Expression Of NOD-like Receptor Protein 3 Research Articles

Tonabersat suppresses priming/activation of the NOD-like receptor protein-3 (NLRP3) inflammasome and decreases renal tubular epithelial-to-macrophage crosstalk in a model of diabetic kidney disease

BackgroundAccompanied by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, aberrant connexin 43 (Cx43) hemichannel-mediated ATP release is situated upstream of inflammasome assembly and inflammation and contributes to multiple secondary complications of diabetes and associated cardiometabolic comorbidities. Evidence suggests there may be a link between Cx43 hemichannel activity and inflammation in the diabetic kidney. The consequences of blocking tubular Cx43 hemichannel-mediated ATP release in priming/activation of the NLRP3 inflammasome in a model of diabetic kidney disease (DKD) was investigated. We examined downstream markers of inflammation and the proinflammatory and chemoattractant role of the tubular secretome on macrophage recruitment and activation.MethodsAnalysis of human transcriptomic data from the Nephroseq repository correlated gene expression to renal function in DKD. Primary human renal proximal tubule epithelial cells (RPTECs) and monocyte-derived macrophages (MDMs) were cultured in high glucose and inflammatory cytokines as a model of DKD to assess Cx43 hemichannel activity, NLRP3 inflammasome activation and epithelial-to-macrophage paracrine-mediated crosstalk. Tonabersat assessed a role for Cx43 hemichannels.ResultsTranscriptomic analysis from renal biopsies of patients with DKD showed that increased Cx43 and NLRP3 expression correlated with declining glomerular filtration rate (GFR) and increased proteinuria. In vitro, Tonabersat blocked glucose/cytokine-dependant increases in Cx43 hemichannel-mediated ATP release and reduced expression of inflammatory markers and NLRP3 inflammasome activation in RPTECs. We observed a reciprocal relationship in which NLRP3 activity exacerbated increased Cx43 expression and hemichannel-mediated ATP release, events driven by nuclear factor kappa-B (NFκB)-mediated priming and Cx43 hemichannel opening, changes blocked by Tonabersat. Conditioned media (CM) from RPTECs treated with high glucose/cytokines increased expression of inflammatory markers in MDMs, an effect reduced when macrophages were pre-treated with Tonabersat. Co-culture using conditioned media from Tonabersat-treated RPTECs dampened macrophage inflammatory marker expression and reduced macrophage migration.ConclusionUsing a model of DKD, we report for the first time that high glucose and inflammatory cytokines trigger aberrant Cx43 hemichannel activity, events that instigate NLRP3-induced inflammation in RPTECs and epithelial-to-macrophage crosstalk. Recapitulating observations previously reported in diabetic retinopathy, these data suggest that Cx43 hemichannel blockers (i.e., Tonabersat) may dampen multi-system damage observed in secondary complications of diabetes.

Betaine alleviates doxorubicin-related cardiotoxicity via suppressing oxidative stress and inflammation via the NLRP3/SIRT1 pathway.

Cardiotoxicity is one of the side effects of the anti-cancer drug doxorubicin (DOX) that limits its clinical application. Betaine (BT) is a natural agent with promising useful effects against inflammation and oxidative stress (OS). We assessed the effects of BT on DOX-induced cardiotoxicity in mice. Forty-two male NMRI mice were assigned to six groups: I: control; II: BT (200mg/kg; orally, alone); III: DOX (2.5mg/kg; six injections (ip)) for two weeks; IV, V, VI: BT (50mg/kg, 100mg/kg, and 200mg/kg; orally, once a day for two weeks, respectively) plus DOX administration. The cardiac enzymes like cardiac troponin-I (cTn-I), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were assessed in serum. Oxidative/inflammatory markers like nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reduced glutathione level (GSH), and glutathione peroxidase (GPx) activities were determined in cardiac tissue. The expressions of NOD-like receptor protein 3 (NLRP3), caspase-1, interleukin (IL)-1β, and silent information regulator 1 (SIRT1) proteins were also evaluated in cardiac tissue. The results indicated that DOX significantly increased LDH, CK-MB, cTn-I, MDA, and NO levels and also the caspase-1, NLRP3, and IL-1β expression. Furthermore, DOX caused a significant reduction in the GSH levels and SOD, CAT, GPX activities, and the expression of SIRT1 protein in heart tissue. However, BT significantly improved all studied parameters. The findings were confirmed by histopathological assessments of the heart. BT can protect against DOX-induced cardiotoxicity by suppressing the activation of NLRP3 and OS by stimulating the SIRT1 pathway.

The critical role of P2XR/PGC-1α signalling pathway in hypoxia-mediated pyroptosis and M1/M2 phenotypic differentiation of mouse microglia.

Microglia are endogenous immune cells in the brain, and their pyroptosis and phenotype dichotomy are proved to play roles in neurodegenerative diseases. We investigated whether and how hypoxia affected pyroptosis and phenotype polarization in mouse microglia. Primary mouse microglia and BV2 microglia were exposed to hypoxia. Pyroptosis and M1/M2 phenotype were assessed by measuring gasdermin D truncation and M1/M2 surface marker expression. Mechanisms including purinergic ionotropic receptor (P2XR), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) and NOD-like receptor protein 3 (NLRP3) inflammasome were investigated. We reported hypoxia (90% N2, 5% O2 and 5% CO2) induced pyroptosis and promoted M1 phenotype polarization in primary mouse microglia and BV2 microglia, and the effect appeared after 6h exposure. Although hypoxia (90% N2, 5% O2 and 5% CO2, 6h) had no effect on P2X1R and P2X7R expression, it increased P2X4R expression and decreased PGC-1α expression. Interestingly, blockade of P2X4R or P2X7R abolished hypoxia-modulated PGC-1α expression, pyroptosis and M1 polarization. PGC-1α overexpression or overactivation alleviated hypoxia-induced pyroptosis and M1 polarization, while PGC-1α knockdown or deactivation promoted pyroptosis and M1 polarization under normoxic situation. Further, hypoxia induced NLRP3 expression and activated caspase-1 and induced the phosphorylation of NF-κB and reduced the phosphorylation of STAT3/6. NLRP3 inhibitor and caspase-1 inhibitor abolished hypoxia-induced pyroptosis, while NF-κB inhibitor and STAT phosphorylation inducer ameliorated hypoxia-induced M1 polarization. In addition, NF-κB activator and STAT3/6 inhibitor caused microglia M1 polarization under normoxic situation. We concluded in cultured mouse microglia, hypoxia may induce pyroptosis via P2XR/PGC-1α/NLRP3/caspase-1 pathway and trigger M1 polarization through P2XR/PGC-1α/NF-κB/STAT3/6 pathway.

Oxymatrine alleviates high-fat diet/streptozotocin-induced non-alcoholic fatty liver disease in C57BL/6 J mice by modulating oxidative stress, inflammation and fibrosis

Non-alcoholic fatty liver disease (NAFLD) represents a complex complication of type 2 diabetes mellitus (T2DM). Oxymatrine (OMT) is an alkaloid extracted from Sophora flavescens with broad pharmacological effects. However, there is currently a lack of research on OMT in the field of NAFLD. The present study aimed to explore the effects and underlying mechanisms of oxymatrine in treating T2DM with NAFLD. The T2DM mice model was induced by high-fat diet (HFD) combined with streptozotocin (STZ) injection in male C57BL/6 J mice. Animals were randomly divided into four groups (n = 8): Control group, DC group, OMT-L group (45 mg/kg i.g.), and OMT-H group (90 mg/kg, i.g.). The drug was administered once a day for 8 weeks. In addition, HepG2 hepatocytes were incubated with palmitic acid (PA) to establish a fatty liver cell model. Treated with OMT, the body weight and fasting blood glucose (FBG) of DC mice were reduced and the liver organ coefficient was significantly optimized. Meanwhile, OMT markedly enhanced the activities of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and also reduced malondialdehyde (MDA) levels. These biochemical alterations were accompanied by noticeable improvements in liver histopathology. Furthermore, OMT down-regulated the expression of NOD-like receptor protein 3 (NLRP3), interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1) and collagen I significantly, highlighting its potential in modulating inflammatory and fibrotic pathways. In conclusion, OMT improved liver impairment effectively in diabetic mice by suppressing oxidative stress, inflammation and fibrosis. These results suggest that OMT may represent a novel therapy for NAFLD with diabetes.

CXCL13/CXCR5 promote chronic postsurgical pain and astrocyte activation in rats by targeting NLRP3.

Chronic postsurgical pain (CPSP) with high incidence negatively impacts the quality of life. X-C motif chemokine 13 (CXCL13) has been associated with postsurgery inflammation and exacerbates neuropathic pain in patients with CPSP. This study was aimed to illustrate the relationship between CXCL13 and nod-like receptor protein-3 (NLRP3), which is also involved in CPSP. A CPSP model was constructed by skin/muscle incision and retraction (SMIR) in right medial thigh, and the rats were divided into three groups: Sham, SMIR, and SMIR + anti-CXCL13 (intrathecally injected with anti-CXCL13 antibody). Then, the paw withdrawal threshold (PWT) score of rats was recorded. Primary rat astrocytes were isolated and treated with recombinant protein CXCL13 with or without NLRP3 inhibitor INF39. The expressions of CXCL13, CXCR5, IL-1β, IL-18, GFAP, NLRP3, and Caspase-1 p20 were detected by real-time quantitative reverse transcription PCR, western blot, ELISA, immunocytochemistry, and immunofluorescence analyses. The anti-CXCL13 antibody alleviated SMIR-induced decreased PWT and increased expression of GFAP, CXCL13, CXCR5, NLRP3, and Caspase-1 p20 in spinal cord tissues. The production of IL-1β, IL-18, and expression of CXCL13, CXCR5, GFAP, NLRP3, and Caspase-1 p20 were increased in recombinant protein CXCL13-treated primary rat astrocytes in a dose-dependent manner. Treatment with NLRP3 inhibitor INF39 inhibited the function of recombinant protein CXCL13 in primary rat astrocytes. The CXCL13/CXCR5 signaling could promote neuropathic pain, astrocytes activation, and NLRP3 inflammasome activation in CPSP model rats by targeting NLRP3. NLRP3 may be a potential target for the management of CPSP.

Expression and diagnostic value of lncRNA MALAT1 and NLRP3 in lower limb atherosclerosis in diabetes

ObjectiveThis study aimed to examine the diagnostic predictive value of long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1(MALAT1) and NOD-like receptor protein 3(NLRP3) expression in patients with type 2 diabetes mellitus(T2DM) and lower extremity atherosclerosis disease (LEAD).MethodsA total of 162 T2DM patients were divided into T2DM with LEAD group (T2DM + LEAD group) and T2DM alone group (T2DM group). The lncRNA MALAT1 and NLRP3 expression levels were measured in peripheral blood, and their correlation was examined. Least absolute shrinkage and selection operator (LASSO) regression model was used to screen for the best predictors of LEAD, and multivariate logistic regression was used to establish a predictive model and construct the nomogram. The effectiveness of the nomogram was assessed using the receiver operating characteristic (ROC) curve, area under the curve (AUC), calibration curve, and decision curve analysis (DCA).ResultsThe levels of the lncRNA MALAT1 and NLRP3 in the T2DM + LEAD group were significantly greater than those in the T2DM group (P <0.001), and the level of the lncRNA MALAT1 was positively correlated with that of NLRP3 (r = 0.453, P<0.001). The results of the LASSO combined with the logistic regression analysis showed that age, smoking, systolic blood pressure (SBP), NLRP3, and MALAT1 were the influencing factors of T2DM with LEAD(P<0.05). ROC curve analysis comparison: The discriminatory ability of the model (AUC = 0.898), MALAT1 (AUC = 0.804), and NLRP3 (AUC = 0.794) was greater than that of the other indicators, and the predictive value of the model was the greatest. Calibration curve: The nomogram model was consistent in predicting the occurrence of LEAD in patients with T2DM (Cindex = 0.898). Decision curve: The net benefit rates obtained from using the predictive models for clinical intervention decision-making were greater than those obtained from using the individual factors within the model.ConclusionMALAT1 and NLRP3 expression increased significantly in T2DM patients with LEAD, while revealing the correlation between MALAT1 and NLRP3. The lncRNA MALAT1 was found as a potential biomarker for T2DM with LEAD.

MicroRNA-7 attenuates secondary brain injury following experimental intracerebral hemorrhage via inhibition of NLRP3

Background and purposeThe pathophysiological mechanisms underlying brain injury resulting from intracerebral hemorrhage (ICH) remain incompletely elucidated, and efficacious therapeutic interventions to enhance the prognosis of ICH patients are currently lacking. Previous research indicates that MicroRNA-7 (miR-7) can suppress the expression of Nod-like receptor protein 3 (NLRP3), thereby modulating neuroinflammation in Parkinson's disease pathogenesis. However, the potential regulatory effects miR-7 on NLRP3 inflammasome after ICH are yet to be established. This study aims to ascertain whether miR-7 mitigates secondary brain injury following experimental ICH by inhibiting NLRP3 and to investigate the underlying mechanisms. MethodsAn ICH model was established by stereotaxically injecting 100 μL of autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. Subsequently, these rats were allocated into three groups: sham, ICH + Vehicle, and ICH + miR-7, each comprising 18 animals. Twelve hours post-modeling, rats received intraventricular injections of 10 μL physiological saline, 10 μL phosphate, and 10 μL phosphate-buffered saline solution containing 0.5 nmol of miR-7 mimics, respectively. Neurological function was assessed on day three post-modeling, followed by euthanasia for brain tissue collection. Brain water content was determined using the dry-wet weight method. The expression of inflammatory cytokines in cerebral tissues surrounding the hematoma was analyzed through immunohistochemistry and Western blot assays. These cytokines were re-evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, bioinformatics tools were employed to predict miR-7′s binding to NLRP3. A wild-type luciferase reporter gene vector and a corresponding mutant vector were constructed, followed by transfection of miR-7 mimics into HEK293T cells to assess luciferase activity. ResultsOur study demonstrates that the administration of miR-7 mimics markedly reduced neurological function scores and attenuated brain edema in rats following ICH. A significant upregulation of NLRP3 expression in microglia/macrophage adjacent to the hematoma was observed, substantially reduced after the treatment with miR-7 mimics. Furthermore, this intervention ameliorated neurodegenerative changes and effectively decreased the protein and mRNA levels of pro-inflammatory cytokines, namely TNF-α, IL-1β, IL-6, and Caspase1, in the cerebral tissues proximate to the hematomas. In addition, miR-7 mimics distinctly inhibited the luciferase activity associated with the wild-type reporter gene, an effect not mirrored in its mutant variant. ConclusionsThe miR-7 suppressed NLRP3 expression in microglia/macrophage to reduce the production of inflammatory cytokines, leading to conducting certain neuroprotection post-ICH in rats.

Effect of electroacupuncture of "Jiaji" (EX-B2) on NOD-like receptor protein 3 mediated pyroptosis in spinal cord tissue of rats with acute spinal cord injury.

To observe the effect of electroacupuncture (EA) stimulation of "Jiaji"(EX-B2) on motor function, histomorphology, and expression of NOD-like receptor protein 3 (NLRP3) and N-terminal domain of gasdermin D (GSDMD-N) in the spinal cord tissue of rats with spinal cord injury (SCI), so as to explore its mechanism underlying improvement of SCI. Forty eight female SD rats were randomly divided into sham surgery (sham), SCI model (model), EA, and NLRP3 agonist (monosodium urate, MSU) combined with Jiaji EA (MSU+EA) groups, with 12 rats in each group which were further divided into 3 d and 7 d subgroups, with 6 rats at each time point. Two EA groups received EA stimulation of EX-B2 with a frequency of 100 Hz, electrical current of 1-2 mA for 30 min, once a day for 3 or 7 days. After 5 min, 6 h, and 24 h of modeling, rats of the MSU+EA group received intraperitoneal injection of MSU (200 μg/kg, 200 μg/mL) . The motor function was evaluated using Basso-Beattie-Bresnahan (BBB) scale, the morphological structure of rat spinal cord tissue was observed by H.E. staining. The expression of pyroptosis related factors NLRP3, cleaved Caspase-1 and GSDMD-N of the spinal cord was observed by using immunohistochemistry and Western blot separately, the expression and localization of Iba-1 and GSDMD-N in the spinal cord tissue were observed using immunofluorescence double staining method. Compared with the sham group, the BBB scores after modeling and on day 3 and 7 were decreased (P<0.05), while the average OD values (immunoactivity) and expression levels of NLRP3, cleaved Caspase-1 and GSDMD-N proteins, and the immunofluorescence intensity of Iba-1/GSDMD-N (co-expression) of the spinal cord tissues on day 3 and 7 were significantly increased in the model group (P<0.05). In comparison with the model group, the BBB scores on day 3 and 7 were obviously increased (P<0.05), while the immunoactivity and expression levels of NLRP3, cleaved Caspase-1 and GSDMD proteins, and the immunofluorescence intensity of Iba-1/GSDMD-N on day 3 and 7 significantly down-regulated in the EA group (P<0.05) but not in the MSU+EA group (P>0.05), suggesting an elimination of the effects of EA after administration of NLRP3 agonist (MSU). H.E. staining showed obvious bleeding area in the spinal cord tissue, loose tissue and inflammatory cell infiltration on day 3 after modeling, and basic absorption of the bleeding, loose tissue, obvious vacuolar changes of the white matter area, loss and contraction of neurons with infiltration of a large number of inflammatory cells, which was milder in the EA group but not in the MSU+EA group. EA of EX-B2 can improve the motor function of SCI rats, which may be related to its functions in inhibiting pyroptosis of microglia mediated by NLRP3/Caspase-1 signaling pathway.

Dopamine Receptor 1 Treatment Promotes Epithelial Repair of Corneal Injury by Inhibiting NOD-Like Receptor Protein 3-Associated Inflammation.

To elucidate the influence of dopamine receptor 1 (DRD1) on the proliferation of mouse corneal epithelial cells (MCECs) under inflammatory conditions. In vitro, immortalized MCECs (iMCECs) were treated with IL-1β, with and without pcDNA3.1_DRD1. Primary MCECs (pMCECs) were exposed to IL-1β, with and without DRD1 agonist (A68930). Cell proliferation was quantified using the Cell Counting Kit-8 (CCK-8) assay and immunofluorescence staining for Ki-67 and p63. Expression levels of NOD-like receptor protein 3 (NLRP3), IL-1β, and IL-6 were assessed. To establish a corneal injury model in mice, a 2-mm superficial keratectomy was performed. Either 0.1% A68930 or PBS was topically administered three times daily to the injured eyes for up to 5days post-injury. Immunofluorescence analysis was employed to evaluate the expression of Ki-67, p63, and CD45 in mouse corneas. Western blotting and real-time quantitative PCR were utilized for quantitative analysis of DRD1, NLRP3, IL-1β, and IL-6 in mouse corneas. Corneal epithelial regeneration was monitored through fluorescein sodium staining for a duration of up to 5days following the injury. Overexpression of DRD1 and A68930 promoted MCEC proliferation and suppressed the expression of NLRP3, IL-1β, and IL-6 in vitro. Topical application of the 0.1% A68930 following mechanical corneal injury in mice led to increased Ki-67 and p63 expression compared to PBS treatment. Furthermore, topical administration of the 0.1% A68930 reduced the expression of CD45, NLRP3, IL-1β, and IL-6. Analysis with fluorescein sodium indicated accelerated corneal epithelial regeneration in the 0.1% A68930 treatment group. DRD1 treatment counteracts NLRP3-associated inflammation and facilitates epithelial repair of corneal injury.

Implication of lncRNA MSTRG.81401 in Hippocampal Pyroptosis Induced by P2X7 Receptor in Type 2 Diabetic Rats with Neuropathic Pain Combined with Depression.

Major depressive disorder (MDD) is a common complication of diabetes and is often observed alongside diabetic neuropathic pain (DNP) as a comorbidity in diabetic patients. Long non-coding RNA (lncRNA) plays an important role in various pathophysiological processes. The P2X7 receptor is responsible for triggering inflammatory responses, such as pyroptosis, linked to pain and depression. The aim of this study was to investigate the effect of lncRNA MSTRG.81401 on hippocampal pyroptosis induced by the P2X7 receptor in diabetic rats with DNP combined with MDD (DNP + MDD). Our results showed that the expression of lncRNA MSTRG.81401 was significantly elevated in the hippocampus of DNP + MDD rats compared with the control group. Following the administration of shRNA targeting lncRNA MSTRG.81401, a notable elevation in mechanical and thermal pain thresholds was observed in rats with comorbid DNP and MDD. Additionally, significant improvements in depression-like behaviors were evident in the open-field test (OFT), sucrose preference test (SPT), and forced swim test (FST). In the DNP + MDD rats, elevated levels in hippocampal P2X7 receptor mRNA and protein were observed, along with increased co-expression of P2X7 and the astrocytic marker glial fibrillary acidic protein (GFAP). Meanwhile, in DNP + MDD rats, the heightened mRNA expression of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), pyroptosis-related protein Gasdermin D (GSDMD), caspase-1, IL-1β, IL-18, and TNF-α was detected, in addition to increased serum levels of IL-1β, IL-18 and TNF-α. After shRNA treatment with lncRNA MSTRG.81401, the above abnormal changes in indicators for pyroptosis and inflammation were improved. Therefore, our study demonstrates that shRNA of lncRNA MSTRG.81401 can alleviate the pain and depression-like behaviors in diabetic rats associated with the comorbidity of DNP and MDD by inhibiting the hippocampal P2X7 receptor-mediated pyroptosis pathway and pro-inflammatory responses. This suggests that the P2X7R/NLRP3/caspase-1 implicated pyroptosis and inflammatory scenario may serve as a potential target for the management of comorbid DNP and MDD in diabetes.

Cathepsin B aggravates atherosclerosis in ApoE-deficient mice by modulating vascular smooth muscle cell pyroptosis through NF-κB / NLRP3 signaling pathway.

Atherosclerosis (AS) is a chronic inflammatory disease involving cell death and inflammatory responses. Pyroptosis, a newly discovered pro-inflammatory programmed cell death process, exacerbates inflammatory responses. However, the roles of cathepsin B (CTSB) in pyroptosis and AS remain unclear. To gain further insight, we fed ApoE-/- mice a high-fat diet to investigate the effects and mechanisms of CTSB overexpression and silencing on AS. We also explored the specific role of CTSB in vascular smooth muscle cells (VSMCs) in vitro. The study revealed that high-fat diet led to the formation of AS plaques, and CTSB was found to increase the AS plaque lesion area. Immunohistochemical and TUNEL/caspase-1 staining revealed the existence of pyroptosis in atherosclerotic plaques, particularly in VSMCs. In vitro studies, including Hoechst 33342/propidium iodide staining, a lactate dehydrogenase (LDH) release assay, detection of protein indicators of pyroptosis, and detection of interleukin-1β (IL-1β) in cell culture medium, demonstrated that oxidized low-density lipoprotein (ox-LDL) induced VSMC pyroptosis. Additionally, CTSB promoted VSMC pyroptosis. Ox-LDL increased the expression of CTSB, which in turn activated the NOD-like receptor protein 3 (NLRP3) inflammasome and promoted NLRP3 expression by facilitating nuclear factor kappa B (NF-κB) p65 nuclear translocation. This effect could be attenuated by the NF-κB inhibitor SN50. Our research found that CTSB not only promotes VSMC pyroptosis by activating the NLRP3 inflammasome, but also increases the expression of NLRP3.

Protective effect of the natural flavonoid naringenin in mouse models of retinal injury

Glaucoma is an eye disease with a high rate of blindness and a complex pathogenesis. Ocular hypertension (OHT) is a critical risk factor, and retinal ischemia/reperfusion (I/R) is an important pathophysiological basis. This study was designed to investigate the retinal neuroprotective effect of oral naringenin in an acute retinal I/R model and a chronic OHT model and the possible mechanism involved. After the I/R and OHT models were established, mice were given vehicle or naringenin (100 mg/kg or 300 mg/kg). Hematoxylin-eosin (HE) staining and immunostaining of RBPMS and glial fibrillary acidic protein (GFAP) were used to evaluate retinal injury. GFAP, CD38, Sirtuin1 (SIRT1), and NOD-like receptor protein 3 (NLRP3) expression levels were measured by Western blotting. In the OHT model, intraocular pressure (IOP) was dynamically maintained at approximately 20–25 mmHg after injury. The retinal structure was damaged, and retinal ganglion cells (RGCs) were lost in both models. Naringenin ameliorated the abovementioned indications but also demonstrated that high concentrations of naringenin significantly inhibited retinal astrocyte activation and inhibited damage-induced increases in the expression of GFAP, NLRP3, and CD38 proteins, while SIRT1 protein expression was upregulated. This study showed for the first time that naringenin can reduce microbead-induced IOP elevation in the OHT model, providing new evidence for the application of naringenin in glaucoma. Naringenin may mediate the CD38/SIRT1 signaling pathway, inhibit astrocyte activation, and ultimately exert an anti-inflammatory effect to achieve retinal neuroprotection.

Taohong Siwu decoction reduces acute myocardial ischemia–reperfusion injury by promoting autophagy to inhibit pyroptosis

Ethnopharmacological relevanceTaohong Siwu decoction (TSD) is a classic traditional Chinese medicine (TCM) prescription used to promote the blood circulation and alleviate blood stasis. TSD consists of Paeonia lactiflora Pall., Conioselinum anthriscoides (H. Boissieu) Pimenov & Kljuykov, Rehmannia glutinosa (Gaertn.) DC., Prunus persica (L.) Batsch, Angelica sinensis (Oliv.) Diels, and Carthamus creticus L. in the ratio of 3:2:4:3:3:2. Studies on the effects of TSD on myocardial ischemia–reperfusion injury (MIRI) from the perspective of autophagy and pyroptosis have not been reported. Aim of the studyInvestigate the effect of TSD on MIRI and explore the underlying mechanisms. Materials and methodsWe searched the main components and corresponding potential targets of TSD on The Pharmacology of Traditional Chinese Medicine Systems database for target prediction. We identified targets for MIRI on Online Mendelian Inheritance in Man and GeneCards databases. The intersection of the compound target and disease target was obtained and a protein–protein interaction network constructed. We undertook enrichment analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The results of network pharmacology were verified by in vivo experiments in mice. ResultsIn mice, TSD significantly reduced the volume of the myocardial infarct, significantly reduced serum levels of cardiac troponin-nI (CTnI), creatine kinase-myocardial band (CK-MB), malonaldehyde (MDA), interleukin (IL)-6, increased the activity of superoxide dismutase (SOD) and IL-10 level, reduced the level of pyroptosis in myocardial tissue, increased the number of autophagosomes, and significantly reduced the fluorescence intensity of apoptosis-associated speck-like protein (ASC), Nod-like receptor protein 3 (NLRP3), and caspase-1. TSD administration increased the protein expression of microtubule-associated protein light chain 3 (LC3), but reduced the protein expression of p62, NLRP3, ASC, caspase-1, cleaved caspase-1, pro-caspase-1, gasdermin D (GSDMD), GSDMD-N-terminal, IL-18, and IL-1β. Administration of 3-Methyladenin could reverse the effect of TSD in inhibiting inflammation and the release of proinflammatory factors. ConclusionTSD treatment alleviated MIRI by promoting autophagy to suppress activation of the NLRP3 inflammasome and reducing the release of proinflammatory factors.

Green Tea Extract Reduced Lipopolysaccharide-Induced Inflammation in L2 Cells as Acute Respiratory Distress Syndrome Model Through Genes and Cytokine Pro-Inflammatory.

Acute Respiratory Distress Syndrome (ARDS) is a severe lung inflammatory condition that has the capacity to impair gas exchange and lead to hypoxemia. This condition is found to have been one of the most prevalent in patients of COVID-19 with a more serious condition. Green tea (Camellia sinensis L.) contains polyphenols that possess many health benefits. The purpose of this study was to assess the anti-inflammatory activities of green tea extract in Lipopolysaccharide (LPS)-induced lung cells as ARDS cells model. In this study, rat lung cells (L2) were induced by LPS to mimic the inflammation observed in ARDS and later treated with green tea extract. Pro-inflammatory cytokines such as Interleukin (IL)-12, C-Reactive Protein (CRP) as well as Tumor Necrosis Factor-α (TNF-α) were investigated using the ELISA method. Gene expression of NOD-Like Receptor Protein 3 (NLRP-3), Receptor for Advanced Glycation End-product (RAGE), Toll-like Receptor-4 (TLR-4), and Nuclear Factor-kappa B (NF-κB) were evaluated by qRTPCR. Apoptotic cells were measured using flow cytometry. The results showed that green tea extract treatment can reduce inflammation by suppressing gene expressions of NF-κB, NLRP-3, TLR-4, and RAGE, as well as pro-inflammatory cytokines such as IL-12, TNF-α, and CRP, an acute phase protein. Apoptosis levels of inflamed cells also found to be lowered when green tea extract was administered; thus, also increasing live cells compared to non-treated cells. These findings could lead to the future development of supplements from green tea to help alleviate ARDS symptoms, especially during critical moments such as the current pandemic.

Echinococcus granulosus cyst fluid inhibits KDM6B-mediated demethylation of trimethylated histone H3 lysine 27 and interleukin-1β production in macrophages

BackgroundEchinococcus granulosus can manipulate its host's immune response to ensure its own survival. However, the effect of histone modifications on the regulation of the NOD-like receptor protein 3 (NLRP3) inflammasome and downstream interleukin-1β (IL-1β) production in response to the parasite is not fully understood.MethodsWe evaluated IL-1β secretion through enzyme-linked immunosorbent assay and assessed reactive oxygen species levels using the dichlorodihydrofluorescein diacetate probe. Western blotting and quantitative real-time polymerase chain reaction were performed to examine the expression of NLRP3 and IL-1β in mouse peritoneal macrophages and Tohoku Hospital Pediatrics-1 cells, a human macrophage cell line. The presence of trimethylated histone H3 lysine 27 (H3K27me3) modification on NLRP3 and IL-1β promoters was studied by chromatin immunoprecipitation.ResultsTreatment with E. granulosus cyst fluid (EgCF) considerably reduced IL-1β secretion in mouse and human macrophages, although reactive oxygen species production increased. EgCF also suppressed the expression of NLRP3 and IL-1β. Mechanistically, EgCF prompted the enrichment of repressive H3K27me3 modification on the promoters of both NLRP3 and IL-1β in macrophages. Notably, the presence of EgCF led to a significant reduction in the expression of the H3K27me3 demethylase KDM6B.ConclusionsOur study revealed that EgCF inhibits KDM6B expression and H3K27me3 demethylation, resulting in the transcriptional inhibition of NLRP3 and IL-1β. These results provide new insights into the immune evasion mechanisms of E. granulosus.Graphical

The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection.

Purinergic receptors and NOD-like receptor protein 3 (NLRP3) inflammasome regulate inflammation and viral infection, but their effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain poorly understood. Here, we report that the purinergic receptor P2X7 and NLRP3 inflammasome are cellular host factors required for SARS-CoV-2 infection. Lung autopsies from patients with severe coronavirus disease 2019 (COVID-19) reveal that NLRP3 expression is increased in host cellular targets of SARS-CoV-2 including alveolar macrophages, type II pneumocytes and syncytia arising from the fusion of infected macrophages, thus suggesting a potential role of NLRP3 and associated signaling pathways to both inflammation and viral replication. In vitro studies demonstrate that NLRP3-dependent inflammasome activation is detected upon macrophage abortive infection. More importantly, a weak activation of NLRP3 inflammasome is also detected during the early steps of SARS-CoV-2 infection of epithelial cells and promotes the viral replication in these cells. Interestingly, the purinergic receptor P2X7, which is known to control NLRP3 inflammasome activation, also favors the replication of D614G and alpha SARS-CoV-2 variants. Altogether, our results reveal an unexpected relationship between the purinergic receptor P2X7, the NLRP3 inflammasome and the permissiveness to SARS-CoV-2 infection that offers novel opportunities for COVID-19 treatment.

The anti-liver fibrosis effect of Tibetan medicine (Qiwei Tiexie capsule) is related to the inhibition of NLRP3 inflammasome activation in vivo and in vitro

Ethnopharmacological relevanceQiwei Tiexie capsule (QWTX) is an improved form of a classical prescription of Tibetan medicine—Qiwei Tiexie pill. It has been employed in the treatment of a variety of chronic liver disorders, including liver fibrosis. Uncertainty still exists regarding the mechanism of QWTX action in liver fibrosis. Aim of the studyConfirm the anti-liver fibrosis effect of QWTX and reveal its mechanism from the perspective of NOD-like receptor protein 3 (NLRP3) inflammasome activation. Materials and methodsIn vivo experiment: A rat model of carbon tetrachloride -induced liver fibrosis was constructed. All rats were randomly divided into six groups: a control group, a model group, a group receiving the positive drug (Biejia Ruangan tablet), and three groups receiving QWTX at high, medium, and low doses. The contents of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBil) were detected in serum. Hematoxylin and eosin staining and Masson's staining were used to assess the histomorphological alteration of the liver. The levels of glutathione peroxidase, hydroxyproline, tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β) in the liver were determined using the corresponding detection kits. Real-time polymerase chain reaction, immunofluorescence, and western blotting were used to determine the expression levels of NLRP3, adaptor protein (ASC), caspase-1, and alpha-smooth muscle actin (α-SMA). In vitro experiment: Four groups of rat hepatic stellate cell line (HSC-T6) cells were created: the control group, the low-dose QWTX group (0.05 mg/mL), the medium-dose QWTX group (0.1 mg/mL), and the high-dose QWTX group (0.2 mg/mL). Cell viability was assessed using a cell counting kit, and the amounts of collagen type I (Col I) and IL-1β in the cell lysate were measured using an enzyme-linked immunosorbent assay kit. The mRNA and protein expression of NLRP3, ASC, caspase-1, and α-SMA were also estimated. ResultsQWTX had an inhibitory effect on liver fibrosis and a negative effect on HSC activation, while it improved liver histopathological injury and abnormal liver function and increased hydroxyproline content and glutathione peroxidase activity in vivo. QWTX decreased the expression of α-SMA, NLRP3, caspase-1, ASC, and IL-1β both in vitro and in vivo. ConclusionsTibetan medicine QWTX had a significant anti-liver fibrosis effect that was related to the inhibition of NLRP3 inflammasome activation in vivo and in vitro.

Connexin-43 hemichannels orchestrate NOD-like receptor protein-3 (NLRP3) inflammasome activation and sterile inflammation in tubular injury

BackgroundWithout a viable cure, chronic kidney disease is a global health concern. Inflammatory damage in and around the renal tubules dictates disease severity and is contributed to by multiple cell types. Activated in response to danger associated molecular patterns (DAMPs) including ATP, the NOD-like receptor protein-3 (NLRP3) inflammasome is integral to this inflammation. In vivo, we have previously observed that increased expression of Connexin 43 (Cx43) is linked to inflammation in chronic kidney disease (CKD) whilst in vitro studies in human proximal tubule cells highlight a role for aberrant Cx43 hemichannel mediated ATP release in tubule injury. A role for Cx43 hemichannels in priming and activation of the NLRP3 inflammasome in tubule epithelial cells remains to be determined.MethodsUsing the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD. The unilateral ureteral obstruction (UUO) mouse model was combined with genetic (tubule-specific Cx43 knockout) and specific pharmacological blockade of Cx43 (Peptide5), to explore a role for Cx43-hemichannels in tubule damage. Human primary tubule epithelial cells were used as an in vitro model of CKD.ResultsIncreased Cx43 and NLRP3 expression correlates with declining glomerular filtration rate and increased proteinuria in biopsies isolated from patients with CKD. Connexin 43-tubule deletion prior to UUO protected against tubular injury, increased expression of proinflammatory molecules, and significantly reduced NLRP3 expression and downstream signalling mediators. Accompanied by a reduction in F4/80 macrophages and fibroblast specific protein (FSP1+) fibroblasts, Cx43 specific hemichannel blocker Peptide5 conferred similar protection in UUO mice. In vitro, Peptide5 determined that increased Cx43-hemichannel activity primes and activates the NLRP3 inflammasome via ATP-P2X7 receptor signalling culminating in increased secretion of chemokines and cytokines, each of which are elevated in individuals with CKD. Inhibition of NLRP3 and caspase 1 similarly decreased markers of tubular injury, whilst preventing the perpetual increase in Cx43-hemichannel activity.ConclusionAberrant Cx43-hemichannel activity in kidney tubule cells contributes to tubule inflammation via activation of the NLRP3 inflammasome and downstream paracrine mediated cell signalling. Use of hemichannel blockers in targeting Cx43-hemichannels is an attractive future therapeutic target to slow or prevent disease progression in CKD.-uWW98vNU7-T3Uscg54sCsVideo

Filamin A facilitates NLRP3 inflammasome activation during arsenic-induced nonalcoholic steatohepatitis.

Prolonged exposure to arsenic can cause nonalcoholic steatohepatitis (NASH). The NOD-like receptor protein 3 (NLRP3) inflammasome plays an essential role in the process of NASH. However, the mechanism by which arsenic promotes NLRP3 expression remains unclear. Three-month NaAsO2 gavage led to the nuclear factor-κB (NF-κB) signaling pathway activation and NASH. Additionally, NaAsO2 upregulated the level of Filamin A (FLNA) and pyroptosis, thereby activating the NLRP3 inflammasome in SD rat liver. Using FLNA siRNA, NASH-associated inflammation and pyroptosis were clearly mitigated by reducing activation of the NLRP3 inflammasome. Furthermore, arsenic treatment facilitated activation of the NF-κB signaling pathway and promoted p-p65 translocation into the nucleus. Chromatin immunoprecipitation (Ch-IP) assay indicated that FLNA promoted p65 binding to the NLRP3 gene and upregulated the transcription of NLRP3, ultimately leading to pyroptosis and NASH. Our findings indicate that FLNA and pyroptosis are strongly associated with NASH induced by NaAsO2. Collectively, the findings of this study indicated that FLNA mediates NF-κB signaling pathway-induced activation of the NLRP3 inflammasome and ultimately activates pyroptosis and NASH upon NaAsO2 exposure. This information may be useful for improving therapeutic strategies against arsenic-induced NASH.

Kynurenic acid inhibits macrophage pyroptosis by suppressing ROS production via activation of the NRF2 pathway.

Macrophage pyroptosis and related inflammatory responses play an important role in periodontitis. Kynurenic acid (KA) is hypothesized to have anti‑inflammatory potential, but whether KA can inhibit macrophage pyroptosis and the underlying mechanisms remain unclear. Lipopolysaccharide (LPS) was used to induce pyroptosis in THP‑1‑derived macrophages. KA or ML385 was used to pretreat macrophages, after which, cell viability, NOD‑like receptor protein 3 (NLRP3) inflammasome‑related protein expression, oxidative stress levels and nuclear factor erythroid 2‑related factor 2 (NRF2) expression were measured. The results showed that KA improved the LPS‑induced decrease in macrophage viability and lactate dehydrogenase release. KA prevented THP‑1 macrophage pyroptosis induced by LPS by reducing the expression of NLRP3, Gasdermin‑D, and Caspase1, and decreased the expression of inflammatory factors. KA suppressed NLRP3 inflammasome activation by inhibiting ROS overproduction and increasing Heme Oxygenase 1 and glutathione levels. Moreover, KA promoted NRF2 translocation from the cytoplasm to the nucleus. In addition, the anti‑pyroptotic and antioxidant effects of KA were reversed by ML385 inhibition of NRF2. In the present study, it was found that KA significantly suppressed macrophage pyroptosis induced by LPS. It was further demonstrated that the anti‑pyroptotic effects of KA were mediated by activation of the NRF2 pathway.