NOD-like Receptor Protein 3 Inflammasome Pathway Research Articles

Histone deacetylase 3-specific inhibitor RGFP966 attenuates oxidative stress and inflammation after traumatic brain injury by activating the Nrf2 pathway.

Oxidative stress (OS) and inflammatory reactions play pivotal roles in secondary brain injury after traumatic brain injury (TBI). Histone deacetylase 3 (HDAC3) controls the acetylation of histones and non-histones, which has a significant impact on the central nervous system's reaction to damage. This research determined the implications of RGFP966, a new and specific inhibitor of HDAC3, for the antioxidant (AO) systems mediated by nuclear factor erythroid2-related factor 2 (Nrf2) and the Nod-like receptor protein 3 (NLRP3) inflammasome in TBI. The study also studied the underlying mechanisms of RGFP966's actions. Our objective was to examine the impacts and underlying RGFP966 mechanisms in TBI. In vitro, a rat cortical neuron OS model was induced by H2O2, followed by the addition of RGFP966 to the culture medium. Neurons were collected after 24h for western blot (WB), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 2'-7'-dichlorodihydrofluorescein diacetate staining. In vivo, RGFP966 (10mg/kg) was administered post-TBI. Brain tissue water content and modified neurological severity scores were assessed 72h post-injury. Cortical tissues surrounding the focal injury were subjected to western blot, TUNEL staining, Nissl staining and immunofluorescence/immunohistochemistry staining, and malondialdehyde level, hindered glutathione content and superoxide dismutase activity were measured. Serum was collected for the enzyme-linked immunosorbent assay. Nrf2-specific shRNA lentivirus was injected into the lateral ventricle of rats for 7days, and cerebral cortex tissue was analyzed by WB and real-time polymerase chain reaction. During in vitro and in vivo experiments, RGFP966 suppressed HDAC3 expression, promoted Nrf2 nuclear translocation, activated downstream AO enzymes, mitigated excessive reactive oxygen species production and alleviated nerve cell apoptosis. RGFP966 effectively reduced brain edema and histological damage and enhanced neurological and cognitive function in rats with TBI. RGFP966 markedly inhibited NLRP3 inflammasome activation mediated by high-mobility group box 1 (HMGB1)/toll-like receptor 4 (TLR4). Nrf2 knockdown in TBI rats attenuated the AO and anti-inflammatory, neuroprotective impacts of RGFP966. Overall, our findings demonstrate that RGFP966 can mitigate the first brain damage and neurological impairments in TBI. The underlying mechanism involves triggering the Nrf2-mediated AO system and negatively regulating the HMGB1/TLR4-mediated NLRP3 inflammasome pathway.

Korean Chestnut Honey Suppresses HSV-1 Infection by Regulating the ROS-NLRP3 Inflammasome Pathway.

Herpes simplex virus 1 (HSV-1) is double-stranded DNA virus that belongs to the Orthoherpesviridae family. It causes serious neurological diseases of the central nervous system, such as encephalitis. The current U.S. Food and Drug Administration (FDA)-approved drugs for preventing HSV-1 infection include acyclovir (ACV) and valacyclovir; however, their long-term use causes severe side effects and often results in the emergence of drug-resistant strains. Therefore, it is important to discover new antiviral agents that are safe and effective against HSV-1 infection. Korean chestnut honey (KCH) has various pharmacological activities, such as antioxidant, antibacterial, and anti-inflammation effects; however, antiviral effects against HSV-1 have not yet been reported. Therefore, we determined the antiviral activity and mechanism of action of KCH after HSV-1 infection on the cellular level. KCH inhibited the HSV-1 infection of host cells through binding and virucidal steps. KCH decreased the production of reactive oxygen species (ROS) and calcium (Ca2+) following HSV-1 infection and suppressed the production of inflammatory cytokines by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) activity. Furthermore, we found that KCH inhibited the expression of the nod-like receptor protein 3 (NLRP3) inflammasome during HSV-1 infection. Taken together, the antiviral effects of KCH occur through multiple targets, including the inhibition of viral replication and the ROS-mediated NLRP3 inflammasome pathway. Our findings suggest that KCH has potential for the treatment of HSV-1 infection and related diseases.

Integrating UHPLC-MS/MS quantitative analysis and exogenous purine supplementation to elucidate the antidepressant mechanism of Chaigui granules by regulating purine metabolism

Chaigui granules (CG) are a compound composed of six herbal medicines with significant antidepressant effects. However, the antidepressant mechanism of CG remains unclear. In the present study, we attempted to elucidate the antidepressant mechanism of CG by regulating purine metabolism and purinergic signaling. First, the regulatory effect of CG on purine metabolites in the prefrontal cortex (PFC) of chronic unpredictable mild stress (CUMS) rats was analyzed by ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) targeted quantitative analysis. Meanwhile, purinergic receptors (P2X7 receptor (P2X7R), A1 receptor (A1R) and A2A receptor (A2AR)) and signaling pathways (nod-like receptor protein 3 (NLRP3) inflammasome pathway and cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway) associated with purine metabolism were analyzed by western blotting and enzyme-linked immunosorbent assay (ELISA). Besides, antidepressant mechanism of CG by modulating purine metabolites to activate purinergic receptors and related signaling pathways was dissected by exogenous supplementation of purine metabolites and antagonism of purinergic receptors in vitro. An in vivo study showed that the decrease in xanthine and the increase in four purine nucleosides were closely related to the antidepressant effects of CG. Additionally, purinergic receptors (P2X7R, A1R and A2AR) and related signaling pathways (NLRP3 inflammasome pathway and cAMP-PKA pathway) were also significantly regulated by CG. The results of exogenous supplementation of purine metabolites and antagonism of purinergic receptors showed that excessive accumulation of xanthine led to activation of the P2X7R-NLRP3 inflammasome pathway, and the reduction of adenosine and inosine inhibited the A1R-cAMP-PKA pathway, which was significantly ameliorated by CG. Overall, CG could promote neuroprotection and ultimately play an antidepressant role by inhibiting the xanthine-P2X7R-NLRP3 inflammasome pathway and activating the adenosine/inosine-A1R-cAMP-PKA pathway.

Fuzheng Huayu recipe alleviates liver fibrosis via inhibiting NLRP3 inflammasome activation in macrophages

Ethnopharmacological relevanceFuzheng Huayu recipe (FZHY) is a commonly used Traditional Chinese Medicine formula for treating liver fibrosis in clinical settings. Despite its widespread use, the specific curative effects and underlying pharmacological mechanisms of FZHY in treating liver fibrosis are not yet fully understood. Aim and studyThis study aims to investigate the antifibrotic mechanism of FZHY treatment by exploring its effects on the activation of NOD-like receptor protein 3 (NLRP3) inflammasome in macrophages. Materials and methodsIn order to investigate the impact of FZHY on the activation and priming of NLRP3 inflammasome in clinical trials and animal experiments using immunohistochemistry and Western blotting. Twenty-four C57BL/6 mice were used to induce liver fibrosis by feeding a diet that contained 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). To study inflammasome function, Lipopolysaccharide (LPS)/adenine triphosphate (ATP) induced NLRP3 inflammasome activation was induced in bone marrow-derived macrophages (BMDMs) isolated from wild mice. The effects of macrophage NLRP3 inflammasome activation on the function of hepatic stellate cells (HSCs) were explored by treating primary HSCs with preconditioned media from BMDMs culture. ResultsFZHY treatment resulted in the downregulation of NLRP3 protein expression and inhibition of its priming and activation in both human fibrotic livers and DDC-induced liver fibrosis. Furthermore, FZHY was observed to block the activation of the NLRP3 inflammasome pathway, which can lead to excessive inflammatory cytokine release in supernatants and cell lysates in response to LPS and ATP. Lastly, treatment with FZHY was able to inhibit the activation of HSCs induced by supernatants from macrophages. ConclusionsFZHY has been shown to potentially prevent NLRP3 inflammasome activation in macrophages which can result in the suppression of HSCs activation. Ultimately, these effects may lead to the improvement of liver fibrosis. The ability of FZHY to act on this novel mechanism represents an important aspect of its therapeutic potential for liver fibrosis.

An HPK1 inhibitor enhanced the tumour response to anti-PD-1 immunotherapy in non-Hodgkin's lymphoma.

Anti-PD-1 immunotherapy has been widely applied in patients with some types of lymphoma. Classical Hodgkin's lymphoma (cHL) is highly sensitive to immunotherapy, but non-Hodgkin's lymphoma (NHL) does not show a good response. Studies have indicated that haematopoietic progenitor kinase 1 (HPK1) suppresses T cells and reduces antitumour immunity. Therefore, HPK1 inhibitors may restore and elicit antitumour immune responses and are promising candidate drug targets for cancer immunotherapy. We first explored the Gene Expression Profile Interactive Analysis (GEPIA) database and predicted that HPK1 expression was increased in diffuse large B-cell lymphoma (DLBCL) and associated with Nod-like receptor protein 3 (NLRP3) expression. We investigated whether an HPK1 inhibitor could enhance the tumour response to anti-PD-1 immunotherapy in NHL and the association between HPK1 and NLRP3 expression. Employing shHPK1 and an inhibitor, we demonstrated that the HPK1 inhibitor increased anti-PD-1-mediated T-cell cytotoxicity in BJAB and WSU-DLCL2 cells cocultured with peripheral blood mononuclear cells (PBMCs). HPK1 inhibitor treatment increased PD-1, PD-L1, Bax, p53 and NK-kB expression but decreased NLRP3 expression, indicating that the HPK1 inhibitor promoted apoptosis and blocked the NLRP3 inflammasome pathway to affect anti-PD-1-mediated T-cell cytotoxicity. Moreover, the HPK1 inhibitor enhanced the efficiency of anti-PD-1 immunotherapy in vivo in a zebrafish xenograft model of NHL. In summary, this study provides evidence that an HPK1 inhibitor enhanced the tumour response to anti-PD-1 immunotherapy in NHL by promoting apoptosis and blocking the NLRP3 pathway. These findings provide a potential therapeutic option for NHL combining HPK1 inhibitor treatment and anti-PD-1 immunotherapy.

Inhibitors of the NLRP3 inflammasome pathway as promising therapeutic candidates for inflammatory diseases (Review).

The inflammasome regulates innate immunity by serving as a signaling platform. The Nod‑like receptor protein 3 (NLRP3) inflammasome, equipped with NLRP3, the adaptor protein apoptosis‑associated speck‑like protein (ASC) and pro‑caspase‑1, is by far the most extensively studied and well‑characterized inflammasome. A variety of stimuli can activate the NLRP3 inflammasome. When activated, the NLRP3 protein recruits the adaptor ASC protein and activates pro‑caspase‑1, resulting in inflammatory cytokine maturation and secretion, which is associated with inflammation and pyroptosis. However, the aberrant activation of the NLRP3 inflammasome has been linked to various inflammatory diseases, including atherosclerosis, ischemic stroke, Alzheimer's disease, diabetes mellitus and inflammatory bowel disease. Therefore, the NLRP3 inflammasome has emerged as a promising therapeutic target for inflammatory diseases. In the present review, systematic searches were performed using 'NLRP3 inhibitor(s)' and 'inflammatory disease(s)' as key words. By browsing the literature from 2012 to 2022, 100 articles were retrieved, of which 35 were excluded as they were reviews, editorials, retracted or unavailable online, and 65 articles were included. According to the retrieved literature, the current understanding of NLRP3 inflammasome pathway activation in inflammatory diseases was summarize, and inhibitors of the NLRP3 inflammasome pathway targeting the NLRP3 protein and other inflammasome components or products were highlighted. Additionally, the present review briefly discusses the current novel efforts in clinical research.

Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans

Ethnopharmacological relevancePulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC.Aim of the study: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. Materials and methodsUltra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1β and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. ResultsAfter administration of BEPD-containing serum, the levels of IL-1β, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. ConclusionsBEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

BAY-117082-driven NLRP3 inflammasome inhibition resolves nitro-glycerine (NTG) neuronal damage in in vivo model of migraine

Migraine is a common neuronal disorder characterized by recurrent episodes of headache associated with a higher prevalence in women than men. Several risk factors have been associated with migraine disease as genetic factors, gender, and age. Although understanding migraine pathophysiology is improved, it has been reported that NOD-like receptor protein 3 (NLRP3) inflammasome pathway overactivation can contribute to migraine progression. Therefore, the aim of this study was to investigate the effect of BAY-117082, an NLRP3 inflammasome inhibitor, in a mouse model of nitroglycerin (NTG)-induced migraine. The in vivo model of migraine was induced by intraperitoneal (i.p) injection of NTG (dose of 10 mg/kg). Mice were treated intraperitoneally with BAY-117082 at doses of 1 mg/kg, 5 mg/kg, and 10 mg/kg, 5 min following NTG injection. After 4 h of NTG injection, the whole brain tissue with the rostral spinal cord were collected and used to perform further analysis. Our results demonstrated that BAY-117082 treatments (5 mg/kg and 10 mg/kg) reduced pain attacks, hyperalgesia and photophobia more in female mice NTG-induced. Moreover, the treatment with BAY-117082 significantly reduced histological damage in the trigeminal nerve nucleus in female mice accordingly to significantly decreased in NLRP3 complex components expression levels such as ASC, IL-1β, IL-18, caspase-1 and TNF-α levels. Additionally, the treatment with BAY-117082 at both higher doses significantly modulated CREB/Erk/Akt pathways strictly correlated to the expression of neurotrophic factors. Taken together, obtained results confer new insight into the role of the NLRP3 inflammasome pathway in migraine pathogenesis, suggesting that BAY-117082 could be considered a novel strategy therapeutics for migraine treatment despite unconventional drug use.

Baicalin Alleviates Contrast-Induced Acute Kidney Injury Through ROS/NLRP3/Caspase-1/GSDMD Pathway-Mediated Proptosis in vitro.

PurposeTo investigate the effect of baicalin on the reactive oxygen species (ROS)/ NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin-D (GSDMD) inflammasome pathway and its related mechanism in regulating pyroptosis of human renal tubular epithelial cells (HK-2) induced by contrast media.MethodsIohexol was used to act on HK-2 cells to establish a renal tubular cell pyroptosis model; and the signal pathway genes were silenced, cytokines were detected by enzyme-linked immunosorbent assay (ELISA), and cell viability, gene expression, and protein expression were evaluated by double fluorescence staining and flow cytometry. To assess the cytotoxicity caused by the contrast agent; cells were pretreated with different concentrations of baicalin; and then the cells were exposed to iohexol again, and the relevant indicators were tested again.ResultsAfter HK-2 cells were exposed to iohexol, the NLRP3 inflammasome pathway markers NLRP3, interleukin (IL)-1β, and IL-18 mRNA levels as well as the protein expression levels of NLRP3, ASC, Caspase-1, and GSDMD were up-regulated. In addition, the effect also significantly increased the IL-18, IL-1β, lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA) release, and cellular ROS levels. The results of Annexin V-FITC/PI flow cytometry showed that the level of apoptosis was increased. However, after the intervention of baicalin, the changes in the above indexes caused by iohexol stimulation of HK-2 cells were inhibited.ConclusionExposure to iohexol can induce pyroptosis of HK-2 cells through the ROS/NLRP3/Caspase-1/GSDMD signaling pathway. Baicalin ameliorated iohexol-induced pyroptosis in HK-2 cells by regulating the NLRP3 inflammasome pathway.

The Protective Effects of Neoastilbin on Monosodium Urate Stimulated THP-1-Derived Macrophages and Gouty Arthritis in Mice through NF-κB and NLRP3 Inflammasome Pathways.

Gouty arthritis (GA) is a frequent inflammatory disease characterized by pain, swelling, and stiffness of joints. Neoastilbin is a flavonoid isolated from the rhizome of Smilax glabra, which possesses various anti-inflammatory effects. However, the mechanism of neoastilbin in treating GA has not yet been clarified. Thus, this study was to investigate the protective effects of neoastilbin in both monosodium urate (MSU) stimulated THP-1-derived macrophages and the animal model of GA by injecting MSU into the ankle joints of mice. The levels of key inflammatory cytokines in MSU stimulated THP-1-derived macrophages were detected by enzyme-linked immunosorbent assay (ELISA) kits. Protein expressions of nuclear factor kappa B (NF-κB) and NOD-like receptor protein 3 (NLRP3) inflammasome pathways were further detected by Western blotting. In addition, swelling degree of ankle joints, the levels of inflammatory factors, infiltration of inflammatory cells and the expressions of related proteins were determined. Swelling degree and histopathological injury in ankle joints of MSU-injected mice were significantly decreased after being treated with neoastilbin. Moreover, neoastilbin significantly diminished the secretion of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), suppressing the activation of NF-κB and NLRP3 inflammasome pathways in both MSU stimulated THP-1-derived macrophages and the mouse model of GA. In summary, neoastilbin could alleviate GA by inhibiting the NF-κB and NLRP3 inflammasome pathways, which provided some evidence for neoastilbin as a promising therapeutic agent for GA treatment.

Effect of BAY‐117082, a NLRP3 inflammasome inhibitor, in a mouse model of nitroglycerin (NTG)‐induced migraine

Migraine is a common brain disorder characterized by recurrent episodes of headache. It is a complex and multifactorial disease with a higher prevalence in females than in males. Several risk factors have been associated to migraine disease as genetic factors, sex and age. The pathophysiology of migraine is still unclear; however, it has been proven that NOD‐like receptor protein 3 (NLRP3) inflammasome pathway overactivation as well as p‐ERK/p‐CREB axis contribute to migraine pathogenesis. Therefore, the aim of this study was to investigate the effect of BAY‐117082, a NLRP3 inflammasome inhibitor and p‐ERK/p‐CREB modulator, in an in vivo model of nitroglycerin (NTG)‐induced migraine.Migraine model was induced by NTG intraperitoneal administration at dose of 10 mg/kg diluted in 0.9% saline. Mice were treated intraperitoneally with BAY‐117082 at doses of 1 mg/kg, 5 mg/kg, and 10 mg/kg, 5 minutes after NTG injection. Mice were sacrificed 4 h following NTG injection; the whole brain with the rostral spinal cord was removed to perform several analysis.Our results demonstrated that, following behavioral tests for pain and photophobia, BAY‐117082 treatment at doses of 5 mg/kg and 10 mg/kg reduced pain attacks induced by NTG more than BAY‐117082 at the dose of 1 mg/kg. Moreover, the treatment with BAY‐117082 at doses of 5 mg/kg and 10 mg/kg significantly reduced histological damage in the trigeminal nerve nucleus and significantly decreased inflammosome activation by reduction of NLRP3, ASC, IL‐1β and TNF‐α expression. Additionally, the treatment with BAY‐117082 at doses of 5 mg/kg and 10 mg/kg significantly modulated p‐ERK/p‐CREB axis activation through the reduction of p‐ERK, p‐AKT, p‐CREB and p‐PI3K expression.Therefore, the obtained results offer new insight into the role of NLRP3 inflammasome pathway and p‐ERK/p‐CREB axis in migraine pathogenesis, suggesting that BAY‐117082 could be considered a novel therapeutic strategy to treat migraine.

Curcumin mitigates aflatoxin B1-induced liver injury via regulating the NLRP3 inflammasome and Nrf2 signaling pathway

Aflatoxins are produced as secondary metabolites by the toxigenic Aspergillus fungi. Among the aflatoxins, aflatoxin B1 (AFB1) is a common contaminant of global concern in human and animal food products. Prolonged exposure to AFB1 may provoke hepatocyte pyroptosis and oxidative stress, which leads to liver damage. Dietary polyphenols could protect the liver from a wide range of toxins. Curcumin, a polyphenolic substance derived from turmeric, is rich in pharmacological activity. The aim of this study was to systematically investigate the protective effects of curcumin against AFB1-induced liver injury in mice and to explore the possible molecular mechanisms. BALB/c mice received oral gavage of AFB1 (0.75 mg/kg) and curcumin (100 or 200 mg/kg) for 30 days. Our data demonstrated that curcumin attenuated AFB1-induced weight loss in mice and rescued liver injury by mitigating the alterations in pathology and liver function with AFB1 exposure. Curcumin reduced the accumulation of AFB1-DNA adducts in the liver and alleviated hepatotoxicity by inhibiting AFB1-induced oxidative stress and potentiating glutathione S-transferase (GST)-mediated phase II detoxification. In addition, curcumin significantly reduced the characteristic indices of AFB1-induced pyroptosis, such as the expression of mRNAs for genes related to NOD-like receptor protein 3 (NLRP3) inflammasome assembly and activation, the expression of key proteins (NLRP3, Caspase-1 and GSDMD). The release of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the serum detected by ELISA was also significantly decreased. Notably, administration of curcumin upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its related downstream antioxidant molecules (SOD, CAT, HO-1, NQO1) and phase II detoxification enzyme-related molecules (GST, GSH, GSS, GCLC, GCLM) in the presence of AFB1 exposure. To summarize, our results indicated that curcumin could modulate the NLRP3 inflammasome and Nrf2 signaling pathways to attenuate AFB1-induced liver pyroptotic damage and oxidative stress.

Mechanism of Huanglian Wendan Decoction in improving impaired glucose tolerance based on skeletal muscle NLRP3/caspase-1/IL-1β, IL-18 pathway

This study investigated the mechanism of improving impaired glucose tolerance(IGT) of rats by Huanglian Wendan Decoction from the perspective of the skeletal muscle Nod-like receptor protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/interleukin-1β(IL-1β), interleukin-18(IL-18) pathway. Healthy male SD rats were fed with the diet containing 45% fat for 20 weeks, accompanied by a high-temperature and high-humidity environment and an inactive lifestyle, for the establishment of the IGT rat model. The rats were divided into the blank control group, model control group, metformin hydrochloride group(positive drug group, 0.05 g·kg~(-1)·d~(-1)) and Huanglian Wendan Decoction group(7.8 g·kg~(-1)·d~(-1)). After continuous intragastric administration for 4 weeks, the obesity and glycemic indexes of all the rats were measured. The fasting serum insulin(FINS) level was determined by ELISA, with the insulin sensitivity index(ISI) and insulin resistance index(IRI) calculated. The mRNA and protein expression le-vels of nuclear factor kappaB(NF-κB), NLRP3, caspase-1, IL-1β and IL-18 in skeletal muscle tissue were detected by real-time polymerase chain reaction(PCR), Western blot and immunofluorescence. Compared with the blank control group, the model control group witnessed significantly increased mRNA and protein expression of NF-κB, NLRP3, caspase-1, IL-1β and IL-18. As revealed by the comparison with the model control group, Huanglian Wendan Decoction could effectively regulate the obesity status, reduce body weight, correct the abnormal levels of 2-hour plasma glucose(2 hPG), insulin resistance index(IRI), insulin sensitivity index(ISI), and lower the mRNA and protein expression of NF-κB, NLRP3, caspase-1, IL-1β and IL-18 in the skeletal muscle tissue of IGT rats. Combined with previous studies, the above results showed that the occurrence and development of IGT was closely related to inflammatory response and the classic pyroptosis pathway in skeletal muscle, such as NLRP3/caspase-1/IL-1β, IL-18. It is inferred that the mechanism of Huanglian Wendan Decoction was to alleviate insulin resistance(IR) and then reverse the course of IGT lies in the regulation of the abnormal insulin receptor signaling pathway based on the NLRP3 inflammasome pathway.

Genetically determined NLRP3 inflammasome activation associates with systemic inflammation and cardiovascular mortality.

Inflammation plays an important role in cardiovascular disease (CVD) development. The NOD-like receptor protein-3 (NLRP3) inflammasome contributes to the development of atherosclerosis in animal models. Components of the NLRP3 inflammasome pathway such as interleukin-1β can therapeutically be targeted. Associations of genetically determined inflammasome-mediated systemic inflammation with CVD and mortality in humans are unknown. We explored the association of genetic NLRP3 variants with prevalent CVD and cardiovascular mortality in 538167 subjects on the individual participant level in an explorative gene-centric approach without performing multiple testing. Functional relevance of single-nucleotide polymorphisms on NLRP3 inflammasome activation has been evaluated in monocyte-enriched peripheral blood mononuclear cells (PBMCs). Genetic analyses identified the highly prevalent (minor allele frequency 39.9%) intronic NLRP3 variant rs10754555 to affect NLRP3 gene expression. rs10754555 carriers showed significantly higher C-reactive protein and serum amyloid A plasma levels. Carriers of the G allele showed higher NLRP3 inflammasome activation in isolated human PBMCs. In carriers of the rs10754555 variant, the prevalence of coronary artery disease was significantly higher as compared to non-carriers with a significant interaction between rs10754555 and age. Importantly, rs10754555 carriers had significantly higher risk for cardiovascular mortality during follow-up. Inflammasome inducers (e.g. urate, triglycerides, apolipoprotein C3) modulated the association between rs10754555 and mortality. The NLRP3 intronic variant rs10754555 is associated with increased systemic inflammation, inflammasome activation, prevalent coronary artery disease, and mortality. This study provides evidence for a substantial role of genetically driven systemic inflammation in CVD and highlights the NLRP3 inflammasome as a therapeutic target.

Exosomes derived from human-induced pluripotent mesenchymal stem cells inhibit the pyrolysis of alveolar macrophages

To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells (iMSC-Exos) on alveolar macrophages (AM) pyroptosis. The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells (iMSC) were extracted by rotating ultrafiltration, and the extracted exosomes were identified by transmission electron microscopy, Western blotting and high-resolution adjustable resistance pulse. The rat alveolar macrophage cells (NR8383 cells) were cultured in vitro and the logarithmic growth phase cells were divided into three groups: the control group was added with an equal volume of phosphate buffered saline (PBS) in the AM supernatant; in LPS/ATP group AM cells were stimulated with 500 μg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis; iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP. The cytotoxic activity was detected by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) analysis, the apoptosis and the expression of caspase-1 were observed by immunofluorescence, the levels of inflammatory factors interleukins (IL-1β and IL-18) released by AM were detected by enzyme linked immunosorbent assay (ELISA), the NOD-like receptor protein 3 (NLRP3) inflammasome pathway and the expression level of pyroptosis related protein gasdermin D (GSDMD) were detected by Western blotting. The extracted exosomes were observed by transmission electron microscopy as round vesicles, expressing exosomal markers CD63 and CD9 showed by Western blotting, high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm, and could be uptaken by AM. Compared with the control group, the cell activity decreased [(0.56±0.05)% vs. (1.06±0.07)%, P < 0.01], the release of necrotic substance LDH increased (U/L: 1 218.86±22.73 vs. 188.30±1.61, P < 0.01), the expression levels of inflammatory factors increased [IL-1β (ng/L): 958.91±32.78 vs. 194.63±5.14, IL-18 (ng/L): 870.89±21.86 vs. 288.85±24.48, both P < 0.01], and the apoptosis rate [(55.35±6.19)% vs. (12.01±1.32)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 41.06±3.65 vs. 2.80±0.54, P < 0.01) elevated in the AM after LPS/ATP stimulation, suggesting that LPS combined with ATP successfully induced alveolar pyroptosis. Compared with the LPS/ATP group, AM pretreated with iMSC-Exos showed increased cell viability [(0.81±0.05)% vs. (0.56±0.05)%, P < 0.01], decreased LDH secretion (U/L: 535.05±42.55 vs. 1 218.86±22.73, P < 0.01), decreased expression of inflammatory factors [IL-1β (ng/L): 381.82±19.50 vs. 958.91±32.78, IL-18 (ng/L): 533.77±31.54 vs. 870.89±21.86, both P < 0.01], and decreased apoptosis rate [(19.74±2.96)% vs. (55.35±6.19)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 12.16±1.31 vs. 41.06±3.65, P < 0.01). At the same time, the expression of NLRP3 inflammasome pathway [NLRP3 protein (NLRP3/β-actin): 0.62±0.06 vs. 1.89±0.11; cleaved caspase-1 protein (cleaved caspase-1/β-actin): 0.42±0.07 vs. 1.22±0.17, both P < 0.01] and pyrolysis-related protein was significantly inhibited [GSDMD protein (GSDMD/β-actin): 0.57±0.05 vs. 1.22±0.05, P < 0.01]. iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP, which may be due to the targeted inhibition of NLRP3 inflammasome pathway, suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.

GABA-mediated activated microglia induce neuroinflammation in the hippocampus of mice following cold exposure through the NLRP3 inflammasome and NF-κB signaling pathways

Chronic cold stress has long-term dramatic effects on the animal immune and neuroendocrine systems. As one of the important regions of the brain, the hippocampus is the main region involved in response to stressors. Nevertheless, the impact to the hippocampus following cold exposure and the underlying mechanism involved are not clear. To evaluate the response of the hippocampus during chronic cold stress, male C57BL/6 mice were exposed to 4 °C, 3 h per day for 1 week, after which neuroinflammation and the molecular and signaling pathways in the hippocampus response to cold stress were investigated. To confirm the potential mechanism, BV2 cells were treated with γ-aminobutyric acid (GABA) and BAY 11-7082 and MCC950, then the activation of microglia and key proteins involved in the regulation of inflammation were measured. We demonstrated that chronic cold stress induced the activation of microglia, the emergence of neuroinflammation, and the impairment of neurons in the hippocampus, which might be the result of GABA-mediated activation of nod-like receptor protein 3 (NLRP3) inflammasome and the nuclear factor kappa B (NF-κB) signaling pathway.

SO003LIFE-LONG NLRP3 INFLAMMASOME-MEDIATED SYSTEMIC INFLAMMATION ASSOCIATES WITH CARDIOVASCULAR MORTALITY: A GENETIC ASSOCIATION STUDY OF &gt;500,000 INDIVIDUALS

Abstract Background and Aims Chronic kidney disease (CKD) represents one of the strongest cardiovascular risk factors. Recently, we have shown that activation of the NOD-like receptor protein-3 (NLRP3) inflammasome mediates progression of kidney injury and CKD-associated vascular disease (Zewinger et al. Nat Immunol 2013). Components of the NLRP3 inflammasome pathway such as interleukin-1β (IL-1β) can therapeutically be targeted. Associations of genetically determined life-long inflammasome-mediated systemic inflammation with CVD and mortality in humans are unknown. Method We explored the association of genetic NLRP3 variants with prevalent CVD and cardiovascular mortality in 3,061 patients enrolled in the LURIC study undergoing coronary angiography in a gene-centric approach. Functional relevance of the rs10754555 NLRP3 variant was studied in freshly isolated human monocytes. Findings were validated in 39,755 participants from eight independent studies (CV risk populations) as well as in 483,258 participants of the UKBiobank study (general population). Results Genetic analyses identified the highly prevalent (MAF 39.9%) intronic NLRP3 variant rs10754555 to affect NLRP3 gene expression. rs10754555 carriers showed significantly higher C-reactive protein and serum amyloid A plasma levels. Expression of NLRP3 mRNA and inflammasome-dependent effector responses of isolated human monocytes from rs10754555 carriers were significantly higher as compared to non-carriers. In homozygous rs10754555 carriers, the prevalence of coronary artery disease (CAD) was significantly higher as compared to non-carriers with a significant interaction between rs10754555 and age in LURIC and in UKBiobank. Importantly, in LURIC and in UKBiobank, homozygous rs10754555 carriers had significantly higher risk for cardiovascular mortality during follow-up (HR 1.37, 95%-CI 1.08-1.74 and HR 1.13, 95%-CI 1.02-1.25, respectively). Consistently, rs10754555 was associated with higher cardiovascular mortality (HR 1.11, 95%-CI 1.05-1.16; P&amp;lt;0.001) in an additive model in patients with prevalent CAD. Conclusion The NLRP3 intronic variant rs10754555 is associated with increased systemic inflammation, inflammasome activation, prevalent CAD and mortality. This study provides evidence for a substantial role of life-long, genetically-driven systemic inflammation in cardiovascular disease and highlights the NLRP3 inflammasome as a therapeutic target in the general population and particular in patients with CKD.

Oridonin Attenuates Myocardial Ischemia/Reperfusion Injury via Downregulating Oxidative Stress and NLRP3 Inflammasome Pathway in Mice.

Oridonin (ORI), the major pharmacological component extracted from a traditional Chinese medicine, possesses a beneficial effect on myocardial ischemia/reperfusion (I/R) injury. However, the underlying molecular mechanism by which ORI effects take place is not completely known. Thus, whether ORI works via downregulating oxidative stress and nod-like receptor protein-3 (NLRP3) inflammasome pathway was investigated in this study. Mice underwent surgery to induce myocardial I/R injury, and some were administered ORI (10 mg/kg/day) pretreatment, while others were not. The myocardial enzymes' levels, infarct area, and inflammatory injury were measured. The activation situation of oxidative stress and NLRP3 inflammasome was also detected. We found that ORI pretreatment significantly alleviated CK-MB and cTnI levels and infarct size induced by I/R. ORI mitigated the inflammatory injury by decreasing the pathological damage and lowering TNF-α, IL-1β, and IL-18 levels. Moreover, the SOD1 and eNOS levels were significantly increased by ORI, while MDA and iNOS levels were relatively decreased. The oxidative stress was reversed using ORI pretreatment. Importantly, NLRP3 inflammasome pathway was also inhibited by ORI, as reflected by the lower protein levels of NLRP3, caspase-1, and IL-1β. In conclusion, ORI alleviates myocardial injury induced by I/R via inhibiting the oxidative stress and NLRP3 inflammasome pathway.

Neferine alleviates memory and cognitive dysfunction in diabetic mice through modulation of the NLRP3 inflammasome pathway and alleviation of endoplasmic-reticulum stress

Accumulating clinical and epidemiological evidence indicates a close relationship between diabetes mellitus and dysfunction in memory and cognition. Neferine (NE) is a unique bis-benzylisoquinoline alkaloid derived from the seed embryo of Nelumbo nucifera (Lotus), an herbal medicine with a long history of use in used in China. NE has been reported to ameliorate diabetes mellitus and exert considerable protective effects on the central nervous system. Thus, this study aimed to investigate the effects of NE on memory and cognitive dysfunction in db/db mouse model of diabetes. First, we found that NE treatments significantly ameliorated behavioral impairment and cognitive dysfunction in the Morris water maze, Y-maze, and fear conditioning test in db/db mice. Additionally, in these diabetic mice, NE decreased fasting glucose and insulin resistance while promoting lipid metabolism. Furthermore, NE treatments alleviated oxidative stress and inhibited inflammatory responses in the hippocampus. Further investigations showed that NE suppressed the NOD-like receptor protein 3 (NLRP3) inflammasome pathway via down-regulating the levels of thioredoxin-interacting protein (TXNIP), NLRP3 inflammasomes, apoptosis-associated speck-like protein containing a CARD (ASC), and mature interleukin-1β (IL-1β) in the hippocampus. Moreover, NE alleviated endoplasmic-reticulum (ER) stress via down-regulating the levels of immunoglobulin heavy-chain-binding protein (GRP78), C/EBP homologous protein (CHOP), proteins kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) in the hippocampus. In conclusion, these results suggest that NE ameliorated memory and cognitive dysfunction, possibly through modulating the NLRP3 inflammasome pathways and alleviating ER stress.

Nicardipine Inhibits Priming of the NLRP3 Inflammasome via Suppressing LPS-Induced TLR4 Expression.

The Nod-like receptor protein 3 (NLRP3) inflammasome is a multi-protein complex composed of NLRP3, pro-caspase-1, and apoptosis-associated speck-like protein that contains a caspase recruitment domain (ASC). After NLRP3 priming by lipopolysaccharide (LPS), the ligand of toll-like receptor 4 (TLR4), activation of the NLRP3 inflammasome triggers caspase-1 maturation, leading to pyroptosis and release of interleukin-1beta (IL-1beta). Expression of TLR4 modulates LPS-triggered inflammatory cascades as well as the NLRP3 signaling. L-type calcium channel antagonists are widely used as anti-hypertensive drugs and also exert anti-inflammatory effects through inhibiting release of cytokines including IL-1beta. However, few studies reveal effects of L-type calcium channel antagonists on the NLRP3 inflammasome. In this study, we investigated the effects of nicardipine and verapamil, both L-type calcium channel antagonists, on the NLRP3 inflammasome using differentiated THP-1 cells. Pyroptosis or levels of IL-1beta and caspase-1 were assayed by flow cytometry or enzyme-linked immunosorbent assay, respectively. ASC oligomerization was assayed by immunofluorescence microscopy. Expression of NLRP3 or TLR4 was assayed by polymerase chain reaction and immunoblotting. Nuclear factor-kappaB (NF-kappaB) pathway was also studied. Our results showed that pyroptosis and IL-1beta release were attenuated by nicardipine, but not verapamil. Nicardipine also mitigated caspase-1 activation, inhibited ASC oligomerization, and reduced NLRP3 expression. Furthermore, nicardipine downregulated phosphorylation or nuclear translocation of NF-kappaB p65, consistent with the inhibitory effect of nicardipine on LPS-induced TLR4 expression. In conclusion, nicardipine exerted anti-inflammatory effects through inhibiting NLRP3 inflammasome pathway. Nicardipine may mitigate NLRP3 priming via inhibiting NF-kappaB activation, mediated by suppressing LPS-induced TLR4 expression.