Protein ASC Research Articles

Abstract TP345: Electronic Cigarette Exposure Worsens Stroke Induced Inflammation in Female Rats

Introduction: The rise in popularity of nicotine-containing electronic cigarettes (ECs) globally necessitates a deeper understanding of their effects on stroke outcomes. This study investigates the influence of EC exposure on post-ischemic inflammation. Methods: Adult Sprague-Dawley rats of both sexes were randomly assigned to either air or EC vapor exposure (5% nicotine Juul pods) for 16 nights, followed by transient middle cerebral artery occlusion (tMCAO; 90 minutes) or sham surgery. Post-surgery, animals were divided into two cohorts. In the first cohort, cortical brain tissue was collected 24 hours after tMCAO or sham surgery for Western blot analysis of inflammasome proteins, including Caspase-1, Interleukin-1β (IL-1β), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and gasdermin-D (GSDMD). The second cohort was sacrificed 21 days post-tMCAO for immunohistochemical analysis of microglial marker ionized calcium-binding adapter molecule-1 (Iba-1). De-ramified microglia were quantified across three serial coronal sections (10 μm) using the optical fractionator probe in StereoInvestigator software, with a counting frame of 100x100 μm and approximately 40 sampling sites per section. Microglial morphology and Sholl analysis were performed by capturing three cortical images per slide, each containing at least three microglia, and across three slides per subject, resulting in 18 images for peri-infarct and contralateral areas. Analysis was conducted using ImageJ software. Results: Western blot analysis revealed a significant increase in Caspase-1 and ASC protein levels in the ipsilateral cortex of EC-exposed female rats compared to males after tMCAO (p<0.05). Quantification of Iba-1 indicated an increase in de-ramified microglia in the cortex of EC-exposed female rats as compared to air-exposed after tMCAO, potentially exacerbating post-stroke infarction. Microglial morphology analysis showed increased cell soma size and decreased branch length in EC-exposed female rats. Sholl analysis demonstrated reduced microglial complexity in EC-exposed group as compared to air-exposed after tMCAO, with a smaller area under the curve and fewer peak intersections in the cortex. Conclusion: EC exposure exacerbates stroke-induced inflammation and cognitive deficits in female rats. Future research should explore whether EC withdrawal mitigates ischemic severity and determine the necessary duration of withdrawal for potential recovery.

Anti-Inflammatory and Anti-Migratory Effects of Morin on Non-Small-Cell Lung Cancer Metastasis via Inhibition of NLRP3/MAPK Signaling Pathway.

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths globally, with a persistently low five-year survival rate of only 14-17%. High rates of metastasis contribute significantly to the poor prognosis of NSCLC, in which inflammation plays an important role by enhancing tumor growth, angiogenesis, and metastasis. Targeting inflammatory pathways within cancer cells may thus represent a promising strategy for inhibiting NSCLC metastasis. This study evaluated the anti-inflammatory and anti-metastatic properties of morin, a bioactive compound derived from a Thai medicinal herb, focusing on its effects on NLRP3 inflammasome-mediated pathways in an in vitro NSCLC model. The A549 and H1299 cell lines were stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to activate the NLRP3 pathway. The inhibition effects exhibited by morin in reducing pro-inflammatory secretion in LPS- and ATP-stimulated NSCLC cells were assessed by ELISA, while wound healing and trans-well invasion assays evaluated its impact on cell migration and invasion. RT-qPCR measurement quantified the expression of inflammatory genes, and zymography and Western blotting were used to examine changes in invasive protein levels, epithelial-to-mesenchymal transition (EMT) markers, and underlying molecular mechanisms. Our findings demonstrated the significant ability of morin to decrease the production of IL-1β, IL-18, and IL-6 in a dose-dependent manner (p < 0.05), as well as suppress NSCLC cell migration and invasion. Morin downregulated invasive proteins (MMP-2, MMP-9, u-PAR, u-PA, MT1-MMP) and EMT markers (fibronectin, N-cadherin, vimentin) (p < 0.01) while also reducing the mRNA levels of NLRP3, IL-1β, IL-18, and IL-6. Mechanistic investigations revealed that morin suppressed NLRP3 inflammasome activity and inactivated MAPK pathways. Specifically, it decreased the expression of NLRP3 and ASC proteins and reduced caspase-1 activity, while reducing the phosphorylation of ERK, JNK, and p38 proteins. Collectively, these findings suggest that morin's inactivation of the NLRP3 inflammasome pathway could offer a novel therapeutic strategy for counteracting pro-tumorigenic inflammation and metastatic progression in NSCLC.

Complex Immunotoxic Effects of T-2 Toxin on the Murine Spleen and Thymus: Oxidative Damage, Inflammasomes, Apoptosis, and Immunosuppression

T-2 toxin (T-2), a highly stable and toxic mycotoxin, poses a significant public health risk as an inevitable environmental pollutant. However, the mechanisms behind its immunotoxic and immunosuppressive effects are not fully understood. For this study, sixty healthy 4-week-old male C57BL/6N mice were divided randomly into four groups and treated for 28 days with T-2 concentrations of 0, 0.5, 1.0, and 2.0mg/kg. Our findings revealed significant damage to the thymus and spleen that was proportional to the dose administered, as evidenced by changes in organ indices and histopathological abnormalities. We observed mitochondrial swelling, chromatin condensation, and nuclear structure disruptions in these organs. Even at low doses (0.5mg/kg), T-2 administration resulted in significant immunosuppression, as evidenced by disturbed blood parameters and altered CD4+/CD8+ ratios. Elevated ROS and MDA levels indicate oxidative damage, whereas SOD, T-AOC, CAT, and GSH levels are reduced in both the thymus and spleen. Furthermore, the levels of NLRP3, ASC, caspase-1, and IL-1β proteins were significantly elevated, indicating the activation of the NLRP3 inflammasome pathway. Additionally, activation of the apoptosis pathway was demonstrated by an increased Bax/Bcl-2 ratio and heightened activation of caspase-3 and -9. Transcriptomic analysis elucidated the pivotal role of mitochondrial pathways in T-2-induced immunotoxicity. This study elucidates the significant immunotoxic effects of T-2 on the murine spleen and thymus, detailing the underlying mechanisms of T-2-induced immunosuppression. The key mechanisms identified include oxidative stress, activation of the NLRP3 inflammasome, apoptosis, and mitochondrial dysfunction. These findings reveal critical pathways through which T-2 impairs immune system functionality and provide a basis for developing targeted therapeutic strategies to mitigate its immunotoxic effects.

NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis.

Inclusion body myositis (IBM) is the most prevalent muscle disease in adults for which no current treatment exists. The pathogenesis of IBM remains poorly defined. In this study, we aimed to explore the interplay between inflammation and mitochondrial dysfunction in IBM. The study population consisted of 38 IBM patients and 22 age- and sex-matched controls without a myopathy. Mean age was 62.9 years (SD = 9) in IBM group and 59.7 (10) in controls. Bulk RNA sequencing, Meso Scale Discovery electrochemiluminescence (ECL), western blotting, histochemistry and immunohistochemistry were performed on frozen muscle samples from the study participants. We demonstrated activation of the NLRP3 inflammasome in IBM muscle samples, with the NLRP3 inflammasome being the most upregulated pathway on RNA sequencing, along with increased expression of NLRP3 and ASC proteins in IBM group. NLRP3 RNA levels most strongly correlated with TLR7 (correlation coefficient ρ = 0.91) and complement activation-related genes, and inversely correlated with several mitochondria-related genes among others. On muscle histopathology, there was increased NRLP3 immunoreactivity in both inflammatory cells and muscle fibres. Mitophagy is critical for removing damaged mitochondria and preventing the formation of a vicious cycle of mitochondrial dysfunction-NLRP3 inflammasome activation. Herein, we showed altered mitophagy, as witnessed by the elevated levels of p-S65-Ubiquitin, a mitophagy marker, in muscle lysates from IBM patients compared to controls (median of 114.3 vs. 81.25 ECL units, p = 0.005). The p-S65-Ubiquitin levels were most significantly elevated in IBM males compared to male controls (136 vs. 83.5 ECL units; p = 0.013), whereas IBM females had milder nonsignificant elevation compared to female controls (97.25 vs. 69 ECL units, p = 0.31). On muscle histopathology, p-S65-Ubiquitin aggregates accumulated in muscle fibres that were mostly Type 2 and devoid of cytochrome-c-oxidase reactivity. NLRP3 RNA levels correlated with p-S65-Ubiquitin levels in both sexes (males: ρ = 0.48, females: ρ = 0.54) but with loss of muscle strength, as reflected by the manual motor test score, only in males (males: ρ = 0.62, females: ρ = -0.14). Lastly, we identified sex-specific molecular pathways in IBM. Females had upregulation of pathways related to response to stress, which could conceivably offset some of the pathomechanisms of IBM, while males had upregulation of pathways related to cell adhesion and migration. There is activation of the NLRP3 inflammasome in IBM, along with altered mitophagy, particularly in males, which is of potential therapeutic significance. These findings suggest sex-specific mechanisms in IBM that warrant further investigation.

Palmatine, an isoquinoline alkaloid from Phellodendron amurense Rupr., ameliorated gouty inflammation by inhibiting pyroptosis via NLRP3 inflammasome.

Palmatine (Pal), derived from Daemonorops margaritae (Hance) Becc and Phellodendron amurense Rupr. is a natural isoquinoline alkaloid widely used in clearing heat and drying dampness, purging the pathogenic fire and removing symptoms, detoxifying toxins and healing sores. Gout is a common metabolic inflammatory disease caused by the deposition of MSU crystals (MSU) in joints and non-articulation structures. Given the multiple toxic side effects of clinical anti-gout medications, there is a need to find a safe and effective alternative. We investigated the therapeutic effects of Pal on MSU crystal-induced acute gouty inflammation, targeting the NLRP3 inflammasome mediated pyroptosis. In vitro, mouse peritoneal macrophages (MPM) and rat articular chondrocytes were stimulated with LPS plus MSU in the presence or absence of Palmatine. In vivo, arthritis models include the acute gouty arthritis model by injecting MSU crystals in the paws of mice and the air pouch acute gout model by injecting MSU crystals into the mouse subcutaneous tissue of the back. Expression of NLRP3 inflammasome activation and NETosis formation was determined by Western blot, ELISA kit, immunohistochemistry, and immunofluorescence. In addition, the anti-cartilage damage of Palmatine on MSU-induced arthritis mice were also evaluated. Pal dose-dependently decreased levels of NLRP3 inflammasome activation related proteins NLRP3, ASC, caspase-1, IL-1β, HMGB1 and Cathepsin B. The NETosis protein levels of caspase-11, histone3, PR3 and PAD4 were remarkably reduced by Pal. Pal effectively blocked the activation of NLRP3 inflammasome, attenuated the caspase-11 mediated noncanonical NLRP3 inflammasome activation and intervened the formation of NETs, thereby inhibiting the pyroptosis. In vivo, Pal attenuated MSU-induced inflammation in gouty arthritis and protect the articular cartilage through inhibiting the pyroptosis of proteins NLRP3, ASC, caspase-1, IL-1β, HMGB1 and Cathepsin B, reducing levels of NETosis relevant proteins caspase-11, histone3, PR3 and PAD4 and up-regulating expression of protein MMP-3. Palmatine ameliorated gouty inflammation by inhibiting pyroptosis via NLRP3 inflammasome.

Acylated Anthocyanins From Black Carrots and Their Related Phenolic Acids Diminish Priming and Activation of the NLRP3 Inflammasome in THP-1 Monocytes.

Excessive activation of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to chronic inflammation. Thus, targeting NLRP3 inflammasome activation by anthocyanins may prevent inflammatory diseases. Therefore, the present study determines the influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins and their related phenolic acids on the NLRP3 inflammasome. THP-1 monocytes are pretreated with a BCE, cyanidin-3-glucoside (C3G), or hydroxycinnamic acids. NLRP3 inflammasome assembly is initiated by priming THP-1 monocytes with lipopolysaccharide and/or activating the NLRP3 inflammasome with nigericin. Flow cytometry is used to assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, as well as ASC and NLRP3 protein expression. Caspase-1 activity is measured using a bioluminescent assay, and cytokine concentrations are determined by enzyme-linked immunosorbent assays (ELISA). C3G and phenolic acids diminish ASC and NLRP3 protein expression. In addition, C3G and phenolic acids attenuate ASC speck formation. Furthermore, the BCE and C3G decline caspase-1 activity. Consistently, IL-1β and IL-18 secretion are reduced upon NLRP3 inflammasome activation. The present study shows that a BCE with high amounts of acylated anthocyanins and their related phenolic acids diminish priming and activation of the NLRP3 inflammasome in THP-1 monocytes.

Early divergent modulation of NLRP2′s and NLRP3′s inflammasome sensors vs. AIM2′s one by signals from Aβ·Calcium-sensing receptor complexes in human astrocytes

Alzheimer’s disease (AD), the most prevalent human dementia, is driven by accruals of extracellular Aβ42 senile patches and intracellular neurofibrillary tangles of hyperphosphorylated Tau (p-Tau) proteins. AD’s concurrent neuroinflammation is prompted by innate immunity-related cytosolic protein oligomers named inflammasomes. Upon proper “first” (priming) and “second” (activating) signals, inflammasomes overproduce proinflammatory Interleukin (IL)-1β, and IL-18 while cleaving pyroptosis-promoting Gasdermin D’s N-terminal fragments. Our earlier studies highlighted that in pure monocultures, exogenous Aβ25-35-treated nonproliferating human cortical astrocytes (HCAs) made and released surpluses of endogenous Aβ42-oligomers (−os) and p-Tau-os, just as alike-treated human cortical neurons did. Aβ25-35-exposed HCAs also over-released NO, VEGFA, and IL-6. Aβ•CaSR (Aβ·Calcium-Sensing Receptor) complexes generated intracellular signals mediating all such neurotoxic effects since CaSR’s negative allosteric modulators (aka NAMs or calcilytics, e.g., NPS2143) fully suppressed them. However, it had hitherto remained unexplored whether signals from Aβ·CaSR complexes also induced the early expression and/or activation of NOD-like 2 (NLRP2) and 3 (NLRP3) and of PYHIN absent in melanoma 2 (AIM2) inflammasomes in monocultured HCAs. To clarify this topic, we used in-situ-Proximity Ligation, qRT-PCR, double antibody arrays, immunoblots, and Caspase 1/4 enzymatic assays. Aβ·CaSR complexes quickly assembled on HCAs surface and issued intracellular signals activating Akt and JAK/STAT axes. In turn, the latter upregulated NLRP2 and NLRP3 PRRs (pattern recognition receptors) yet downregulated AIM2. These effects were specific, being significantly hindered by NPS2143 and inhibitors of PI3K (LY294002), AMPKα (Dorsomorphin), mTOR (Torin1), and JAK/TYK (Brepoticinib). A wide-spectrum inhibitor, Bay11-7082, intensified the Aβ·CaSR/Akt/JAK/STAT axis-driven opposite control of NLRP3’s and AIM2’s PRR proteins without affecting NLRP2 PRR upregulation. However, the said effects on the PRRs proteins vanished within 24-h. Moreover, Aβ·CaSR signals neither concurrently changed ASC, pro-IL-1β, and Gasdermin-D (holo- and fragments) protein levels and Caspases 1 and 4 enzymatic activities nor induced pyroptosis. Therefore, Aβ·CaSR cues acted as “first (priming) signals” temporarily increasing NLRP2 and NLRP3 PRRs expression without activating the corresponding inflammasomes. The neatly divergent modulation of NLRP3’s vs. AIM2’s PRR proteins by Aβ·CaSR cues and by Bay11-7082 suggests that, when bacterial or viral DNA fragments are absent, AIM2 might play “anti-inflammasomal” or other roles in HCAs. However, Bay11-7082’s no effect on NLRP2 PRR overexpression also reveals that CaSR’s downstream mechanisms controlling inflammasomes’ sensors are quite complex in HCAs, and hence, given AD’s impact on human health, well worth further studies.

Potential Impact of Bioactive Compounds as NLRP3 Inflammasome Inhibitors: An Update.

The inflammasome NLRP3 comprises a caspase recruitment domain, a pyrin domain containing receptor 3, an apoptosis-linked protein like a speck containing a procaspase-1, and an attached nucleotide domain leucine abundant repeat. There are a wide variety of stimuli that can activate the inflammasome NLRP3. When activated, the protein NLRP3 appoints the adapter protein ASC. Adapter ASC protein then recruits the procaspase-1 protein, which causes the procaspase- 1 protein to be cleaved and activated, which induces cytokines. At the same time, abnormal activation of inflammasome NLRP3 is associated with many diseases, such as diabetes, atherosclerosis, metabolic syndrome, cardiovascular and neurodegenerative diseases. As a result, a significant amount of effort has been put into comprehending the mechanisms behind its activation and looking for their specific inhibitors. In this review, we primarily focused on phytochemicals that inhibit the inflammasome NLRP3, as well as discuss the defects caused by NLRP3 signaling. We conducted an in-depth research review by searching for relevant articles in the Scopus, Google Scholar, and PubMed databases. By gathering information on phytochemical inhibitors that block NLRP3 inflammasome activation, a complicated balance between inflammasome activation or inhibition with NLRP3 as a key role was revealed in NLRP3-driven clinical situations.

Short-term respiratory cadmium exposure partially activates pulmonary NLRP3 inflammasome by inducing ferroptosis in mice

Cadmium (Cd) is a common environmental metal. Previous studies indicated that long-term respiratory Cd exposure caused lung injury and airway inflammation. The purpose of this study was to evaluate whether short-term respiratory Cd exposure induces pulmonary ferroptosis and NLRP3 inflammasome activation. Adult C57BL/6J mice were exposed to Cd by inhaling CdCl2 aerosol (0, 10, or 100 ppm) for 5 days. Serum and lung Fe2+ contents were elevated in Cd-exposed mice. Oxidized AA metabolites, the major oxidized lipids during ferroptosis, were upregulated in Cd-exposed mouse lungs. Pulmonary MDA content and 4-HNE-positive cells were increased in Cd-exposed mice. ACSL4 and COX-2, two lipoxygenases, were upregulated in Cd-exposed mouse lungs. Further analyses found that phosphorylated NF-kB p65 was elevated in Cd-exposed mouse lungs. Innate immune receptor protein NLRP3 and adapter protein ASC were upregulated in Cd-exposed mouse lungs. Caspase-1 was activated and IL-1β and IL-18 were upregulated in Cd-exposed mouse lungs. Fer-1, a specific inhibitor of ferroptosis, attenuated Cd-induced elevation of pulmonary NLRP3 and ASC, caspase-1 activation, and IL-1β and IL-18 upregulation. Finally, mitoquinone (MitoQ), a mitochondria-target antioxidant, suppressed Cd-caused ferroptosis and NLRP3 inflammasome activation. Our results demonstrate that ferroptosis might partially mediate Cd-evoked activation of NLRP3 inflammasome in the lungs.

Linoleyl acetate and mandenol alleviate HUA-induced ED via NLRP3 inflammasome and JAK2/STAT3 signalling conduction in rats.

Hyperuricemia (HUA) is characterized by elevated blood uric acid levels, which can increase the risk of erectile dysfunction (ED). Clinical studies have demonstrated satisfactory efficacy of a traditional Chinese medicine formula QYHT decoction in improving ED. Furthermore, the main monomeric components of this formula, linoleyl acetate and mandenol, demonstrate promise in the treatment of ED. This study established an ED rat model induced by HUA and the animals were administered with linoleyl acetate and mandenol. HE and TUNEL were performed to detect tissue changes, ELISA to measure the levels of serum testosterone (T), MDA, NO, CRP, and TNF-α and qPCR and WB to assess the expression levels of NLRP3, ASC, Caspase-1, JAK2, and STAT3 in whole blood. The findings showed that linoleyl acetate and mandenol improved kidney tissue morphology, reduced cell apoptosis in penile tissue, significantly increased T and NO levels, while substantially decreasing levels of MDA, CRP, and TNF-α. Meanwhile, the expression of NLRP3, ASC, and Caspase-1 mRNAs and proteins was markedly reduced, and the phosphorylation of JAK2 and STAT3 was inhibited. These findings were further validated through faecal microbiota transplantation results. Taken together, linoleyl acetate and mandenol could inhibit NLRP3 inflammasome activation, reduce inflammatory and oxidative stress responses, suppress the activity of JAK-STAT signalling pathway, ultimately providing a potential treatment for HUA-induced ED.

Discovery of natural product derivative triptolidiol as a direct NLRP3 inhibitor by reducing K63-specific ubiquitination.

NLRP3 is up-regulated in inflammatory and autoimmune diseases. The development of NLRP3 inhibitors is challenged by the identification of compounds with distinct mechanisms of action avoiding side effects and toxicity. Triptolide is a natural product with multiple anti-inflammatory activities, but a narrow therapeutic window. Natural product triptolide derivatives were screened for NLRP3 inhibitors in human THP-1 and mouse bone marrow-derived macrophages. The efficacy of potent NLRP3 inhibitors was evaluated in LPS-induced acute lung injury and septic shock models. Triptolidiol was identified as a selective inhibitor of NLRP3 with high potency. Triptolidiol inactivated the NLRP3 inflammasome in human THP-1 and mouse primary macrophages primed with LPS. Triptolidiol specifically inhibited pro-caspase 1 cleavage downstream of NLRP3, but not AIM2 or NLRC4 inflammasomes. Based on the structure-activity relationship study, the C8-β-OH group was critical for its binding to NLRP3. Triptolidiol exhibited a submicromolar KD for NLRP3, binding to residue C280. This binding prevented the interaction of NLRP3 with NEK7, the key regulator of NLRP3 inflammasome oligomerization and assembly, but not with the inflammasome adaptor protein ASC. Triptolidiol decreased the K63-specific ubiquitination of NLRP3, leading NLRP3 to a "closed" inactive conformation. Intraperitoneal administration of triptolidiol significantly attenuated LPS-induced acute lung injury and lethal septic shock. Triptolidiol is a novel NLRP3 inhibitor that regulates inflammasome assembly and activation by decreasing K63-linked ubiquitination. Triptolidiol has novel structural features that make it distinct from reported NLRP3 inhibitors and represents a viable therapeutic lead for inflammatory diseases.

Nrf2 Deficiency Exacerbates Parkinson's Disease by Aggravating NLRP3 Inflammasome Activation in MPTP-Induced Mouse Models and LPS-Induced BV2 Cells.

Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopamine neurons. Microglia-mediated neuroinflammation drives disease progression and becomes a critical factor in neuronal degeneration. Recent studies have found that nuclear factor-erythroid 2-related-2 (Nrf2) expression levels are reduced during aging and neurodegenerative diseases, but its regulatory mechanism on microglia-induced neuroinflammation has not been fully elucidated. In vivo, we used the intraperitoneal injection of the neurotoxic drug neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish an animal model of PD and, at the same time, administered Nrf2 inhibitors ML385 and dimethyl fumarate to regulate Nrf2 protein levels. In vitro, we used si-RNA to knock out the Nrf2 gene to intervene in BV2 cells and used lipopolysaccharide (LPS) to stimulate and induce the cell model. The study found that inhibition of Nrf2 expression aggravated the motor defects of PD mice, accompanied by a significant loss of dopaminergic neurons in the substantia nigra and striatum of the brain. In addition, after inhibition of Nrf2, the malondialdehyde (MDA) level in the substantia nigra of the midbrain of mice increased, and the levels of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) decreased, accompanied by the proliferation of microglia and astrocytes. In addition, the activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, the assembly of apoptosis-associated speck-like protein containing a CARD (ASC) protein in microglia, and the release of downstream inflammatory factors caspase-1 and interleukin (IL)-1β, were aggravated. At the cellular level, it was found that knocking out the expression of Nrf2 would aggravate the activation of NLRP3 inflammasomes and the assembly of ASC in LPS-induced BV2 cells. Inhibited Nrf2 activity can reduce the downstream antioxidant enzyme HO-1 and antioxidant levels, induce NLRP3 inflammasome activation and ASC protein assembly in microglia, and ultimately aggravate PD inflammatory response and dopamine neuron degeneration.

Britannilactone 1-O-acetate induced ubiquitination of NLRP3 inflammasome through TRIM31 as a protective mechanism against reflux esophagitis-induced esophageal injury

BackgroundReflux esophagitis (RE) is a disease in which inflammation of the esophageal mucosa owing to the reflux of gastric contents into the esophagus results in cytokine damage. Britannilactone 1-O-acetate (Brt) has anti-inflammatory effects, significantly inhibiting the activation of the NLRP3 inflammasome, leading to a decrease in inflammatory factors including IL-1 β, IL-6, and TNF-α. However, the mechanism underlying its protective effect against RE-induced esophageal injury remains unclear. In the present study, we investigated the protective mechanism of TRIM31 against NLRP3 ubiquitination-induced RE both in vivo and in vitro.MethodsA model of RE was established in vivo in rats by the method of “4.2 mm pyloric clamp + 2/3 fundoplication”. In vitro, the mod was constructed by using HET-1A (esophageal epithelial cells) and exposing the cells to acid, bile salts, and acidic bile salts. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used to screen the concentration of administered drugs, and the viability of HET-1A cells in each group. HE staining was used to assess the degree of pathological damage in esophageal tissues. Toluidine blue staining was used to detect whether the protective function of the esophageal epithelial barrier was damaged and restored. The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-1 β, IL-6, and TNF-α factors in serum. Immunohistochemistry (IHC) was used to detect the expression level of NLRP3 in esophageal tissues. The molecular docking and Co-immunoprecipitation assay (Co-IP assay) were used to detect the TRIM31 interacts with NLRP3. Western blotting detected the Claudin-4, Claudin-5, The G-protein-coupled receptor calcium-sensitive receptor (CaSR), NLRP3, TRIM31, ASC, C-Caspase1, and Caspase1 protein expression levels.ResultsBrt could alleviate RE inflammatory responses by modulating serum levels of IL-1 β, IL-6, and TNF-α. It also activated the expression of NLRP3, ASC, Caspase 1, and C-Caspase-1 in HET-1A cells. Brt also attenuated TRIM31/NLRP3-induced pathological injury in rats with RE through a molecular mechanism consistent with the in vitro results.ConclusionsBrt promotes the ubiquitination of NLRP3 through TRIM31 and attenuates esophageal epithelial damage induced by RE caused by acidic bile salt exposure. This study provides valuable insights into the mechanism of action of Brt in the treatment of RE and highlights its promising application in the prevention of NLRP3 inflammatory vesicle-associated inflammatory pathological injury.Graphical

Mycobacterium intracellulare mediates macrophage pyroptosis by activating AIM2 and NLRP3 inflammasomes.

Clinically, the incidence of nontuberculous mycobacteria (NTM) lung disease is on the rise, and Mycobacterium intracellulare (M. intracellulare) has attracted much attention as a common opportunistic pathogen in clinical practice. So it is very important to study its immunopathogenic mechanism. In this study, the mechanism of M. intracellulare induced pyroptosis of macrophage was investigated. As shown in Fig.1, the secretion of IL-1β and IL-18 in J774A.1 cells increased with time after M. intracellulare infection and was affected by caspase-1 activation and K + efflux, while caspase-1 was significantly expressed in infected cells. Further from Fig.2, NLRP3,AIM2,ASC proteins were significantly expressed in J774A.1 cells after infection, indicating that the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasome were involved in the infection process. In addition, when caspase-1 activity and K + efflux were inhibited, the expression of related proteins was significantly reduced. It indicates that the activation of NLRP3 and AIM2 is regulated by caspase-1 and K+. Figure3, the percentage of dead cells with cell membrane damage increases after infection and cleavage of GSDMD proteins occurs. In summary, infection of J774A.1 cells with M. intracellulare induces pyroptosis, and this process is mediated by caspase-1. Our study provides information for further understanding of the molecular mechanism of M. intracellulare infection.

NLRP3 inflammasomes as a target for hesperidin and diosmin flavonoids in varicose vein disease and its complications

Varicose vein disease of the lower extremities is an inflammatory disorder with abnormal structure and functional activity of the venous valves, venous walls and cells, as well as with abnormal activity of the infiltrating leukocytes. The abnormally changed varicose vein are characterized by increased venous pressure, blood accumulation and congestion, ischemia, and metabolic and energy turnover derangement, which result in clinical manifestations of complications, such as pain, edema and formation of trophic ulcers. For many years, flavonoids have been used to treat varicose veins. The most effective flavonoids used in varicose vein disease and its complications, are hesperidin and diosmin, as well as their combinations. The review sets forth the state-of-the-art knowledge on the universal inflammatory processes playing a leading role in the pathophysiology of many cardiovascular disorders, including venous ones. In the recent years, it has been found that one of the main causes of inflammation is the formation of intracellular protein complexes (inflammasomes), which both produce a set of proinflammatory cytokines and are responsible for their excretion from the cells. In addition, inflammasomes control the development of regulated necrosis (pyroptosis) that takes part in the process of ulceration. The inflammasome activity can be modified by various mechanisms, of which the gene independent synthesis of the inflammasomal proteins has been recognized as the leading one. It has been shown that flavonoids inhibit the activation of a key factor NF-kappa B and suppress the synthesis of proteins, including NOD-like receptor protein 3 (NLRP3) inflammasome components, decrease the expression of NLRP3 receptor, protein ASC and caspase 1, as well as diminish interleukin 1 beta, interleukin 6 and tumor necrosis factor-alpha expression. Thus, this is the explanation of positive effects observed with the use of hesperidin, diosmin and their combination in clinical practice.

Exploration on the protective effect of liraglutide on renal injury in diabetic nephropathy rats based on ROS-NLRP3 inflammasomes

Objective: To investigate the effect of liraglutide on diabetic nephropathy in rats and its regulatory mechanism. Methods: The diabetic nephropathy rat model was constructed with high-glucose-high-fat diet in combination with STZ, and was randomly divided into normal saline group and liraglutide group. The rats in liraglutide group were given sc 200 μg/kg of liraglutide, and the rats in normal saline group were given sc 20 mg/kg of normal saline twice a day for 4 weeks. The normal control group was not treated with any treatment. The biochemical indexes such as rat body weight, 24-hour urine total protein quantification (UTP), fasting blood glucose (FPG), triglyceride (TG), cholesterol (TC), blood urea nitrogen (BUN) and serum creatinine (Scr) were measured. HE staining, Masson staining and PAS staining were used to observe the pathologically morphological changes in renal tissues. Western-blot was used to detect the expression of NLRP3 inflammasome-related protein in renal tissues. Elisa was used to measure the serum levels of interleukin-18 (IL-18) and IL-1β. SPSS 26.0 statistical software and Graph Prism 9.0 software were used for analysis and mapping. The t-test was used for the comparison of measurement data between two groups, one-way ANOVA was used for the comparison among multiple groups, and Tukey’s test was used for the comparison in the same group. Results: Compared with the normal saline group, FBG, UTP, BUN, Scr, TC and TG in the liraglutide group were significantly decreased (p &lt; .01), the glomerular basement membrane was slightly thickened, with the tubular lumen slightly dilated and the lesion damage alleviated; the expression levels of NLRP3, ASC and caspase-1 inflammasome-related proteins were decreased (p &lt; .01), and the levels of IL-18 and IL-1β were decreased (p &lt; .01).Conclusion: Liraglutide can inhibit the activation of NLRP3 inflammasome mediated by oxidative stress in renal tissues through ROS-NLRP3 inflammasome pathway, thereby inhibiting the inflammation, and finally playing an anti-diabetic nephropathy renal injury role.

The Inflammasome Adaptor Protein ASC in Plasma as a Biomarker of Early Cognitive Changes.

Dementia is a group of symptoms including memory loss, language difficulties, and other types of cognitive and functional impairments that affects 57 million people worldwide, with the incidence expected to double by 2040. Therefore, there is an unmet need to develop reliable biomarkers to diagnose early brain impairments so that emerging interventions can be applied before brain degeneration. Here, we performed biomarker analyses for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and amyloid-β 42/40 (Aβ42/40) ratio in the plasma of older adults. Participants had blood drawn at baseline and underwent two annual clinical and cognitive evaluations. The groups tested either cognitively normal on both evaluations (NN), cognitively normal year 1 but cognitively impaired year 2 (NI), or cognitively impaired on both evaluations (II). ASC was elevated in the plasma of the NI group compared to the NN and II groups. Additionally, Aβ42 was increased in the plasma in the NI and II groups compared to the NN group. Importantly, the area under the curve (AUC) for ASC in participants older than 70 years old in NN vs. NI groups was 0.81, indicating that ASC is a promising plasma biomarker for early detection of cognitive decline.

Structural characterization of the glucan from Gastrodia elata Blume and its ameliorative effect on DSS-induced colitis in mice

The polysaccharide glucan was extracted from Gastrodia elata Blume, and its structural characterizations and beneficial effects against acute dextran sulfate sodium (DSS)-induced ulcerative colitis were investigated. The results showed that a polysaccharide GP with a molecular weight of 811.0 kDa was isolated from G. elata Blume. It had a backbone of α-D-1,4-linked glucan with branches of α-d-glucose linked to the C-6 position. GP exhibited protective effects against DSS-induced ulcerative colitis, and reflected in ameliorating weight loss and pathological damages in mice, increasing colon length, inhibiting the expression of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), decreasing the levels of inflammatory related proteins NLRP3 and ASC, and elevating the anti-inflammatory cytokine interleukin-10 (IL-10) level in mouse colon tissues. GP supplementation also reinforced the intestinal barrier by promoting the expression of ZO-1, Occludin, and MUC2 of colon tissues, and positively regulated intestinal microbiota. Thus, GP treatment possessed a significant improvement in ulcerative colitis in mice, and it was expected to be developed as a functional food.

Puerarin inhibits Staphylococcus aureus-induced endometritis through attenuating inflammation and ferroptosis via regulating the P2X7/NLRP3 signalling pathway.

Endometritis is one of the important causes of infertility. Puerarin (PU) can inhibit oxidative stress and reduce inflammation; however, it is unclear whether PU has a protective effect on the endometritis. In our study, we used Staphylococcus aureus to induce mouse endometritis. The PU group (100 mg/kg PU) and the S. aureus + PU group received daily intraperitoneal injection of PU (25, 50 or 100 mg/kg PU). The results showed that S. aureus significantly increased the levels of MPO, TNF-α, IL-1β and IL-6 in uterine tissue, and increased the expression of p-p65 and p-IκBα proteins in uterine tissue to induce endometritis in mice (p < 0.05). Furthermore, it has been found that S. aureus promotes the occurrence of ferroptosis by reducing GSH and ATP content, increasing MDA and iron content and reducing GPX4 and SLC7A11 protein expression levels (p < 0.05). S. aureus significantly increase the expression of NLRP3, ASC, caspase-1 and P2X7 proteins in uterine tissue (p < 0.05). However, PU obviously reduced the inflammatory response and reversed the changes of ferroptosis and the expression of P2X7 receptor/NLRP3 pathway associated proteins of the uterus induced by S. aureus (p < 0.05). Taken together, these findings emphasize the protective effect of PU on endometritis by regulating the P2X7 receptor/NLRP3 signalling pathway and inhibiting ferroptosis.

Ginsenoside Rb2 Inhibits the Pyroptosis in Myocardial Ischemia Progression Through Regulating the SIRT1 Mediated Deacetylation of ASC.

Myocardial ischemic (MI) injury is a common cardiovascular disease, and the potential therapeutic effects of ginsenoside Rb2 (Rb2) have been lately the focus of interest. Therefore, this study aimed to investigate the effects of Rb2 on pyroptosis of cardiomyocytes in MI progression. An in vitro MI model was developed by subjecting rat's cardiomyocytes (H9c2) to hypoxia/reoxygenation (H/R). The cell viability was determined by CCK-8 assay, while cell death was analyzed by propidium iodide staining. Similarly, pyroptosis-related protein levels and acetylation levels of apoptosis-associated speck-like protein containing a CARD (ASC) were detected by western blotting, and the relationship between Sirtuin 1 (SIRT1) and ASC was confirmed by co-immunoprecipitation (Co-IP) assay. Moreover, hematoxylin-eosin (H&E) and triphenyl tetrazolium chloride staining were used to study pathological structure and infarct size. It was found that post-Rb2 treatment significantly increased the cell viability and decreased the cell death and lactic dehydrogenase release, while the increased gasdermin D-N, NOD-like receptor thermal protein domain-associated protein 3, ASC, and cleaved-caspase-1 protein levels were significantly decreased in H/R-stimulated H9c2 cells. Moreover, the acetylation levels of H92c cells were decreased post-Rb2 treatment via increasing SIRT1 levels, while knocking down SIRT1, translated into an increase in ASC acetylation levels, leading to the increase in ASC protein stability and expressions. Additionally, the Rb2 effects on pyroptosis in H/R-stimulated H92c cells were reversed by overexpressing ASC, while reduced myocardial tissue damage was observed in MI rats following in vivo Rb2 treatment. Rb2 treatment inhibited pyroptosis in MI progression by decreasing the ASC levels. Mechanistically, Rb2 treatment increased the SIRT1 levels, further increasing the acetylation levels of ASC and decreasing the protein stability of ASC.