NOD-like Receptor Pyrin Domain Containing 3 Research Articles

Antagonistic effect of selenium on mercuric chloride in the central immune organs of chickens: The role of microRNA-183/135b-FOXO1/TXNIP/NLRP3 inflammasome axis.

Mercury (Hg) is a persistent environmental and industrial pollutant that accumulated in the body and induces oxidative stress and inflammation damage. Selenium (Se) has been reported to antagonize immune organs damage caused by heavy metals. Here, we aimed to investigate the prevent effect of Se on mercuric chloride (HgCl2 )-induced thymus and bursa of Fabricius (BF) damage in chickens. The results showed that HgCl2 caused immunosuppression by reducing the relative weight, cortical area of the thymus and BF, and the number of peripheral blood lymphocytes. Meanwhile, HgCl2 induced oxidative stress and imbalance in cytokines expression in the thymus and BF. Further, we found that thioredoxin-interacting protein (TXNIP) and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome mediated HgCl2 -induced oxidative stress and inflammation. Mechanically, the targeting and inhibitory effect of microRNA (miR)-135b/183 on forkhead box O1 (FOXO1) were an upstream event for HgCl2 -activated TXNIP/NLRP3 inflammasome pathway. Most importantly, Se effectively attenuated the aforementioned damage in the thymus and BF caused by HgCl2 and inhibited the TXNIP/NLRP3 inflammasome pathway by reversing the expression of FOXO1 through inhibiting miR-135b/183. In conclusion, the miR-135b/183-FOXO1/TXNIP/NLRP3 inflammasome axis might be a novel mechanism for Se to antagonize HgCl2 -induced oxidative stress and inflammation in the central immune organs of chickens.

NLRP3-Inflammasome Gene Variations in the Risk of Type 2 Diabetes.

Inflammation is the natural immunological response of an organism against any harmful, foreign or destructive effect to heal and repair damaged tissue. The nod-like receptor pyrin domain-containing-3 (NLRP3) inflammasome is one of the main components of the inflammatory mechanism and is associated with many inflammatory diseases, but it is also closely related to metabolic abnormalities, such as type 2 diabetes mellitus (T2DM), insulin resistance and obesity. NLRP3 activates inflammation and causes interleukin-1β release, exogenous and endogenous danger signals, as well as insulin resistance. In this direction, we focus on the gene structure of NLRP3 in diabetes and accordingly, we aim to determine the relationship between eight gene variations in the NLRP3 gene and T2DM. We investigated the rs10802501, rs10733113, rs10754558, rs10925026, rs10925027, rs35829419, rs4612666 and rs4925659 single-nucleotide polymorphisms of NLRP3 gene using the Sequenom MassARRAY system in 100 T2DM patients and 100 control individuals. There were no significant differences between T2DM risk and the genotype frequencies of rs10802501, rs10733113, rs35829419 and rs10925026 variants (p > 0.05). However, significant genotype frequencies were determined for rs10925027 (p = 0.0013) and rs4925659 (p < 0.001). For the risk allele G of the rs10754558 variant, significant differences were found in the heterozygous and dominant model (p = 0.036, p = 0.033). The genotype distribution of the rs4612666 variant was significant only in the heterozygous model (p = 0.047). In this hospital-based case-control study, rs10925027, rs4925659 and rs10754558 variants were found to be closely related to T2DM risk. The rs10925027 CC genotype, rs4925659 GG genotype, rs10754558 GG and GC+GG genotypes of the NLRP3 were determined as important risk factors for the T2DM.

Potential role of BAY11-7082, a NF-κB blocker inhibiting experimental autoimmune encephalomyelitis in C57BL/6J mice via declining NLRP3 inflammasomes.

Multiple sclerosis (MS) is an inflammatory autoimmune demyelinating disease of the central nervous system. NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is implicated in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which NLRP3 inflammasome is involved in the development of MS and EAE is not clear. NF-kappaB (NF-κB) is associated with the activity of NLRP3 inflammasomes, but the role of NF-κB is controversial. We sought to demonstrate that both NF-κB and NLRP3 contribute to development of MS and EAE, and NF-κB pathway is positively correlated with NLRP3 activation in EAE. The inhibitor of NF-κB and NLRP3, BAY11-7082, can prevent and treat EAE. BAY11-7082 (5 and 20mg/kg/i.p.) was intraperitoneally administered to EAE mice at the time of second injection of pertussis toxin (BAY11-7082 prevention group) or at the onset of symptoms (BAY11-7082 treatment group). mRNA expressions of NLRP3 were determined by qPCR. Protein expressions of NLRP3, NF-κB p65, and phosphorylated p65 were determined by western blotting. Serum levels of inflammatory cytokines were measured by cytometric bead array. Mice treated with BAY11-7082 (both prevention and treatment groups) showed lower clinical scores and attenuated pathological changes. NLRP3 inflammasome and activity of NF-κB in spinal cord of EAE mice was higher than that in control group. However, the level of NLRP3 inflammasome decreased in BAY11-7082 prevention and treatment groups. BAY11-7082 is a promising therapeutic agent for MS. NLRP3 activation in EAE maybe related with NF-κB pathway.

SIRT1 Promotes M2 Microglia Polarization via Reducing ROS-Mediated NLRP3 Inflammasome Signaling After Subarachnoid Hemorrhage.

Mounting evidence has suggested that modulating microglia polarization from pro-inflammatory M1 phenotype to anti-inflammatory M2 state might be a potential therapeutic approach in the treatment of subarachnoid hemorrhage (SAH) injury. Our previous study has indicated that sirtuin 1 (SIRT1) could ameliorate early brain injury (EBI) in SAH by reducing oxidative damage and neuroinflammation. However, the effects of SIRT1 on microglial polarization and the underlying molecular mechanisms after SAH have not been fully illustrated. In the present study, we first observed that EX527, a potent selective SIRT1 inhibitor, enhanced microglial M1 polarization and nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation in microglia after SAH. Administration of SRT1720, an agonist of SIRT1, significantly enhanced SIRT1 expression, improved functional recovery, and ameliorated brain edema and neuronal death after SAH. Moreover, SRT1720 modulated the microglia polarization shift from the M1 phenotype and skewed toward the M2 phenotype. Additionally, SRT1720 significantly decreased acetylation of forkhead box protein O1, inhibited the overproduction of reactive oxygen species (ROS) and suppressed NLRP3 inflammasome signaling. In contrast, EX527 abated the upregulation of SIRT1 and reversed the inhibitory effects of SRT1720 on ROS-NLRP3 inflammasome activation and EBI. Similarly, in vitro, SRT1720 suppressed inflammatory response, oxidative damage, and neuronal degeneration, and improved cell viability in neurons and microglia co-culture system. These effects were associated with the suppression of ROS-NLRP3 inflammasome and stimulation of SIRT1 signaling, which could be abated by EX527. Altogether, these findings indicate that SRT1720, an SIRT1 agonist, can ameliorate EBI after SAH by shifting the microglial phenotype toward M2 via modulation of ROS-mediated NLRP3 inflammasome signaling.

Caspase-6 promotes activation of the caspase-11-NLRP3 inflammasome during gram-negative bacterial infections

The innate immune system acts as the first line of defense against infection. One key component of the innate immune response to gram-negative bacterial infections is inflammasome activation. The caspase-11 (CASP11)-nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is activated by cytosolic lipopolysaccharide, a gram-negative bacterial cell wall component, to trigger pyroptosis and host defense during infection. Although several cellular signaling pathways have been shown to regulate CASP11-NLRP3 inflammasome activation in response to lipopolysaccharide, the upstream molecules regulating CASP11 activation during infection with live pathogens remain unclear. Here, we report that the understudied caspase-6 (CASP6) contributes to the activation of the CASP11-NLRP3 inflammasome in response to infections with gram-negative bacteria. Using in vitro cellular systems with bone marrow-derived macrophages and 293T cells, we found that CASP6 can directly process CASP11 by cleaving at Asp59 and Asp285, the CASP11 auto-cleavage sites, which could contribute to the activation of CASP11 during gram-negative bacterial infection. Thus, the loss of CASP6 led to impaired CASP11-NLRP3 inflammasome activation in response to gram-negative bacteria. These results demonstrate that CASP6 potentiates activation of the CASP11-NLRP3 inflammasome to produce inflammatory cytokines during gram-negative bacterial infections.

PFOS facilitates liver inflammation and steatosis: An involvement of NLRP3 inflammasome-mediated hepatocyte pyroptosis.

Perfluorooctane sulfonate (PFOS) is a fluorinated organic pollutant with substantial accumulation in mammalian liver tissues. However, the impact of chronic PFOS exposure on liver disease progression and the underlying molecular mechanisms remain elusive. Herein, we for the first time revealed that micromolar range of PFOS exposure initiates the activation of NLR pyrin domain containing 3 (NLRP3) inflammasome to drive hepatocyte pyroptosis. We showed that 5mg/kg/day PFOS exposure may exacerbated liver inflammation and steatosis in high-fat diet (HFD)-fed mice with concurrently elevated expression of NLRP3 and caspase-1. PFOS exposure resulted in viability impairment and LDH release in BRL-3A rat liver cells. 25-100 μM concentrations of PFOS exposure activated the NLRP3 inflammasome, leading to consequent GSDMD cleavage, IL-1β release and the initiation of pyroptosis in a dose-dependent manner, whereas treatment with 10μM NLRP3 inhibitor MCC950 abrogated this effect. Moreover, pretreatment of 5mM ROS scavenger N-acetyl-L-cysteine (NAC) ameliorated PFOS-induced NLRP3 inflammasome activation and pyroptosis. Collectively, our data highlight a pivotal role of pyroptotic death in PFOS-mediated liver inflammation and metabolic disorder.

The Protective Activity of Penehyclidine Hydrochloride against Renal Ischemia/Reperfusion-Mediated NLRP3 Inflammasome Activation is Induced by SIRT1

Background: The activation of alveolar macrophages (AMs) modulated via leucine-rich repeat (NLR) pyrin domain containing 3 (NLRP3) inflammasome activation is key to the progression of renal ischemia/reperfusion (rI/R)-mediated acute lung injury (ALI). Sirtuin-1 (SIRT1) can attenuate NLRP3 inflammasome activation during I/R stress and may be an important mechanism underlying ALI pathogenesis. Penehyclidine hydrochloride (PHC), an anticholinergic drug, exerts protective effects against rI/R-mediated ALI. This study aimed to decipher the effects of PHC on SIRT1 activation and the underlying mechanism of the protective activity of PHC against rI/R-mediated ALI.Materials and methods: We used an ALI rat model and the rat AMs cell line NR8383 to assess the degree of lung injury in vivo and in vitro.Results: The results show that PHC attenuates rI/R-mediated lung injury indices, myeloperoxidase, and apoptosis in vivo. It decreases the rI/R-mediated release of prostaglandin E2 and nitric oxide, mitochondrial reactive oxygen species production, and the activity of NADPH oxidase-4 in vitro. PHC ameliorates the rI/R-induced activation of the thioredoxin-interacting protein, caspase 1 (P10 unit), and NLRP3 inflammasome, along with reduced activation of interleukin-1β and interleukin-18 in vitro. We show that PHC alleviates the rI/R-induced reduction of SIRT1 and the depletion of SIRT1 eliminates the ameliorating activity of PHC on the NLRP3 inflammasome activation in vitro. Conclusions: In summary, the findings suggest that PHC ameliorates the rI/R-mediated ALI through the SIRT1-mediated NLRP3 inflammasome activation.

RGFP966 is protective against lipopolysaccharide-induced depressive-like behaviors in mice by inhibiting neuroinflammation and microglial activation

Depression is a prevalent mental disorder. However, its pathophysiological mechanism has still remained elusive, and a limited number of effective treatments have been presented. Recent studies have shown that neuroinflammation and microglial activation are involved in the pathogenesis of depression. Histone deacetylase 3 (HDAC3) has neurotoxic effects on several neuropathological conditions. The inhibition of HDAC3 has been reported to induce anti-inflammatory and antioxidant effects. RGFP966 is a highly selective inhibitor of HDAC3. This study aimed to investigate the antidepressant effect of RGFP966 on lipopolysaccharide (LPS)-induced depressive-like behaviors in mice and to explore its possible mechanism. Adult male C57BL/6J mice were utilized in this study. The LPS and RGFP966 were injected intraperitoneally daily for 5 days. The behavior tests were performed to elucidate the depression-like behaviors. Western blot, ELISA and immunofluorescence staining were used to study the HDAC3/TLR4/NLRP3 pathway-related proteins. The results of behavioral tests showed that RGFP966 could improve the LPS-induced depressive-like behaviors in mice. The results of Western blotting showed that RGFP966 treatment downregulated the expression levels of toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) (P < 0.05). Furthermore, the results of immunofluorescence staining showed that RGFP966 treatment inhibited microglial activation in the hippocampus of mice (P < 0.01). These findings suggested that RGFP966 could effectively ameliorate LPS-induced depressive-like behaviors in mice by inhibiting neuroinflammation and microglial activation. The anti-inflammatory mechanism of RGFP966 might be related to the inhibition of the HDAC3/TLR4/NLRP3 signaling pathway. Therefore, inhibition of HDAC3 using RGFP966 could serve as a potential treatment strategy for depression.

Microglial NLRP3 Inflammasome Activation upon TLR2 and TLR5 Ligation by Distinct α-Synuclein Assemblies.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder and is characterized by the formation of cellular inclusions inside neurons that are rich in an abnormal form of the protein α-synuclein (α-syn). Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of signaling transduction pathways. Here, we studied activation of primary microglia isolated from wild-type mouse by distinct α-syn forms and their clearance. Internalization of α-syn monomers and oligomers efficiently activated the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome via TLR2 and TLR5 ligation, thereby acting on different signaling checkpoints. We found that primary microglia effectively engulf α-syn but hesitate in its degradation. NLRP3 inhibition by the selective inhibitor CRID3 sodium salt and NLRP3 deficiency improved the overall clearance of α-syn oligomers. Together, these data show that distinct α-syn forms exert different microglial NLRP3 inflammasome activation properties, thereby compromising its degradation, which can be prevented by NLRP3 inhibition.

The miR-223-3p Regulates Pyroptosis Through NLRP3-Caspase 1-GSDMD Signal Axis in Periodontitis.

Salivary exosomes contain various components and may play important roles in oral diseases. The purpose of this study was to verify the possible function of miR-223-3p from salivary exosomes in periodontitis. We isolated the salivary exosomes and found that the miR-223-3p content of salivary exosomes from periodontitis was less than the healthy control. Furthermore, we performed dual-luciferase reporter assay and real-time PCR to verify that (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) was the target of miR-223-3p. When we knocked down the miR-223-3p expression in THP-1-derived macrophages, the expression of NLRP3 and the downstream inflammatory mediators interleukin-1β (IL-1β) and IL-6 were upregulated. By using integrated bioinformatics analysis, we found that pyroptosis and cytokine secretion participated in inflammatory gingival tissues. In addition, NLRP3, and the pyroptosis executioner, gasdermin D (GSDMD) was highly active in inflammatory gingival tissues compared with healthy controls by western blotting and immunohistochemistry. In summary, we speculated that miR-223-3p in salivary exosomes might regulate GSDMD-mediated pyroptosis by targeting NLRP3 in periodontitis. Detection of miR-223-3p expression in salivary exosomes could be used as an important non-invasive method to diagnose and evaluate the severity of periodontitis.

Role of tumor necrosis factor receptor‑associated factor 6 in pyroptosis during acute pancreatitis.

Acute pancreatitis (AP) is hypothesized to be related to the activation of an inflammatory response induced by pyroptosis. The aim of the present study was to investigate the potential role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in pyroptosis in an AP rat model and the human pancreatic ductal epithelial HPDE6C7 cell line. In vivo, AP was induced by intraperitoneal injection of caerulein (CAE) in rats. The rats were sacrificed at 24 or 48 h after the final CAE injection. In vitro, HPDE6C7 cells were treated with CAE for 12, 24 and 48 h. Moreover, TRAF6 was overexpressed and treated with CAE for 48 h. Histopathological changes of pancreatic, serum and supernatant inflammatory cytokines and pyroptosis-related mRNA and protein expression levels were determined by histopathological scores, ELISA, reverse transcription-quantitative PCR and western blotting. In addition, pyroptosis morphological changes were also determined by Hoechst/PI staining in HPDE6C7 cells. Results showed that AP was observed in the CAE-induced rat model, and that serum IL-1β and IL-18 levels, and TRAF6, NLR pyrin domain containing 3 (NLRP3), caspase-1 and caspase-3 mRNA and protein expression levels were increased. Similar in HPDE6C7 cells, CAE treatment caused supernatant IL-1β level, NLRP3 and caspase-1 mRNA expression levels to significantly increase. After TRAF6 overexpression and CAE treatment, supernatant IL-1β level, caspase-1 protein expression level, and NLRP3 and caspase-3 mRNA and protein expression levels were also significantly increased. Furthermore, cells exhibited red fluorescence in Hoechst/PI staining, which can be used as a method of detecting pyroptosis activation. The results also showed that the red fluorescence was stronger after CAE treatment or TRAF6 overexpression plus CAE treatment. In conclusion, TRAF6 and caspase-1/3 signaling pathways were involved in the pathogenesis of CAE-induced AP in rats. Pyroptosis was activated by CAE and TRAF6 overexpression via the caspase-1/3 signaling pathways in HPDE6C7 cells.

The therapeutic effect of Apocynin against hyperoxy and Inflammation-Induced lung injury

Lung damage due to hyperoxia and inflammation are important causes of bronchopulmonary dysplasia (BPD). We aimed to investigate the beneficial effects of Apocynin (Apo) on rat pups exposed to hyperoxia and inflammation. Forty-eight rat pups were randomly divided into 3 groups as hyperoxia (95% O2) + lipopolysaccharide (LPS), hyperoxia + LPS + Apo treated and control (21% O2). Rat pups in the Apo group received Apo at a daily dose of 40 mg/kg. Histopathological (Hematoxylin-Eosin, Masson trichrome), immunochemical (surfactant B and C protein staining) evaluations and biochemical studies incluiding, total antioxidant status (TAS), total oxidant status (TOS), OSI (oxidant stress index), AOPP (advanced protein degradation product), Lipid hydroperoxide (LPO), 8-OHdG, NADPH oxidase activity (NOX), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF- α), interleukin-1 beta (IL-1β), IL-18, IL-6, caspase-1 and 3, nuclear factor erythroid 2-related factor 2 (NFR2), Nod-like receptor pyrin domain-containing 3 (NLRP3) activities were studied. After Apo treatment, AOPP, LPO, 8-OHdG, NOX, TOS, OSI levels decreased; SOD, CAT, GSH and TAS levels increased (P < 0.05). Apo reduced inflammatory cell infiltration and proinflammatory cytokines with reduction in NLRP3 inflammasome in addition to increased Nrf2 levels. Moreover, caspase-1 and 3 levels decreased with Apo (P < 0.05). Apo was found to provide preventive and therapeutic effects by reducing oxidant stress, blocking inflammation and increasing antioxidant status. Beyond anti-oxidative effects, Apo also have anti-inflammatory effects by suppressing NLRP3 inflammasome activation and inducing Nrf2 as well. Therefore, Apo might be a potential option in the treatment of BPD.

Inhibition of microRNA-297 alleviates THLE-2 cell injury induced by hypoxia/reoxygenation by inhibiting NLRP3 inflammasome activation via sirtuin 3.

It has been acknowledged that microRNAs (miRNAs/miRs) assume a critical role in hypoxia/reoxygenation (H/R) - induced hepatocyte injury. Therefore, cell experiments were performed in this study to investigate the mechanism of miR-297 in H/R-induced hepatocyte injury with the involvement of sirtuin 3 (SIRT3) and NOD-like receptor pyrin domain containing 3 (NLRP3). Initially, transformed human liver epithelial-2 (THLE-2) cells were utilized for H/R challenge. After miR-297 antagomir and NLRP3 adenovirus vector delivery, THLE-2 cell proliferation and apoptosis were measured by MTT, EdU, and TUNEL assays, respectively. Enzyme-linked immunosorbent assay was conducted to evaluate the levels of apoptosis-related indicators (Bax and Bcl-2) and inflammation-related indicators (interleukin 6 (IL-6) and IL-10), Western blot analysis to detect NLRP3, and cleaved caspase-1 expression. The binding relation between miR-297 and SIRT3 was examined using dual-luciferase assay. The results showed that miR-297 antagomir repressed the apoptosis and inflammation induced by H/R treatment in THLE-2 cells. Mechanistically, miR-297 antagomir diminished the extent of IκBα and nuclear factor-kappa B (NF-κB) phosphorylation and NLRP3 activation in H/R-induced THLE-2 cells by targeting SIRT3. Furthermore, NLRP3 overexpression normalized the promoting effects of miR-297 antagomir on proliferation and its inhibitory effects on apoptosis and inflammation in H/R-induced THLE-2 cells. In summary, our results elucidated that miR-297 antagomir repressed H/R-induced THLE-2 cell injury via SIRT3 promotion and NLRP3 inactivation.

Sinapic Acid Controls Inflammation by Suppressing NLRP3 Inflammasome Activation.

A natural phenolic acid compound, sinapic acid (SA), is a cinnamic acid derivative that contains 3,5-dimethoxyl and 4-hydroxyl substitutions in the phenyl ring of cinnamic acid. SA is present in various orally edible natural herbs and cereals and is reported to have antioxidant, antitumor, anti-inflammatory, antibacterial, and neuroprotective activities. Although the anti-inflammatory function of SA has been reported, the effect of SA on the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome has not been explored. In the present study, to elucidate the anti-inflammatory mechanism of SA, we examined whether SA modulates the NLRP3 inflammasome. We found that SA blocked caspase-1 activation and IL-1β secretion by inhibiting NLRP3 inflammasome activation in bone marrow-derived macrophages (BMDMs). Apoptosis-associated speck-like protein containing CARD (ASC) pyroptosome formation was consistently blocked by SA treatment. SA specifically inhibited NLRP3 activation but not the NLRC4 or AIM2 inflammasomes. In addition, SA had no significant effect on the priming phase of the NLRP3 inflammasome, such as pro-IL-1β and NLRP3 inflammasome expression levels. Moreover, we found that SA attenuated IL-1β secretion in LPS-induced systemic inflammation in mice and reduced lethality from endotoxic shock. Our findings suggest that the natural compound SA has potential therapeutic value for the suppression of NLRP3 inflammasome-associated inflammatory diseases.

Dietary supplementation of camel whey protein attenuates heat stress-induced liver injury by inhibiting NLRP3 inflammasome activation through the HMGB1/RAGE signalling pathway

• The HMGB1/RAGE/NLRP3 signaling pathway is involved in HS-induced liver injury. • CWP inhibited HS-induced liver injury by reducing activity of the NLRP3 inflammasome. • CWP inhibited the activation of NLRP3 inflammasome by restoring HMGB1 nuclear localization. • The combined application of CWP and anti-inflammatory agent completely blocked HS-induced liver injury. Inflammasomes play an important role in promoting heat stress (HS) induced liver injury. Dietary supplementation of camel whey protein (CWP) has been shown to alleviate HS-induced tissue damage. However, whether and how dietary CWP supplementation can reduce HS-induced liver injury remains unclear. Thus, we evaluated the ability of rats supplemented with CWP to resist HS-induced liver injury. To induce liver injury, Sprague-Dawley rats were exposed to HS. The mechanism of action was confirmed using an antagonist of high mobility group box 1 (HMGB1) and the NLR pyrin domain containing 3 (NLRP3) inhibitor before HS. Histological changes in the livers of the HS rats were visualised using haematoxylin and eosin staining. The protein expression of HMG1, receptor for advanced glycation end products (RAGE), NLRP3, Caspase-3, and Bcl-2 were detected using immunohistochemistry or western blotting. The content of IL-1β, the activation of Caspase-1, and the level of alanine transaminase (ALT) were determined using commercial kits. We confirmed that HS activated NLRP3 in the liver, as evidenced by enhanced Caspase-1 activity and increased IL-1β content. Inhibition of NLRP3 activation reversed abnormal expression and nuclear translocation of Caspase-3 and Bcl-2, and alleviated apoptosis, necrosis, and subsequent liver injury. HS-induced hepatocyte NLRP3 activation is dependent on elevated extracellular HMGB1 levels. Glycyrrhizic acid, an antagonist of HMGB1, which is also an anti-inflammatory agent, inhibited HS-induced NLRP3 activation. Interestingly, CWP also reversed HS-induced abnormal expression of HMGB1, RAGE, NLRP3, IL-1β, ALT, Bcl-2, and Caspase-3, inhibited Caspase-1 activity, and alleviated apoptosis and liver histological changes. More importantly, CWP combined with glycyrrhizic acid completely prevented HS-induced hepatocyte apoptosis and injury. These results indicate that CWP ameliorates HS-induced liver injury by inhibiting the HMGB1/RAGE/NLRP3 axis. Dietary supplementation with CWP may be an effective strategy to prevent serious complications from HS.

Association of Three Functional Polymorphisms in the NLRP3 Gene with Susceptibility to Rheumatoid Arthritis in the Iranian Population.

Rheumatoid arthritis (RA) is a complex systemic autoimmune disorder with multifactorial nature. Numerous previous studies have shown that several genes are involved in the pathogenesis and increased risk of RA. The Nod-like receptor pyrin domain containing 3 (NLRP3) is involved in the regulation of innate immunity and its upregulation has previously been reported in RA. To evaluate the correlation between 3 functional polymorphisms of NLRP3 and its gene expression and RA risk. One hundred and fourteen patients with RA and 120 healthy participants were recruited to this case-control study. Genotyping of rs4612666 (intronic variant), rs10754558 (3UTR variant), and rs6672995 (downstream variant) were performed applying the real‑time polymerase chain reaction high‑resolution melting (HRM) method. Based on logistic regression analysis, subjects with CC genotype and C allele in rs4612666 had increased risk of RA (OR for CC genotype= 3.10; 95%CI [1.78-8.26]/ OR for C allele= 2.00; 95%CI [1.45-3.10]). Furthermore, in the patient groups, there was a significant relationship between the concentration of C-reactive protein (CRP) and rs4612666 and rs10754558 polymorphism (p < 0.05). Besides, our results revealed no significant association between the genotype and allele frequency of rs10754558 and rs6672995 and the risk of RA (P> 0.05). Our findings propose a significant association between rs4612666 polymorphism and increased risk of RA in the Iranian population. Moreover, rs4612666 and rs10754558 were correlated with disease activity.

Cerebral-Cardiac Syndrome and Diabetes: Cardiac Damage After Ischemic Stroke in Diabetic State.

Cerebral-cardiac syndrome (CCS) refers to cardiac dysfunction following varying brain injuries. Ischemic stroke is strongly evidenced to induce CCS characterizing as arrhythmia, myocardial damage, and heart failure. CCS is attributed to be the second leading cause of death in the post-stroke stage; however, the responsible mechanisms are obscure. Studies indicated the possible mechanisms including insular cortex injury, autonomic imbalance, catecholamine surge, immune response, and systemic inflammation. Of note, the characteristics of the stroke population reveal a common comorbidity with diabetes. The close and causative correlation of diabetes and stroke directs the involvement of diabetes in CCS. Nevertheless, the role of diabetes and its corresponding molecular mechanisms in CCS have not been clarified. Here we conclude the features of CCS and the potential role of diabetes in CCS. Diabetes drives establish a “primed” inflammatory microenvironment and further induces severe systemic inflammation after stroke. The boosted inflammation is suspected to provoke cardiac pathological changes and hence exacerbate CCS. Importantly, as the key element of inflammation, NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome is indicated to play an important role in diabetes, stroke, and the sequential CCS. Overall, we characterize the corresponding role of diabetes in CCS and speculate a link of NLRP3 inflammasome between them.

Inhibition of heat shock protein family A member 8 attenuates spinal cord ischemia\u2013reperfusion injury via astrocyte NF-\u03baB/NLRP3 inflammasome pathway

BackgroundAstrocyte over-activation and extensive neuron loss are the main characteristic pathological features of spinal cord ischemia–reperfusion injury (SCII). Prior studies have placed substantial emphasis on the role of heat shock protein family A member 8 (HSPA8) on postischemic myocardial inflammation and cardiac dysfunction. However, it has never been determined whether HSPA8 participates in astrocyte activation and thus mediated neuroinflammation associated with SCII.MethodsThe left renal artery ligation-induced SCII rat models and oxygen–glucose deprivation and reoxygenation (OGD/R)-induced rat primary cultured astrocytes were established. The lentiviral vector encoding short hairpin RNA targeting HSPA8 was delivered to the spinal cord by intrathecal administration or to culture astrocytes. Then, the spinal neuron survival, gliosis, and nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome and its related pro-inflammatory cytokines were analyzed.ResultsSCII significantly enhanced the GFAP and HSPA8 expression in the spinal cord, resulting in blood–brain barrier breakdown and the dramatical loss of spinal neuron and motor function. Moreover, injury also increased spinal nuclear factor-kappa B (NF-κB) p65 phosphorylation, NLRP3 inflammasome-mediated caspase-1 activation, and subsequent interleukin (IL)-1β as well as IL-18 secretion. Silencing the HSPA8 expression efficiently ameliorated the spinal cord tissue damage and promoted motor function recovery after SCII, through blockade of the astrocyte activation and levels of phosphorylated NF-κB, NLRP3, caspase-1, IL-1β, and IL-18. Further in vitro studies confirmed that HSPA8 knockdown protected astrocytes from OGD/R-induced injury via the blockade of NF-κB and NLRP3 inflammasome activation.ConclusionOur findings indicate that knockdown of HSPA8 inhibits spinal astrocytic damage after SCII, which may provide a promising therapeutic strategy for SCII treatment.

Genotype\u2013phenotype associations of polymorphisms within the gene locus of NOD-like receptor pyrin domain containing 3 in Swiss inflammatory bowel disease patients

BackgroundGenetic variations within the regulatory region of the gene encoding NOD-like receptor pyrin domain containing 3 (NLRP3) have been associated with Crohn’s Disease (CD). NLRP3 is part of the NLRP3-inflammasome that mediates the maturation of IL-1β and IL-18. Carrying the major allele of the single nucleotide polymorphisms (SNPs) rs10733113, rs4353135 and rs55646866 is associated with an increased risk for CD. We here studied the impact of these polymorphisms on clinical characteristics in patients of the Swiss IBD Cohort Study (SIBDCS).MethodsWe included 981 Crohn’s disease (CD) patients and 690 ulcerative colitis (UC) patients of the SIBDCS. We analyzed whether three CD-associated NLRP3 polymorphisms have an impact on the clinical disease course in these patients.ResultsIn CD patients presence of the major allele (G) of rs10733113 was associated with less surgeries and lower maximal CDAI and a similar trend was observed for rs55646866 and rs4353135. Presence of the major allele of all three SNPs was negatively correlated to maximal CDAI. In UC patients homozygous genotype for the major allele (CC) for rs55646866 was associated with a higher age at diagnosis and a higher MTWAI index. Homozygous genotype for the major allele of all three polymorphisms was associated with a higher number of ambulatory visits and longer hospital stays.ConclusionsIn CD patients presence of the major allele of all three polymorphisms was associated with markers of a less severe disease course, while in UC the homozygous genotype for all major alleles suggested a more severe disease activity.

MiR-124-3p Inhibits Microglial Secondary Inflammation After Basal Ganglia Hemorrhage by Targeting TRAF6 and Repressing the Activation of NLRP3 Inflammasome.

Objectives: Intracerebral hemorrhage (ICH) represents a serious central nervous system emergency with high morbidity and mortality, and the basal ganglia is the most commonly affected brain region. Differentially expressed microRNAs (miRs) have recently been highlighted to serve as potential diagnostic biomarkers and therapeutic targets for ICH. This study investigated the mechanism of miR-124-3p in microglial secondary inflammation after ICH.Methods: In this study, 48 patients with primary basal ganglia ICH and 48 healthy volunteers were selected and venous blood was collected from all patients on the second morning of admission (within 24 h of stroke onset). The expression of miR-124-3p in serum was detected by RT-qPCR. Three months after ICH, the patients were assessed by modified Rankin Scale (mRS), and the correlation between miR-124-3p expression and mRS score was analyzed by Pearson. The inflammatory response of microglia was induced by lipopolysaccharide (LPS) to establish the cell model of microglial inflammation. miR-124-3p expression patterns were detected in the serum of ICH patients and healthy volunteers, normal microglia, and LPS-induced microglia. The miR-124-3p mimic was transfected into LPS-induced microglia, followed by measurement of the inflammatory factors, apoptosis rate, and cell viability. The target gene of miR-124-3p was predicted and verified. The expression patterns of tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected. pcDNA3.1 and pcDNA3.1-TRAF6 were transfected into LPS-induced HMC3 cells, and nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain-containing 3 (NLRP3) expression patterns were determined. Lastly, the effects of TRAF6 overexpression on apoptosis, cell viability, and inflammation in HMC3 cells were measured.Results: miR-124-3p was downregulated in the serum of basal ganglia ICH patients and LPS-induced microglia, and miR-124-3p expression was negatively correlated with mRS. Overexpression of miR-124-3p reduced the inflammatory factors and apoptosis rate and promoted cell activity in LPS-induced microglia. miR-124-3p was found to target TRAF6. Overexpression of TRAF6 enhanced the expression of NLRP3 inflammasome, inflammatory factors and apoptosis rate, and reduced cell viability.Conclusion: Our findings indicate that miR-124-3p repressed the activation of NLRP3 inflammasome by targeting TRAF6, thus inhibiting microglial secondary inflammation after ICH in basal ganglia.