NLRP3 Inflammasome Formation Research Articles

Forsythoside B Ameliorates Neuroinflammation via Inhibiting NLRP3 Inflammasome of Glial Cells in Experimental Autoimmune Encephalomyelitis Mice.

Neuroinflammation mediated by glial cells plays a crucial role in demyelination in experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis (MS) model. Forsythoside B (FTS·B), a natural phenylethanoid glycoside isolated from the dried fruits and leaves of Forsythia suspensa (Thunb.) Vahl, has been found to have antioxidant, anti-apoptotic, and anti-inflammatory properties. However, there is currently no report or research on the effectiveness of FTS·B treatment for EAE. The aim of this study was to investigate the neuroprotective properties of (FTS·B) on EAE and reveal its potential mechanisms. Myelin oligodendrocyte glycoprotein-induced EAE mice were randomly categorized into the control, EAE model, and FTS·B treatment groups. Behavioral testing, pathology, immunohistochemistry, immunofluorescence staining, and western blot analysis of spinal cord tissue were used to determine the effects and mechanisms of FTS·B on EAE in mice. We found that FTS·B treatment could significantly alleviate and reduce the clinical symptoms and morbidity of EAE, respectively. In addition, FTS·B administration reduced inflammatory response and demyelination by inhibiting glial cell activation in the spinal cord of EAE mice. Further experiments confirmed that FTS·B inhibited the formation of NLRP3 inflammasome in microglia and astrocytes, thereby suppressing neuroinflammation and GSDMD-mediated pyroptosis. Altogether, these results suggest that FTS·B treatment attenuates central neuroinflammation and pyroptosis by inhibiting NLRP3 inflammasome of glial cells in EAE mice.

Denfivontinib activates effector T-cells through NLRP3-inflammasome, yielding potent anticancer effects by combination with pembrolizumab.

Various combination therapies have been investigated to overcome the limitations of using immune checkpoint inhibitors. However, determining the optimal combination therapy remains challenging. To overcome the therapeutical limitation, we conducted a translational research to elucidate the mechanisms by which AXL inhibition enhances the anti-tumor effects when combined with anti-PD-1 antibody therapy. Herein, we demonstrated improved antitumor effects through combination treatment with denfivontinib and pembrolizumab which resulted in enhanced differentiation into effector CD4+ and CD8+ memory T cells, accompanied by an increase in IFN-γ expression in the YHIM-2004 xenograft model derived from patients with NSCLC. Concurrently, a reduction in the number of immunosuppressive M2 macrophages and myeloid-derived suppressor cells was observed. Mechanistically, denfivontinib potentiated the NOD-like receptor pathway, thereby facilitating the NLRP3 inflammasome formation. This leads to macrophage activation via the NF-kB signaling pathway activation. We have confirmed that the positive interaction between macrophages and T cells arises from the enhanced antigen-presenting machinery of activated macrophages. Furthermore, the observed tumor effects in AXL knock-out mice confirmed that AXL inhibition by denfivontinib enhances the anti-tumor effects, thus opening new avenues for therapeutic interventions aimed at overcoming limitations in immunotherapy. To demonstrate the extent to which our findings reflect clinical results, we analyzed bulk-RNA sequencing data from 21 NSCLC patients undergoing anti-PD-1 immunotherapy. The NLRP3 inflammasome score influenced enhanced immune responses in patient data undergoing anti-PD-1 immunotherapy, suggesting a role for NLRP3 inflammasome in activating immune responses during treatment.

The role of NLRP3 and NLRP12 inflammasomes in glioblastoma.

Glioblastoma (GBM) is the deadliest malignant brain tumor, with a survival of less than 14 months after diagnosis. The highly invasive nature of GBM makes total surgical resection challenging, leading to tumor recurrence and declined survival. The heterocellular composition of the GBM reprograms its microenvironment, favoring tumor growth, proliferation, and migration. The innate immune cells in the GBM tumor microenvironment, including microglia, astrocytes, and macrophages, express pattern recognition receptors such as NLRs (Nucleotide-binding domain and leucine-rich repeat-containing) that sense pathogen- and damage-associated molecular patterns initiating inflammation. Upon activation, NLRP3 promotes inflammation by NLRP3 inflammasome formation. Auto-proteolytic cleavage and activation of Caspase-1 within the inflammasome leads to caspase-1-mediated cleavage, activation, and conversion of pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, leading to pyroptosis. In contrast, NLRP12 downregulates inflammatory responses in microglia and macrophages by regulating the NF-κB pathway. NLRP3 and NLRP12 have been implicated in the disease pathophysiology of several cancers with cell-context-dependent, pro- or anti-tumorigenic roles. In this review, we discuss the current literature on the mechanistic roles of NLRP3 and NLRP12 in GBM and the gaps in the scientific literature in the context of GBM pathophysiology with potential for targeted therapeutics.

Tanshinone IIA Regulates NRF2/NLRP3 Signal Pathway to Restrain Oxidative Stress and Inflammation in Uric Acid-Induced HK-2 Fibrotic Models.

This study aims to investigate the function and potential mechanism of Tanshinone IIA in uric acid-induced HK-2 fibrosis models. An in vitro model of fibrosis was constructed using uric acid stimulation. RT-qPCR and Western blot were used to evaluate the levels of inflammatory cytokines. The detection of ROS and ELISA assay were used to analyze the changes in oxidative stress. Tanshinone IIA inhibited the increase in inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-18 and the formation of NLRP3 inflammasome induced by uric acid stimulation. In addition, Tanshinone IIA treatment reduced the production of ROS and MDA, promoting the expression of SOD and CAT, thereby protecting HK-2 cells from oxidative stress damage. Besides, the expression of TGF-β, FN, and COL-1 was significantly reduced by the treatment of Tanshinone IIA. Mechanistically, Tanshinone IIA inhibited the expression of inflammatory cytokines and the formation of the NLRP3 inflammasome by targeting NRF2. Tanshinone IIA exerts a protective role in uric acid-induced HK-2 fibrosis models by targeting the NRF2-NLRP3 signaling pathway to reduce the occurrence of inflammation and oxidative stress.

MIR181A1HG is a novel long noncoding RNA regulating vascular inflammation and atherosclerosis

Abstract Background Endothelial cell (EC) dysfunction and vascular inflammation are critical in the initiation and progression of atherosclerosis. Long noncoding RNAs (lncRNAs) plays a critical role in vascular pathology, but relatively little is known about their involvement in controlling vascular inflammation. MIR181A1HG is a lncRNA locating juxta-position with miR-181a1 and miR-181b1, both involved in vascular inflammation. However, the role of MIR181A1HG in vascular inflammation is still unknow. Purpose The study aims to investigate the role of MIR181A1HG in regulating vascular inflammation and atherosclerosis. Methods We examined expression of MIR181A1HG in both human and mouse atherosclerotic lesions. Loss-of-function and gain-of-function studies, multiple RNA–protein interaction assays luciferase reporter and chromatin immunoprecipitation assays were used to investigate the role and molecular mechanisms of MIR181A1HG in the vascular inflammation and atherosclerosis. The single-cell RNA-sequencing were used to analyze atherosclerotic phenotypes of MIR181A1HG-/-ApoE-/- mice. Results MIR181A1HG was abundant in ECs and significantly increased in both humans and mouse atherosclerotic lesions. Deficiency of MIR181A1HG effectively suppressed NLRP3 inflammasome, ECs activation, and atherosclerotic lesion formation. In contrast, EC-specific MIR181A1HG overexpression promoted NLRP3 inflammasome, ECs activation, and atherosclerotic lesion formation, which were reversed by pharmacological inhibition of NLRP3 (MCC950). MIR181A1HG functioned by regulating NLRP3 inflammation, ECs activation though decoying Foxp1 away from the promoters of targets genes, which is independently of miR181a1/b1 cluster. EC-specific Foxp1 silencing reversed in vivo anti-atherosclerotic effect mediated by MIR181A1HG-deletion.

Benzoylmesaconine mitigates NLRP3 inflammasome-related diseases by reducing intracellular K+ efflux and disrupting NLRP3 inflammasome assembly

BackgroundBenzoylmesaconine (BMA), a major alkaloid derived from the traditional Chinese medicine Aconitum carmichaeli Debx, exhibits potent anti-inflammatory properties. However, the precise mechanism underlying its action remains unclear. PurposeThis study aimed to investigate the inhibitory mechanism of BMA on the NLRP3 inflammasome and assess its therapeutic efficacy in NLRP3-related metabolic diseases. MethodsA classic NLRP3 inflammasome-activated bone marrow-derived macrophage (BMDM) model was established to evaluate BMA's effects on NLRP3 upstream and downstream protein expression, as well as pyroptosis. Two distinct animal disease models, MSU-induced gouty arthritis and DSS-induced colitis, were utilized to validate BMA's anti-inflammatory activity in vivo. ResultsIn vitro findings revealed that BMA can suppress NLRP3 inflammasome activation by inhibiting interleukin-1β (IL-1β) secretion and GSDMD-N protein expression. This mechanism involved blocking intracellular K+ efflux and interfering with the formation of NLRP3 inflammasomes. In vivo studies demonstrated that BMA significantly alleviated inflammatory symptoms in MSU-induced acute gout and DSS-induced colitis models. ConclusionThese findings suggest that BMA effectively inhibits the activation of the NLRP3 signaling pathway through dual mechanisms: reducing intracellular K+ efflux and disrupting NLRP3 inflammasome assembly. This multifaceted action highlights the therapeutic potential of BMA for NLRP3-related diseases.

A carbamazepine metabolite activates NLRP3 and controls skin homing of CD8+ T-cells in SJS/TEN

BackgroundStevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe adverse drug reactions with extensive keratinocyte death. Carbamazepine (CBZ), the most commonly implicated drug in SJS/TEN, is metabolized by the cytochrome P450 enzyme 3A4 (CYP3A4) into carbamazepine-10,11-epoxide (CBZE) in the liver. While CD8+ cytotoxic T cells play an important role in SJS/TEN, the underlying mechanism of exuberant immune response by CD8+ T cells in these conditions remains incompletely understood. ObjectivesTo examine the expression of NLRP3 inflammasome and their skin migration in CBZE-induced SJS/TEN. MethodsThe expression of the NLRP3 inflammasome complex in skin lesions, sera, and blister fluids of SJS/TEN patients were analyzed by immunohistochemistry and enzyme-linked immunosorbent assay. NLRP3 formation and CD8+ T cell activation status and their functions were examined by immunoblotting, immunofluorescence, and chemotaxis assays. ResultsThe expression of the NLRP3 inflammasome complex was greatly increased in skin lesions of SJS/TEN patients. Moreover, IL-1β and IL-18 levels in sera and blister fluids of SJS/TEN patients were approximately 3-fold higher than those in healthy individuals, with a linear correlation between IL-1β levels and disease activity. CBZE induced NLRP3 inflammasome formation, upregulated CXCL9/CXCL10 levels, and activated CD8+ cytotoxic T cell functions via IL-1β/IL-1R or IL-18/IL-18R signaling in SJS/TEN keratinocytes, which promoted CD8+ cytotoxic T cell migration in SJS/TEN patients. ConclusionThis study showed that CBZE promoted NLRP3 inflammasome formation and strengthened the activation and function of CD8+ cytotoxic T cells in the skin, which contributed to the initiation and progression of SJS/TEN.

Protective effects of tetramethylpyrazine on myocardial ischemia/reperfusion injury involve NLRP3 inflammasome suppression by autophagy activation

Tetramethylpyrazine (TMP) belongs to the active ingredients of the traditional Chinese medicine Chuanxiong, which has a certain protective effect in myocardial ischemia–reperfusion (I/R) injury. It can improve postoperative cardiac function and alleviate ventricular remodeling in acute myocardial infarction patients. However, its specific protective mechanism is still unclear. In this study, a certain concentration of TMP was introduced into I/R mice or H9C2 cells after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment to observe the effects of TMP on cardiomyocyte activity, cytotoxicity, apoptosis, autophagy, pyroptosis, and NLRP3 inflammasome activation. The results displayed that TMP intervention could reduce OGD/R and I/R-induced cardiomyocyte apoptosis, accelerate cellular activity and autophagy levels, and ameliorate myocardial tissue necrosis in I/R mice in a dose-dependent manner. Further, TMP prevented the formation of NLRP3 inflammasomes to suppress pyroptosis by increasing the level of cardiomyocyte autophagy after I/R and OGD/R modelling, the introduction of chloroquine to suppress autophagic activity in vivo and in vitro was further analyzed to confirm whether TMP inhibits NLRP3 inflammasome activation and pyroptosis by increasing autophagy, and we found the inhibitory effect of TMP on NLRP3 inflammasomes and its protective effect against myocardial injury were blocked when autophagy was inhibited with chloroquine. In conclusion, this experiment demonstrated that TMP unusually attenuated I/R injury in mice, and this protective effect was achieved by inhibiting the activation of NLRP3 inflammasomes through enhancing autophagic activity.

Inhibitors of NLRP3 Inflammasome Formation: A Cardioprotective Role for the Gasotransmitters Carbon Monoxide, Nitric Oxide, and Hydrogen Sulphide in Acute Myocardial Infarction.

Myocardial ischaemia reperfusion injury (IRI) occurring from acute coronary artery disease or cardiac surgical interventions such as bypass surgery can result in myocardial dysfunction, presenting as, myocardial "stunning", arrhythmias, infarction, and adverse cardiac remodelling, and may lead to both a systemic and a localised inflammatory response. This localised cardiac inflammatory response is regulated through the nucleotide-binding oligomerisation domain (NACHT), leucine-rich repeat (LRR)-containing protein family pyrin domain (PYD)-3 (NLRP3) inflammasome, a multimeric structure whose components are present within both cardiomyocytes and in cardiac fibroblasts. The NLRP3 inflammasome is activated via numerous danger signals produced by IRI and is central to the resultant innate immune response. Inhibition of this inherent inflammatory response has been shown to protect the myocardium and stop the occurrence of the systemic inflammatory response syndrome following the re-establishment of cardiac circulation. Therapies to prevent NLRP3 inflammasome formation in the clinic are currently lacking, and therefore, new pharmacotherapies are required. This review will highlight the role of the NLRP3 inflammasome within the myocardium during IRI and will examine the therapeutic value of inflammasome inhibition with particular attention to carbon monoxide, nitric oxide, and hydrogen sulphide as potential pharmacological inhibitors of NLRP3 inflammasome activation.

Role of Cathelicidins in Atherosclerosis and Associated Cardiovascular Diseases

Cathelicidins (human LL-37 and rat CRAMP) are multifunctional peptides involved in various cardiovascular conditions. This review integrates the recent findings about the functional involvement of LL-37/CRAMP across atherosclerosis, acute coronary syndrome, myocardial infarction, heart failure, diabetic cardiomyopathy, and platelet aggregation/thrombosis. In atherosclerosis, LL-37 interacts with scavenger receptors to modulate lipid metabolism and binds with mitochondrial DNA and lipoproteins. In acute coronary syndrome, LL-37 influences T cell responses and mitigates calcification within atherosclerotic plaques. During myocardial infarction and ischaemia/reperfusion injury, LL-37/CRAMP exhibits dual roles: protecting against myocardial damage through the AKT and ERK1/2 signalling pathways, while exacerbating inflammation via TLR4 and NLRP3 inflammasome activation. In heart failure, LL-37/CRAMP attenuates hypertrophy and fibrosis via NF-κB inhibition and the activation of the IGFR1/PI3K/AKT and TLR9/AMPK pathways. Moreover, in diabetic cardiomyopathy, these peptides alleviate oxidative stress and fibrosis by inhibiting TGFβ/Smad and AMPK/mTOR signalling and provide anti-inflammatory effects by reducing NF-κB nuclear translocation and NLRP3 inflammasome formation. LL-37/CRAMP also modulates platelet aggregation and thrombosis through the FPR2 and GPVI receptors, impacting apoptosis, autophagy, and other critical cellular processes. This comprehensive overview underscores LL-37/CRAMP as a promising therapeutic target in cardiovascular diseases, necessitating further elucidation of its intricate signalling networks and biological effects for clinical translation.

Defect in Migration of HSPCs in Nox-2 Deficient Mice Explained by Impaired Activation of Nlrp3 Inflammasome and Impaired Formation of Membrane Lipid Rafts.

NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.

Abstract Tu011: Targeted chelation therapy decreases Cryopyrin/NLRP3 expression and acts as senomorphic in Chronic Kidney Disorder induced Vascular Calcification.

Background: The high phosphate (P) microenvironment caused by mineral imbalance in chronic kidney disease (CKD) has been reported to induce osteogenic changes in healthy Vascular Smooth Muscle Cells (VSMCs), leading to the calcification of arteries. We have previously shown that EDTA therapy can reverse calcification of the arteries in a rat model of CKD. This study investigated whether global mineral imbalance observed during CKD can initiate senescent changes in the aorta. We focused on NLRP3 as an intracellular source of SASP and evaluated if chelation therapy with EDTA has senotherapeutic potential. Methods: We used an early-stage adenine diet-based rodent model of CKD to evaluate whether senescent cells accumulate prior to vascular calcification. For validation studies, we adopted an ex-vivo approach, wherein we cultured aortic rings under high phosphate conditions for different durations to simulate the early and late stages of vascular calcification. Further, to test the efficacy of chelation therapy, EDTA loaded- human serum albumin nanoparticles tagged with anti-elastin antibody- Flexibzumab (EL-EDTA -NPs) were successfully targeted to the site of calcification in the aorta in a progressive CKD rodent model. Results: Using a flow-cytometry-based approach to detect senescent cells based on SA-βGal activity, we evidence the presence of senescent cells that accumulate in the aorta prior to calcific changes as observed using micro-CT and histopathology in the in vivo model of vascular calcification. Further, we show that targeted chelation therapy with EDTA NPs acts as senomorphic and decreases NLRP3 inflammasome formation. The EL-EDTA-NPs decreased mineral deposits in arteries and thus altered the local immune microenvironment, secretion of growth factors, and proteases within the aortic tissue, identified as SASP. Conclusion: For the first time, the current study reports that the accumulation of senescent cells occurs before calcification and provides an opportunity to analyze the potential role of senescence in the pathological progression of vascular calcification. Additionally, these findings emphasize the role of altered Ca and Pi microenvironment observed during CKD in initiating senescent changes via NLRP3 activation in the aorta. Furthermore, these findings highlight the potential of a targeted EDTA delivery approach to modulate tissue micro-environment selectively by decreasing Ca deposition and SASP in the aorta.

LPS-LBP complex induced endothelial cell pyroptosis in aortic dissection is associated with gut dysbiosis

Acute aortic dissection (AAD) is the most severe traumatic disease affecting the aorta. Pyroptosis-mediated vascular wall inflammation is a crucial trigger for AAD, and the exact mechanism requires further investigation. In this study, our proteomic analysis showed that Lipopolysaccharide (LPS)-binding protein (LBP) was significantly upregulated in the plasma and aortic tissue of patients with AAD. Further, 16S rRNA sequencing of stool samples suggested that patients with AAD exhibit gut dysbiosis, which may lead to an impaired intestinal barrier and LPS leakage. By comparing with control mice, we found that LBP, including Pyrin Domain Containing Protein3 (NLRP3), the CARD-containing adapter apoptosis-associated speck-like protein (ASC), and Cleaved caspase-1, were upregulated in the AAD aorta, whereas gut intestinal barrier-related proteins were downregulated. Moreover, treated with LBPK95A (an LBP inhibitor) attenuated the incidence of AAD, the expression levels of pyroptosis-related factors, and the extent of vascular pathological changes compared to those in AAD mice. In addition, LPS and LBP treatment of human umbilical vein endothelial cells (HUVECs) activated TLR4 signaling and intracellular reactive oxygen species (ROS) production, which stimulated NLRP3 inflammasome formation and mediated pyroptosis in endothelial cells. Our findings showed that gut dysbiosis mediates pyroptosis by the LPS-LBP complex, thus providing new insights into developing AAD.

Yiqi Jiedu Huayu decoction inhibits precancerous lesions of chronic atrophic gastritis by inhibiting NLRP3 inflammasome-mediated pyroptosis.

Chronic atrophic gastritis (CAG) is a prevalent chronic gastritis usually accompanied by precancerous lesions such as intestinal metaplasia and dysplasia. The increasing application of traditional Chinese medicine in CAG treatment has shown promising results with low side effects and significant efficacy. To investigate the pharmacological effects of Yiqi Jiedu Huayu decoction (YJHD) on precancerous lesions of CAG. A CAG rat model was established by Helicobacter pylori bacteria solution combined with N-methyl-N'-nitro-N-nitrosoguanidine. Histopathological measurements were conducted by hematoxylin-eosin and alcian blue and periodic acid-Schiff staining. Serum levels of inflammatory factors and gastric mucosal-related factors were examined using enzyme-linked immunosorbent assay. Protein and mRNA levels were quantified via western blot and quantitative real-time polymerase chain reaction analysis, respectively. Molecular interaction was verified by chromatin immunoprecipitation (ChIP) assay. YJHD greatly attenuated pathological changes in the gastric mucosa and precancerous lesions in CAG rats. Meanwhile, YJHD treatment reduced serum levels of inflammatory factors [interleukin (IL)-6, tumor necrosis factor-α and C-reactive protein] and increased serum levels of gastric mucosal-related factors (gastrin, pepsin, somatostatin and prostaglandin E2) in CAG rats. In addition, YJHD administration suppressed NLRP3 inflammasome-mediated cell pyroptosis, as well as the activation of TLR4/NF-κB and IL-6/STAT3 signaling pathways. Mechanically, ChIP experiments confirmed that NLRP3 transcription was regulated by TLR4/NF-κB and IL-6/STAT3 signaling. Taken together, YJHD alleviated NLRP3 inflammasome formation and pyroptosis of epithelial cells in CAG, potentially through the inactivation of TLR4/NF-κB and IL-6/STAT3 pathways.

Asprosin promotes vascular inflammation via TLR4-NFκB-mediated NLRP3 inflammasome activation in hypertension

Objectiveand design Mild vascular inflammation promotes the pathogenesis of hypertension. Asprosin, a newly discovered adipokine, is closely associated with metabolic diseases. We hypothesized that asprosin might led to vascular inflammation in hypertension via NLRP3 inflammasome formation. This study shows the importance of asprosin in the vascular inflammation of hypertension. MethodsPrimary vascular smooth muscle cells (VSMCs) were obtained from the aorta of animals, including spontaneously hypertensive rats (SHR), Wistar–Kyoto rats (WKY), NLRP3−/− and wild-type mice. Studies were performed in VSMCs in vitro, as well as WKY and SHR in vivo. ResultsAsprosin expressions were up-regulated in VSMCs and media of arteries in SHR. Asprosin overexpression promoted NLRP3 inflammasome activation via Toll-like receptor 4 (TLR4), accompanied with activation of NFκB signaling pathway in VSMCs. Exogenous asprosin protein showed similar roles in promoting NLRP3 inflammasome activation. Knockdown of asprosin restrained NLRP3 inflammasome and p65-NFκB activation in VSMCs of SHR. NLRP3 inhibitor MCC950 or NFκB inhibitor BAY11-7082 attenuated asprosin-caused VSMC proliferation and migration. Asprosin-induced interleukin-1β production, proliferation and migration were attenuated in NLRP3−/− VSMCs. Local asprosin knockdown in common carotid artery of SHR attenuated inflammation and vascular remodeling. ConclusionsAsprosin promoted NLRP3 inflammasome activation in VSMCs by TLR4-NFκB pathway, and thereby stimulates VSMCs proliferation, migration, and vascular remodeling of SHR.

Extreme Heat Exposure Induced Acute Kidney Injury through NLRP3 Inflammasome Activation in Mice.

Climate change has resulted in a marked increase in heat extremes that carry a severe risk for morbidity and mortality. Kidney is sensitive to heat stimulation, and acute kidney injury (AKI) is the early event. In this study, we investigated the adverse effects of heat extremes and their underlying mechanism. A total of 16 wild-type C57BL/6N mice were randomly divided into groups of control (exposed to 22 ± 0.5 °C) and heat (exposed to 39.5 ± 0.5 °C until the core body temperature reached the maximum). First, extreme heat exposure induced AKI evidenced by kidney dysfunction and morphological impairment. In addition, heat exposure suppressed expression of molecules for mitochondrial energetics and fatty acid beta-oxidation and disturbed the balance of oxidative stress in the kidney. Moreover, heat exposure enhanced the protein levels in the upstream signaling pathway for NLRP3 inflammasome formation, followed by NLRP3 inflammasome activation and inflammatory cytokine production. These findings demonstrated that acute extreme heat exposure may induce AKI through the NLRP3 inflammasome formation and activation.

Intracerebroventricular infusion of secretoneurin inhibits neuronal NLRP3-Apoptosis pathway and preserves learning and memory after cerebral ischemia

Transient global cerebral ischemia (GCI) results in delayed neuronal death, primarily apoptosis, in the hippocampal CA1 subregion, which leads to severe cognitive deficits. While therapeutic hypothermia is an approved treatment for patients following cardiac arrest, it is associated with various adverse effects. Secretoneurin (SN) is an evolutionarily conserved neuropeptide generated in the brain, adrenal medulla and other endocrine tissues. In this study, SN was infused into the rat brain by intracerebroventricular injection 1 day after GCI, and we demonstrated that SN could significantly preserve spatial learning and memory in the Barnes maze tasks examined on days 14–17 after GCI. To further investigate underlying pathways involved, we demonstrated that, on day 5 after GCI, SN could significantly inhibit GCI-induced expression levels of Apoptosis Inducing Factor (AIF) and cleaved-PARP1, as well as neuronal apoptosis and synaptic loss in the hippocampal CA1 region. Additionally, SN could attenuate GCI-induced activation of both caspase-1 and caspase-3, and the levels of pro-inflammatory cytokines IL-1β and IL-18 in the CA1 region. Mechanically, we observed that treatment with SN effectively inhibited NLRP3 protein elevation and the bindings of NLRP3-ASC and ASC-caspase-1 in hippocampal neurons after GCI. In summary, our data indicate that SN could effectively attenuate NLRP3 inflammasome formation, as well as the activation of caspase-1 and -3, the production of pro-inflammatory cytokines, and ultimately the neuronal apoptotic loss induced by GCI. Potential neuronal pyroptosis, or caspase-1-dependent cell death, could also be involved in ischemic neuronal death, which needs further investigation.

Quercetin, a natural flavonoid, protects against hepatic ischemia–reperfusion injury via inhibiting Caspase-8/ASC dependent macrophage pyroptosis

IntroductionHepatic ischemia–reperfusion injury (IRI) is an inevitable adverse event following liver surgery, leading to liver damage and potential organ failure. Despite advancements, effective interventions for hepatic IRI remain elusive, posing a significant clinical challenge. The innate immune response significantly contributes to the pathogenesis of hepatic IRI by promoting an inflammatory cytotoxic cycle. We have reported that blocking GSDMD-induced pyroptosis in innate immunity cells protected hepatic IRI from inflammatory injury. However, the search for effective pyroptosis inhibitors continues. ObjectivesThis study aims to evaluate whether quercetin, a natural flavonoid, can inhibit GSDMD-induced pyroptosis and mitigate hepatic IRI. MethodsWe established the hepatic IRI murine model and cellular pyroptosis model to evaluate the efficacy of quercetin. ResultsQuercetin effectively alleviated hepatic IRI-induced tissue necrosis and inflammation. We found that during hepatic IRI, the cleavage of GSDMD occurred in hepatic macrophages, but not in other non-parenchymal cells. Quercetin inhibited the cleavage of GSDMD in macrophages. Moreover, we found that quercetin blocked the ASC assembly to inhibit the formation of NLRP3 inflammasomes and AIM2 inflammasomes, suppressing macrophage pyroptosis. Co-immunoprecipitation experiments confirmed that quercetin inhibited the interaction between ASC and Caspase-8, which is the mechanism of ASC complex and inflammasome formation. Overexpression of Caspase-8 abolished the anti-pyroptosis effect of quercetin in NLRP3 and AIM2 inflammasome signaling. Furthermore, we found that the hepatoprotective activity of quercetin was reduced in myelocytic GSDMD-deficient mice. ConclusionOur findings suggest that quercetin has beneficial effects on hepatic IRI. Quercetin could attenuate hepatic IRI and target inhibition of macrophage pyroptosis via blocking Caspase-8/ASC interaction. We recommend that quercetin might serve as a targeted approach for the prevention and personalized treatment of hepatic IRI in perioperative patients.

MORIN ATTENUATES INFLAMMASOME-DEPENDENT MYOCARDIAL INJURY IN DIABETIC RATS: MODULATION OF THE SIGNALING PATHWAY

Objective: Diabetes is a risk factor that predisposes to atherosclerotic cardiovascular diseases. The risk of myocardial infarction in diabetes is higher and this warrants investigation into exploring new drugs that can attenuate myocardial injury. In diabetes, high blood glucose levels lead to vascular inflammation which accelerates atherosclerosis. Morin, a plant flavonoid, was found effective for septic shock, cancer, cardiovascular, and renal complications and acts as an agonist for insulin secretion. This study aims to evaluate the mechanism involved in the cardioprotective action of Morin in diabetic rats. Design and method: In male Wistar rats, streptozotocin (70 mg/kg; i.p.) was administered to induce diabetes. Diabetes-confirmed rats (blood glucose >400mg/dl) were divided into 4 groups (n=8), i.e., Diabetic-control; Diabetes+Isoproterenol (ISO); Diabetes+ISO+Morin and Diabetes+Morin, in addition to this, one control group was also included in the study. Morin was orally administered at the dose of 40 mg/kg for 28 days and on the 27th and 28th day of morin administration, ISO was administered to designate groups at the dose of 85mg/kg s.c., to induce myocardial injury. After 24 hours of the second dose of ISO administration, the rat was sacrificed after collection of blood, and heart was excised to do the biochemical, histopathological and molecular investigations. Results: ROS rush following ISO administration leads to the activation of the intrinsic and extrinsic pathways of apoptosis. Morin significantly reduced oxidative stress, cardiac injury markers levels, inflammation, and apoptosis. In addition, it also reduced serum insulin and blood glucose levels. Histopathology showed cardio-protection with morin. Akt/eNOS, Nrf2/HO-1, MAPK signaling pathways, and Insulin signal transduction pathways were positively modulated by Morin pre-treatment. It also significantly modulated NLRP3 inflammasome formation. Conclusions: Morin attenuated oxidative stress and inflammation and also modified the activity of various molecular pathways to mitigate cardiomyocyte damage during ISO-induced MI in diabetic rats.

Nattokinase attenuates endothelial inflammation through the activation of SRF and THBS1

Natto contains a potent fibrinolytic enzyme called nattokinase (NK), which has thrombolytic, antihypertensive, antiatherosclerotic and lipid-lowering effects. Although NK has been recognized for its beneficial effect on humans with atherosclerotic cardiovascular disease (ASCVD), the underlying mechanisms involved in vascular inflammation-atherosclerosis development remain largely unknown. The current study aimed to explore the effects of NK on gene regulation, autophagy, necroptosis and inflammasome in vascular inflammation. The transcriptional profiles of NK in endothelial cells (ECs) by RNA sequencing (RNA-seq) revealed that NK affected THBS1, SRF and SREBF1 mRNA expression. In Q-PCR analysis, SRF and THBS1 were upregulated but SREBF1 was unaffected in ECs treated with NK. NK treatment induced autophagy and inhibited NLRP3 inflammasome and necroptosis in ECs. Furthermore, the inhibition of SRF or THBS1 by siRNA suppressed autophagy and enhanced the NLRP3 inflammasome and necroptosis. In a mouse model, NK reduced vascular inflammation by activating autophagy and inhibiting NLRP3 inflammasome and necroptosis. Our findings provide the first evidence that NK upregulates SRF and THBS1 genes, subsequently increasing autophagy and decreasing necroptosis and NLRP3 inflammasome formation to reduce vascular inflammation. Therefore, NK could serve as nutraceuticals or adjuvant therapies to reduce vascular inflammation and possible atherosclerosis progression.