Expression Of NOD-like Receptor Protein 3 Research Articles

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.

Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1β and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1β and IL-18 in the serum and spleen index(P<0.01), significantly lowered arthritis score and degree of paw edema(P<0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P<0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

Astrocytic Kir4.1 regulates NMDAR/calpain signaling axis in lipopolysaccharide-induced depression-like behaviors in mice

The activation of Nod-like receptor protein 3 (NLRP3) inflammasome propagates pro-inflammatory signaling cascades linking to depression-like behaviors. However, the signaling pathway contributing to NLRP3 inflammasome activation and depression-like behaviors is still not clear. In this study, we evidenced that lipopolysaccharide (LPS) injection (i.p.) triggered depression-like behaviors, promoted the expression of Kir4.1, p-GluN2B and calpain-1, and activated NLRP3 inflammasome. The blockage of N-methyl-d-aspartate receptors (NMDAR) by memantine reduced LPS-induced depression-like behaviors, NLRP3 inflammasome and astrocyte activation, and calpain-1 expression. Additionally, memantine also inhibited LPS-induced reduction of postsynaptic density protein 95 (PSD-95) and Arc expression. Specific reduction of Kir4.1 in astrocytes attenuated LPS-induced expression of NLRP3 and calpain-1, and phosphorylation of GluN2B. Interestingly, LPS-induced expression of calpain-1 largely co-localized with GFAP, indicating the specific function of calpain-1 in astrocytes. Together, these data indicate that astrocytic Kir4.1 could regulate NMDAR/calpain-1 signaling axis, contributing to depression-like behaviors, likely through regulating NLRP3 inflammasome activation.

Selenomethionine alleviates LPS-induced JNK/NLRP3 inflammasome-dependent necroptosis by modulating miR-15a and oxidative stress in chicken lungs.

Selenium (Se) was involved in many physiological processes in humans and animals. microRNAs (miRNAs) also played important roles in lung diseases. However, the regulatory mechanism of miRNA in chicken lungs and the mechanism of lipopolysaccharide (LPS)-induced pneumonia remained unclear. To further study these mechanisms, we established a supplement of selenomethionine (SeMet) and/or LPS-treated chicken model and a cell model of LPS and/or high and low expression of miR-15a in chicken hepatocellular carcinoma (LMH) cells. We detected the expression of some selenoproteins, p-c-Jun N-terminal kinase (JNK), nod-like receptor protein 3 (NLRP3), caspase1, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL), miR-15a, and oxidative stress kits. Additionally, we observed the morphology of lungs by H.E. staining in vitro. The results indicated that necroptosis occurred in LPS-treated chicken and LMH cells. Moreover, LPS stimulation inhibited miR-15a, and increased the expression of JNK, NLRP3, caspase1, RIPK1, RIPK3, and MLKL. We also found that LPS treatment not only increased the content of H2O2 and MDA in the lungs but also increased the activities of iNOS and CAT and the content of GSH decreased. Conclusion: SeMet could reduce the oxidative damage and activate NLRP3 inflammasome reaction by stimulating miR-15a/JNK, thus reduced the pulmonary necroptosis induced by LPS.

NLRP3 Overexpression Associated With Poor Prognosis and Presented as an Effective Therapeutic Target in Osteosarcoma.

Despite the development of diagnostic and treatment strategies, the survival outcome of patients with osteosarcoma remains poor. Nod-like receptor protein 3 (NLRP3) plays a crucial role in the inflammasome pathway, which is related to the progression of various tumors. However, the effect of NLRP3 on osteosarcoma has not yet been well explored. Our study aimed to investigate the role of NLRP3 in the malignant biological behavior of osteosarcoma as well as its therapeutic value. Immunohistochemistry was applied to investigate the NLRP3 expression in osteosarcoma and osteochondroma specimens. Cell Counting Kit-8, colony formation, wound healing, transwell, and flow cytometry assays were used to explore the contribution of NLRP3 to the proliferation, migration, invasion, apoptosis and cell cycle distribution of osteosarcoma cells in vitro. Western blot was performed to evaluate the expression of NLRP3 and the related proteins in osteosarcoma cell lines after the blockade of NLRP3 using CY-09 and lentivirus intervention. Furthermore, tumor formation assay was used to analyze the effect of NLRP3 on the growth of osteosarcoma in vivo. The results showed that the NLRP3 protein was overexpressed in osteosarcoma, which was independently correlated with the poor prognosis of patients. Moreover, NLRP3 suppression by the inhibitor of CY-09 or lentivirus-induced gene knockdown inhibited the cell proliferation, migration, invasion and promoted the cell apoptosis and G1 cell cycle arrest in osteosarcoma via targeting the inflammasome pathway. Our in vivo results confirmed that the inhibition of NLRP3 suppressed the tumor formation of osteosarcoma. In conclusion, NLRP3 may be regarded as an independent prognostic biomarker and a potential therapeutic target for osteosarcoma.

Activation of NOD-like receptor protein 3 inflammasome mediates inflammatory response and apoptosis in septic intestinal injury model

To investigate the expression of NOD-like receptor protein 3 (NLRP3) inflammasome in intestinal injury models with different severity of sepsis and the inflammatory response and apoptosis mediated by NLRP3 inflammasome. Human colorectal adenocarcinoma cells (Caco-2) were cultured in vitro. The logarithmic growth phase cells were divided into blank control group (normal culture in complete medium) and lipopolysaccharide (LPS) 1, 2 and 4 mg/L groups (complete medium containing 1, 2 and 4 mg/L LPS, respectively). The supernatant were collected at 6, 12 and 24 hours, and the levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β, IL-18) were detected by enzyme linked immunosorbent assay (ELISA). The apoptotic level of cells was detected by flow cytometry. The cells were harvested, and the real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of NLRP3 and silent information regulator 1 (SIRT1). Western blotting was used to detect the protein expressions of NLRP3, SIRT1, caspase-1 and apoptosis-associated speck-like protein (ASC). ELISA results showed that the levels of IL-6, TNF-α, IL-1β, and IL-18 in cell supernatant of LPS groups increased in a dose-dependent and time-dependent manner as compared with the blank control group during the same intervention period. The increase was most significant in LPS 4 mg/L group at 24 hours [IL-6 (ng/L): 3.55±0.06 vs. 0.67±0.09, TNF-α (ng/L): 15.37±0.19 vs. 5.04±0.14, IL-1β (ng/L): 2.26±0.10 vs. 0.56±0.09, IL-18 (ng/L): 433.92±22.55 vs. 93.55±21.13, all P < 0.05]. The results of the apoptotic test showed that, compared with the blank control group, the apoptotic rate of LPS groups increased in a dose-dependent and time-dependent manner, and the apoptotic rate of LPS 4 mg/L group increased most significantly at 24 hours [(14.83±3.73)% vs. (5.87±1.17)%, P < 0.05]. RT-qPCR results showed that the expression level of NLRP3 mRNA was increased, while the expression level of SIRT1 mRNA was decreased with the increase of LPS intervention dose and the prolonging of intervention time. At 24 hours, there were significant differences between LPS 4 mg/L group and blank control group [NLRP3 mRNA (2-ΔΔCt): 8.20±2.82 vs. 1.00±0.36, SIRT1 mRNA (2-ΔΔCt): 0.58±0.01 vs. 1.03±0.06, both P < 0.05]. Western blotting showed that compared with the blank control group, the protein expression levels of NLRP3, caspase-1 and ASC in LPS groups were significantly increased, while the protein expression levels of SIRT1 were significantly decreased. During each intervention period, with the increase of LPS dose, the expressions of NLRP3, caspase-1 and ASC protein increased gradually, while the expression of SIRT1 protein decreased gradually. At 24 hours, the difference between the LPS 4 mg/L group and the blank control group was significant [NLRP3 protein (NLRP3/β-actin): 1.48±0.03 vs. 0.90±0.12, caspase-1 protein (caspase-1/β-actin): 1.18±0.11 vs. 0.72±0.09, ASC protein (ASC/β-actin) : 1.09±0.01 vs. 0.82±0.03, SIRT1 protein (SIRT1/β-actin): 0.48±0.03 vs. 0.76±0.05, all P < 0.05]. In vitro, in the sepsis induced intestinal inflammation model, with the increase of LPS intervention dose and the prolongation of intervention time, intestinal inflammatory response and cell apoptosis showed an increasing trend, which may be related to the up-regulation of NLRP3 inflammasome and its downstream products ASC and caspase-1, and to the down-regulation of SIRT1 expression.

Extracellular vesicles derived from M2 microglia reduce ischemic brain injury through microRNA-135a-5p/TXNIP/NLRP3 axis

Accumulating evidences have suggested that extracellular vesicles (EVs) are crucial players in the pathogenesis of ischemic brain injury. This study was designed to explore the specific functions of M2 phenotype microglia-derived EVs in ischemic brain injury progression. The expression of microRNA-135a-5p (miR-135a-5p) in M2 microglia-derived EVs was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), followed by the identification of expression relationship among miR-135a-5p, thioredoxin-interacting protein (TXNIP), and nod-like receptor protein 3 (NLRP3) by dual luciferase reporter gene assay. After construction of an oxygen-glucose deprivation/reperfusion (OGD/R) cell model, the effects of miR-135a-5p on the biological characteristics of HT-22 cells were assessed by cell counting kit 8 (CCK-8) assay and flow cytometry. Finally, a mouse model of transient middle cerebral artery occlusion (tMCAO) was established and cerebral infarction volume was determined by triphenyltetrazolium chloride (TTC) staining and the expression of IL-18 and IL-1β in the brain tissue was determined by enzyme-linked immunosorbent assay (ELISA). We found that M2 microglia-derived EVs had high expression of miR-135a-5p, and that miR-135a-5p in M2 microglia-derived EVs negatively regulated the expression of NLRP3 via TXNIP. Overexpression of miR-135a-5p promoted the proliferation but inhibited the apoptosis of neuronal cells, and inhibited the expression of autophagy-related proteins. M2 microglia-derived EVs delivered miR-135a-5p into neuronal cells to inhibit TXNIP expression, which further inhibited the activation of NLRP3 inflammasome, thereby reducing neuronal autophagy and ischemic brain injury. Hence, M2 microglia-derived EVs are novel therapeutic targets for ischemic brain injury treatment.M2 microglia-derived EVs inhibit neuronal autophagic activity. Mechanistically, M2 microglia-derived EVs deliver miR-135a-5p into neuronal cells to inhibit TXNIP expression, which further inhibits the activation of NLRP3 inflammasomes, thereby reducing neuronal autophagy and ischemic brain injury. Hence, M2 microglia-derived EVs can serve as novel therapeutic targets for ischemic brain injury.

Sodium Sulfite-Induced Mast Cell Pyroptosis and Degranulation.

Sodium sulfite, a common food additive, has been proved to cause allergic reaction. Pyroptosis is an inflammatory form of programmed cell death with plasma membrane lysis. In this study, we found that sodium sulfite triggered pyroptosis, which depended on reactive oxygen species (ROS)/NOD-like receptor protein 3 (NLRP3) in RBL-2H3 mast cells. Sodium sulfite increased the generation of ROS and the expression of NLRP3, caspase-1, gasdermin D N-terminal (GSDMD-N), interleukin-1β (IL-1β), and interleukin-18 (IL-18). The ROS scavenger N-acetyl-L-carnosine (NAC) and the NLRP3 inhibitor MCC950 reversed these effects. Furthermore, using a lactate dehydrogenase kit, propidium iodide staining, scanning electron microscopy, colocalization of GSDMD-N with histamine, and neutral red staining, we found that sodium sulfite notably induced cell membrane rupture. Because β-Hexosaminidase and histamine play a key role in allergic reactions, we detected the release of β-Hexosaminidase and histamine. The data showed that the release of β-Hexosaminidase and histamine induced by sodium sulfite was increased with dose independence, which were inhibited after treatment with NAC or MCC950. Overall, evidence suggested that pyroptosis induced by sodium sulfite may rupture the cell membrane and result in degranulation of mast cells. Our study may provide new insights for the mechanism by which sodium sulfite induces mast cell death and sensitization.

Salidroside protects endothelial cells against LPS-induced inflammatory injury by inhibiting NLRP3 and enhancing autophagy

BackgroundSalidroside (SAL) is a bioactive compound extracted from Rhodiola rosea with various biological properties. This study was designed to explore the functions of SAL on the endothelial damage induced by lipopolysaccharide (LPS) and its related mechanisms.MethodsHuman umbilical vein endothelial cells (HUVECs) were pretreated with SAL (0, 10, 25, 50, 100 μM), and then incubated with LPS (10 μg/mL). Cell viability was evaluated by MTT assay, cell injury by lactate dehydrogenase (LDH) release, and inflammatory cytokines release by ELISA assay. Oxidative stress was evaluated by malondialdehyde (MDA) and superoxide dismutase (SOD) in cell lysate. Apoptosis was detected by flow cytometry and caspase-3 activity. Western blot were performed to determine expression levels of autophagy and NOD-like receptor protein 3 (NLRP3) related proteins.ResultsSAL at 50 μM concentration showed no toxicity on HUVECs, but attenuated LPS-induced injury, as evidenced by increased cell viability, reduction in LDH level and inflammatory cytokines in culture media. SAL also reduced MDA level and increased SOD activity in HUVECs, and inhibited apoptosis rate and caspase-3 activity. (P < 0.05). Moreover, LPS enhanced HUVECs autophagy, and SAL pretreatment further enhanced autophagy, with increased Beclin-1 protein and decreased P62 protein. SAL also attenuated LPS-induced activation of NLRP3 inflammasome, reduced the protein expression of NLRP3-related proteins, including ASC and caspase-1. Autophagy inhibition by 3-MA markedly reversed SAL-modulated changes in cell viability and NLRP3 expression in LPS-stimulated HUVECs.ConclusionSAL protects endothelial cells against LPS-induced injury through inhibition of NLRP3 pathways and enhancing autophagy.

NLRP3 induces the autocrine secretion of IL-1β to promote epithelial–mesenchymal transition and metastasis in breast cancer

Tumor metastasis is a leading cause of mortality in patients with breast cancer (BC). As a predominant component of inflammasome, Nod-like receptor protein 3 (NLRP3) was found to be required for tumor progression, while the role of NLRP3 in BC metastasis remains largely undefined. In current study, we found that invasive BC had aberrant upregulation of NLRP3 expression, especially in the claudin-low subtype. And higher expression of NLRP3 predicted poor survival of BC patients. Further investigation suggested that NLRP3 promotes the migration and invasion, as well as the metastasis of BC cells. Moreover, we revealed that NLRP3 induces the autocrine secretion of IL-1β to promote epithelial–mesenchymal transition via a Caspase-1-dependent manner. Hence, this study suggested that upregulation of NLRP3 in BC induces the autocrine secretion of IL-1β and promotes EMT and metastasis of BC cells.

Altered inflammasome activation in neonatal encephalopathy persists in childhood

SummaryNeonatal encephalopathy (NE) is characterized by altered neurological function in term infants and inflammation plays an important pathophysiological role. Inflammatory cytokines interleukin (IL)‐1β, IL‐1ra and IL‐18 are activated by the nucleotide‐binding and oligomerization domain (NOD)‐, leucine‐rich repeat domain (LRR)‐ and NOD‐like receptor protein 3 (NLRP3) inflammasome; furthermore, we aimed to examine the role of the inflammasome multiprotein complex involved in proinflammatory responses from the newborn period to childhood in NE. Cytokine concentrations were measured by multiplex enzyme‐linked immunosorbent assay (ELISA) in neonates and children with NE in the absence or presence of lipopolysaccharide (LPS) endotoxin. We then investigated expression of the NLRP3 inflammasome genes, NLRP3, IL‐1β and ASC by polymerase chain reaction (PCR). Serum samples from 40 NE patients at days 1 and 3 of the first week of life and in 37 patients at age 4–7 years were analysed. An increase in serum IL‐1ra and IL‐18 in neonates with NE on days 1 and 3 was observed compared to neonatal controls. IL‐1ra in NE was decreased to normal levels at school age, whereas serum IL‐18 in NE was even higher at school age compared to school age controls and NE in the first week of life. Percentage of LPS response was higher in newborns compared to school‐age NE. NLRP3 and IL‐1β gene expression were up‐regulated in the presence of LPS in NE neonates and NLRP3 gene expression remained up‐regulated at school age in NE patients compared to controls. Increased inflammasome activation in the first day of life in NE persists in childhood, and may increase the window for therapeutic intervention.

Effect of astragaloside IV on the expression of NOD-like receptor protein 3 inflammasome in neonatal rats with hypoxic-ischemic brain damage

OBJECTIVE To study the effect of astragaloside IV (AS-IV) on NOD-like receptor protein 3 (NLRP3) inflammasome in neonatal rats with hypoxic-ischemic brain damage (HIBD). METHODS A total of 24 Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group, an HIBD group, and an AS-IV treatment group, with 8 rats in each group. After 24 hours of modeling, brain tissue was collected for hematoxylin-eosin staining, yo-PRO-1 staining, and EthD-2 immunofluorescent staining in order to observe the cerebral protection effect of AS-IV in vivo. HT22 cells were used to prepare a model of oxygen-glycogen deprivation (OGD), and a concentration gradient (50-400 μmol/L) was established for AS-IV. CCK-8 assay was used to measure the viability of HT22 cells. RT-PCR and Western blot were used to observe the effect of different concentrations of AS-IV on the mRNA and protein expression of NLRP3, gasdermin D (GSDMD), caspase-1, and interleukin-1β (IL-1β). RESULTS Yo-Pro-1 and EthD-2 staining showed that compared with the sham-operation group, the HIBD group had an increase in pyroptotic cells with a small number of necrotic cells, and the AS-IV group had reductions in both pyroptotic and necrotic cells. Compared with the sham-operation group, the HIBD group had significantly higher protein expression levels of NLRP3, IL-1β, caspase-1, and GSDMD (P < 0.05). Compared with the HIBD group, the AS-IV group had significant reductions in the protein expression levels of NLRP3, caspase-1, and GSDMD (P < 0.05). HT22 cell experiment showed that compared with the OGD group, the AS-IV group had inhibited mRNA and protein expression of NLRP3, GSDMD, caspase-1, and IL-1β, with the best therapeutic effect at the concentration of 200 μmol/L (P < 0.05). CONCLUSIONS AS-IV may alleviate HIBD in neonatal rats by inhibiting the expression of NLRP3, GSDMD, caspase-1, and IL-1β.

Baicalein Exerts Anti-Neuroinflammatory Effects by Inhibiting the TLR4-ROS /NF-κB-NLRP3 Inflammasome

Emerging evidence indicates that NOD-like receptor protein 3 (NLRP3) inflammasome-induced inflammation plays a critical role in the pathogenesis of Parkinson’s disease (PD). Baicalein has been considered as a possible option for PD treatment based on its anti-neuroinflammatory effects. However, no studies have elucidated the precise mechanisms underlying the anti-neuroinflammatory activity of baicalein, particularly inflammasome-mediated effects. In this present study, rotenone-induced PD mice and BV2 microglia were used to investigate the anti-neuroinflammatory effects of baicalein and explore its underlying mechanism in vivo and in vitro. The results demonstrated that baicalein alleviated motor impairments and attenuated several inflammatory responses in rotenone-induced PD mice. Also, baicalein inhibited the expression of NLRP3 and activated caspase-1 in brain tissues. Correspondingly, baicalein prominently suppressed the inflammatory response in BV2 microglia induced by rotenone. Furthermore, in vitro data showed that baicalein suppressed the expression of NLRP3 and activated caspase-1 by abrogating the upregulation of ROS, as well as by inhibiting the TLR4/NF-κB signaling cascade. Overall, the results of the present study indicated that baicalein exerted anti-neuroinflammatory effects partly by inhibiting activation of the NLRP3 inflammasome, and targeting NLRP3 inflammasome signaling offers a novel therapeutic strategy for PD treatment.

Gut microbiota dysbiosis promotes age-related atrial fibrillation by lipopolysaccharide and glucose-induced activation of NLRP3-inflammasome.

Ageing is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). The gut microbiota dysbiosis is involved in age-related diseases. However, whether the aged-associated dysbiosis contributes to age-related AF is still unknown. Direct demonstration that the aged gut microbiota is sufficient to transmit the enhanced AF susceptibility in a young host via microbiota-intestinal barrier-atria axis has not yet been reported. This study aimed to determine whether gut microbiota dysbiosis affects age-related AF. Herein, by using a faecal microbiota transplantation (FMT) rat model, we demonstrated that the high AF susceptibility of aged rats could be transmitted to a young host. Specially, we found the dramatically increased levels of circulating lipopolysaccharide (LPS) and glucose led to the up-regulated expression of NOD-like receptor protein (NLRP)-3 inflammasome, promoting the development of AF, which depended on the enhanced atrial fibrosis in recipient host. Inhibition of inflammasome by a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, resulted in a lower atrial fibrosis and AF susceptibility. Then, we conducted cross-sectional clinical studies to explore the effect of ageing on the altering trends with glucose levels and circulating LPS among clinical individuals in two China hospitals. We found that both of serum LPS and glucose levels were progressively increased in elderly patients as compared with those young. Furthermore, the ageing phenotype of circulating LPS and glucose levels, intestinal structure and atrial NLRP3-inflammasome of rats were also confirmed in clinical AF patients. Finally, aged rats colonized with youthful microbiota restored intestinal structure and atrial NLRP3-inflammasome activity, which suppressed the development of aged-related AF. Collectively, these studies described a novel causal role of aberrant gut microbiota in the pathogenesis of age-related AF, which indicates that the microbiota-intestinal barrier-atrial NLRP3 inflammasome axis may be a rational molecular target for the treatment of aged-related arrhythmia disease.

Microglial TLR4-induced TAK1 phosphorylation and NLRP3 activation mediates neuroinflammation and contributes to chronic morphine-induced antinociceptive tolerance

Background and purposeThe aim of this work was to investigate the role and signal transduction of toll-like receptor 4 (TLR4), TGF-β-activated kinase 1 (TAK1) and nod-like receptor protein 3 (NLRP3) in microglial in the development of morphine-induced antinociceptive tolerance. MethodsTLR4 and NLRP3 knockout mice and 5Z-7-oxozeaeno (a selective inhibitor against TAK1 activity) were used to observe their effect on the development of morphine tolerance. Intrathecal injections of morphine (0.75 mg/kg once daily for 7 days) were used to establish anti-nociceptive tolerance, which was measured by the tail-flick test. Spinal TLR4, TAK1, and NLRP3 expression levels and phosphorylation of TAK1 were evaluated by Western blotting and immunofluorescence. ResultsRepeated treatment with morphine increased total expression of spinal TLR4, TAK1, and NLRP3 and phosphorylation of TAK1 in wild-type mice. TLR4 knockout attenuated morphine-induced tolerance and inhibited the chronic morphine-induced increase in NLRP3 and phosphorylation of TAK1. Compared with controls, mice that received 5Z-7-oxozeaenol showed decreased development of morphine tolerance and inhibition on repeated morphine-induced increase of NLRP3 but not TLR4. NLRP3 knockout mice showed resistance to morphine-induced analgesic tolerance with no effect on chronic morphine-induced expression of TLR4 and TAK1. TLR4, TAK1, and NLRP3 were collectively co-localized together and with the microglia marker Iba1. ConclusionsMicroglial TLR4 regulates TAK1 expression and phosphorylation and results in NLRP3 activation contributes to the development of morphine tolerance through regulating neuroinflammation. Targeting TLR4-TAK1-NLRP3 signaling to regulate neuro-inflammation will be alternative therapeutics and strategies for chronic morphine-induced antinociceptive tolerance.

Effect of acupuncture on hippocampal inflammation in rats with intrauterine distress induced hypoxic-ischemic brain injury

To observe the effects of acupuncture on the behavior, serum inflammatory cytokines, hippocampal Nod-like receptor protein 3(NLRP3) inflammasome-related proteins and microglia activation in rats with intrauterine distress induced ischemia-hypoxic brain injury (HIBD), so as to explore the mechanism of acupuncture underlying treatment of HIBD by regulating hippocampal inflammation. The HIBD model was established by clipping bilateral uterine arteries for 10 min and delaying caesarean delivery. HIBD rats were randomly divided into model group and acupuncture group according to body weight and litter factors, with 12 rats in each group. Twelve rats delivered normally were regarded as the normal group. Acupuncture was applied to bilateral "Benshen"(GB13) for 10 min, once a day for 14 consecutive days. H.E. staining was used to observe the pathological changes of hippocampus. The three chamber sociability test was used to determine the rat's social ability. The serum IL-1β and IL-18 levels, the hippocampal expressions of NLRP3, apoptosis associated speck like protein containing CARD(ASC) and cysteinyl aspartate specific proteinase-1(Caspase-1), andionized calcium bindingadaptor molecule-1(IBA-1) were detected by ELISA，Western blot and Immunofluorescence, separately. Compared with the normal group, The hippocampal neurons in the model group are arranged loosely, and the cells are solidified and shrunk and the whole cells are deeply stained. The time of exploring the stranger rat in the model group was significantly reduced (P<0.01). The contents of IL-1β and IL-18, the expressions of NLRP3, ASC, Caspase-1 and IBA-1 of the model group were higher than those of the normal group (P<0.01, P<0.05, P<0.001). Following acupuncture intervention, the cell structure of hippocampal neurons was improved, and there were fewer cases of cell solidification, atrophy, and deep staining. In comparison with the model group, the time of exploring stranger rat in the acupuncture group was significantly increased (P<0.01). The contents of IL-1β and IL-18, the expressions of NLRP3, ASC and Caspase-1, and IBA-1 of the acupuncture group were significantly down-regulated than those of the model group (P<0.01, P<0.05). Acupuncture can effectively improve the social behavior deficits of HIBD rats，which may be related to inhibition of the expression of inflammatory response proteins NLRP3, ASC and Caspase-1 in the hippocampus, decrease the activation of microglia in the hippocampus, and reduce serum IL-1β and IL-18, thereby alleviating inflammation.

Mechanism of Ginkgolide B Amelioration of Neuropathic Pain Induced by Chronic Constriction Injury

Activation of NOD-like receptor protein 3 (NLRP3) inflammasome is implicated in the pathogenesis of neuropathic pain. Ginkgolide B contributes to the suppression of NLRP3 inflammasome activation to prevent hypoxic-ischemic brain injury. However, the role of ginkgolide B on neuropathic pain has not been reported yet. We have shown that administration of ginkgolide B lowered pain threshold measured by paw-withdrawal threshold and paw-withdrawal latency in rats subjected to chronic constriction injury. Nerve fibers in rats postchronic constriction injury were swollen, and the fibrous structure was disordered. Treatment with ginkgolide B attenuated the nerve fiber swelling and reduced the disordered fibrous structure. Ginkgolide B dosage dependently attenuated chronic constriction injury-induced increase of proinflammatory cytokines. Protein expression of NLRP3 and its downstream targets (caspase 1 and IL-1β) were increased by chronic constriction injury and reduced by ginkgolide B. Lastly, ginkgolide B counteracted with the promotive effects of chronic constriction injury on protein expression of TLR4 and p-NF-κB. In conclusion, ginkgolide B demonstrated anti-inflammatory and antinociceptive effects in rats' model with neuropathic pain by suppression of TLR4-NF-κB-mediated NLRP3 inflammasome activation.

Melatonin Ameliorates Hemorrhagic Transformation via Suppression of ROS-Induced NLRP3 Activation after Cerebral Ischemia in Hyperglycemic Rats.

Melatonin is a strong antioxidant which beneficially protects against middle cerebral artery occlusion (MCAO) followed by hemorrhagic transformation in rats; protection includes the reduction of neurological deficits, infarction, and hematoma volume. The molecular mechanisms underlying these neuroprotective effects in the MCAO model have not been clearly identified. This study examined the influence and involved mechanism of melatonin on inflammation in hemorrhagic transformation following hyperglycemia MCAO rat model. Compared with the MCAO group, MCAO+dextrose (DX) group showed worse neurological function and higher infarction and hematoma volume. Interestingly, the protein expression of Nod-like receptor protein 3 (NLRP3) inflammasome increased in the MCAO+DX group compared with the MCAO group, which indicated that NLRP3 inflammasome may be involved in the DX-induced hemorrhagic transformation following MCAO. Then, three dosages of melatonin were intraperitoneally injected 2 h after MCAO induction. Melatonin treatment attenuated inflammatory response by inhibiting the reactive oxygen species (ROS) and NLRP3 inflammasome, alleviating neuronal injury, and reducing infarction and hematoma volume, finally improving neurological score. Melatonin also repressed cortical levels of proinflammatory cytokine IL-1β, which were increased 24 h after hyperglycemia MCAO. In order to identify the potential mechanisms, we further revealed that nigericin administration reversed the neuroprotective effect of melatonin by promoting NLRP3 inflammasome activation. In general, this present study reveals that melatonin prevents the occurrence of hyperglycemia-enhanced hemorrhagic transformation, and this effect might be beneficial to attenuate neurological dysfunction via suppressing the inflammatory response after MCAO which possibly associated with the inhibition of the ROS/NLRP3 inflammasome pathway.

NF-κB and NLRP3 gene expression changes during warm hepatic ischemia-reperfusion in rats with and without silibinin

Aim: This research examined silibinin's anti-inflammatory outcomes on the NOD-like receptor protein-3 (NLRP3) and NF-κB gene expression, which plays a notable role in inciting inflammatory pathways. Background: Hepatic ischemia-reperfusion (I/R) is a common phenomenon in many clinical cases, including liver surgery and transplantation. Inflammatory mediators are vital contributors to the expansion of hepatic damage after I/R injury (I/RI), and therefore, targeting inflammation is a considerable candidate for the management of hepatic I/RI and its complications. Methods: Thirty-two male Wistar rats were divided equally into four groups: 1) Control (Vehicle) group, in which rats underwent laparotomy and received normal saline; 2) SILI group, in which rats underwent laparotomy, and 30 mg/kg silibinin was injected intraperitoneal (IP); 3) I/R group, in which rats underwent I/R and received normal saline; and 4) I/R + SILI group, who encountered I/R after laparotomy and received silibinin. After one hour of ischemia and three hours of reperfusion, blood and liver tissue samples were assembled for future biochemical, histological, and gene expression studies. Results: In vivo analysis attested that serum AST and ALT activities were significantly lessened by silibinin in the SILI + I/R group (p <0.001). Silibinin ameliorated inflammatory liver tissue injuries, including neutrophil and macrophage infiltration, hepatocyte degeneration, cytoplasmic vacuolation, vascular endothelial damages, and sinusoid dilation observed in the I/R group. During I/R, NLRP3 and NF-κB gene expression showed a significant increment compared to the control group (p <0.001), which could be alleviated by silibinin (p <0.01). Conclusion: The results evidence that adjusting the expression of NLRP3 and NF-κB genes during I/R is probably one of the mechanisms of the anti-inflammatory effects of silibinin. Keywords: Ischemia, NF-Κb, NLRP3, Reperfusion, Silibinin. (Please cite as: Zarpou S, Mosavi H, Bagheri A, Malekzadeh Shafaroudi M, Khonakdar-Tarsi A. NF-κB and NLRP3 gene expression changes during warm hepatic ischemia-reperfusion in rats with and without silibinin. Gastroenterol Hepatol Bed Bench 2021;14(3):267-275).

Bone marrow-derived mesenchymal stem cells microvesicles stabilize atherosclerotic plaques by inhibiting NLRP3-mediated macrophage pyroptosis.

Rupture of atherosclerotic plaques constitutes the major cause of thrombosis and acute ischemic coronary syndrome. Bone marrow-derived mesenchymal stem cells microvesicles (BMSCs-MVs) are reported to promote angiogenesis. This study investigated the role of BMSCs-MVs in stabilizing atherosclerotic plaques. BMSCs-MVs in mice were isolated and identified. The mouse model of atherosclerosis was established, and mice were injected with BMSCs-MVs via the tail vein. The macrophage model with high glucose and oxidative damage was established and then incubated with BMSCs-MVs. Nod-like receptor protein 3 (NLRP3) expression, pyroptosis-related proteins, and inflammatory factors were detected. Actinomycin D was used to inhibit the secretion of BMSCs-MVs to verify the source of microRNA-223 (miR-223). The binding relationship between miR-223 and NLRP3 was predicted and verified. BMSCs-MVs with knockdown of miR-223 were cocultured with bone marrow-derived macrophageswith knockdown of NLRP3, and then levels of miR-223, NLRP3, pyroptosis-related proteins, and inflammatory factors were detected. BMSCs-MVs could reduce the vulnerability index of atherosclerotic plaques and intima-media thickness in mice, and inhibit pyroptosis and inflammation. BMSCs-MVs inhibited pyroptosis and inflammatory factors in macrophages. BMSCs-MVs carried miR-223 to inhibit NLRP3 expression and reduce macrophage pyroptosis, thereby stabilizing the atherosclerotic plaques.