Inhibition of SIRT7 overcomes sorafenib acquired resistance by suppressing ERK1/2 phosphorylation via the DDX3X-mediated NLRP3 inflammasome in hepatocellular carcinoma.

AIMS: Sirtuin 7 (SIRT7) plays an important role in tumor development, and has been characterized as a potent regulator of cellular stress. However, the effect of SIRT7 on sorafenib acquired resistance remains unclear and a possible anti-tumor mechanism beyond this process in HCC has not been clarified. We examined the therapeutic potential of SIRT7 and determined whether it functions synergistically with sorafenib to overcome chemoresistance. METHODS: Cancer Genome Atlas-liver HCC data and unbiased gene set enrichment analyses were used to identify SIRT7 as a potential effector molecule in sorafenib acquired resistance. Two types of SIRT7 chemical inhibitors were developed to evaluate its therapeutic properties when synergized with sorafenib. Mass spectrometry was performed to discover a direct target of SIRT7, DDX3X, and DDX3X deacetylation levels and protein stability were explored. Moreover, an in vivo xenograft model was used to confirm anti-tumor effect of SIRT7 and DDX3X chemical inhibitors combined with sorafenib. RESULTS: SIRT7 inhibition mediated DDX3X depletion can re-sensitize acquired sorafenib resistance by disrupting NLRP3 inflammasome assembly, finally suppressing hyperactive ERK1/2 signaling in response to NLRP3 inflammasome-mediated IL-1ß inhibition. CONCLUSIONS: SIRT7 is responsible for sorafenib acquired resistance, and its inhibition would be beneficial when combined with sorafenib by suppressing hyperactive pro-cell survival ERK1/2 signaling.

Tangeretin Attenuates Cerebral Ischemia-Reperfusion-Induced Neuronal Pyroptosis by Inhibiting AIM2 Inflammasome Activation via Regulating NRF2.

Pyroptosis is closely involved in the pathopoiesis of cerebral ischemia and reperfusion (I/R) injury which seriously dangers human's life. Studies report that tangeretin (TANG), which is enriched in the peel of Citrus reticulata, has neuroprotective effects. Here, we explored whether absent in melanoma 2 (AIM2) inflammasome-mediated pyroptosis is involved in the cerebral I/R injury and the protective mechanism of TANG against cerebral I/R injury. In this study, we found that TANG treatment effectively alleviated I/R-induced brain injury and inhibited neuronal pyroptosis in an in vivo mice model with middle cerebral artery occlusion/reperfusion (MCAO/R) injury and in an in vitro hippocampal HT22 cell model with oxygen-glucose deprivation and reoxygenation (OGD/R) injury. Furthermore, we found TANG inhibited cerebral I/R-induced neuronal AIM2 inflammasome activation in vivo and in vitro via regulating nuclear factor E2-related factor 2 (NRF2). Moreover, administration of ML385, a chemical inhibitor of NRF2, notably blocked the neuroprotective effects of TANG against cerebral I/R injury. In conclusion, TANG attenuates cerebral I/R-induced neuronal pyroptosis by inhibiting AIM2 inflammasome activation via regulating NRF2. These findings indicate TANG is a potential therapeutic agent for cerebral I/R injury.

Lipopolysaccharide (LPS) stimulation of Pancreatic Ductal Adenocarcinoma (PDAC) and macrophages activates the NLRP3 inflammasome that influences the levels of pro-inflammatory cytokines in a co-culture model.

Modified macrophages, tumor-associated macrophages (TAMs), are key contributors to the survival, growth, and metastatic behavior of pancreatic ductal adenocarcinoma (PDAC) cells. Central to the role of inflammation and TAMs lies the NLRP3 inflammasome. This study investigated the effects of LPS-stimulated inflammation on cell proliferation, levels of pro-inflammatory cytokines, and the NLRP3 inflammasome pathway in a co-culture model using PDAC cells and macrophages in the presence or absence of MCC950, a NLRP3-specific inhibitor. The effects of LPS-stimulated inflammation were tested on two PDAC cell lines (Panc 10.05 and SW 1990) co-cultured with RAW 264.7 macrophages. Cell proliferation was determined using the MTT assay. Levels of pro-inflammatory cytokines, IL-1ß, and TNF-α were determined by ELISA. Western blot analyses were used to examine the expression of NLRP3 in both PDAC cells and macrophages. The co-culture and interaction between PDAC cell lines and macrophages led to pro-inflammatory microenvironment under LPS stimulation as evidenced by high levels of secreted IL-1ß and TNF-α. Inhibition of the NLRP3 inflammasome by MCC950 counteracted the effects of LPS stimulation on the regulation of the NLRP3 inflammasome and pro-inflammatory cytokines in PDAC and macrophages. However, MCC950 differentially modified the viability of the metastatic vs primary PDAC cell lines. LPS stimulation increased PDAC cell viability by regulating the NLRP3 inflammasome and pro-inflammatory cytokines in the tumor microenvironment of PDAC cells/macrophages co-cultures. The specific inhibition of the NLRP inflammasome by MCC950 effectively counteracted the LPS-stimulated inflammation.

Interplay between inflammasomes and PD-1/PD-L1 and their implications in cancer immunotherapy.

The inflammasomes play crucial roles in inflammation and cancer development, while the PD-1/PD-L1 pathway is critical for immune suppression in the tumor microenvironment (TME). Recent research indicates a reciprocal regulatory relationship between inflammasomes and PD-1/PD-L1 signaling in cancer development and PD-1 blockade treatment. By activating in diverse cells in tumor tissues, inflammasome upregulates PD-L1 level in the TME. Moreover, the regulation of PD-1/PD-L1 activity by inflammasome activation involves natural killer cells, tumor-associated macrophages and myeloid-derived suppressor cells. Conversely, PD-1 blockade can activate the inflammasome, potentially influencing treatment outcomes. The interplay between inflammasomes and PD-1/PD-L1 has profound and intricate effects on cancer development and treatment. In this review, we discuss the crosstalk between inflammasomes and PD-1/PD-L1 in cancers, exploring their implications for tumorigenesis, metastasis and immune checkpoint inhibitor (ICI) resistance. The combined therapeutic strategies targeting both inflammasomes and checkpoint molecules hold promising potential as treatments for cancer.

Formononetin ameliorates the LPS-induced inflammatory response and apoptosis of neuronal cells via NF-κB/NLRP3 signaling pathway.

The objective of this study was to investigate the impact of formononetin on cellular apoptosis and inflammatory responses following spinal cord injury (SCI), as well as the underlying mechanisms involved. In this study, PC12 cells were treated with lipopolysaccharide (LPS) and different concentrations of Formononetin (FT) (50 µM, 100 µM, 200 µM). To confirm the effect of nuclear factor-κB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3) signaling pathways, the cells in the phorbol-12-myristate-13-acetate (PMA) group were treated with 0.1 µmol/L PMA (NF-κB/NLRP3 signaling pathway activators). The lactate dehydrogenase (LDH) concentration and cell viability, proliferating cell nuclear antigen (PCNA) fluorescence intensity, and cell apoptosis were determined using an LDH kit, Cell Counting Kit-8 (CCK-8), immunofluorescence, and flow cytometry assays, respectively. Tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-16 (IL-6) expression levels were detected by quantitative ELISA assay. The expression of proteins related to the NF-κB/NLRP3 signaling pathway was detected by western blotting. Our results showed that LPS increased LDH levels in PC12 cells, suggesting that inflammation caused PC12 cell damage. However, the PC12 cell damage was decreased by methylprednisolone. Formononetin promotes cell survival and proliferation, and prevents apoptosis in a concentration-dependent manner. Formononetin reduced the TNF-α, IL-1ß, and IL-6 levels in the LPS-treated model. Moreover, formononetin decreased the levels of p-p65 NF-κB and NLRP3 in PC12 cells. We conclude that formononetin ameliorated the inflammatory response and apoptosis in LPS-induced inflammatory injury in neuronal cells via the NF-κB/NLRP3 signaling pathway.

Effect of NLRP3 deficiency on cytotoxic and IL-1ß-producing activities of synthetic candidalysin peptide.

OBJECTIVES: Candidalysin (CL), a hydrophobic peptide toxin secreted by Candida albicans, is a key virulence factor that contributes to cytolysis, tissue damage, and immune activation. CL is thought to exert some of its biological activities, including IL-1ß production, through the activation of the NLRP3-inflammasome pathway. To date, the mechanism by which CL affects human NLRP3 is not fully understood. We investigated specific activities of synthetic CL peptides using human-derived NLRP3-deficient cells. METHODS: Two distinct synthetic CL peptide solutions were prepared: CLd, with CL completely solubilized as nanoparticles in dimethyl sulfoxide, and CLw, with CL partly solubilized in water, and including insoluble microparticles. THP-1 human monocytic cells and NLRP3-deficient THP-1 cells were differentiated into macrophages and stimulated with these peptide solutions. Cell membrane damage, lactate dehydrogenase release, IL-1ß production, and caspase-1 activation in stimulated cells were subsequently evaluated. RESULTS: Both CLd and CLw exhibited cytotoxic activities independent of NLRP3. Importantly, CLd induced IL-1ß production and caspase-1 activation in an NLRP3-independent manner, whereas these activities in CLw-stimulated cells were entirely NLRP3-dependent, suggesting that the NLRP3-dependent response might be triggered by insoluble microparticles. CONCLUSIONS: Our results demonstrate that inherent CL activities can cause cell damage and IL-1ß production in an NLRP3-independent manner. Our research advances the elucidation of the role of NLRP3 in CL biological activity, underscoring the necessity for further exploration of the precise mechanisms underlying the NLRP3-independent effects of CL and providing novel insights into the complexity of host-pathogen interactions.

Inhibition of the uric acid efflux transporter ABCG2 enhances stimulating effect of soluble uric acid on IL-1ß production in murine macrophage-like J774.1 cells.

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1ß secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1ß production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1ß production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1ß, mature IL-1ß, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1ß, mature IL-1ß and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1ß and mature IL-1ß in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1ß production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1ß production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1ß transcription in macrophage-like J774.1 cells.

Knockout of Erbin promotes pyroptosis via regulating NLRP3/caspase-1/Gasdermin D pathway in sepsis-induced acute kidney injury.

BACKGROUND: This study aims to investigate the correlation between Erbin and sepsis, and the role of Erbin on the pyroptosis pathway in acute kidney injury caused by sepsis and NLRP3/caspase-1/Gasdermin D pathway. METHODS: In the study, lipopolysaccharide (LPS) treatment or cecal ligation and puncture (CLP) surgery on mice were used to stimulate the in vitro and in vivo sepsis-induced renal injury model. The male C57BL/6 of wild-type mice (WT) and Erbin-knockout mice (Erbin-/-, EKO) were randomly divided into four groups (WT + Sham, WT + CLP, EKO + Sham, EKO + CLP). Inflammatory cytokine, renal function, pyroptotic cell numbers and the levels of protein and mRNA expression of pyroptosis, including the NLRP3 (all P < 0.05), were analyzed and found increase in Erbin-/- mice with CLP and LPS-induced HK-2 cells. RESULTS: The inhibited of Erbin shows a renal damaged effect by promoting NLRP3 inflammasome-mediated pyroptosis in SI-AKI. CONCLUSION: This study demonstrated a novel mechanism by which Erbin regulates NLRP3 inflammasome-mediated pyroptosis in SI-AKI.

The role of pyroptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the main histologic type of liver cancer. It accounts for the majority of all diagnoses and deaths due to liver cancer. The induction of tumor cell death is an effective strategy to control tumor development. Pyroptosis is an inflammatory programmed cell death caused by microbial infection, accompanied by activation of inflammasomes and release of pro-inflammatory cytokines, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18). The cleavage of gasdermins (GSDMs) promotes the occurrence of pyroptosis leading to cell swelling, lysis, and death. Accumulating evidence has indicated that pyroptosis influences the progression of HCC by regulating immune-mediated tumor cell death. Currently, some researchers hold the view that inhibition of pyroptosis-related components may prevent the incidence of HCC, but more researchers have the view that activation of pyroptosis exerts a tumor-inhibitory effect. Growing evidence indicates that pyroptosis can prevent or promote tumor development depending on the type of tumor. In this review, pyroptosis pathways and pyroptosis-related components were discussed. Next, the role of pyroptosis and its components in HCC was described. Finally, the therapeutic significance of pyroptosis in HCC was discussed.

Oridonin alleviates the inhibitory effect of lipopolysaccharide on the proliferation and osteogenic potential of periodontal ligament stem cells by inhibiting endoplasmic reticulum stress and NF-κB/NLRP3 inflammasome signaling.

BACKGROUND: The aim of this study was to investigate the protective effect and mechanism of oridonin in an in vitro lipopolysaccharide (LPS)-induced human periodontal ligament stem cells (hPDLSCs) model of periodontitis. METHODS: Primary hPDLSCs were isolated and cultured, and then the expression of surface antigens CD146, STRO-1 and CD45 of hPDLSCs was detected by flow cytometry. The mRNA expression level of Runx2, OPN, Col-1, GRP78, CHOP, ATF4 and ATF6 in the cells was tested by qRT-PCR. MTT was taken to determine the cytotoxicity of oridonin at different concentrations (0-4 µM) on hPDLSCs. Besides, ALP staining, alizarin red staining and Oil Red O staining were utilized to assess the osteogenic differentiation (ALP concentration, mineralized calcium nodule formation) and adipogenic differentiation abilities of the cells. The proinflammatory factors level in the cells was measured by ELISA. The protein expression level of NF-κB/NLRP3 pathway-related proteins and endoplasmic reticulum (ER) stress-related markers in the cells were detected by Western blot. RESULTS: hPDLSCs with positive CD146 and STRO-1 expression and negative CD45 expression were successfully isolated in this study. 0.1-2 µM of oridonin had no significant cytotoxicity on the growth of hPDLSCs, while 2 µM of oridonin could not only greatly reduce the inhibitory effect of LPS on the proliferation and osteogenic differentiation of hPDLSCs cells, but also inhibit LPS-induced inflammation and ER stress in hPDLSCs cells. Moreover, further mechanism research showed that 2 µM of oridonin suppressed NF-κB/NLRP3 signaling pathway activity in LPS-induced hPDLSCs cells. CONCLUSIONS: Oridonin promotes proliferation and osteogenic differentiation of LPS-induced hPDLSCs in an inflammatory environment, possibly by inhibiting ER stress and NF-κB/NLRP3 pathway. Oridonin may have a potential role in the repair and regeneration of hPDLSCs.

[Methylophiopogonanone a Inhibits LPS/ATP-Induced Macrophage Pyroptosis via ROS/NLRP3 Pathway].

As a byproduct of mitochondrial respiration or metabolism, reactive oxygen species (ROS) can act as a signaling molecule to activate NLR family pyrin domain containing 3 (NLRP3) inflammasome, thereby triggering immune response. NLRP3 inflammasome acts as a sensor of various danger signals and is central to the control of pyroptosis occurrence. Macrophage pyroptosis is closely related to atherosclerosis, arthritis, pulmonary fibrosis and other inflammatory diseases. Methylophiopogonanone A (MO-A) is a main homoisoflavonoid in Chinese herb Ophiopogonis Radix, which has antioxidant effect. However, it is not clear whether MO-A can alleviate macrophage pyroptosis by inhibiting oxidative stress. Here we have shown that MO-A increases the activities of superoxide dismutase (SOD) and catalase (CAT), inhibits the production of ROS, reduces the activation of NLRP3 inflammasome and the release of lactate dehydrogenase (LDH), and inhibits pyroptosis in macrophages induced by lipopolysaccharides (LPS) and adenosine triphosphate (ATP). These effects can be reversed by the ROS promoter H2O2. Therefore, MO-A can inhibit macrophage pyroptosis through the ROS/NLRP3 pathway and may be considered as a candidate drug for the treatment of inflammatory diseases.

NLRX1 increases human retinal pigment epithelial autophagy and reduces H2O2-induced oxidative stress and inflammation by suppressing FUNDC1 phosphorylation and NLRP3 activation.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of impaired vision as well as some earlier effects, such as reading and face recognition. Oxidative damage and inflammation of retinal pigment epithelial (RPE) cells are major causes of AMD. Additionally, autophagy in RPE cells can lead to cellular homeostasis under oxidative stress. Nucleotide-binding oligomerization domain (NOD)-like receptor X1 (NLRX1) is a mysterious modulator of the immune system function which inhibits inflammatory response, attenuates reactive oxygen species (ROS) production, and regulates autophagy. This study attempted to explore the role of NLRX1 in oxidative stress, inflammation, and autophagy in AMD. METHODS: An in vitro model of AMD was built in human retinal pigment epithelial cell line 19 (ARPE-19) treated with H2O2. The cell viability, NLRX1 expressions, levels of superoxide dismutase (SOD), glutathione (GHS), and ROS, concentrations of interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), IL-6, and monocyte chemoattractant protein-1 (MCP-1), expressions of NLRX1, p62, LC3-II/LC3-I, FUNDC1, and NOD-like receptor protein 3 (NLRP3) inflammasome were expounded by cell counting kit-8, colorimetric, enzyme-linked immunosorbent serologic assay (ELISA), and Western blot assay. RESULTS: H2O2 treatment notably reduced the relative protein expression of NLRX1. Meanwhile, H2O2 incubation decreased cell viability, diminished SOD and GSH concentrations, accompanied with the increased level of ROS, enhanced IL-1ß, TNF-α, IL-6, and MCP-1 concentrations, and aggrandized the relative protein expression of p62 with reduced LC3-II/LC3-I ratio. Moreover, these results were further promoted with knockdown of NLRX1 and reversed with overexpression. Mechanically, silencing of NLRX1 further observably enhanced the relative levels of -phosphorylated FUNDC1/FUNDC1, and NLRP3 inflammasome-related proteins, while overexpression of NLRX1 exhibited inverse results in the H2O2-induced ARPE-19 cells. CONCLUSION: NLRX1 suppressed H2O2-induced oxidative stress and inflammation, and facilitated autophagy by suppressing FUNDC1 phosphorylation and NLRP3 activation in ARPE-19 cells.

Tacrolimus prevents complement-mediated Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis of mesenchymal stem cells from immune thrombocytopenia.

The abnormal immunomodulatory functions of mesenchymal stem cells (MSCs) have been implicated in the development of immune thrombocytopenia (ITP). Recent studies have suggested important effects of complement on immune cell function. However, whether complement modulates bone marrow MSCs function in ITP is poorly defined. Tacrolimus has recently been applied to the treatment of autoimmune diseases. Here, we explored whether impaired ITP-MSCs could be targeted by tacrolimus. Our results showed that the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome was activated in ITP MSCs with complement deposition (MSCs-C+ ) and initiated caspase-1-dependent pyroptosis. Transcriptome sequencing results showed abnormal fatty acid metabolism in MSCs-C+ . Enhanced fatty acid ß-oxidation and reactive oxygen species production activated the NLRP3 inflammasome. Adipocytes derived from MSCs-C+ secreted less adiponectin. Adiponectin promoted the differentiation of megakaryocytes and inhibited the destruction of platelets. Tacrolimus inhibited NLRP3 inflammasome activation and MSCs-C+ pyroptosis in vitro and in vivo. Tacrolimus plus danazol elicited a higher sustained response than danazol monotherapy in corticosteroid-resistant patients with ITP. Our findings demonstrate that the activation of the NLRP3 inflammasome in ITP MSCs mediated by complement could be inhibited by tacrolimus, which might be a potential new therapy for ITP.

Triclosan Reprograms Immunometabolism and Activates the Inflammasome in Human Macrophages.

To gather enough energy to respond to harmful stimuli, most immune cells quickly shift their metabolic profile. This process of immunometabolism plays a critical role in the regulation of immune cell function. Triclosan, a synthetic antibacterial component present in a wide range of consumer items, has been shown to cause immunotoxicity in a number of organisms. However, it is unclear whether and how triclosan impacts immunometabolism. Here, human macrophages were used as model cells to explore the modulatory effect of triclosan on immunometabolism. Untargeted metabolomics using integrated liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) revealed that triclosan changed the global metabolic profile of macrophages. Furthermore, Seahorse energy analysis and 13C isotope-based metabolic flux analysis revealed that triclosan decreased mitochondrial respiratory activity and promoted a metabolic transition from oxidative phosphorylation to glycolysis. Triclosan also polarizes macrophages to the proinflammatory M1 phenotype and activates the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing receptor 3 (NLRP3) inflammasome, which is consistent with triclosan-induced metabolic phenotypic modifications. Collectively, these findings showed that triclosan exposure at micromolar concentrations caused metabolic reprogramming in macrophages, which triggered an inflammatory response. These findings are important for understanding the immunotoxicity caused by triclosan, which is necessary for determining the risk posed by triclosan in the environment.

Total saponins of Aralia elata (Miq.) Seem. alleviate myocardial ischemia-reperfusion injury by promoting NLRP3-inflammasome inactivation via PI3K/Akt signaling.

Total saponins of Aralia elata (Miq.) Seem. (TSAE) have been shown to play a significant role in cardiovascular protection, anti-tumor, liver protection, anti-oxidant stress, and anti-inflammation. However, the specific mechanisms of TSAE in myocardial ischemia-reperfusion injury (MIRI) remain largely elusive. Hearts from male Wistar rats were used to establish the isolated heart MIRI model. Using a multichannel physiological recorder, the whole course heart rate (HR), left ventricular development pressure (LVDP), and maximum rise/decrease rate of left ventricular pressure (±dp/dtmax ) were recorded. 2,3,5-triphenyl-2H-tetrazolium chloride staining observed the infarct area, while hematoxylin & eosin staining detected pathological changes in myocardial tissue. Creatine kinase, lactate dehydrogenase, total superoxide dismutase, and malondialdehyde concentrations were determined by enzyme-linked immunosorbent assay. Immunohistochemistry, quantitative PCR, and western blot assay were used to assess the amounts of IL-18 and IL-1ß, NLR family protein (NLRP3) inflammasome- and apoptosis-related proteins, respectively. Treatment with TSAE or MCC950 (NLRP3-specific inhibitor) significantly reduced the myocardial infarction area, alleviated pathological changes in myocardial tissues, enhanced LVDP and ±dp/dtmax levels, prevented myocardial oxidative damage, and inhibited NLRP3 inflammasome formation. In addition, TSAE enhanced Akt and GSK3ß phosphorylation, and LY29004 co-reperfusion markedly diminished the protective role of TSAE reperfusion on cardiac function, oxidative damage, and inflammatory responses. Collectively, TSAE treatment exhibited a protective effect on I/R-triggered inflammatory responses, cell necrosis, and oxidative stress injury by stimulating PI3K/Akt signaling-mediated NLRP3 inflammasome inhibition.

Effects of glucose on the proliferation of human umbilical cord blood hematopoietic stem cells.

Hematopoietic stem cells (HSCs) can be isolated through umbilical cord blood (UCB), which can be used for HSC transplantation. Despite many advantages, the low number of UCB CD34+ cells lead to delayed engraftment. Ex-vivo CD34+HSC expansion is a potentially safe approach to increasing CD34+ cell numbers. The NLR family of pyrin domain-containing 3 (NLRP3) is an intracellular protein that plays an essential role in the innate immune response. Several blood cell types, HSCs and progenitor cells (HSPCs) express the NLRP3 inflammasome complex genes and participate in the development and proliferation of HSPCs. In this study, magnetic-activated cell sorting (MACS) beads isolated CD34+HSCs. The cell purity was evaluated by flow cytometry. CD34+ cells, under the influence of different doses of glucose, MCC950 were cultured for seven days. The qRT-PCR was used to evaluate gene expression. The results showed that in the culture medium treated with glucose concentrations, the expression of the NLRP3 inflammasome complex genes and the amount of CD34+ cells increased by more than 50%. In contrast, genes expression and the number of CD34+ cells in the culture medium treated with MCC950 decreased. UCB is a source of new therapeutic methods. This study demonstrates the relationship between glucose and the activation of the NLRP3 inflammasome. Based on these results, glucose causes the expansion of CD34+HSCs through its effect on HSCs in simultaneous culture.

Aryl-, halogenated- and alkyl- organophosphate esters induced oxidative stress, endoplasmic reticulum stress and NLRP3 inflammasome activation in HepG2 cells.

Organophosphate esters (OPEs) are a group of extensively used man-made chemicals with diverse substituents that are ubiquitously detected in human-related samples including serum, breastmilk, food and house dust. The understanding of their toxicological effects and potential mechanisms on hepatocytes is still limited. In this study, nine most frequently detected OPEs were selected and divided into three subgroups (aryl-, halogenated- and alkyl-OPEs) based on their substituents. The cytotoxicity, apoptosis, oxidative stress, endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation induced by OPEs were evaluated in human hepatocellular carcinomas HepG2 cells. All OPEs induced apoptosis likely through a caspase-dependent apoptotic pathway. The activities of anti-oxidative enzyme SOD and CAT exhibited sensitive responses after OPEs treatment for 6 h. The OPEs induced ROS overproduction, DNA damage, endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation varied among aryl-, halogenated- and alkyl-OPEs. Halogenated- and alkyl- OPEs induced overproduction of ROS and DNA damage, and elevated ER stress and NLRP3 inflammasome activation are observed aryl-OPEs induced cytotoxicity.

Exogenous 8-hydroxydeoxyguanosine attenuates doxorubicin-induced cardiotoxicity by decreasing pyroptosis in H9c2 cardiomyocytes.

Doxorubicin (DOX), which is widely used in cancer treatment, can induce cardiomyopathy. One of the main mechanisms whereby DOX induces cardiotoxicity involves pyroptosis through the NLR family pyrin domain containing 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Increased NAPDH oxidase (NOX) and oxidative stress trigger pyroptosis. Exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases reactive oxygen species (ROS) production by inactivating NOX. Here, we examined whether 8-OHdG treatment can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. Exposure to DOX increased the peroxidative glutathione redox status and NOX1/2/4, toll-like receptor (TLR)2/4, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, while an additional 8-OHdG treatment attenuated these effects. Furthermore, DOX induced higher expression of NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a c-terminal caspase recruitment domain (ASC), and pro-caspase-1. Moreover, it increased caspase-1 activity, a marker of pyroptosis, and interleukin (IL)-1ß expression. All these effects were attenuated by 8-OHdG treatment. In addition, the expression of the cardiotoxicity markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was increased by DOX, whereas the increase of ANP and BNP induced by DOX treatment was reversed by 8-OHdG. In conclusion, exogenous 8-OHdG attenuated DOX-induced pyroptosis by decreasing the expression of NOX1/2/3, TLR2/4, and NF-κB. Thus, 8-OHdG may attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis.

M2 Macrophage-Derived Exosomes Regulate Myocardial Ischemia-Reperfusion And Pyroptosis Via ROS/NLRP3 Pathway.

OBJECTIVE: To evaluate whether M2 macrophage-derived exosomes protect against MI/R injury and reveal the protective mechanism of exosomes [Kourembanas 2015]. METHODS: I/R model injury was induced by temporary left anterior descending coronary artery occlusion in Sprague-Dawley (SD) rats, macrophages isolated from bone marrow-derived macrophages (BMDMs) were induced to M2 polarization, and H9C2 cells subjected to hypoxia/reperfusion (H/R) were used to establish an in vitro model. I/R-induced rats and H/R-induced H9C2 cells were treated with M2-exos in vivo and in vitro, respectively. Masson staining was performed to observe myocardial fibrosis in rats. Immunohistochemical (IHC) staining of myocardial tissues showed the expression of NLRP3 inflammasome activation and pyrolysis. Exosomes derived from IL-4-treated macrophages (M2-exos) were detected by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western bolt. Western bolt was performed to determine the protein level, including NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, cleaved IL-1ß, gasdermin D (GSDMD), and N-terminus of gasdermin D (GSDMD-N). RESULTS: Activity of NLRP3 inflammasome and existence of pyroptosis in the rats subjected to MI/R were significantly higher than those in the control (P < 0.05). Moreover, we confirmed the accumulation of ROS during I/R injury in cardiomyocytes. M2-exos protected against I/R injury and reduced activity of NLRP3 inflammasome and existence of pyroptosis, accompanied with attenuating oxidative stress. In vitro studies showed similar effects, H9c2 cells co-cultured with M2-exos could attenuated H/R-induced cell injury, while M2-exos suppressed the expression of NLRP3 inflammasome and pyroptosis (P < 0.05).

Daptomycin exerts differential immunomodulatory effects on host responses against methicillin-resistant Staphylococcus aureus biofilms.

Daptomycin (DAP) is indicated for difficult-to-treat Gram-positive infections, especially those caused by methicillin-resistant Staphylococcus aureus (MRSA). Exposure of S. aureus to subinhibitory antimicrobial concentrations (sub-MICs) has been shown to alter cell morphology and biofilm formation. This study aimed to investigate the influence of DAP biofilm sub-MICs on the damage caused by human polymorphonuclear neutrophils (PMNs) against MRSA biofilms and the potential immunomodulatory activity of DAP on human monocytes (MNCs) exposed to MRSA biofilms. DAP activity against biofilms and the impact of DAP on PMN-induced biofilm damage were evaluated by the XTT reduction assay, whereas pathogen recognition, signal transduction and cytokine modulation of DAP on MNCs in response to MRSA biofilms were assessed by RT-PCR and ELISA methodology. The MIC50 of DAP to MRSA biofilms was 16-32 mg/L. Pre-treatment of MRSA with 1, 2 or 4 mg/L DAP caused a synergistic effect on PMN-mediated biofilm damage, being dependent on the effector-to-target ratio. MNCs responded to MRSA biofilms and DAP through Toll like receptor 2 (TLR2) upregulation and increased NLRP3 inflammasome production. DAP caused 2.5-fold greater TLR2 mRNA levels than those caused by MRSA biofilms. A predominantly inflammatory response was induced by either component, causing the release of significantly increased IFN-γ, TNF-α, IL-8 and IL-6 levels by MNCs exposed to the combination treatment. MRSA biofilms alone or combined with DAP caused low amounts of IL-10 production, but increased IL-1ß levels. DAP may condition MNCs towards an inflammatory response through TLR2 engagement and NLRP3 inflammasome activation, possibly controlling biofilm-associated pathogenicity.