy receptor is impaired in PE. A wider comprehension of the inflammatory molecular mechanisms involved in the pathogenesis of the obstetric syndromes might lead to the identification of new potential therapeutic targets.

Application of Advanced Microscopic Methods to Study the Interaction of Carboxylated Fluorescent Nanodiamonds with Membrane Structures in THP-1 Cells: Activation of Inflammasome NLRP3 as the Result of Lysosome Destabilization.

Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.

Hepatic nuclear factor kappa B signaling pathway and NLR family pyrin domain containing 3 inflammasome is over-activated in ketotic dairy cows.

Ketosis is an important metabolic disease that can negatively affect the production efficiency of dairy cows. Earlier studies have revealed metabolic and inflammatory alterations in the blood associated with ketosis; however, a link between ketosis and hepatic inflammation has not been well documented. The objective of this study was to investigate whether the nuclear factor kappa B (NF-κB) signaling pathway and NLR family pyrin domain containing 3 (NLRP3) inflammasome were activated in the liver of ketotic cows. Liver and blood samples were collected from healthy (n = 15, control group) and ketotic (n = 15, ketosis group) cows that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Results showed that serum levels of fatty acids, ß-hydroxybutyrate (BHB), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were higher and glucose was lower in ketotic cows. Concentrations of serum proinflammatory cytokines IL18, tumor necrosis factor (TNF)-α, and IL1B were greater and the anti-inflammatory cytokine IL10 was lower in the ketosis group. Cows with ketosis had triacylglycerol accumulation in the liver. Upregulation of phosphorylated (p)-NF-κB and p-inhibitor of κB (IκB)α protein abundance in cows with ketosis indicated that the hepatic NF-κB signaling pathway was overactivated. The mRNA abundance of TNFA, inducible nitric oxide synthase (NOS2), IL18, and IL1B were greater and IL10 was lower in ketotic cows. More importantly, the mRNA and protein abundance of NLRP3 and caspase-1 (CASP1) along with CASP1 activity were greater in the liver of cows with ketosis. Overall, the data indicate that the onset of ketosis is accompanied by activation of the NF-κB signaling pathway and NLRP3 inflammasome, resulting in a state of inflammation.

Zika virus induces inflammasome activation in the glial cell line U87-MG.

In the last years, neurological complications related to Zika virus (ZIKV) infection have emerged as an important threat to public health worldwide. ZIKV infection has been associated to neurological disorders such as congenital microcephaly in newborns and Guillain-Barré syndrome, myelopathy and encephalitis in adults. ZIKV is characterized by neurotropism and neurovirulence. Several studies have identified microglial nodules, gliosis, neuronal and glial cells degeneration and necrosis in the brain of ZIKV infected infants, suggesting that ZIKV could play a role in these neurological disorders through neuroinflammation and microglial activation. Little information is available about neuroinflammation and ZIKV-related neurological disorders. Therefore, we investigated if ZIKV is able to infect a glial cell line (U87-MG) and how the glial cell line responds to this infection in terms of inflammation (IL-1ß, NLRP-3 and CASP-1), oxidative stress (SOD2 and HemeOX) and cell death. We observed a significant increase of ZIKV load in both cells and supernatants after 72 h, compared to 48 h of infection. We found that ZIKV infection induces an increase of IL-1ß, NLRP-3 and CASP-1 genes expression. Significant increase of IL-1ß and unchanged pro-IL-1ß protein levels have also been detected. Moreover, we observed SOD2 and HemeOX increased gene expression mainly after 72 h post ZIKV infection. Subsequently, we found a decrease of U87-MG cell viability, after both 48 h and 72 h of ZIKV infection. Our results show that U87-MG cells are susceptible to ZIKV infection. ZIKV is able to successfully replicate in infected cells causing oxidative stress, NLRP3 inflammasome activation and subsequent release of mature IL-1ß; this process culminates in cell death. Thus, considering the central role of neuroinflammation in neurological disorders, it is important to comprehend every aspect of this mechanism in order to better understand the pathogenesis of ZIKV infection and to identify possible strategies to fight the virus by rescuing cell death.

Genetic polymorphisms of IL-18 rs1946518 and IL-1ß rs16944 are associated with prognosis and survival of acute myeloid leukemia.

BACKGROUND: Though the pathogenesis of AML is still unknown, accumulating evidence revealed that immune response plays a vital part in it. NLRP3 inflammasome as a component of immune system has been found related to several cancers. The single nucleotide polymorphisms (SNPs) of NLRP3 inflammasome genes may be related to pathogenesis and prognosis of AML. METHODS AND RESULTS: We determined polymorphisms of NLRP3 (rs35829419), CARD8 (rs2043211), IL-1ß (rs16944), IL-18 (rs1946518) and NF-κB -94 ins/del ATTG in de novo AML patients to find out whether they play roles in the susceptibility and severity of AML. In our study, 383 AML cases and 300 randomly selected healthy individuals were examined for the polymorphisms and expression of NLRP3 genes. IL-1ß (rs16944) polymorphism in different risk AML subgroups was found statistically different, with more GA genotype in favorable-risk cytogenetics group. We also demonstrated that the bone marrow blasts of patients carrying IL-18 (rs1946518) GG or GT genotype were higher than patients of TT genotype. IL-18 plasma level of patients with IL-18 (rs1946518) GT or TT genotype was higher than GG genotype. Moreover, the GT genotype of IL-18 (rs1946518) led to statistically poorer AML-specific survival. CONCLUSION: IL-1ß (rs16944) and IL-18 (rs1946518) may be served as potential predictors for AML.

ALA-PDT promotes IL-1ß secretion from human SZ95 sebocytes via activation of the NLRP3 inflammasome.

BACKGROUND: 5-Aminolevulinic acid photodynamic therapy (ALA-PDT) is an effective treatment for pilosebaceous inflammatory diseases, such as acne vulgaris. In this study, we explored ALA-PDT's mechanisms against acne in vitro. METHODS: We treated human SZ95 sebocytes with ALA (0.2 mM) and subjected them to varied PDT doses (0, 5, 10, 20 J/cm²) over 12 h. We assessed cell viability post-treatment using the Annexin V FITC/PI apoptosis kit. ROS accumulation in the sebocytes was detected with a DCFDA probe. We quantified NLRP3 and caspase-1 mRNA via quantitative PCR and determined IL-1ß release following ALA-PDT by ELISA. Western blotting helped identify the levels of proteins associated with pyroptosis (NLRP3, caspase-1, and IL-1ß). To elucidate the mechanisms, we re-evaluated these parameters after administering various concentrations of NAC antioxidants (0, 0.4, 2, 10 mM) and the caspase inhibitor Z-VAD-FMK (0, 5, 10, 20 µM). RESULTS: Increasing PDT dose inversely affected SZ95 sebocyte survival, with a corresponding rise in ROS and pyroptosis-related proteins (NLRP3, caspase-1, and IL-1ß). Furthermore, NAC and Z-VAD-FMK modulated the expression and secretion of these molecules in a dose-responsive manner. CONCLUSION: Our findings suggest ALA-PDT's potential mechanism of action on sebaceous glands could involve ROS induction, leading to NLRP3 inflammasome assembly, thereby heightening caspase-1 activation and IL-1ß secretion. This cascade may amplify the local inflammatory response to break chronic inflammation in acne vulgaris treatment.

Sodium houttuyfonate plays a protective role in the asthmatic airway by alleviating the NLRP3-related pyroptosis and Th1/Th2 immune imbalance.

BACKGROUND: Houttuynia cordata is an herbal compound that grows in China and exhibits anti-inflammatory, antiviral, and antioxidant properties. Additionally, pyroptosis is mediated by the activated NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome after stimulation by various inflammatory factors in asthma. OBJECTIVE: To investigate the effect of sodium houttuyfonate on NLRP3 inflammasome-related pyroptosis and Th1/Th2 immune imbalance in asthma. METHODS: Asthmatic mice model were made, sodium houttuyfonate was injected intraperitoneally to treat the asthmatic mice. Airway reactivity, cell classification and counting in the bronchoalveolar lavage fluid were measured. Hematoxylin-eosin and periodic acid-Schiff staining were used to analyze airway inflammation and mucus hypersecretion. Beas-2b cells were cultured, LPS, NLRP3 antagonist (Mcc950) and sodium houttuyfonate were used to intervene the Beas-2b cells, NLRP3, ASC, caspase-1, GSDMD, IL-1ß, and IL-18 expression in the lung tissue and cells were analyzed using immunohistochemistry and western blot, while qRT- PCR was performed to analyze the mRNA contents in the pulmonary and the cells, respectively. Th1 and Th2 cytokines (IL-4 and IFN-γ) were detected with ELISA and the proportions of Th1 and Th2 in splenocyte were detected by flow cytometry. RESULTS: Airway reactivity decreased in sodium houttuyfonate group when compared with asthmatic group mice. In the BALF, the numbers of leukocytes, eosinophils, neutrophils, lymphocytes, and macrophages were significantly lower in sodium houttuyfonate group mice than in asthmatic group mice. The proportion of TH1/TH2 cells in spleen cells and IFN-γ /IL-4 in plasma increased in sodium houttuyfonate treatment group when compared with asthma group. Immunohistochemistry, western blot and RT-PCR showed that the expressions of NLRP3, ASC, caspase-1, GSDMD, IL-1ß and IL-18 were decreased in the lung tissue of mice after treated with sodium houttuyfonate when compared with those in the asthma group. However, sodium houttuyfonate combined with dexamethasone induced a stronger effect on NLRP3-related pyroptosis and Th1/Th2 immune imbalance compared to sodium houttuyfonate or dexamethasone alone. Beas-2b cells were cultured in vitro, sodium houttuyfonate can alleviate LPS-induced ASC, casepase-1, GSDMD, IL-18 and IL-1ß increasing, especially in SH (10 µg/ml) treated group, but the effect less than Mcc950. CONCLUSIONS: Sodium houttuyfonate can alleviated NLRP3-related pyroptosis and Th1/Th2 immune imbalance to reduce asthma airway inflammation and airway reactivity.

NLRP3 inflammasome activation in human peripheral blood mononuclear cells induced by venoms secreted PLA2s.

Bothropic venoms contains high amount of secreted phospholipases A2 (sPLA2s) that play a significant role in leukocyte activation and inflammation. Monocytes and lymphocytes are highly functional immune system cells that mediate and provide efficient responses during the inflammation. NLRP3 inflammasome is a multiprotein complex found in immune system cells that is triggered by pathogen- and damage-associated molecular patterns, PAMPs and DAMPs, respectively. PLA2s' effect on human peripheral blood mononuclear cells (PBMCs) is still incompletely understood. PBMCs were isolated by density gradient and incubated with RPMI (control), LPS, BthTX-I (PLA2-Lys49) or BthTX-II (PLA2-Asp49) isolated from Bothrops jararacussu venom, to evaluate viability, and the results showed that there was no cell death. RT-qPCR and immunoblot were used to assess the gene and protein expression of NLRP3 components. Results indicated that there was substantial amplification of ASC, Caspase-1, IL-6, and IL-1ß in 1 h and NLRP3 in 2 h. Protein expression was measured, and the results revealed substantial expression of the NLRP3 inflammasome complex after 4 h. IL-1ß and LDH was quantified in the supernatant of the cells. Taken together, the findings demonstrate that BthTX-I and BthTX-II activate the NLRP3 inflammasome complex in human PBMCs and contribute to the inflammatory response seen in envenoming.

Downstream Signaling of Inflammasome Pathway Affects Patients' Outcome in the Context of Distinct Molecular Breast Cancer Subtypes.

Inflammasomes are protein complexes involved in the regulation of different biological conditions. Over the past few years, the role of NLRP3 in different tumor types has gained interest. In breast cancer (BC), NLRP3 has been associated with multiple processes including epithelia mesenchymal transition, invasion and metastization. Little is known about molecular modifications of NLRP3 up-regulation. In this study, in a cohort of BCs, the expression levels of NLRP3 and PYCARD were analyzed in combination with CyclinD1 and MYC ones and their gene alterations. We described a correlation between the NLRP3/PYCARD axis and CyclinD1 (p < 0.0001). NLRP3, PYCARD and CyclinD1's positive expression was observed in estrogen receptor (ER) and progesterone receptor (PgR) positive cases (p < 0.0001). Furthermore, a reduction of NLRP3 and PYCARD expression has been observed in triple negative breast cancers (TNBCs) with respect to the Luminal phenotypes (p = 0.017 and p = 0.0015, respectively). The association NLRP3+/CCND1+ or PYCARD+/CCND1+ was related to more aggressive clinicopathological characteristics and a worse clinical outcome, both for progression free survival (PFS) and overall survival (OS) with respect to NLRP3+/CCND1− or PYCARD+/CCND1− patients, both in the whole cohort and also in the subset of Luminal tumors. In conclusion, our study shows that the NLRP3 inflammasome complex is down-regulated in TNBC compared to the Luminal subgroup. Moreover, the expression levels of NLRP3 and PYCARD together with the alterations of CCND1 results in Luminal subtype BC'ss poor prognosis.

Caloric restriction in mice improves short-term recognition memory and modifies the neuroinflammatory response in the hippocampus of male adult offspring.

Restrictive diets (RD) can influence the inflammatory phenotype of dams and their offspring. Thus, this study aimed to evaluate the effects of caloric restriction on the neuroinflammatory profile in the hippocampus and the short-term recognition memory of male offspring from RD-fed dams. Mice dams received standard diet ad libitum (CONT) or restrictive diet (RD; 30% reduction of CONT consumption) during pregnancy and lactation. Male pups were weaned at 21 days and randomly divided into two groups that received CONT or RD; groups were named according to maternal/offspring diets: CONT/CONT, CONT/RD, RD/CONT, and RD/RD. At 90 days old, short-term memory was assessed by the object recognition test (ORT); the inflammatory state of the hippocampus was analyzed by gene expression of sirtuin-1 (Sirt1) and inflammasome Nlrp3; and by protein expression of toll-like receptor-4 (TLR-4) and zonula occludens-1 (ZO-1). Our results showed an improvement in short-term memory in RD-fed offspring. The expression of Sirt1 was higher in RD/CONT compared to CONT/CONT and decreased in RD/RD compared to CONT/RD. Nlrp3 gene expression showed an offspring effect, being decreased in RD-fed mice. TLR-4 expression was higher in RD/CONT compared to CONT/CONT, similarly to ZO-1 expression. However, ZO-1 also showed a maternal diet effect and increased expression in the offspring of RD dams. Our findings demonstrate that caloric restriction improved short-term recognition memory. However, a restrictive diet should be applied with caution; depending on the offspring's diet, it may not benefit the neuroinflammatory phenotype or cognition.

Physalin B ameliorates inflammatory responses in lipopolysaccharide-induced acute lung injury mice by inhibiting NF-κB and NLRP3 via the activation of the PI3K/Akt pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalin B (PB) is an active constituent of Physalis alkekengi L. var. Franchetii, which is a traditional medicine for clearing heat and detoxification, resolving phlegm, and diuresis. It has been commonly applied to treat sore throat, phlegm-heat, cough, dysuria, pemphigus, and eczema. AIM OF STUDY: Physalin B has shown efficacy as an anti-acute lung injury (ALI) agent previously; however, its mechanisms of action remain unclear. In the present study, we established a lipopolysaccharide-induced septic ALI model using BALB/c mice to further confirm the therapeutic potential of PB and to assess the underlying molecular mechanisms. MATERIALS AND METHODS: We used 75% ethanol and macroporous resin for extraction, separation, and enrichment of PB. The LPS-induced ALI mouse model was used to determine anti-inflammatory effects of PB. The severity of acute lung injury was evaluated by hematoxylin and eosin staining, wet/dry lung ratio, and myeloperoxidase (MPO) activity in lung tissue. An automatic analyzer was used to measure the arterial blood gas index. Protein levels of pro-inflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung tissue was measured using an ELISA. Quantitative RT-PCR was used to measure changes in RNA levels of pro-inflammatory cytokines in the lungs. A fluorometric assay kit was used for determination of apoptosis-related factors to assess anti-apoptotic effects of PB. Western blotting was used to assess levels of key pathway proteins and apoptosis-related proteins. Connections between the pathways were tested through inhibitor experiments. RESULTS: Pretreatment with PB (15 mg kg-1 d-1, i.g.) significantly reduced lung wet/dry weight ratios and MPO activity in blood and BALF of ALI mice, and it alleviated LPS-induced inflammatory cell infiltration in lung tissue. The levels of pro-inflammatory factors TNF-α, IL-6, and IL-1ß and their mRNA levels in blood, BALF, and lung tissue were reduced following PB pretreatment. PB pretreatment also downregulated the apoptotic factors caspase-3, caspase-9, and apoptotic protein Bax, and it upregulated apoptotic protein Bcl-2. The NF-κB and NLRP3 pathways were inhibited through activation of the PI3K/Akt pathway due to PB pretreatment, whereas administration of PI3K inhibitors increased activation of these pathways. CONCLUSIONS: Taken together, our results suggest that the anti-ALI properties of PB are closely associated with the inactivation of NF-κB and NLRP3 by altering the PI3K/Akt pathway. Furthermore, our findings provide a novel strategy for application of PB as a potential agent for treating patients with ALI. To the best of our knowledge, this is the first study to elucidate the underlying mechanism of action of PB against ALI.

Sodium Danshensu protects against oxygen glucose deprivation/reoxygenation-induced astrocytes injury through regulating NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome and tuberous sclerosis complex-2 (TSC2)/mammalian target of rapamycin (mTOR) pathways.

Background: Cerebral ischemic stroke is a serious condition with high incidence, mortality, and associated disability. Currently, effective therapeutic options are available for ischemic stroke are limited. Accumulating evidence indicates that sodium Danshensu, mono sodium compound derived from Salvia miltiorrhiza, plays protective roles in ischemic stroke. However, the underlying protective mechanism of sodium Danshensu in cerebral ischemic stroke remains unknown. Methods: In the current study, we explored the role and mechanism of sodium Danshensu on astrocytes exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), which mimics the process of ischemia-reperfusion. The impact of sodium Danshensu on cell viability and apoptosis after OGD/R were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-dophenyl tetrazolium bromide (MTT) assay and flow cytometry. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were used to detect the expression of target messenger RNA (mRNA) and proteins associated with apoptosis and autophagy. The release of lactate dehydrogenase (LDH) was determined, and the production of proinflammatory cytokines were detected using enzyme-linked immunosorbent assay (ELISA) kits. Results: It was found that sodium Danshensu could significantly increase cell viability and decrease LDH release and apoptosis. Besides, it inhibited the production of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. Sodium Danshensu also dose-dependently decreased protein and mRNA levels of nucleotide binding oligomerization NOD-like receptor pyrin domain containing 3 (NLRP3) and high mobility group box 1 (HMGB1), which play a crucial role in promoting ischemic stroke-induced cell injury. Moreover, sodium Danshensu dose-dependently upregulated Beclin 1 expression, downregulated P62 protein expression, and further increased LC3B-II/LC3B-I ratio through inducing autophagy in astrocytes. Additionally, we noticed that sodium Danshensu dose-dependently increased tuberous sclerosis complex-2 (TSC2) protein expression, while significantly reduced the levels of mammalian target of rapamycin (mTOR) in the presence of OGD/R insult. Conclusions: These findings suggest that sodium Danshensu protects against OGD/R-induced injury by modulating the NLRP3 inflammasome and TSC2/mTOR pathways.