Nod-like Receptor Protein Research Articles

Based on SIRT1/NF-κB/NLRP3 Signal Pathway to Explore the Effect of Yiqi Huatan Huoxue Recipe on Inflammatory Injury in AIT Mice by Pyroptosis.

Autoimmune thyroiditis (AIT) is the most common autoimmune thyroid disease. In recent decades, its incidence and prevalence have sharply increased. Yiqi Huatan Huoxue recipe is a traditional Chinese medicine formula we use to treat AIT. Its clinical efficacy is clear, but the specific mechanism remains unclear. This study aims to explore whether pyroptosis mediated by the SIRT1/NF-κB/NLRP3 signaling pathway is one of the therapeutic mechanisms of Yiqi Huatan Huoxue recipe. Forty 8-week-old female NOD.H-2h4 mice were randomly divided into four groups: the normal group (NG), model group (MG), Yiqi Huatan Huoxue recipe group (YG), and western medicine group (selenium yeast tablet, SeG). The normal group was gavaged with distilled water, while the remaining groups were gavaged with 0.05% sodium iodide (NaI) solution for 8 weeks. After the AIT animal model formed naturally, the mice were euthanized by gavage after 8 weeks. Hematoxylin-eosin staining was used to observe thyroid tissue changes, and enzymelinked immunosorbent assay (ELISA) was used to detect serum anti-thyroglobulin antibodies (TGAb) and mouse anti-thyroid peroxidase antibodies (TPOAb). Real-time quantitative PCR (qRT-PCR), Western blot, and immunohistochemistry were used to detect the expression of sirtuin 1 (SIRT1), nuclear factor κB p65 (NF-κB p65), nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase- 1, gasdermin D (GSDMD), and interleukin (IL)-1β in thyroid tissue. Compared with the NG group, the thyroid structure of rats in the MG group was severely damaged, with significant lymphocyte infiltration, significantly increased serum TGAb and TPOAb levels, and significantly increased expression levels of SRIT1, NF-κB p65, NLRP3, ASC, Caspase-1, GSDMD, IL-1β mRNA, and protein. Compared with the MG group, the thyroid structure damage and lymphocyte infiltration in rats of each treatment group were improved, and the serum TGAb, TPOAb, SRIT1, NF-κB p65, NLRP3, ASC, Caspase-1, GSDMD, IL-1β mRNA, and protein expression levels were significantly reduced. Yiqi Huatan Huoxue recipe can alleviate thyroid structural damage in AIT mice, and its mechanism may be related to the upregulation of SIRT1, NF-κB deacetylation, and inhibition of NLRP3-mediated pyroptosis.

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.

Activation of pregnane X receptor protects against cholestatic liver injury by inhibiting hepatocyte pyroptosis.

Our previous study shows that activation of pregnane X receptor (PXR) exerts hepatoprotection against lithocholic acid (LCA)-induced cholestatic liver injury. In this study we investigated whether PXR activation could inhibit hepatocyte pyroptosis, as well as the underlying mechanisms. Male mice were treated with mouse PXR agonist pregnenolone 16α-carbonitrile (PCN, 50 mg·kg-1·d-1, i.p.) for 7 days, and received LCA (125 mg/kg, i.p., bid) from D4, then sacrificed 12 h after the last LCA injection. We showed that LCA injection resulted in severe cholestatic liver injury characterized by significant increases in gallbladder size, hepatocellular necrosis, and neutrophil infiltration with a mortality rate of 68%; PCN treatment significantly inhibited hepatocyte pyroptosis during LCA-induced cholestatic liver injury, as evidenced by reduced serum lactic dehydrogenase (LDH) levels, TUNEL-positive cells and hepatocyte membrane damage. Furthermore, PXR activation suppressed both the NOD-like receptor protein 3 (NLRP3) inflammasome-induced canonical pyroptosis and the apoptosis protease activating factor-1 (APAF-1) pyroptosome-induced non-canonical pyroptosis. Inhibition of the nuclear factor kappa B (NF-κB) and forkhead box O1 (FOXO1) signaling pathways was also observed following PXR activation. Notably, dual luciferase reporter assay showed that PXR activation inhibited the transcriptional effects of NF-κB on NLRP3, as well as FOXO1 on APAF-1. Our results demonstrate that PXR activation protects against cholestatic liver injury by inhibiting the canonical pyroptosis through the NF-κB-NLRP3 axis and the non-canonical pyroptosis through the FOXO1-APAF-1 axis, providing new evidence for PXR as a prospective anti-cholestatic target.

New applications of Clioquinol in the treatment of inflammation disease by directly targeting arginine 335 of NLRP3

The NOD-like receptor protein 3 (NLRP3) inflammasome is essential in innate immune-mediated inflammation, with its overactivation implicated in various autoinflammatory, metabolic, and neurodegenerative diseases. Pharmacological inhibition of NLRP3 offers a promising treatment strategy for inflammatory conditions, although no medications targeting the NLRP3 inflammasome are currently available. This study demonstrates that clioquinol (CQ), a clinical drug with chelating properties, effectively inhibits NLRP3 activation, resulting in reduced cytokine secretion and cell pyroptosis in both human and mouse macrophages, with a half maximal inhibitory concentration (IC50) of 0.478 μM. Additionally, CQ mitigates experimental acute peritonitis, gouty arthritis, sepsis, and colitis by lowering serum levels of interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α). Mechanistically, CQ covalently binds to Arginine 335 (R335) in the NACHT domain, inhibiting NLRP3 inflammasome assembly and blocking the interaction between NLRP3 and its component protein. Collectively, this study identifies CQ as an effective natural NLRP3 inhibitor and a potential therapeutic agent for NLRP3-driven diseases.

Hyperglycemia induces microglial pyroptosis by increasing oxygen extraction rate: Implication in neurological impairment during ischemic stroke.

Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO2), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO2) were measured. To investigate the significance of the NOD‑like receptor protein 3 (NLRP3) inflammasome, NLRP3‑/‑ mice were used, and the expression levels of NLRP3, caspase‑1, full‑length gasdermin D (GSDMD‑FL), GSDMD‑N domain (GSDMD‑N), IL‑1β and IL‑18 were evaluated. In addition, Z‑YVAD‑FMK, a caspase‑1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO2 and OCR, and decreased PbtO2 in response to high glucose treatment. Furthermore, hyperglycemia‑induced microglial pyroptosis was confirmed by detection of increased levels of caspase‑1, GSDMD‑N, IL‑1β and IL‑18 and a decreased level of GSDMD‑FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase‑1 also reduced the expression levels of GSDMD‑N, IL‑1β and IL‑18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.

The Protective Effects of Ruscogenin Against Lipopolysaccharide-Induced Myocardial Injury in Septic Mice.

Sepsis-induced myocardial dysfunction commonly occurs in individuals with sepsis and is a severe complication with high morbidity and mortality rates. This study aimed to investigate the effects and potential mechanisms of the natural steroidal sapogenin ruscogenin (RUS) against lipopolysaccharide (LPS)-induced myocardial injury in septic mice. We found that RUS effectively alleviated myocardial pathological damage, normalized cardiac function, and increased survival in septic mice. RNA sequencing demonstrated that RUS administration significantly inhibited the activation of the NOD-like receptor signaling pathway in the myocardial tissues of septic mice. Subsequent experiments further confirmed that RUS suppressed myocardial inflammation and pyroptosis during sepsis. In addition, cultured HL-1 cardiomyocytes were challenged with LPS, and we observed that RUS could protect these cells against LPS-induced cytotoxicity by suppressing inflammation and pyroptosis. Notably, both the in vivo and in vitro findings indicated that RUS inhibited NOD-like receptor protein-3 (NLRP3) upregulation in cardiomyocytes stimulated with LPS. As expected, knockdown of NLRP3 blocked the LPS-induced activation of inflammation and pyroptosis in HL-1 cells. Furthermore, the cardioprotective effects of RUS on HL-1 cells under LPS stimulation were abolished by the novel NLRP3 agonist BMS-986299. Taken together, our results suggest that RUS can alleviate myocardial injury during sepsis, at least in part by suppressing NLRP3-mediated inflammation and pyroptosis, highlighting the potential of this molecule as a promising candidate for sepsis-induced myocardial dysfunction therapy.

Novel genetic variants in the NLRP3 inflammasome-related PANX1 and APP genes predict survival of patients with hepatitis B virus-related hepatocellular carcinoma.

The nod-like receptor protein 3 (NLRP3) is one of the most characterized inflammasomes involved in the pathogenesis of several cancers, including hepatocellular carcinoma (HCC). However, the effects of genetic variants in the NLRP3 inflammasome-related genes on survival of hepatitis B virus (HBV)-related HCC patients are unclear. We performed multivariable Cox proportional hazards regression analysis to evaluate associations between 299 single-nucleotide polymorphisms (SNPs) in 16 NLRP3 inflammasome-related genes and overall survival (OS) of 866 patients with HBV-related HCC. We further performed expression quantitative trait loci (eQTL) analysis using the data from the GTEx project and 1000 Genomes projects, and performed differential expression analysis using the TCGA dataset to explore possible molecular mechanisms underlying the observed associations. We found that two functional SNPs (PANX1 rs3020013 A > G and APP rs9976425 C > T) were significantly associated with HBV-related HCC OS with the adjusted hazard ratio (HR) of 0.83 [95% confidence interval (CI) = 0.73-0.95, P = 0.008], and 1.26 (95% CI = 1.02-1.55, P = 0.033), respectively. Moreover, the eQTL analysis revealed that the rs3020013 G allele was correlated with decreased mRNA expression levels of PANX1 in both normal liver tissues (P = 0.044) and whole blood (P < 0.001) in the GTEx dataset, and PANX1 mRNA expression levels were significantly higher in HCC samples and associated with a poorer survival of HCC patients. However, we did not observe such correlations for APP rs9976425. These results indicated that SNPs in the NLRP3 inflammasome-related genes may serve as potential biomarkers for HBV-related HCC survival, once replicated by additional larger studies.

AMPK Attenuation of β-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of β-Arrestin-1-ser330.

β-adrenergic receptor (β-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of β-AR remains unclear. Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates β-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the β-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between β-adrenergic insult and β-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from β-arrestin-1-S330A/S330D mutation and β-adrenergic insult. Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to β-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted β-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing β-arrestin-1-S330D (active form) inhibited the β-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. β-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the β-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. AMPK phosphorylation of β-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting β-AR/cAMP/PKA activation. Subsequently, β-arrestin-1 Ser330 phosphorylation blocks β-AR-induced cardiac inflammasome activation and remodeling.

Oncoprotein-induced transcript 3 protein-enriched extracellular vesicles promotes NLRP3 ubiquitination to alleviate acute lung injury after cardiac surgery

Acute lung injury (ALI) including acute respiratory distress syndrome (ARDS) is a major complication and increase the mortality of patients with cardiac surgery. We previously found that the protein cargoes enriched in circulating extracellular vesicles (EVs) are closely associated with cardiopulmonary disease. We aimed to evaluate the implication of EVs on cardiac surgery-associated ALI/ARDS. The correlations between “oncoprotein-induced transcript 3 protein (OIT3) positive” circulating EVs and postoperative ARDS were assessed. The effects of OIT3-overexpressed EVs on the cardiopulmonary bypass (CPB) -induced ALI in vivo and inflammation of human bronchial epithelial cells (BEAS-2B) were detected. OIT3 enriched in circulating EVs is reduced after cardiac surgery with CPB, especially with postoperative ARDS. The “OIT3 positive” EVs negatively correlate with lung edema, hypoxemia and CPB time. The OIT3-overexpressed EVs can be absorbed by pulmonary epithelial cells and OIT3 transferred by EVs triggered K48- and K63-linked polyubiquitination to inactivate NOD-like receptor protein 3 (NLRP3) inflammasome, and restrains pro-inflammatory cytokines releasing and immune cells infiltration in lung tissues, contributing to the alleviation of CPB-induced ALI. Overexpression of OIT3 in human bronchial epithelial cells have similar results. OIT3 promotes the E3 ligase Cbl proto-oncogene B associated with NLRP3 to induce the ubiquitination of NLRP3. Immunofluorescence tests reveal that OIT3 is reduced in the generation from the liver sinusoids endothelial cells (LSECs) and secretion in liver-derived EVs after CPB. In conclusion, OIT3 enriched in EVs is a promising biomarker of postoperative ARDS and a therapeutic target for ALI after cardiac surgery.

Oxycodone attenuates lipopolysaccharide-induced myocardial injury by inhibiting inflammation, oxidation and pyroptosis via Nrf2/HO-1 signalling pathway.

Myocardial injury and cardiovascular dysfunction are the most common complications of sepsis, and effective therapeutic candidate is still lacking. This study aims to investigate the protective effect of oxycodone in myocardial injury of lipopolysaccharide-induced sepsis and its related signalling pathways. Wild-type and nuclear factor erythroid 2-related factor 2 (Nrf2)-knockout mice, as well as H9c2 cardiomyocytes cultures treated with lipopolysaccharide (LPS) were used as models of septic myocardial injury. H9c2 cardiomyocytes culture showed that oxycodone protected cells from pyroptosis induced by LPS. Mice model confirmed that oxycodone pretreatment significantly attenuated myocardial pathological damage and improved cardiac function demonstrated by increased ejection fraction (EF) and fractional shortening (FS), as well as decreased cardiac troponin I (cTnI) and creatine kinase isoenzymes MB (CK-MB). Oxycodone also reduced the levels of inflammatory factors and oxidative stress damage induced by LPS, which involves pyroptosis-related proteins including: Nod-like receptor protein 3 (NLRP3), Caspase 1, Apoptosis-associated speck-like protein contain a CARD (ASC), and Gasdermin D (GSDMD). These changes were mediated by Nrf2 and heme oxygenase-1 (HO-1) because Nrf2-knockout mice or Nrf2 knockdown in H9c2 cells significantly reversed the beneficial effect of oxycodone on oxidative stress, inflammatory responses and NLRP3-mediated pyroptosis. Our findings yielded that oxycodone therapy reduces LPS-induced myocardial injury by suppressing NLRP3-mediated pyroptosis via the Nrf2/HO-1 signalling pathway in vivo and in vitro.

Changes and clinical significance of NLRP3 inflammasomes and related factors in patients with spinal fracture complicated with acute spinal cord injury

To investigate the changes and clinical significance of NOD like receptor protein 3 (NLRP3) inflammasomes and related factors in patients with spinal fractures complicated with acute spinal cord injury (SCI). Eighty-six spinal fracture patients complicated with acute SCI admitted to hospital from June 2019 to March 2022 were selected as SCI group, There were 48 males and 38 females, with an average age of (43.48±6.58) years old. And 100 healthy volunteers who underwent physical examination during the same time were selected as control group, including 56 males patients and 44 females patients, with an average age of (45.13±6.43) years old. Peripheral blood mononuclear cell (PBMC) were collected, and the mRNA expressions of NLRP3 and Caspase-1 were detected. Serum was collected and the levels of interleukin (IL)- 1β, IL-18 were detected. According to Frankel's grade, the SCI group was divided into complete injury patients and incomplete injury patients, and according to the Japanese Orthopedic Society (JOA) grade, the SCI group was divided into good prognosis group and poor prognosis group. The difference of NLRP3, Caspase-1, IL-1β, IL-18 among groups were compared, the influencing factors for poor prognosis in SCI patients was analyzed by Logistic regression. The mRNA expression levels of NLRP3 (1.41±0.33) and Caspase-1 (1.44±0.35) in PBMC and the levels of IL-1β(45.34±13.22) pg·ml-1, IL-18(40.95±8.77) pg·ml-1 in serum of SCI group were higher than those of the control group[(1.00±0.19), (1.00±0.16), (16.58±4.24) pg·ml-1, (12.57±3.68) pg·ml-1] (P<0.05). The mRNA expression levels of NLRP3(1.63±0.34) and Caspase-1 (1.67±0.27) in PBMC and the levels of IL-1β(51.09±11.10) pg·ml-1, IL-18 (47.65±7.93) pg·ml-1 in serum of patients with complete injury in the SCI group were higher than those of patients with incomplete injury [(1.31±0.27), (1.34±0.33), (42.85±13.36) pg·ml-1, (38.05±7.48) pg·ml-1](P<0.05). The mRNA expression levels of NLRP3 (1.66±0.31) and Caspase-1 (1.72±0.31)in PBMC and the levels of IL-1β(51.21±11.31) pg·ml-1, IL-18 (45.70±7.25) pg·ml-1 in serum, the proportion of complete injury(21 patients), and the proportion of spinal cord edema or bleeding of patients(15 patients) with poor prognosis in the SCI group were higher than those of patients with good prognosis[(1.28±0.26), (1.37±0.36), (42.79±13.25) pg·ml-1、(38.90±8.63) pg·ml-1, 5、20 cases](P<0.05). Complete injury and the mRNA expression of NLRP3 in PBMC were the influencing factors for poor prognosis in the SCI group (P<0.05). The activation of NLRP3 inflammasomes in patients with spinal fractures complicated with acute SCI is associated with worsening injury and poor prognosis, and NLRP3 expression can serve as a marker for evaluating prognosis.

Novel insights into the molecular mechanisms underlying anti-nociceptive effect of myricitrin against reserpine-induced fibromyalgia model in rats: Implication of SIRT1 and miRNAs

Ethnopharmacological relevanceManilkara zapota (L.) P. Royen, also termed sapodilla or chikoo, is a significant plant in ethnomedicine because of its long history of traditional medical applications. In diverse cultures, sapodilla is believed to protect against oxidative stress, inflammation, and some chronic diseases because of its high antioxidant content. The naturally occurring antioxidant myricitrin (MYR) flavonoid is primarily found in the leaves and other plant parts of sapodilla and it is well-known for having therapeutic qualities and possible health advantages. Aim of the studyTo appraise the possible impact of MYR on a rat model of reserpine-induced fibromyalgia (FM) and explore its mechanism of action. Materials and methodsIsolation and identification of MYR with more than 99% purity from Manilkara zapota leaves were primarily done and confirmed through chromatographic and spectrophotometric techniques. To develop FM model, reserpine (RSP) was injected daily (1 mg/kg, s.c.) for three successive days. Then, MYR (10 mg/kg, i.p.) and pregabalin (PGB, 30 mg/kg, p.o.) were given daily for another five days. Behavioral changes were assessed through open field test (OFT), hot plate test, and forced swimming test (FST). Further analyses of different brain parameters and signaling pathways were performed to assess monoamines levels, oxidative stress, inflammatory response, apoptotic changes as well as silent information regulator 1 (SIRT1) and micro RNAs (miRNAs) expressions. ResultsFrom High-Performance Liquid Chromatography (HPLC) analysis, the methanol extract of sapodilla leaves contains 166.17 μg/ml of MYR. Results of behavioral tests showed a significant improvement in RSP-induced nociceptive stimulation, reduced locomotion and exploration and depressive-like behavior by MYR. Biochemical analyses showed that MYR significantly ameliorated the RSP-induced imbalance in brain monoamine neurotransmitters. In addition, MYR significantly attenuated oxidative stress elicited by RSP via up-regulating nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein expressions, enhancing superoxide dismutase (SOD) and catalase (CAT) activities, and reducing malondialdehyde (MDA) content in brain. The RSP-provoked inflammatory response was also diminished by MYR treatment as shown by a significant decreased NOD-like receptor protein 3 (NLRP3) inflammasome expression along with reduced levels of interleukin 1 beta (IL-1β) and nuclear factor-κB (NF-κB). Furthermore, the anti-apoptotic activity of MYR was demonstrated by a marked rise in Bcl-2-associated X protein (BAX)/B cell lymphoma-2 (Bcl-2) ratio by lowering Bcl-2 while increasing BAX levels. In addition, MYR treatment significantly boosted the expression of SIRT1 deacetylase in RSP-treated animals. Interestingly, molecular docking showed the ability of MYR to form a stable complex in the binding site of SIRT1. Regarding miRNAs, MYR effectively ameliorated RSP-induced changes in miR-320 and miR-107 gene expressions. ConclusionOur findings afford new insights into the anti-nociceptive profile of MYR in the RSP-induced FM model in rats. The underlying mechanisms involved direct binding and activation of SIRT1 to influence different signaling cascades, including Nrf2 and NF-κB/NLRP3 together with modulation of miRNAs. However, more in-depth studies are needed before proposing MYR as a new clinically relevant drug in the management of FM.

TMEM106B Knockdown Exhibits a Neuroprotective Effect in Parkinson's Disease via Decreasing Inflammation and Iron Deposition.

Parkinson's disease (PD) is closely related to iron accumulation and inflammation. Emerging evidence indicates that TMEM106B plays an essential role in PD. But whether TMEM106B could act on neuroinflammation and iron metabolism in PD has not yet been investigated. The aim of this study was to investigate the pathological mechanisms of inflammation and iron metabolism of TMEM106B in PD. 1-methyl-4-phenylpyridinium (MPP+)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced SH-SY5Y cells and mice were treated with LV-shTMEM106B and AAV-shTMEM106B to construct PD cellular and mouse models. Pole tests and open-field test (OFT) were performed to evaluate the locomotion of the mice. Immunohistochemistry and iron staining were used to detect TH expression and iron deposition in the SN. Iron staining was used to measure the levels of iron. Western blotting was used to detect the expression of inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6)), NOD-like receptor protein 3 (NLRP3) inflammasome, divalent metal transporter 1 (DMT1), and Ferroportin1 (FPN1)). Knockdown of TMEM106B improved motor ability and rescued dopaminergic (DA) neuron loss. TMEM106B knockdown attenuated the increases of TNF-α, IL-6, NLRP3 inflammasome, and DMT1 expression in the MPP+ and MPTP-induced PD models. Furthermore, TMEM106B knockdown also increases the expression of FPN1. This study provides the first evidence that knockdown of TMEM106B prevents dopaminergic neurodegeneration by modulating neuroinflammation and iron metabolism.

Linoleic acid-derived diol 12,13-DiHOME enhances NLRP3 inflammasome activation in macrophages.

12,13-dihydroxy-9z-octadecenoic acid (12,13-DiHOME) is a linoleic acid diol derived from cytochrome P-450 (CYP) epoxygenase and epoxide hydrolase (EH) metabolism. 12,13-DiHOME is associated with inflammation and mitochondrial damage in the innate immune response, but how 12,13-DiHOME contributes to these effects is unclear. We hypothesized that 12,13-DiHOME enhances macrophage inflammation through effects on NOD-like receptor protein 3 (NLRP3) inflammasome activation. To test this hypothesis, we utilized human monocytic THP1 cells differentiated into macrophage-like cells with phorbol myristate acetate (PMA). 12,13-DiHOME present during lipopolysaccharide (LPS)-priming of THP1 macrophages exacerbated nigericin-induced NLRP3 inflammasome activation. Using high-resolution respirometry, we observed that priming with LPS+12,13-DiHOME altered mitochondrial respiratory function. Mitophagy, measured using mito-Keima, was also modulated by 12,13-DiHOME present during priming. These mitochondrial effects were associated with increased sensitivity to nigericin-induced mitochondrial depolarization and reactive oxygen species production in LPS+12,13-DiHOME-primed macrophages. Nigericin-induced mitochondrial damage and NLRP3 inflammasome activation in LPS+12,13-DiHOME-primed macrophages were ablated by the mitochondrial calcium uniporter (MCU) inhibitor, Ru265. 12,13-DiHOME present during LPS-priming also enhanced nigericin-induced NLRP3 inflammasome activation in primary murine bone marrow-derived macrophages. In summary, these data demonstrate a pro-inflammatory role for 12,13-DiHOME by enhancing NLRP3 inflammasome activation in macrophages.

Molecular mechanism of HDAC6-mediated pyroptosis in neurological function recovery after cardiopulmonary resuscitation in rats

Brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is the leading cause of neurological dysfunction and death. This study aimed to explore the mechanism of histone deacetylase 6 (HDAC6) in neurofunctional recovery following CA/CPR in rats. A rat model was established by CA/CPR treatment. Adenovirus-packaged sh-HDAC6 was injected into the tail vein. To evaluate the neurofunction of rats, survival time, neurofunctional scores, serum NSE/S100B, and brain water content were measured and Morris water maze test was performed. HDAC6, microRNA (miR)-138-5p, Nod-like receptor protein 3 (NLRP3), and pyroptotic factor levels were determined by real-time quantitative polymerase chain reaction or Western blot assay. HDAC6 and H3K9ac enrichment on miR-138-5p promoter were examined by chromatin immunoprecipitation. miR-138-5p-NLRP3 binding was analyzed by dual-luciferase reporter assay. NLRP3 inflammasome was activated with nigericin sodium salt. After CPR treatment, HDAC6 was highly expressed, while miR-138-5p was downregulated. HDAC6 downregulation improved neurofunction and reduced pyroptosis. HDAC6 enrichment on the miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p, and promoting NLRP3-mediated pyroptosis. Downregulating miR-138-5p partially reversed the protective effect of HDAC6 inhibition after CPR. In Conclusion, HDAC6 enrichment on miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p expression and promoting NLRP3-mediated pyroptosis, worsening neurological dysfunction in rats after CPR.

Identification of novel NLRP3 inhibitors as therapeutic options for epilepsy by machine learning-based virtual screening, molecular docking and biomolecular simulation studies

The NOD-Like Receptor Protein-3 (NLRP3) inflammasome is a key therapeutic target for the treatment of epilepsy and has been reported to regulate inflammation in several neurological diseases. In this study, a machine learning-based virtual screening strategy has investigated candidate active compounds that inhibit the NLRP3 inflammasome. As machine learning-based virtual screening has the potential to accurately predict protein-ligand binding and reduce false positives outcomes compared to traditional virtual screening. Briefly, classification models were created using Support Vector Machine (SVM), Random Forest (RF), and K-Nearest Neighbor (KNN) machine learning methods. To determine the most crucial features of a molecule's activity, feature selection was carried out. By utilizing 10-fold cross-validation, the created models were analyzed. Among the generated models, the RF model obtained the best results as compared to others. Therefore, the RF model was used as a screening tool against the large chemical databases. Molecular operating environment (MOE) and PyRx software's were applied for molecular docking. Also, using the Amber Tools program, molecular dynamics (MD) simulation of potent inhibitors was carried out. The results showed that the KNN, SVM, and RF accuracy was 0.911 %, 0.906 %, and 0.946 %, respectively. Moreover, the model has shown sensitivity of 0.82 %, 0.78 %, and 0.86 % and specificity of 0.95 %, 0.96 %, and 0.98 % respectively. By applying the model to the ZINC and South African databases, we identified 98 and 39 compounds, respectively, potentially possessing anti-NLRP3 activity. Also, a molecular docking analysis produced ten ZINC and seven South African compounds that has comparable binding affinities to the reference drug. Moreover, MD analysis of the two complexes revealed that the two compounds (ZINC000009601348 and SANC00225) form stable complexes with varying amounts of binding energy. The in-silico studies indicate that both compounds most likely display their inhibitory effect by inhibiting the NLRP3 protein.

Unveiling the Potential of Sulfur-Containing Gas Signaling Molecules in Acute Lung Injury: A Promising Therapeutic Avenue.

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), are pulmonary conditions that cause significant morbidity and mortality. The common etiologies of these conditions include pneumonia, pulmonary contusion, fat embolism, smoke inhalation, sepsis, shock, and acute pancreatitis. Inflammation, oxidative stress, apoptosis, and autophagy are key pathophysiological mechanisms underlying ALI. Hydrogen sulfide (H2S) and sulfur dioxide (SO2) are sulfur-containing gas signaling molecules that can mitigate these pathogenic processes by modulating various signaling pathways, such as toll-like receptor 4 (TLR4)/nod-like receptor protein 3 (NLRP3), extracellular signal-regulating protein kinase 1/2 (ERK1/2), mitogen-activated protein kinase (MAPK), phosphatidyl inositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), and nuclear factor kappa B (NF-κB), thereby conferring protection against ALI. Given the limited clinical effectiveness of prevailing ALI treatments, investigation of the modulation of sulfur-containing gas signaling molecules (H2S and SO2) in ALI is imperative. This article presents an overview of the regulatory pathways of sulfur-containing gas signaling molecules in ALI animal models induced by various stimuli, such as lipopolysaccharide, gas inhalation, oleic acid, and ischemia-reperfusion. Furthermore, this study explored the therapeutic prospects of diverse H2S and SO2 donors for ALI, stemming from diverse etiologies. The aim of the present study was to establish a theoretical framework, in order to promote the new treatment of ALI.

Ligustrazine alleviates the progression of coronary artery calcification by inhibiting caspase-3/GSDME mediated pyroptosis.

Coronary artery calcification (CAC) is an early marker for atherosclerosis and is mainly induced by the osteoblast-like phenotype conversion of vascular smooth muscle cells (VSMCs). Recent reports indicate that NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis plays a significant role in the calcification of vascular smooth muscle cells (VSMCs), making it a promising target for treating calcific aortic valve disease (CAC). Ligustrazine, or tetramethylpyrazine (TMP), has been found effective in various cardiovascular and cerebrovascular diseases and is suggested to inhibit NLRP3-mediated pyroptosis. However, the function of TMP in CAC is unknown. Herein, influences of TMP on β-glycerophosphate (β-GP)-stimulated VSMCs and OPG-/- mice were explored. Mouse Aortic Vascular Smooth Muscle (MOVAS-1) cells were stimulated by β-GP with si- caspase-3, si- Gasdermin E (GSDME) or TMP. Increased calcification, reactive oxygen species (ROS) level, Interleukin-1beta (IL-1β) and Interleukin-18 (IL-18) levels, lactate dehydrogenase (LDH) release, enhanced apoptosis, and activated cysteine-aspartic acid protease-3 (caspase-3)/GSDME signaling were observed in β-GP-stimulated MOVAS-1 cells, which was sharply alleviated by si-caspase-3, si-GSDME or TMP. Furthermore, the impact of TMP on the β-GP-induced calcification and injury in MOVAS-1 cells was abolished by raptinal, an activator of caspase-3. Subsequently, OPG-/- mice were dosed with TMP or TMP combined with raptinal. Calcium deposition, increased nodules, elevated IL-1β and IL-18 levels, upregulated CASP3 and actin alpha 2, smooth muscle (ACTA2), and activated caspase-3/GSDME signaling in OPG-/- mice were markedly alleviated by TMP, which were notably reversed by the co-administration of raptinal. Collectively, TMP mitigated CAC by inhibiting caspase-3/GSDME mediated pyroptosis.

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

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

Sevoflurane impedes neuropathic pain by maintaining endoplasmic reticulum stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

To investigate the effect of sevoflurane on neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve in mice, and to elucidate its mechanism by animal experiments. Thirty-two C57BL/6 mice were randomly divided into four groups: Sham group, Model group, Control group and Sevoflurane group. First, a mouse model of neuropathic pain was established. Then, the mice in each group were killed on Day 14 after operation to harvest the enlarged lumbosacral spinal cord. In contrast with the Model group, the Sevoflurane group displayed a significantly increased paw withdrawal mechanical threshold (PWMT) and significantly prolonged paw withdrawal thermal latency (PWTL) from Day 5 after operation. The morphological changes of lumbosacral spinal cord were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy. Pathological results showed that sevoflurane reduced nuclear pyknosis in lumbosacral spinal cord tissue, with a large number of mitochondrial crista disappearance and mitochondrial swelling. The results of Western blotting showed that sevoflurane significantly decreased the protein expressions of phosphorylated phospholipase Cγ (p-PLCγ), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) and phosphorylated inositol 1,4,5-triphosphate receptor (p-IP3R), and reduced the protein expressions of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78) and GRP94, oxidative stress-related proteins P22 and P47 and inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α). Sevoflurane inhibits neuropathic pain by maintaining ER stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.