Dexmedetomidine alleviates hepatic ischaemia-reperfusion injury via the PI3K/AKT/Nrf2-NLRP3 pathway

Hepatic ischaemia-reperfusion (I/R) injury constitutes a tough difficulty in liver surgery. Dexmedetomidine (Dex) plays a protective role in I/R injury. This study investigated protective mechanism of Dex in hepatic I/R injury. The human hepatocyte line L02 received hypoxia/reoxygenation (H/R) treatment to stimulate cell model of hepatic I/R. The levels of pyroptosis proteins and inflammatory factors were detected. Functional rescue experiments were performed to confirm the effects of miR-494 and JUND on hepatic I/R injury. The levels of JUND, PI3K/p-PI3K, AKT/p-AKT, Nrf2, and NLRP3 activation were detected. The rat model of hepatic I/R injury was established to confirm the effect of Dex in vivo. Dex reduced pyroptosis and inflammation in H/R cells. Dex increased miR-494 expression, and miR-494 targeted JUND. miR-494 inhibition or JUND upregulation reversed the protective effect of Dex. Dex repressed NLRP3 inflammasome by activating the PI3K/AKT/Nrf2 pathway. In vivo experiments confirmed the protective effect of Dex on hepatic I/R injury. Overall, Dex repressed NLRP3 inflammasome and alleviated hepatic I/R injury via the miR-494/JUND/PI3K/AKT/Nrf2 axis.     

The combination of Panax ginseng and Angelica sinensis alleviates ischemia brain injury by suppressing NLRP3 inflammasome activation and microglial pyroptosis

BackgroundThe combination of Panax ginseng and Angelica sinensis (CPA) has been used to treat stroke for one thousand years and demonstrated clinically to have satisfied effects. However, the underlying mechanism remains unknown.PurposeWe investigate whether CPA has neuroprotective effects via suppressing Nod-like receptor protein 3 (NLRP3) inflammasome and microglial pyroptosis against ischemic injury in transient middle cerebral artery occlusion (MCAO) rats.MethodsMale rats were divided randomly into sham operated, MCAO, MCC950 (NLRP3-specific inhibitor) and CPA groups. Neurological deficits, glucose uptake, infarct size, activation of NLRP3 inflammasomes, microglial pyroptosis and related signaling pathways were detected. BV-2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used in in vitro experiments.ResultsCompared with sham rats, elevated level of proinflammatory interleukin-1β (IL-1β) in plasma, neurological function deficit, reduced glucose uptake in ipsilateral hemisphere, obvious infarct size, the activation of NLRP3 inflammasomes and enhanced microglial pyroptosis were presented in MCAO rats. The administrations of MCC950 and CPA respectively reversed the results. In vitro OGD/R induced the release of lactate dehydrogenase, promoted NLRP3 inflammasomes activation and pyroptosis in BV-2 cells, which was significantly suppressed by treatment with ginsenoside Rd (Rd) and Z-ligustilide (LIG). Mechanistically, OGD/R induced high expression of dynamin-related protein 1 (Drp1) and mitochondrial fission, as well as NLRP3 inflammasomes activation and pyroptosis in BV-2 cells, which was attenuated by treatment with Rd and LIG. Moreover, the increased expression of Drp1 was validated in MCAO rats, and also abolished by MCC950 or CPA treatments.ConclusionCPA treatment attenuates cerebral injury via inhibition of NLRP3 inflammasomes activation and microglial pyroptosis after stroke, which at least partially involved in the amelioration of Drp1-mediated mitochondrial fission.     

Piperine protects against pyroptosis in myocardial ischaemia/reperfusion injury by regulating the miR-383/RP105/AKT signalling pathway

miRNA-mediated pyroptosis play crucial effects in the development of myocardial ischaemia/reperfusion (I/R) injury (MIRI). Piperine (PIP) possesses multiple pharmacological effects especially in I/R condition. This study focuses on whether PIP protects MIRI from pyroptosis via miR-383-dependent pathway. Rat MIRI model was established by 30 minutes of LAD ligation and 4 hours of reperfusion. Myocardial enzymes, histomorphology, structure and function were detected to evaluate MIRI. Recombinant adenoviral vectors for miR-383 overexpression or miR-383 silencing or RP105 knockdown were constructed, respectively. Luciferase reporter analysis was used to confirm RP105 as a target of miR-383. Pyroptosis-related markers were measured by Western blotting assay. The results showed that I/R provoked myocardial injury, as shown by the increases of LDH/CK releases, infarcted areas and apoptosis as well as worsened function and structure. Pyroptosis-related mediators including NLRP3, cleaved caspase-1, cleaved IL-1β and IL-18 were also reinforced after MIRI. However, PIP treatment greatly ameliorated MIRI in parallel with pyroptotic repression. In mechanistic studies, MIRI-caused elevation of miR-383 and decrease of RP105/PI3K/AKT pathway were reverted by PIP treatment. Luciferase reporter assay confirmed RP105 as a miR-383 target. miR-383 knockdown ameliorated but miR-383 overexpression facilitated pyroptosis and MIRI. Moreover, the anti-pyroptotic effect from miR-383 silencing was verified to be relied on the RP105/PI3K/AKT signalling pathway. Additionally, our present study further indicated the miR-383/RP105/AKT-dependent approach resulting from PIP administration against pyroptosis in MIRI. Therefore, PIP treatment attenuates MIRI and pyroptosis by regulating miR-383/RP105/AKT pathway, and it may provide a therapeutic manner for the treatment of MIRI.     

LncRNA-HOTAIR promotes endothelial cell pyroptosis by regulating the miR-22/NLRP3 axis in hyperuricaemia

Long non-coding RNA (lncRNA) plays an important role in the renal inflammatory response caused by hyperuricaemia. However, the underlying molecular mechanisms through which lncRNA is involved in endothelial injury induced by hyperuricaemia remain unclear. In this study, we investigated the regulatory role of lncRNA-HOTAIR in high concentration of uric acid (HUA)–induced renal injury. We established hyperuricaemia mouse model and an in vitro uric acid (UA)–induced human umbilical vein endothelial cell (HUVEC) injury model. In HUA-treated HUVECs and hyperuricaemia mice, we observed increased HOTAIR and decreased miR-22 expression. The expression of pyroptosis-associated protein (NLRP3, Caspase-1, GSDMD-N, GSDMD-FL) was increased. The release of LDH, IL-1β and IL-18 in cell supernatants and the sera of model mice was also increased. The proliferation of HUVECs stimulated by HUA was significantly inhibited, and the number of TUNEL-positive cells in hyperuricaemia mouse kidney was increased. Bioinformatics analysis and luciferase reporter and RIP assays confirmed that HOTAIR promoted NLRP3 inflammasome activation by competitively binding miR-22. In gain- or loss-of-function experiments, we found that HOTAIR and NLRP3 overexpression or miR-22 knock down activated the NLRP3 inflammasome and promoted pyroptosis in HUA-treated HUVECs, while NLRP3 and HOTAIR knockdown or a miR-22 mimic exerted the opposite effects. Furthermore, in vivo experiments validated that HOTAIR knockdown alleviated renal inflammation in hyperuricaemia mice. In conclusion, we demonstrated that in hyperuricaemia, lncRNA-HOTAIR promotes endothelial cell pyroptosis by competitively binding miR-22 to regulate NLRP3 expression.     

NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model

Background

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved.

Methods

Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation.

Results

Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β.

Conclusion

NLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.     

Aesculin suppresses the NLRP3 inflammasome-mediated pyroptosis via the Akt/GSK3β/NF-κB pathway to mitigate myocardial ischemia/reperfusion injury

BackgroundAesculin (AES), an effective component of Cortex fraxini, is a hydroxycoumarin glucoside that has diverse biological properties. The nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing 3 (NLRP3) inflammasome has been heavily interwoven with the development of myocardial ischemia/reperfusion injury (MIRI). Nevertheless, it remains unclear whether AES makes a difference to the changes of the NLRP3 inflammasome in MIRI.PurposeWe used rats that were subjected to MIRI and neonatal rat cardiomyocytes (NRCMs) that underwent oxygen-glucose deprivation/restoration (OGD/R) process to investigate what impacts AES exerts on MIRI and the NLRP3 inflammasome activation.MethodsThe establishment of MIRI model in rats was conducted using the left anterior descending coronary artery ligation for 0.5 h ischemia and then untying the knot for 4 h of reperfusion. After reperfusion, AES were administered intraperitoneally using 10 and 30 mg/kg doses. We evaluated the development of reperfusion ventricular arrhythmias, hemodynamic changes, infarct size, and the biomarkers in myocardial injury. The inflammatory mediators and pyroptosis were also assessed. AES at the concentrations of 1, 3, and 10 μM were imposed on the NRCMs immediately before the restoration process. We also determined the cell viability and cell death in the NRCMs exposed to OGD/R insult. Furthermore, we also analyzed the levels of proteins that affect the NLRP3 inflammasome activation, pyroptosis, and the AKT serine/threonine kinase (Akt)/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor-kappa B (NF-κB) signaling pathway via western blotting.ResultsWe found that AES notably attenuated reperfusion arrhythmias and myocardia damage, improved the hemodynamic function, and ameliorated the inflammatory response and pyroptosis of cardiomyocytes in rats and NRCMs. Additionally, AES reduced the NLRP3 inflammasome activation in rats and NRCMs. AES also enhanced the phosphorylation of Akt and GSK3β, while suppressing the phosphorylation of NF-κB. Moreover, the allosteric Akt inhibitor, MK-2206, abolished the AES-mediated cardioprotection and the NLRP3 inflammasome suppression.ConclusionsThese findings indicate that AES effectively protected cardiomyocytes against MIRI by suppressing the NLRP3 inflammasome-mediated pyroptosis, which may relate to the upregulated Akt activation and disruption of the GSK3β/NF-κB pathway.     

Neuroprotective Effects of Cactus Polysaccharide on Oxygen and Glucose Deprivation Induced Damage in Rat Brain Slices

1. The neuroprotective effect of cactus polysaccharide (CP) on oxygen and glucose deprivation (OGD) and reoxygenation (REO)-induced damage in the cortical and hippocampal slices of rat brain was investigated. 2. Cell viability was evaluated by using the 2, 3, 5-triphenyl tetrazolium chloride (TTC) method. The fluorescence of propidium iodide (PI) staining was used for quantification of cellular survival, and lactate dehydrogenase (LDH) activity in incubation medium was assessed by LDH assay to evaluate the degree of injury. 3. The OGD ischemic condition significantly decreased cellular viability and increased LDH release in the incubation medium. CP (0.2 mg/l∼2 mg/l) protected brain slices from OGD injury in a dosage dependent manner as demonstrated by increased A 490 value of TTC, decreased PI intensity and LDH release. At the above concentration, CP also prevented the increase of nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity induced by OGD. 4. CP can protect the brain slices (cortical and hippocampus) against injury induced by OGD. Its neuroprotective effect may be partly mediated by the NO/iNOS system induced by OGD insult. Xianju Huang and Qin Li have contributed equally to this article.     

白藜芦醇对氧糖剥夺／再灌注损伤的PCI2细胞的保护作用及其作用机制

目的：探讨白藜芦醇对氧糖剥夺／再灌注（OGD／R）损伤的PCI2细胞的保护作用及其机制。方法：体外培养PCI2细胞,分为对照组,白藜芦醇组,OGD／R组及OGD／R＋白藜芦醇组。以改良的噻唑蓝法测定细胞活性,采用AnnexinV—FITC／PI双染法检测细胞的凋亡率,用双氯罗丹明（DHR）检测细胞内活性氧簇（Ros）的水平,采用蛋白印迹法（westemblot）分析SIRTl的蛋白表达情况。结果：与对照组相比,经过OGD／R损伤后,细胞活力显著降低。而在OGD／R的同时给予10μmol／L的白藜芦醇处理。可以明显提高细胞活力。流式细胞仪检测发现,10μmol／L的白藜芦醇可以显著地减少OGD／R引起的细胞凋亡,抑制细胞内的ROS产生。westemblot的结果提示,与对照组比较,白藜芦醇可提高SIRTl的蛋白表达水平。结论：白藜芦醇可以通过抑制ROS的产生和上调SIRTl的表达等机制而发挥其对抗氧糖剥夺／再灌注损伤的神经保护性作用。     

DHA/AA alleviates LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3 to inhibit inflammasome complexes assembly

Pyroptosis is a novel type of programmed cell death associated with the pathogenesis of many inflammatory diseases. Docosahexaenoic acid (DHA) and Arachidonic acid (AA) is widely involved in inflammatory pathological processes. However, the effect and mechanism of DHA and AA on pyroptosis in Kupffer cells are poorly understood. The present study demonstrated that DHA and AA ameliorated lipopolysaccharide (LPS)-induced Kupffer cells pyroptosis by reversing the increased expression of NLRP3 inflammasome complex, GSDMD, IL-1β, IL-18, and PI-stained positive rate. Next, the study revealed that GPR120 silencing eliminated the anti-pyroptosis of DHA and AA in LPS-induced Kupffer cells, suggesting that DHA and AA exerted their effect through GPR120 signaling. Importantly, GPR120 endocytose and binds to NLRP3 under LPS stimulation. Furthermore, co-immunoprecipitation showed that DHA and AA promoted the interaction between GPR120 and NLRP3 in LPS-exposed Kupffer cells, thus inhibiting the self-assembly of NLRP3 inflammasome complex. Finally, the study verified that DHA and AA alleviated hepatic injury through inhibiting Kupffer cells pyroptosis in vivo. The findings indicated that DHA and AA alleviated LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3, it might become a potential therapeutic approach hepatic injury.Subject terms: Cell death, Immunology

DHA/AA alleviated LPS-induced Kupffer cells pyroptosis via GPR120 interaction with NLRP3 to inhibit NLRP3 inflammasome assembly.     

Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

Objective

Recent studies indicate that the innate immune system is not only triggered by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As triggers for the innate immune NLRP3 inflammasome protein complex appear to overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NLRP3 inflammasome is involved in IPC and acute I/R injury of the heart.

Principal Findings

Baseline cardiac performance and acute I/R injury were investigated in isolated, Langendorff-perfused hearts from wild-type (WT), ASC^−/− and NLRP3^−/− mice. Deletion of NLRP3 inflammasome components ASC^−/− or NLRP3^−/− did not affect baseline performance. The deletions exacerbated I/R-induced mechanical dysfunction, but were without effect on I/R-induced cell death. When subjected to IPC, WT and ASC^−/− hearts were protected against I/R injury (improved function and less cell death). However, IPC did not protect NLRP3^−/− hearts against I/R injury. NLRP3^−/− hearts had significantly decreased cardiac IL-6 levels with a trend towards lower IL-1β levels at end reperfusion, suggesting abrogation of IPC through diminished IL-6 and/or IL-1β signaling. Subsequent experiments showed that neutralising IL-6 using an antibody against IL-6 abrogated IPC in WT hearts. However, inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) did not abrogate IPC in WT hearts. Analysis of survival kinases after IPC demonstrated decreased STAT3 expression in NLRP3^−/− hearts when compared to WT hearts.

Conclusions

The data suggest that the innate immune NLRP3 protein, in an NLRP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6/STAT3 dependent mechanism.     

Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome activation

Coronary microembolization (CME), a common reason for periprocedural myocardial infarction (PMI), bears very important prognostic implications. However, the molecular mechanisms related to CME remain largely elusive. Statins have been shown to prevent PMI, but the underlying mechanism has not been identified. Here, we examine whether the NLRP3 inflammasome contributes to CME-induced cardiac injury and investigate the effects of statin therapy on CME. In vivo study, mice with CME were treated with 40 mg/kg/d rosuvastatin (RVS) orally or a selective NLRP3 inflammasome inhibitor MCC950 intraperitoneally (20 mg/kg/d). Mice treated with MCC950 and RVS showed improved cardiac contractile function and morphological changes, diminished fibrosis and microinfarct size, and reduced serum lactate dehydrogenase (LDH) level. Mechanistically, RVS decreased the expression of NLRP3, caspase-1, interleukin-1β, and Gasdermin D N-terminal domains. Proteomics analysis revealed that RVS restored the energy metabolism and oxidative phosphorylation in CME. Furthermore, reduced reactive oxygen species (ROS) level and alleviated mitochondrial damage were observed in RVS-treated mice. In vitro study, RVS inhibited the activation of NLRP3 inflammasome induced by tumor necrosis factor α plus hypoxia in H9c2 cells. Meanwhile, the pyroptosis was also suppressed by RVS, indicated by the increased cell viability, decreased LDH and propidium iodide uptake in H9c2 cells. RVS also reduced the level of mitochondrial ROS generation in vitro. Our results indicate the NLRP3 inflammasome-dependent cardiac pyroptosis plays an important role in CME-induced cardiac injury and its inhibitor exerts cardioprotective effect following CME. We also uncover the anti-pyroptosis role of RVS in CME, which is associated with regulating mitochondrial ROS.Subject terms: Cell death, Cardiovascular diseases     

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways.     

Echovirus 11 infection induces pyroptotic cell death by facilitating NLRP3 inflammasome activation

Echovirus 11 (ECHO 11) is a positive-strand RNA virus belonging to the genus Enterovirus of the family Picornaviridae. ECHO 11 infections can cause severe inflammatory illnesses in neonates, including severe acute hepatitis with coagulopathy. The activation of NLRP3 inflammasome is important for host defense against invading viruses, which also contributes to viral pathogenicity. However, whether and how ECHO 11 induces NLRP3 inflammasome activation remains unclear. In this study, we isolated a clinical strain of ECHO 11 from stools of an ECHO 11-infected newborn patient with necrotizing hepatitis. This virus shared 99.95% sequence identity with the previously published ECHO 11 sequence. The clinically isolated ECHO 11 can efficiently infect liver cells and strongly induces inflammation. Moreover, we showed that ECHO 11 induced IL-1β secretion and pyroptosis in cells and mouse bone marrow-derived macrophages (BMDMs). Furthermore, ECHO 11 infection triggered NLRP3 inflammasome activation, as evidenced by cleavages of GSDMD, pro-IL-1β and pro-caspase-1, and the release of LDH. ECHO 11 2B protein was required for NLRP3 inflammasome activation via interacting with NLRP3 to facilitate the inflammasome complex assembly. In vivo, expression of ECHO 11 2B also activated NLRP3 inflammasome in the murine liver. Besides, 2Bs of multiple EVs can also interact with NLRP3 and induce NLRP3 inflammasome activation. Together, our findings demonstrate a mechanism by which ECHO 11 induces inflammatory responses by activating NLRP3 inflammasome, providing novel insights into the pathogenesis of ECHO 11 infection.     

Gastrodin Alleviates Cerebral Ischaemia/Reperfusion Injury by Inhibiting Pyroptosis by Regulating the lncRNA NEAT1/miR-22-3p Axis

Cerebral ischaemia/reperfusion (I/R) injury-induced irreversible brain injury is a major cause of mortality and functional impairment in ageing people. Gastrodin (GAS), derived from the traditional Chinese herbal medicine Tianma, has been reported to inhibit the progression of stroke, but the mechanism whereby GAS modulates the progression of cerebral I/R remains unclear. The middle cerebral artery occlusion method was used as a model of I/R in vivo. Rats were pretreated with GAS by intraperitoneal injection 7 days before I/R surgery and were then treated with GAS for 7 days after I/R surgery. Additionally, an oxygen–glucose deprivation/reoxygenation model using neuronal cells was established in vitro to simulate I/R injury. 2,3,5-Triphenyltetrazolium chloride and Nissl staining were used to evaluate infarct size and neuronal damage, respectively. Lactate dehydrogenase release and cell counting kit-8 assays were used to assess neuronal cell viability. Enzyme-linked immunosorbent assay, qPCR, flow cytometry and western blotting were performed to analyse the expression levels of inflammatory factors (IL-1β, IL-18), lncRNA NEAT1, miR-22-3p, NLRP3 and cleaved caspase-1. Luciferase reporter experiments were performed to verify the association between lncRNA NEAT1 and miR-22-3p. The results indicated that GAS could significantly improve the neurological scores of rats and reduce the area of cerebral infarction. Meanwhile, GAS inhibited pyroptosis by downregulating NLRP3, inflammatory factors (IL-1β, IL-18) and cleaved caspase-1. In addition, GAS attenuated I/R-induced inflammation in neuronal cells through the modulation of the lncRNA NEAT1/miR-22-3p axis. GAS significantly attenuated cerebral I/R injury via modulation of the lncRNA NEAT1/miR-22-3p axis. Thus, GAS might serve as a new agent for the treatment of cerebral I/R injury.

     

Plasma-derived exosomes contribute to pancreatitis-associated lung injury by triggering NLRP3-dependent pyroptosis in alveolar macrophages

Progression of acute pancreatitis (AP) into a severe form usually results in a life-threatening condition with multiple organ dysfunction, and in particular acute lung injury (ALI), often contributes to the majority of AP-associated deaths. Increasing evidence has shown that uncontrolled activation of the immune system with rapid production of inflammatory cytokines play a dominant role in this process. As an intracellular inflammatory signaling platform, the NOD-like receptor protein 3 (NLRP3) inflammasome, is recently reported to be involved in the pathogenesis of AP progression, however, the relationship between NLRP3 inflammasome activation and AP-associated lung injury remains unclear yet. Here, we show that NLRP3 inflammasome activation and subsequent pyroptosis in alveolar macrophages (AMs) is responsible for the lung injury secondary to AP. In addition, plasma-derived exosomes from AP mice is capable of triggering NLRP3-dependent pyroptosis in AMs. Inhibition of exosome release or uptake in vivo by inhibitors substantially suppresses AMs pyroptosis and thereby alleviates AP-induced pulmonary lesion. Collectively, the current work reveals for the first time the involvement of NLRP3-dependent pyroptosis induced by plasma exosomes in the pathogenesis of AP-induced ALI, suggesting that the exosome-mediated NLRP3 inflammatory pathway is a potential therapeutic target for the treatment of lung injury during AP.     

Beclin-1 overexpression regulates NLRP3 activation by promoting TNFAIP3 in microvascular injury following myocardial reperfusion

Innate immune response contributes significantly to ischemia reperfusion (I/R) injury. Targeting innate immunity seems to be a promising method for protecting the microvascular injury in ST-elevation myocardial infarction (STEMI) patients following myocardial I/R injury (MI/R). NLRP3 inflammasome is a central part of the innate immune system involved in the pathophysiological process of MI/R. However, the mechanisms regulating NLRP3 activation are yet to be clarified. Recently, autophagy has been related to the regulation of NLRP3 activation. Thus, how Beclin-1/Becn1 overexpression influences NLRP3 activation in microvascular endothelial cells (CMECs) after MI/R is yet to be investigated. The present study showed that Becn1 overexpression exhibits a significant increase in NLRP3 and IL-1β in CMEC responses to MI/R. Interestingly, Becn1 overexpression promoted TNFAIP3 expression, which restricted NLRP3 activation in vitro and in vivo. The current study also showed that inflammatory cells (CD68) and B (CDB220) lymphocytes were decreased in transgenic mice with overexpression of Beclin-1 (BECN1-Tg) in the spleen and heart. These findings highlighted Becn1 as a prospective target for treating NLRP3 mediated microvascular injury following MI/R.     

Naoxintong attenuates Ischaemia/reperfusion Injury through inhibiting NLRP3 inflammasome activation

Naoxintong (NXT) is a Chinese Materia Medica standardized product extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinensis, Astragali Radix. Naoxintong is clinically effective in treating ischaemia heart disease. Nucleotide‐binding oligomerization domain‐Like Receptor with a Pyrin domain 3 (NLRP3) inflammasome has been critically involved in myocardial ischaemia/reperfusion (I/R) injury. Here, we have been suggested that NXT might attenuate myocardial I/R injury via suppression of NLRP3 inflammasome activation. Male C57BL6 mice were subjected to myocardial I/R injury via 45 min. coronary ligation and release for the indicated times. Naoxintong (0.7 g/kg/day) and PBS were orally administrated for 2 weeks before surgery. Cardiac function assessed by echocardiography was significantly improved in the NXT group compared to PBS group at day 2 after myocardial I/R. NLRP3 inflammasome activation is crucially involved in the initial inflammatory response after myocardial I/R injury, leading to cleaved caspase‐1, mature interleukin (IL)‐1β production, accompanying by macrophage and neutrophil infiltration. The cardioprotective effect of NXT was associated with a diminished NLRP3 inflammasome activation, decreased pro‐inflammatory macrophage (M1 macrophages) and neutrophil infiltration after myocardial I/R injury. In addition, serum levels of IL‐1β, indicators of NLRP3 inflammasome activation, were also significantly suppressed in the NXT treated group after I/R injury. Naoxintong exerts cardioprotive effects at least partly by suppression of NLRP3 inflammasome activation in this I/R injury model.     

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.