NLRP3 Signaling Pathway Research Articles

Inflammasome NLRP3 Potentially Links Obesity-Associated Low-Grade Systemic Inflammation and Insulin Resistance with Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common form of neurodegenerative dementia. Metabolic disorders including obesity and type 2 diabetes mellitus (T2DM) may stimulate amyloid β (Aβ) aggregate formation. AD, obesity, and T2DM share similar features such as chronic inflammation, increased oxidative stress, insulin resistance, and impaired energy metabolism. Adiposity is associated with the pro-inflammatory phenotype. Adiposity-related inflammatory factors lead to the formation of inflammasome complexes, which are responsible for the activation, maturation, and release of the pro-inflammatory cytokines including interleukin-1β (IL-1β) and interleukin-18 (IL-18). Activation of the inflammasome complex, particularly NLRP3, has a crucial role in obesity-induced inflammation, insulin resistance, and T2DM. The abnormal activation of the NLRP3 signaling pathway influences neuroinflammatory processes. NLRP3/IL-1β signaling could underlie the association between adiposity and cognitive impairment in humans. The review includes a broadened approach to the role of obesity-related diseases (obesity, low-grade chronic inflammation, type 2 diabetes, insulin resistance, and enhanced NLRP3 activity) in AD. Moreover, we also discuss the mechanisms by which the NLRP3 activation potentially links inflammation, peripheral and central insulin resistance, and metabolic changes with AD.

Nicorandil inhibits TLR4/MyD88/NF-κB/NLRP3 signaling pathway to reduce pyroptosis in rats with myocardial infarction.

Pyroptosis is an inflammatory cell death that regulates cardiomyocyte loss after myocardial infarction. Reports indicate that nicorandil has a strong anti-inflammatory effect and protects the myocardium from myocardial infarction. However, its relationship with pyroptosis is largely unreported. Here, we investigated to influence and mechanism of action of nicorandil on cardiomyocyte pyroptosis. Forty Sprague Dawley rats were randomly assigned to sham, MI, MI + nicorandil, and MI + nicorandil + TAK242 groups (10 per group). Myocardial infarction modeling was performed through ligation of the anterior descending branch of the left coronary artery. The function of cardiac was evaluated through echocardiography, detection of myocardial adenine nucleotides, cTnI, LDH, TTC, and HE staining. Moreover, we used qRT-PCR, immunohistochemistry, and Western blotting to examine the expression of pyroptosis-related molecules and the inflammasome pathway of TLR4/MyD88/NF-κB/NLRP3. Myocardial infarction caused the activation of GSDMD, aggravated myocardial injury, and triggered cardiac dysfunction. Myocardial infarction induced pyroptotic cell death, manifested as upregulation in mRNA and protein levels associated with pyroptosis, including caspase-1 cleavage and increased expression of IL-1β and IL-18. These changes were mitigated by nicorandil. The achieved data implicate that myocardial infarction induces pyroptosis via the TLR4/MyD88/NF-κB/NLRP3 pathway, which can be inhibited by nicorandil pretreatment. Therefore, nicorandil exerts cardioprotective effects by activating KATP channels, and at least in part through inhibition of the TLR4/MyD88/NF-κB/NLRP3 pathway to reduce myocardial infarction-induced pyroptosis. As such, it is a potential therapy for ischemic heart disease.

Targeting a novel hsa_circ_0000520/miR-556-5p/NLRP3 pathway-mediated cell pyroptosis and inflammation attenuates ovalbumin (OVA)-induced allergic rhinitis (AR) in mice models.

The circRNAs-miRNAs-mRNAs competing endogenous RNA (ceRNA) networks involve in regulating the development of various inflammation-associated diseases, including allergic rhinitis (AR), and the present study aimed to identify novel AR-associated ceRNA networks. The mRNA and protein levels of the associated genes were, respectively, examined by real-time qPCR and western blot analysis. The targeting sites in miR-556-5p and NLRP3 were validated by performing dual-luciferase reporter gene system assay. ELISA was used to measure inflammatory cytokines secretion, and CCK-8 assay was conducted to determine cell proliferation. Here, we first identified a hsa_circ_0000520/miR-556-5p/NLRP3 signaling cascade triggered epithelium pyroptosis and inflammation to regulate the development of AR in cellular and mice models. Specifically, the pyroptosis-associated biomarkers (NLRP3, ASC, IL-1β and IL-18) and pro-inflammatory cytokines (OVA-specific IgE, TNF-α, IL-4 and IL-5) were upregulated in the nasal subjects collected from AR patients and ovalbumin (OVA)-induced AR mice models, compared to their normal counterparts. Next, using the ceRNA networks analysis software, we screened out a hsa_circ_0000520/miR-556-5p axis that potentially regulated NLRP3 in the human nasal epithelial cell line. Mechanistically, miR-556-5p targeted both hsa_circ_0000520 and 3' untranslated region (3'UTR) of NLRP3, and knock-down of hsa_circ_0000520 inactivated NLRP3-mediated epithelium pyroptosis through miR-556-5p in a ceRNA-dependent manner. Furthermore, we proved that both hsa_circ_0000520 ablation and miR-556-5p overexpression suppressed NLRP3-mediated cell pyroptosis to attenuate AR in mice models. Taken together, we evidenced that targeting the hsa_circ_0000520/miR-556-5p/NLRP3 signaling pathway was a novel AQ1strategy to ameliorate AR progression; however, future clinical data are still required to validate our preliminary results.

Metformin protects against neuroinflammation through integrated mechanisms of miR-141 and the NF-ĸB-mediated inflammasome pathway in a diabetic mouse model

The brain responds to diabetic stress by inducing the inflammatory response. Under normal circumstances this process is tightly regulated. However, uncontrolled inflammatory responses lead to compromised function and eventual neurodegeneration. The microRNA (miR)-200 family, specifically miR-141, is differentially expressed in diseased states including cognitive decline, thereby triggering changes in downstream genes. We hypothesised that Metformin (MF) regulates the miR-141/protein phosphatase 2A (PP2A) axis, and associated NF-ĸB-mediated inflammasome expression in diabetic mice brain. Diabetes was induced by intraperitoneal injection of Streptozotocin (STZ), thereafter mice were treated with MF (20 mg/kg BW). Whole brain tissue was harvested for further analysis. In silico analysis showed that Sirt1 and PP2A are prediction targets of miR-141. Selected protein and gene expressions were established through western blotting and qPCR, respectively. Diabetic mice brain tissue demonstrated overexpression of miR-141 and related pro-inflammatory factors as well as decreased PP2A gene expression. MF was able to counteract this by regulating expression of miR-141, PP2A, and p-tau at Ser396 protein expressions. Further experimentation revealed MF's inhibitory action on the inflammasome system by regulating the expression of the upstream controller NLRP3, related cytokines and NF-κB signalling pathway. Collectively, we demonstrate that MF promotes neuroprotection in diabetic mice by dampening inflammatory responses through its inhibitory effects on various signalling pathways. CategoriesInflammation and Immunopharmacology, Metabolic Disorders and Endocrinology, Neuropharmacology.

Suppression of NLRP3 inflammasome by Platycodin D via the TLR4/MyD88/NF-κB pathway contributes to attenuation of lipopolysaccharide induced acute lung injury in rats

Acute lung injury (ALI) is a common clinical condition with a high mortality rate and no specific treatment is available. An excessive inflammatory response contributes to the development of ALI and accelerates its progression, and the NLRP3 inflammasome and NF-κB signaling pathways are key players in inflammation. Platycodin D has been reported to have anti-oxidant and anti-stress properties in various diseases. However, the effects of PLD in ALI has not been clearly demonstrated. The aim of this study was to investigate the therapeutic effects of PLD on ALI and its possible mechanism. Our study found that PLD pre-treatment attenuated lung histopathological injury in LPS-induced SD rats and reduced the levels of inflammatory cytokines and lung wet/dry ratio in bronchoalveolar lavage fluid (BALF). In addition, PLD modulate LPS-induced production of MDA, MPO, GSH, GSH-Px and CAT in lung tissue. In addition, PLD suppressed the activation of NLRP3 inflammatory microsomes and the NF-κB signaling pathway. Thus, our results suggest that PLD are protective against LPS-induced ALI by inhibiting NLRP3 and NF-κB signaling pathway.

Protective Effect of Sirtuin 3 on CLP-Induced Endothelial Dysfunction of Early Sepsis by Inhibiting NF-κB and NLRP3 Signaling Pathways.

It has been revealed that widespread vascular endothelial dysfunction occurs in septic shock, ultimately resulting in multiple organ failure. The mitochondrial deacetylase sirtuin 3 (SIRT3) is essential in the regulation of metabolism, anti-inflammation, and anti-oxidation. The purpose of this study is to investigate whether SIRT3 is associated with the pathological progression of endothelial dysfunction in sepsis. Septic shock model was induced by cecal ligation and puncture (CLP) surgery on wild-type C57BL/6 mice. We activated and inhibited the function of SIRT3 with honokiol (HKL) and 3-TYP, respectively, and then biochemical, inflammatory, and endothelial function parameters of vascular tissue and survival were determined after CLP. CLP significantly activated NF-κB and NLRP3 pathways and decreased survival rate, endothelium-dependent relaxation function, and expression of Ser1177 phosphorylation of endothelial nitric oxide synthase (p-eNOS). The activation of SIRT3 significantly attenuated the increases of NF-κB and NLRP3 pathways and the declines of p-eNOS, endothelium-dependent relaxation function, and survival rate in septic mice. However, it presented exactly opposite results if SIRT3 was suppressed. We suggested that SIRT3 had a critical protective effect against vascular inflammation and endothelial dysfunction in early sepsis. Our data support a potential therapeutic target in vascular dysfunction and septic shock.

Glaesserella parasuis induces inflammatory response in 3D4/21 cells through activation of NLRP3 inflammasome signaling pathway via ROS

Glaesserella parasuis (G. parasuis) is an important pathogenic bacterium that can cause Glässer’s disease, and it has resulted in tremendous economic losses to the global swine industry. The intensive pulmonary inflammatory response caused by G. parasuis infection is the main cause of lung injury and death in pigs. However, the exact mechanism by which it causes severe pulmonary inflammation is not fully understood yet. In this study, severe pneumonia was observed in piglets infected with G. parasuis; and an infection cell model was established using porcine alveolar macrophages cell line 3D4/21, which was determined to be susceptible to G. parasuis infection in vitro. G. parasuis infection of 3D4/21 cells induced upregulation of proinflammatory cytokines TNF-α, IL-1β, IL-18 and production of intracellular reactive oxygen species (ROS). The expression of IL-1β related to activation of the NLRP3 inflammasome signaling pathway, which had not been shown before in G. parasuis infection. Furthermore, it was first found that release of intracellular ROS, which was mediated by NADPH oxidase in 3D4/21 cells, was found crucial for the activation of the NLRP3 signaling pathway and promoted the expression of proinflammatory cytokines, such as TNF-α and IL-1. In general, this study explored the specific mechanism of severe pulmonary inflammation caused by G. parasuis infection, and provides a foundation for further elucidating the pathogenic mechanism of G. parasuis.

Dexmedetomidine attenuates lipopolysaccharide-induced acute liver injury in rats by inhibiting caveolin-1 downstream signaling pathway

Objective: The aim of the present study is to investigate the anti-injury and anti-inflammatory effects of dexmedetomidine (Dex) in acute liver injury induced by lipopolysaccharide (LPS) in Sprague–Dawley rats and its possible mechanism.Methods: The acute liver injury model of male rats was established by injecting LPS into tail vein. The mean arterial pressure (MAP) of rats was recorded at 0–7 h, and lactic acid was detected at different time points. Wet/dry weight ratio (W/D) was calculated. Pathological changes of rat liver were observed by HE staining. ALT and AST levels in serum were detected. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) in liver tissue homogenate and the levels of IL-1β and IL-18 in serum were detected by ELISA. Protein levels of Caveolin-1 (Cav-1), TLR-4 and NLRP3 in liver tissue were tested by immunohistochemistry method. The expression of Cav-1, TLR-4 and NLRP3 mRNA in liver tissue was detected by quantitative polymerase chain reaction (qPCR) to explore its related mechanism.Results: Compared with NS group, serum lactic acid, W/D of liver tissue, MPO, SOD, IL-1β and IL-18 were significantly increased and MAP decreased significantly in LPS group and D+L group. However, compared with NS group, D group showed no significant difference in various indicators. Compared with LPS group, MPO, SOD, IL-1β and IL-18 were significantly decreased and MAP was significantly increased in D+L group. D+L group could significantly increase the level of Cav-1 protein and decrease the level of TLR-4 and NLRP3 protein in liver tissue caused by sepsis. The expression of Cav-1 mRNA was significantly up-regulated and the expression of TLR-4 and NLRP3 mRNA was inhibited in D+L group.Conclusion: Dex pretreatment protects against LPS-induced actue liver injury via inhibiting the activation of the NLRP3 signaling pathway by up-regulating the expression of Cav-1 by sepsis.

Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL-1β release.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.

Low-dose Diosbulbin-B (DB) activates tumor-intrinsic PD-L1/NLRP3 signaling pathway mediated pyroptotic cell death to increase cisplatin-sensitivity in gastric cancer (GC)

BackgroundEmerging evidences suggests that Diosbulbin-B (DB) is effective to improve cisplatin (DDP)-sensitivity in gastric cancer (GC), but its molecular mechanisms were not fully delineated, and this study managed to investigate this issue.MethodsGenes expressions were determined by Real-Time qPCR and Western Blot at transcriptional and translational levels. Cell proliferation and viability were evaluated by cell counting kit-8 (CCK-8) and trypan blue staining assay. Annexin V-FITC/PI double staining assay was used to examine cell apoptosis. The Spheroid formation assay was used to evaluated cell stemness. The xenograft tumor-bearing mice models were established, and the tumors were monitored and the immunohistochemistry (IHC) was employed to examine the expressions and localization of Ki67 protein in mice tumor tissues.ResultsLow-dose DB (12.5 μM) downregulated PD-L1 to activate NLRP3-mediated pyroptosis, and inhibited cancer stem cells (CSCs) properties, to sensitize cisplatin-resistant GC (CR-GC) cells to cisplatin. Mechanistically, the CR-GC cells were obtained, and either low-dose DB or cisplatin alone had little effects on cell viability in CR-GC cells, while low-dose DB significantly induced apoptotic cell death in cisplatin treated CR-GC cells. In addition, low-dose DB triggered cell pyroptosis in CR-GC cells co-treated with cisplatin, which were abrogated by silencing NLRP3. Next, CSCs tended to be enriched in CR-GC cells, instead of their parental cisplatin-sensitive GC (CS-GC) cells, and low-dose DB inhibited spheroid formation and stemness biomarkers (SOX2, OCT4 and Nanog) expressions to eliminate CSCs in CR-GC cells, which were reversed by upregulating programmed death ligand-1 (PD-L1). Furthermore, we proved that PD-L1 negatively regulated NLRP3 in CR-GC cells, and low-dose DB activated NLRP3-mediated pyroptotic cell death in cisplatin treated CR-GC cells by downregulating PD-L1. Also, low-dose DB aggravated the inhibiting effects of cisplatin on tumorigenesis of CR-GC cells in vivo.ConclusionsCollectively, low-dose DB regulated intrinsic PD-L1/NLRP3 pathway to improve cisplatin-sensitivity in CR-GC cells, and this study provided alternative therapy treatments for GC.

Mangiferin inhibited neuroinflammation through regulating microglial polarization and suppressing NF-κB, NLRP3 pathway.

Inflammation plays important roles in the progress of neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Microglia is responsible for the homeostasis of the central nervous system (CNS), and involved in the neuroinflammation. Therefore, it could be potential in treatment of neurodegenerative diseases to suppress the microglia-mediated neuroinflammation. Mangiferin, a major glucoside of xanthone in Anemarrhena Rhizome, has anti-inflammatory, anti-diabetes, and anti-oxidative properties. However, the effect of mangiferin on the inflammatary responses of microglia cells are still poorly understand. In this study, we investigated the mechanism by which mangiferin inhibited inflammation in LPS-induced BV2 microglia cells. BV2 cells were pretreatment with mangiferin followed by LPS stimulation. In vitro assays, NO and cytokines production were quantified. Western blot and immunocytochemistry were used to examine the effect of mangiferin on the polarization of BV2 cells and signaling pathway. The results showed that mangiferin treatment significantly reduced NO, IL-1β, IL-6 and TNF-α production, also reduced the mRNA and protein of iNOS and COX-2, promoted the polarization of inflammatory toward anti-inflammatory, and inhibited activation of NF-κB and NLRP3 inflammasome. These data suggest that mangiferin has an anti-neuroinflammatory property via regulating microglia macrophage polarization and suppressing NF-κB and NLRP3 signaling pathway, and may act as a potential natural therapeutic candidate for neuroinflammatory diseases.

Toxic Shock Syndrome Toxin 1 Induces Immune Response via the Activation of NLRP3 Inflammasome.

Staphylococcus aureus is a Gram-positive opportunistic pathogen which causes infections in a variety of vertebrates. Virulence factors are the main pathogenesis of S. aureus as a pathogen, which induce the host’s innate and adaptive immune responses. Toxic shock syndrome toxin 1 (TSST-1) is one of the most important virulence factors of S. aureus. However, the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) in TSST-1-induced innate immune response is still unclear. Here, purified recombinant TSST-1 (rTSST-1) was prepared and used to stimulate mouse peritoneal macrophages. The results showed that under the action of adenosine-triphosphate (ATP), rTSST-1 significantly induced interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) production in mouse macrophages and the production was dose-dependent. In addition, rTSST-1+ATP-stimulated cytokine production in macrophage depends on the activation of toll like receptor 4 (TLR4), but not TLR2 on the cells. Furthermore, the macrophages of NLRP3−/− mice stimulated with rTSST-1+ATP showed significantly low levels of IL-1β production compared to that of wild-type mice. These results demonstrated that TSST-1 can induce the expression of inflammatory cytokines in macrophages via the activation of the TLR4 and NLRP3 signaling pathways. Our study provides new information about the mechanism of the TSST-1-inducing host’s innate immune responses.

Phytochemical Constituents of Citrus hystrix DC. Leaves Attenuate Inflammation via NF-κB Signaling and NLRP3 Inflammasome Activity in Macrophages.

Citrus hystrix DC. (CH) is found in many countries in Southeast Asia. This plant has been reported for anti-microbial, anti-cancer and anti-inflammatory bioactivities. However, the anti-inflammatory and anti-inflammasome properties of the leaves remain poorly understood. This study aimed to investigate the effect of CH leaves on NLRP3 and NF-κB signaling pathways. CH leaves were sequentially extracted using hexane, ethyl acetate and 95% ethanol to give three crude extracts. An active compound, lupeol was fractionated from the ethanolic extract using chromatographic techniques, and its structure was identified and confirmed by spectroscopic methods. Anti-inflammatory activities were observed on both lipopolysaccharide-stimulated and NLRP3 adenosine triphosphate-induced macrophages. The release of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). Real-time qRT-polymerase chain reaction (PCR) was used to measure inflammatory-associated gene expression. NF-κB protein expressions were investigated using the immunoblotting technique. The active fraction of ethanolic CH leaves and lupeol significantly reduced the release of pro-inflammatory cytokines and suppressed the expression of both inflammasome genes and NF-κB proteins. The ethanolic extract of CH leaves and lupeol showed potent anti-inflammatory activities by targeting NF-κB and NLRP3 signaling pathways.

NLRP3 Inflammasome Blockade Reduces Cocaine-Induced Microglial Activation and Neuroinflammation.

Cocaine use disorder is a major health crisis that is associated with increased oxidative stress and neuroinflammation. While the role of NLRP3 inflammasome in mediating neuroinflammation is well-recognized, whether cocaine induces this response remains unexplored. Based on the premise that cocaine induces both reactive oxygen species (ROS) as well as microglial activation, we hypothesized that cocaine-mediated microglial activation involves both ROS and NLRP3 signaling pathways. We examined activation of the NLRP3 pathway in microglia exposed to cocaine, followed by validation in mice administered either cocaine or saline for 7 days, with or without pretreatment with the NLRP3 inhibitor, MCC950, and in postmortem cortical brain tissues of chronic cocaine-dependent humans. We found that microglia exposed to cocaine exhibited significant induction of NLRP3 and mature IL-1β expression. Intriguingly, blockade of ROS (Tempol) attenuated cocaine-mediated priming of NLRP3 and microglial activation (CD11b). Blockade of NLRP3 by both pharmacological (MCC950) as well as gene silencing (siNLRP3) approaches underpinned the critical role of NLRP3 in cocaine-mediated activation of inflammasome and microglial activation. Pretreatment of mice with MCC950 followed by cocaine administration for 7 days mitigated cocaine-mediated upregulation of mature IL-1β and CD11b, in both the striatum and the cortical regions. Furthermore, cortical brain tissues of chronic cocaine-dependent humans also exhibited upregulated expression of the NLRP3 pathway mediators compared with non-cocaine dependent controls. Collectively, these findings suggest that cocaine activates microglia involving the NLRP3 inflammasome pathway, thereby contributing to neuroinflammation. NLRP3 can thus be considered as a potential therapeutic target for alleviating cocaine-mediated neuroinflammation.

Milk-derived extracellular vesicles alleviate ulcerative colitis by regulating the gut immunity and reshaping the gut microbiota.

Rationale: Bovine milk constitutes an essential part of human diet, especially for children, due to its enrichment of various nutrients. We recently developed an effective protocol for the isolation of extracellular vesicles from milk (mEVs) and discovered that mEVs contained large amounts of immune-active proteins and modulated the gut immunity and microbiota in healthy mice. Here, we aimed to explore the therapeutic effects of mEVs on inflammatory bowel disease.Methods: MicroRNAs and protein content in mEVs were analyzed by RNA sequencing and proteomics, respectively, followed by functional annotation. Ulcerative colitis (UC) was induced by feeding mice with dextran sulfate sodium. Intestinal immune cell populations were phenotyped by flow cytometry, and the gut microbiota was analyzed via 16S rRNA sequencing.Results: We showed that abundant proteins and microRNAs in mEVs were involved in the regulation of immune and inflammatory pathways and that oral administration of mEVs prevented colon shortening, reduced intestinal epithelium disruption, inhibited infiltration of inflammatory cells and tissue fibrosis in a mouse UC model. Mechanistically, mEVs attenuated inflammatory response via inhibiting TLR4-NF-κB signaling pathway and NLRP3 inflammasome activation. Furthermore, mEVs were able to correct cytokine production disorder and restore the balance between T helper type 17 (Th17) cells and interleukin-10+Foxp3+ regulatory T (Treg) cells in the inflamed colon. The disturbed gut microbiota in UC was also partially recovered upon treatment with mEVs. The correlation between the gut microbiota and cytokines suggests that mEVs may modulate intestinal immunity via influencing the gut microbiota.Conclusions: These findings reveal that mEVs alleviate colitis by regulating intestinal immune homeostasis via inhibiting TLR4-NF-κB and NLRP3 signaling pathways, restoring Treg/Th17 cell balance, and reshaping the gut microbiota.

Attenuation of kirenol inflammationinduced by ischemic/reperfusion cerebral infarction stroke via TLR4/ NLRP3 signaling pathway: An in vivo approach

Background: Ischemia/reperfusion (I/R)-induced stroke is a long-lasting disability. Numerous reports have demonstrated that inflammation is the major cause of ischemic cerebral injury. Therefore, it is important to develop an effective anti-inflammatory agent for the attenuation of I/R-induced brain injury. Objective: In this study, we examined the therapeutic role of kirenol against I/R-induced neuronal damage by inhibiting inflammation in Sprague-Dawley (SD) rats. Materials and Methods: I/R was induced in SD rats and subsequently were administered with 10 and 20 mg/kg of kirenol. Then, we assessed the neurological score, brain water content, and infarct size. The level of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione, and acetylcholinesterase, as well as the levels of malondialdehyde, was measured by using standard methods. The level of tumor necrosis factor (TNF)-α; interleukin (IL)-1 β, IL-4, IL-6, and IL-10; and vascular endothelial growth factor was measured using standard kits. The targeted messenger RNA expression (NLRP3, NLRP4, TLR-4, TNF-α, caspase-1, ASC, and IL-1β) was quantified by polymerase chain reaction technique. Histopathological analysis of the brain tissue was performed. Results: According to our results, kirenol decreased the neurological deficit score, ameliorated the motor function, suppressed oxidative stress, reduced inflammation, and mediated the inhibition of TLR4/NLRP3-mediated inflammatory pathway. Conclusion: In conclusion, these findings demonstrate the protective effects of kirenol against I/R-induced cerebral injury. The mechanism of action is associated with the inhibition of inflammation through halting the TLR4/NLRP3 signaling pathway. In summary, kirenol is a potentially new compound which can be used to improve therapeutic strategies for stroke treatments.

P136 Relationship between cystic fibrosis disease severity and susceptibility to COVID-19 infection

Objective: Since cytokine storm and hyperinflammation play a key role on CF disease severity, severe CF patients should be considered to have an increased risk of developing severe symptoms of COVID-19. In this preliminary study, we evaluated whether there was a relationship between clinical severity of CF siblings and their susceptibility to COVID-19 infection. Method: In our preliminary study, we used a targeted transcriptomic approach (CF Profiler Array) obtained from nasal samples of three families who had CF siblings harbouring same mutation but showing different severity of CF phenotype. The siblings were classified as severe or mild CF according to recurrent lung infection, hepatic involvement, and FEV1. Results: In severe CF patients (n = 4) compared to mild patients (n = 3) CXCL1 (FC:-3.53), CXCL2 (FC:-2,16), CXCL8 (FC:-5.41), IL1B (FC:-2.61), SERPINA1 (FC:-2.54), TNFSF10 (FC:-1.73) were found to be downregulated. CXCL1, CXCL2, CXCL8 play critical role during infection control in neutrophiles that release other chemotactic mediators and recruit leukocytes. Additionally IL1B and TNFSF10 also affect activation of leukocytes. In the case of COVID-19 infection, the expression of these genes increases and leads to a cytokine storm. However our results show that CXCL1, CXCL2, CXCL8, IL1B genes which have a function in IL-17, NFKB, NLRP3 signaling pathways are downregulated in severe CF patients. Significant evidence supports the role of IL-1B, NLRP3-dependent inflammasome activation which is a central mediator of severe COVID-19 in the pathogenesis of acute lung injury. However, downregulation of inflammatory pathways is detected in severe forms of CF. Conclusion: The results of our preliminary study strengthens the hypothesis that severe forms of CF may constitute an advantage to mild forms of CF in susceptibility to COVID-19 and CXC inhibitors may be a promising therapeutic option for COVID-19 in the future. Further analyses should be performed with larger sample sizes.

Effects of ergosterone on lipopolysaccharideinduced acute lung injury and nucleo side-binding oligomerization domain, leucine-rich repeats and pyrin domain containing protein 3 inflammatory signaling pathway in mice

Ergosterone has been found to have a diversity of pharmacological effects, but the effect on lipopolysaccharide (LPS) induced Acute Lung Injury (ALI) is not very clear. This study is aimed at assessing the action of ergosterone on LPS-induced ALI. Mice on the 7th day of ergosterone prophylactic treatment were intranasally administered with an appropriate amount of LPS one hour before administration. After 12 hours, W/D ratio, TNF-α, IL-6, IL-1β, P-selectin, ICAM-1, NO, SOD and MDA levels were tested by ELISA in the obtained tissues. Besides, western blot was used to detect NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β leves. The results indicated that ergosterone pretreatment apparently brought down W/D ratio, TNF-α, IL-6, IL-1β, NO and MDA in lung tissues, and significantly increased SOD levels. Furthermore, ergosterone significantly inhibited the activation of NLRP3 induced by LPS. The results indicated that ergosterone has a significant protective action on ALI induced by LPS, and may inhibit the activation of the NLRP3 signaling pathway.

Ozone exposure promotes pyroptosis in rat lungs via the TLR2/4-NF-κB-NLRP3 signaling pathway

ObjectiveOzone has become a major air pollutant in recent years, which leading to a variety of lung diseases. This study aimed to explore the mechanisms of pyroptosis and related signaling pathways in ozone-induced lung injury. MethodsWe exposed 120 Wistar rats to ozone, 20 in each group (half male and half female). Ozone exposure concentrations were 0, 0.12, 0.5, 1.0, 2.0 and 4.0 ppm for 6 h. At the same time, we isolated and cultured type I alveolar epithelial cells, then intervened with high mobility group box 1 protein (HMGB1), hyaluronic acid (HA) and Toll-like receptors 2/4 (TLR2/4) inhibitor. In animal experiments, histopathological experiments, TUNEL, ELISA and biochemical indicators were performed. RT-qPCR and western blot experiments assay were used to detect the expression changes of key factors in relevant signal pathways in vivo and in vitro. ResultsAfter acute ozone exposure, the levels of lung cell injury indicators in bronchoalveolar lavage fluid (BALF), as well as the levels of inflammatory factors in BALF, blood, and lung tissue were significantly increased. Male rats were more sensitive to ozone exposure. Low-concentration ozone exposure caused mild interstitial inflammation in rat lung tissue. Severe inflammation and pulmonary edema appeared with increases in concentration. ELISA results in BALF showed that HMGB1 and HA expressions increased gradually with the increase of ozone exposure concentration. RT-qPCR and Western blot showed that when ozone concentrations increased above 0.5 ppm, the expression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3), cleaved caspase-1, and N-gasdermin D (N-GSDMD) in the lung tissue increased significantly, suggesting that ozone exposure induces pyroptosis. At the same time, it was found that ozone exposure activated the nuclear factor kappa B (NF-κB) signal pathway, and increased the mRNA expressions of Toll-like receptors TLR2/4. The results of cell experiments showed that after the addition of HMGB1 and HA, the expression of NF-κB and pyroptosis related indexes increased in type I alveolar epithelial cells, while the corresponding expression decreased after the addition of TLR2/4 inhibitors. ConclusionOzone exposure causes lung injury in a dose- and gender-dependent manner, and is more severe in males. When injured, the levels of HMGB1 and HA in BALF increased, which interact with TLR 2/4 to activate the downstream NF-κB signaling pathway. Further activating the NLRP3 inflammasome complex and regulating the ozone-induced pyroptosis.

Dendorbium Nobile Lindl. Alkaloids Suppress NF-kB and NLRP3 Signaling Pathways to Attenuate Lipopolysaccharideinduced Neuroinflammation

The important immune cells in the brain are called microglia acting as the central junction between neuroinflammation and neurodegenerative diseases. In patients of cognitive disorders and Alzheimer’s disease (AD) animal models, amoebic morphology and inflammatory pathways are activated to release numerous cells in the inflammatory factors by active microglia.