Protein Expression Of NLRP3 Research Articles

Unraveling the blood microbiome: novel insights into inflammasome responses in Crohn's disease.

Crohn's disease (CD), an inflammatory bowel disease with unknown etiology, is influenced by genetic, environmental, and immunological factors. This study aimed to analyze the blood microbiome and inflammasome responses, emphasizing NLRP3 protein expression and IL-1β and IL-18 plasma levels, between Crohn's patients and healthy subjects. A total of 40 volunteers were included in this study. The 16S rRNA technique was used to sequence the V3-V4 regions of the blood sample. NLRP3 protein levels in plasma were ascertained through Western Blot, and IL-1β and IL-18 plasma profiles were examined using ELISA. Analysis highlighted five unique phyla in patients' plasma, emphasizing the role of the blood microbiome in CD. Compared to controls, Crohn's patients exhibited elevated NLRP3 protein expression. Plasma IL-1β levels were diminished in patients ( P = 0.0041), whereas IL-18 levels were comparably higher ( P = 0.8209). In patients with CD, the presence of Staphylococcus sciuri in blood samples highlights its potential role in the disease's onset. The study also underscored the interplay between dietary habits, specifically increased meat consumption, and the progression of CD. Our pioneering research discerns the variations in the blood microbiome and inflammasome responses between Crohn's patients and healthy individuals. Significant microbiome alterations and the detection of the Staphylococcus sciuri pathogen in Crohn's patients were notable. The pronounced NLRP3 protein in patients suggests its potential as a diagnostic biomarker. Future explorations into IL-1β and IL-18 pathways promise to unveil innovative insights into CD.

Cytosolic mtDNA-cGAS-STING axis contributes to sepsis-induced acute kidney injury via activating the NLRP3 inflammasome.

NLRP3 inflammasome activation is significantly associated with sepsis-induced acute kidney injury (S-AKI). Cytosolic DNA derived from damaged mitochondria has been reported to activate NLRP3 inflammasome via upregulating the cyclic GMP-AMP synthase (cGAS)-the stimulator of interferon genes (STING) axis in nucleus pulposus cell and cardiomyocytes. However, the regulatory effect of mitochondria DNA (mtDNA)-cGAS-STING axis on the NLRP3 inflammasome in S-AKI remains unclear. In the current study, we established an in vivo model of S-AKI by intraperitoneally injecting male C57BL/6J mice with lipopolysaccharide (LPS). Next, selective cGAS inhibitor RU.521, and STING agonist DMXAA were intraperitoneally injected in the mice; then, blood urea nitrogen (BUN), serum creatinine (CRE), urinary kidney injury molecular-1 (KIM-1), pathological changes, and infiltrated neutrophils were detected to assess kidney injury. We also performed western blot and immunofluorescence assays to evaluate STING, cGAS, TBK-1, p-TBK-1, IRF3, p-IRF3, NF-kB, p-NF-kB, NLRP3, cleaved caspase-1, caspase-1, GSDMD-N, and GSDMD expression levels in kidney tissues. IL-18 and IL-1β in renal tissue were identified by ELISA. In vitro, we treated HK-2 cells with LPS to establish a cell model of S-AKI. Furthermore, ethidium bromide (EtBr) was administered to deplete mitochondria DNA (mtDNA). LPS-induced cytotoxicity was evaluated by LDH release assay. Protein expression of cGAS, STING, and NLRP3 in was quantified by western blot. Cytosolic mtDNA was detected by immunofluorescence and q-PCR. Released IL-1β and IL-18 in HK-2 supernatants were detected by ELISA. LPSinjection induced S-AKI in mice, as evidenced by neutrophil infiltration, tubular vacuolation, and increased levels of serum creatinine (CRE), blood urea nitrogen (BUN), and urinary KIM-1. In addition, LPS activated the cGAS-STING axis and NLRP3 inflammasome in vivo, illustrated by increased phosphorylation levels of TBK-1, IRF3, and NF-kB protein, increased ratio of cleaved caspase-1 to caspase-1 and GSDMD-N to GSDMD, and increased IL-1β and IL-18 levels. Moreover, the cGAS inhibitor RU.521 effectively attenuated NLRP3 inflammasome and S-AKI; however, these effects were abolished by treatment with the STING agonist DMXAA. Furthermore, cytosolic release of mtDNA and activation of the cGAS-STING-NLRP3 axis were observed in LPS-treated HK-2 cells. Inhibiting mtDNA replication by Ethidium Bromide (EtBr) treatment reduced cytosolic mtDNA accumulation and downregulated the cGAS-STING-NLRP3 axis, ameliorating the cytotoxicity induced by LPS. This study demonstrated that the cGAS-STING axis was triggered by cytosolic mtDNA and participated in the development of S-AKI by activating NLRP3 inflammasome. Reducing cytosolic mtDNA accumulation or inhibiting the cGAS-STING axis may be potential therapeutic targets for S-AKI.

Jianpi Antai formula prevents miscarriage by repressing M1 polarization of decidual macrophages through ubiquitination of NLRP3 mediated by MARCH7

Ethnopharmacological relevanceJianpi Antai Formula (JAF) is an ancient formula from He's gynecology, which has been used clinically for more than 30 years and has significant therapeutic effects on spontaneous abortion (SA). Both macrophage polarization and NLRP3 inflammasome correlate with the occurrence of SA in women with recurrent or threatened miscarriage. Whether JAF prevent SA via mediating activation of decidual macrophage (dMφ) and ubiquitination-associated degradation of NLRP3 remains uncertain. Aim of the studyThis study aimed to clarify the effects of JAF on pregnancy outcomes and dMφ polarization at the maternal-fetal interface in an SA mouse model, and use in vivo and invitro methods to explore whether JAF can inhibit M1 polarization of dMφ by up-regulating MARCH7-mediated NLRP3 ubiquitination, thereby preventing SA. Materials and methodsThe CBA/J × DBA/2 mating method was used to establish an SA model and the dMφs of SA mice were isolated and cultured. Th1-, Th2-, Th17- and Treg-related cytokine levels were evaluated using ELISA. qRT-PCR was used to detect the levels of M1/M2 macrophage-related cytokine mRNA in the decidua, and western blotting was used to detect the expression of NLRP3 inflammasome-related proteins in the decidua and placenta. The expression of M1/M2 markers of dMφ was detected using flow cytometry, ASC speck formation was observed using immunofluorescence, and the ubiquitination level of MARCH7-NLRP3 was detected using co-immunoprecipitation. ResultsJAF increased the survival rate of fetuses and the levels of estradiol and progesterone in SA model mice. It also reduced the serum Th1 and Th17-associated cytokine levels and decidual M1 macrophage-associated cytokine levels, while elevating the M2 macrophages in SA mice. NLRP3, caspase-1, ASC, and IL-1β protein expression in the decidua and placenta were also reduced. si-MARCH7 transfection reversed the effect of JAF on inhibiting the formation of the NLRP3 inflammasome and the activation of macrophages in dMφs of SA mice. ConclusionJAF could effectively prevent and treat SA by repressing M1 polarization of dMφs through NLRP3 ubiquitination and pyroptosis inhibition, which were mediated by MARCH7.

Alpha-lipoic acid (ALA) ameliorates early brain injury after subarachnoid hemorrhage in Sprague-Dawley (SD) rats via inhibiting STING-NLRP3 inflammatory signaling.

Neuroinflammation is intimately associated with poor prognosis in patients with subarachnoid hemorrhage (SAH). Alpha-lipoic acid (ALA), a disulfide antioxidant, has been shown to be neuroprotective in an in vivo model of neurological injury; however, the role of ALA in SAH has never been evaluated. In this study, the Sprague-Dawley rats SAH model was induced by endovascular perforation method. ALA was transplanted intravenously into rats, and SR-717, a stimulator of interferon genes (STING) agonist, was injected intraperitoneally. The effects of ALA on early brain injury were assayed by neurological score, hematoxylin and eosin staining and Nissl staining. Immunohistochemistry staining and Western blotting were used to analyze various proteins. ALA significantly reduced STING- NLRP3 protein expression and decreased cell death, which in turn mitigated the neurobehavioral dysfunction following SAH. Furthermore, coadministration of ALA and SR-717 promoted STING-NLRP3 signaling pathway activation following SAH, which reversed the inhibitory effect of ALA on STING-NLRP3 protein activation and increased the neurological deficits. In conclusion, ALA may be a promising therapeutic strategy for alleviating early brain injury after SAH.

Naringin's Alleviation of the Inflammatory Response Caused by Actinobacillus pleuropneumoniae by Downregulating the NF-κB/NLRP3 Signalling Pathway.

Actinobacillus pleuropneumoniae (APP) is responsible for causing Porcine pleuropneumonia (PCP) in pigs. However, using vaccines and antibiotics to prevent and control this disease has become more difficult due to increased bacterial resistance and weak cross-immunity between different APP types. Naringin (NAR), a dihydroflavonoid found in citrus fruit peels, has been recognized as having significant therapeutic effects on inflammatory diseases of the respiratory system. In this study, we investigated the effects of NAR on the inflammatory response caused by APP through both in vivo and in vitro models. The results showed that NAR reduced the number of neutrophils (NEs) in the bronchoalveolar lavage fluid (BALF), and decreased lung injury and the expression of proteins related to the NLRP3 inflammasome after exposure to APP. In addition, NAR inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in porcine alveolar macrophage (PAMs), reduced protein expression of NLRP3 and Caspase-1, and reduced the secretion of pro-inflammatory cytokines induced by APP. Furthermore, NAR prevented the assembly of the NLRP3 inflammasome complex by reducing protein interaction between NLRP3, Caspase-1, and ASC. NAR also inhibited the potassium (K+) efflux induced by APP. Overall, these findings suggest that NAR can effectively reduce the lung inflammation caused by APP by inhibiting the over-activated NF-κB/NLRP3 signalling pathway, providing a basis for further exploration of NAR as a potential natural product for preventing and treating APP.

Modified Gexia-Zhuyu Tang inhibits gastric cancer progression by restoring gut microbiota and regulating pyroptosis

BackgroundGexia-Zhuyu Tang (GZT), a traditional Chinese medicine formula, is used to treat a variety of diseases. However, its roles in gastric cancer (GC) remain unclear.ObjectiveThe aim of this study was to explore the roles and underlying molecular mechanisms of modified GZT in GC.MethodsThe effects of modified GZT on GC were investigated by constructing mouse xenograft models with MFC cell line. The fecal samples from low-dose, high-dose, and without modified GZT treatment groups were collected for the 16S rRNA gene sequencing and fecal microbiota transplantation (FMT). Histopathological alterations of mice were evaluated using the hematoxylin–eosin (HE). Immunohistochemical (IHC) analysis with Ki67 and GSDMD was performed to measure tissue cell proliferation and pyroptosis, respectively. Proteins associated with pyroptosis, invasion, and metastasis were detected by Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to assess inflammation-related factors levels.ResultsModified GZT inhibited GC tumor growth and reduced metastasis and invasion-related proteins expression levels, including CD147, VEGF, and MMP-9. Furthermore, it notably promoted caspase-1-dependent pyroptosis, as evidenced by a dose-dependent increase in TNF-α, IL-1β, IL-18, and LDH levels, along with elevated protein expression of NLRP3, ASC, and caspase-1. Additionally, modified GZT increased species abundance and diversity of the intestinal flora. FMT assay identified that modified GZT inhibited GC tumor progression through regulation of intestinal flora.ConclusionsModified GZT treatment may promote pyroptosis by modulating gut microbiota in GC. This study identifies a new potential approach for the GC clinical treatment.

Polygalasaponin F ameliorates middle cerebral artery occlusion-induced focal ischemia / reperfusion injury in rats through inhibiting TXNIP/NLRP3 signaling pathway

BackgroundPolygalasaponin F (PGSF), an oleanane triterpenoid saponin extracted from Polygala japonica, has been demonstrated with neuroprotective effect. However, the therapeutic effects and mechanisms of PGSF on focal ischemia remain unknown; MethodsIn this study, male Sprague Dawley (SD) rats aged 6–8 weeks were initially selected to establish a rat model of middle cerebral artery occlusion (MCAO) to evaluate the therapeutic effect of PGSF intervention and to investigate the impact of PGSF on the thioredoxin-interacting protein/NOD-, LRR-, and pyrin domain-containing protein 3 (TXNIP/NLRP3) inflammatory pathway. Secondly, brain neuron cells were isolated, and the cells received oxygen-glucose deprivation/reoxygenation (OGD/R) culture to establish the cell injury model in vitro. The mechanism of PGSF on the TXNIP/NLRP3 pathway was further validated; ResultsOur results showed that PGSF treatment reduced neurological scores, brain tissue water content and infarct volume and ameliorated the pathological changes in cerebral cortex in MCAO-induced focal ischemia rats. The TNF-α, IL-1β and IL-6 levels decreased in MCAO-induced focal ischemia rats after PGSF treatment. Moreover, PGSF down-regulated the protein expressions of TXNIP, NLRP3, ASC, cleaved caspase-1, IL-1β, and IL-18 in MCAO-induced focal ischemia rats. Meanwhile, PGSF treatment inhibited apoptosis, and reduced the levels of ROS, inflammatory cytokine and TXNIP/NLRP3 pathway-related proteins (TXNIP, NLRP3, ASC, cleaved caspase-1, IL-1β, and IL-18) in OGD/R-induced neuronal injury cells. Finally, PGSF treatment also disrupted the interaction between NLRP3 and TXNIP in vitro; ConclusionsOur study demonstrated the therapeutic effects of PGSF on MCAO-induced focal ischemia rats. Moreover, the neuroprotective mechanism of PGSF on focal ischemia was associated with the inhibition of TXNIP/NLRP3 signaling pathway.

Coumarin and eugenol ameliorate LPS-induced inflammation in RAW 264.7 cells via modulating the NLRP3 inflammasome pathway

Objective: To investigate the underlying mechanism of anti-inflammatory action of coumarin and eugenol in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Methods: RAW 264.7 cells were treated with 2.5 μg/mL of LPS, 50 μM of coumarin, and 50 μM eugenol for 24 h. The viability of the cells was assessed using MTT assay. The production of nitric oxide was determined using Griess reagent and DCFH-DA was used to measure the production of reactive oxygen species. The protein expression of NLRP3, IL-1β, NF-κB, and cyclooxygenase 2 was assessed using Western blot analysis. Results: Coumarin and eugenol showed anti-inflammatory effects against LPS-induced inflammatory response by ameliorating the expression of NLRP3 inflammasome and NF-κB, which further led to a subsequent reduction in IL-1β, nitric oxide, and reactive oxygen species. Conclusions: Coumarin and eugenol exert their anti-inflammatory activities by modulating the NLRP3 inflammasome pathway and NF-κB. These compounds may have promising therapeutic applications for the treatment of various inflammatory diseases.

Remimazolam attenuates inflammation in bronchopneumonia through the inhibition of NLRP3 activity by PDPK1 ubiquitination.

Bronchopneumonia is the most common pneumonia in childhood. Therefore, we tested the effects of Remimazolam presented Bronchopneumonia and its possible mechanisms. Phillygenin increased survival rate, reduced W/D ratio, and lung injury score, and inhibited IL-1β, IL-6, TNF-α, and INF-γ levels in mice model of bronchopneumonia. Remimazolam induced PDPK1 and p-AKT protein expressions, and suppressed NLRP3 protein expression in lung tissue of mice model. Invitro model, Remimazolam also induced PDPK1 and p-AKT protein expressions, and suppressed NLRP3 protein expression. Remimazolam also inhibited inflammation levels invitro model. PDPK1 inhibitor, PHT-427 (100 mg/kg) reduced survival rate, increased W/D ratio and lung injury score, and promoted inflammation levels in mice model of bronchopneumonia by treated with Remimazolam. PHT-427 suppressed PDPK1 and p-AKT protein expressions and induced NLRP3 protein expression in mice model of bronchopneumonia by treated with Remimazolam. Remimazolam interlinked PDPK1 protein. Remimazolam increased the expressions of PDPK1 and p-AKT invitro model. Remimazolam reduced PDPK1 ubiquitination invitro model.

Megalobrama amblycephala IL-22 attenuates Aeromonas hydrophila induced inflammation, apoptosis and tissue injury by regulating the ROS/NLRP3 inflammasome axis.

Mammalian interleukin-22 (IL-22) attenuates organismal injury by inhibiting reactive oxygen species (ROS) and impeding the NLRP3 inflammasome activation. However, the role of fish IL-22 in this process remains unclear. We characterized MaIL-22, an IL-22 homolog in blunt snout bream (Megalobrama amblycephala). Despite its low sequence identity, it shares conserved structures and close evolutionary relationships with other teleost IL-22s. Furthermore, Aeromonas hydrophila (A. hydrophila) infection leads to tissue injury in M. amblycephala immune organs and concomitantly altered Mail-22 mRNA expression, suggesting that MaIL-22 was involved in the antimicrobial immune response. To explore MaIL-22's biological functions, we produced recombinant MaIL-22 (rMaIL-22) protein and demonstrated it significantly enhanced the survival of M. amblycephala post-A. hydrophila infection. To unravel its protective mechanisms, we explored the ROS/NLRP3 inflammasome axis and its downstream signaling responses. The results showed that rMaIL-22 treatment significantly elevated antioxidant enzyme (T-SOD, CAT and GSH-PX) activities to inhibit MDA activity and scavenge ROS in visceral tissues. Meanwhile, rMaIL-22 impeded the activation of NLRP3 inflammasome by suppressing NLRP3 protein and mRNA expression. This indicated that rMaIL-22 contributed to inhibit A. hydrophila-induced activation of the ROS/NLRP3 inflammasome axis. Consistent with these findings, rMaIL-22 treatment attenuated the expression of proinflammatory cytokines (il-1β, tnf-α and il-6) and proapoptotic genes (caspase-3 and caspase-8) while promoting antiapoptotic genes (bcl-2b and mcl-1a) expression, ultimately mitigating tissue injury in visceral tissues. In conclusion, our research underscores MaIL-22's key role in microbial immune regulation, offering insights for developing IL-22-targeted therapies and breeding programs.

Naringin alleviates pneumonia caused by Klebsiella pneumoniae infection by suppressing NLRP3 inflammasome

Klebsiella pneumoniae (Kpn) is an important pathogen of hospital-acquired pneumonia, which can lead to sepsis and death in severe cases. In this study, we simulated pneumonia induced by Kpn infection in mice to investigate the therapeutic effect of naringin (NAR) on bacterial-induced lung inflammation. Mice infected with Kpn exhibited increases in white blood cells (WBC) and neutrophils in the peripheral blood and pathological severe injury of the lungs. This injury was manifested by increased expression of the inflammatory cytokines interleukin (IL)− 18, IL-1β, tumor necrosis factor-α (TNF-α) and IL-6, and elevated the expression of NLRP3 protein. NAR treatment could decrease the protein expression of NLRP3, alleviate lung inflammation, and reduce lung injury in mice caused by Kpn. Meanwhile, molecular docking results suggest NAR could bind to NLRP3 and Surface Plasmon Resonance (SPR) analyses also confirm this result. In vitro trials, we found that pretreated with NAR not only inhibited nuclear translocation of nuclear factor (NF)-κB protein P65 but also attenuated the protein interaction of NLRP3, caspase-1 and ASC and inhibited the assembly of NLRP3 inflammasome in mice AMs. Additionally, NAR could reduce intracellular potassium (K+) efflux, inhibiting NLRP3 inflammasome activation. These results indicated that NAR could protect against Kpn-induced pneumonia by inhibiting the overactivation of the NLRP3 inflammasome signaling pathway. The results of this study confirm the efficacy of NAR in treating bacterial pneumonia, refine the mechanism of action of NAR, and provide a theoretical basis for the research and development of NAR as an anti-inflammatory adjuvant.

Peony Pollen Protects against Primary Dysmenorrhea in Mice by Inhibiting Inflammatory Response and Regulating the COX2/PGE2 Pathway.

Peony pollen contains multiple nutrients and components and has been used as a traditional Chinese medicine with a long history, but the effect of the treatment of primary dysmenorrhea remains to be clarified. The aim of this study is to investigate the therapeutic effect of peony pollen on primary dysmenorrhea mice and the potential mechanism. A uterus contraction model in vitro and primary dysmenorrhea mice were used to evaluate the treatment effect of peony pollen on primary dysmenorrhea. The primary dysmenorrhea mice were treated with 62.5 mg/kg, 125 mg/kg, or 250 mg/kg of peony pollen, and the writhing response, latency period, histopathological changes in the uterus, prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) levels, and infiltration of neutrophils and macrophages were investigated. Protein expression of interleukin 1 β (IL-1β), interleukin 6 (IL-6), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cyclooxygenase-2 (COX-2), microsomal prostaglandin-E synthase 1 (mPGEs-1), BCL2-Associated X (Bax), B-cell lymphoma-2 (BCL-2), caspase-3, and cleaved caspase-3 were detected by Western blot, and the oxidative stress related marker malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and reactive oxygen species (ROS) were evaluated. Peony pollen could attenuate spontaneous or oxytocin-induced uterus contractions in vitro. Moreover, peony pollen decreased the writhing times, prolonged the writhing latency, and reduced the pathological damage of uterine tissues. Furthermore, the inflammatory cell infiltration and the protein expression of IL-1β, IL-6, and NLRP3 were decreased. The COX-2/PGE2 pathway was inhibited; oxidative stress and apoptosis in the uterus also improved in the uterus of primary dysmenorrhea mice. Peony pollen exerts a positive effect on primary dysmenorrhea by inhibiting the inflammatory response and modulating oxidative stress and apoptosis by regulating the COX-2/PGE2 pathway.

Morinda officinalis polysaccharides inhibit the expression and activity of NOD-like receptor thermal protein domain associated protein 3 in inflammatory periodontal ligament cells by upregulating silent information regulator sirtuin 1.

This study aims to investigate the effect of morinda officinalis polysaccharides (MOP) in inflammatory microenvironment on the expression of silent information regulator sirtuin 1 (SIRT1) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in periodontal ligament cells. Thirty rats were randomly divided into control group (n=6) and model group (n=24). The model group used orthodontic wire ligation to establish periodontitis, and six rats from each group were killed after 3 weeks. The successful modeling was confirmed by Micro-CT. The remaining rats in the model group were randomly divided into natural recovery group, normal saline (NS) group, and MOP group. In the MOP group, MOP [200 mg/(kg·3d), 50 µL for 4 weeks] was injected into the palatal side of the left maxillary first molar of the rats, while the NS group was injected with equal volume of NS. The natural recovery group did not undergo any treatment. The left maxilla tissues of the rats were collected, and pathological changes in perio-dontal ligament cells were observed by hematoxylin-eosin (HE) staining. The expression of SIRT1 and NLRP3 was detected by immunohistochemistry. Cultivate periodontal ligament fibroblasts in vitro and detect the effect of MOP on cell activity using CCK-8. The 4th generation cells were divided into control group, inflammation group (10 µg/mL lipopolysaccharide), and experimental group (5 µmol/L MOP, 5 µmol/L MOP+10 µg/mL lipopolysaccharide). The expression of SIRT1 and NLRP3 was detected by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot analyses. The acetylation of NLRP3 and the contents of interleukin (IL)-1β and IL-18 were detected by immunoprecipitation and enzyme-linked immunosorbent assay, respectively. Statistical analysis of data was conducted using Prism 9.0 software. In the vivo experiments, the expression of NLRP3 and SIRT1 in the MOP group decreased significantly compared with that in the natural recovery group and NS group, while the expression of SIRT1 increased (P<0.05) and inflammatory cell infiltration decreased. In the in vitro experiments, the expression of NLRP3 mRNA and protein in the inflammation group increased (P<0.05), while the expression of SIRT1 significantly decreased (P<0.01); MOP upregulated the expression of SIRT1 in inflammatory cells (P<0.05), reduced the expression of NLRP3 and its acetylation level significantly (P<0.05), suppressed the content of IL-1β and IL-18 in the supernatant (P<0.01). The SIRT1 expression decreased, and that of NLRP3 expression increased in inflammatory periodontal ligament cells. MOP intervention promoted SIRT1 expression, resulting in the inhibition of NLRP3. Meanwhile, the acetylation level of NLRP3 reduced through deacetylation, leading to the decreased activity of NLRP3. Thus, MOP acted as inflammatory suppressor.

Shenfu Injection improves chronic heart failure by regulating pyroptosis based on NLRP3/caspase-1 pathway

This study investigated the mechanisms and targets of Shenfu Injection in the intervention in chronic heart failure(CHF) through the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/caspase-1 signaling pathway. A CHF model was induced in rats by subcutaneous injection of isoproterenol. Model rats were randomly divided into a model group, a Shenfu Injection group, and a MCC950(NLRP3 inhibitor) group, and a blank group was also set up as a control. After 15 days of treatment, echocardiography was performed to measure cardiac function parameters [left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS)]. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), interleukin(IL)-1β, and IL-18. Hematoxylin-eosin(HE) and Masson staining were used to observe morphological changes in myocardial tissues, and Western blot was used to measure the expression levels of NLRP3/caspase-1 pathway-related proteins [NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), IL-1β, and IL-18]. The study found that isoproterenol-induced CHF in rats resulted in decreased cardiac function, worsened myocardial fibrosis, increased expression levels of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 in myocardial tissues, elevated serum inflammatory factors, and induced myocardial cell pyroptosis. Following Shenfu Injection intervention, the Shenfu Injection group showed significantly improved LVEF and LVFS, a significant decrease in NT-proBNP, a marked downregulation of NLRP3, ASC, caspase-1, GSDMD-N, IL-1β, and IL-18 protein expression levels, reduced serum inflammatory factors IL-1β and IL-18 expression in CHF rats, and a decrease in the rate of TUNEL-positive cells. Shenfu Injection can significantly improve cardiac function in CHF, inhibit myocardial fibrosis, and alleviate the progression of myocardial cell pyroptosis through the inhibition of the NLRP3/caspase-1 pathway.

A Network Pharmacology Approach to Understand the Action Mechanisms of the TangShenKangPing Decoction for Diabetic Nephropathy Treatment

ABSTRACT Background: TangShenKangPing decoction (TSKPD) has been used to treat patients with diabetic nephropathy (DN) for more than 10 years. However, its active ingredients and their pharmacological mechanisms of action remain unclear. In this study, we aimed to identify the key targets, major active ingredients, and pathways of TSKPD using network pharmacology. Methods: Human phenotypic disease, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed and 37 candidates targets of 40 active TSKPD ingredients were identified. Enrichment analyses revealed that TSKPD reduced podocyte apoptosis via vitamin metabolic processes, regulated the inflammatory response via the advanced glycation end product–receptor for AGE (AGE-RAGE) signaling, and reduced abnormal angiogenesis via vascular endothelial growth factor (VEGF) signaling in patients with DN. Furthermore, we verified the therapeutic roles and action mechanism of TSKPD in db/db mice with DN. The medicine was administered via gavage for 12 weeks. Fasting blood glucose, blood lipid, pro-inflammatory cytokine, and 24 h urinary albumin levels and pathological alterations in the renal tissues were evaluated. KHDRBS1, NLRP3 and VEGF relative mRNA and protein expression levels in renal tissues were determined using reverse transcription-quantitative olymerase chain reaction (RT-qPCR) and western blotting, respectively. Results: Treatment with TSKPD decreased proteinuria and lipid levels in the serum, significantly decreased the kidney weight, ameliorated renal histopathological alterations, and reduced pro-inflammatory cytokine expression and oxidative stress in db/db mice. Conclusion: TSKPD exerts therapeutic effects by regulating multiple factors, reducing oxidative stress and inflammation, and protecting the podocytes.

Hypoxia Inducible Factor-1α Through ROS/NLRP3 Pathway Regulates the Mechanism of Acute Ischemic Stroke Microglia Scorching Mechanism.

In vitro experiments explored how the hypoxia-induced factor-1α (HIF-1α) regulates the regulation of pyroptosis in microglial cells (BV 2) in acute ischemic stroke through ROS/NLRP3 pathway. The microglia acute phase oxygen-glucose deprivation/reoxygenation (OGD/R) was established, CCK-8 was applied to determine the optimal timing of intervention modeling. HIF-1α was overexpressed with stabilizer GF-4592 and HIF-1α small molecule interfering RNA (HIF-1α-siRNA), which was divided into group A (blank group), group B (OGD/R model group), group C (model+FG-4592 intervention group), group D (model+siRNA negative control group) and group E (model+HIF-1α-siRNA group). Cell proliferation of different groups was measured by CCK-8 assay. Pyroptosis and intracellular ROS levels were measured by flow cell technology. IL-18, IL-1β levels were measured by ELISA. HIF-1α, GSDMD-D, GSDMD-N, clean-Caspase-1 and NLRP3 protein expression levels were measured by Western blot. On the above experiments, ROS and NLRP3 response experiments were performed to explore how HIF-1α regulates pyroptosis through ROS/NLRP3 pathway. Hypoxia for 6 h then reoxygenation for 12 h was the optimal intervention time. Compared with groups B and D, cell proliferation in group C was significantly enhanced, pyroptosis, intracellular levels of ROS, IL-18, IL-1β and the expression of GSDMD-D, GSDMD-N, clean-Caspase-1, and NLRP3 proteins were significantly decreased in group C (P < 0.05). However, in group E, the performance of these test indicators were exactly the opposite, and the difference was statistically significant (P < 0.05). Through ROS and NLRP3 response experiments, it was found that HIF-1α Inhibition of Pyroptosis by inhibiting ROS/NLRP3 pathway. Overexpression of HIF-1α factor can inhibit microglia pyroptosis. HIF-1α factor has an inhibitory effect on the ROS/NLRP 3 pathway, which can inhibit the pyroptotic process in microglia.

KLF2 Regulates NLRP3 Inflammasome Priming and Activation in Murine Myeloid Cells

Introduction: Krüppel-like factor-2 (KLF2) is a critical nodal transcription factor and a tonic repressor of myeloid cell activation. Loss of KLF2 is associated with a pro-inflammatory and pro-thrombotic state. Mice deficient in myeloid-KLF2 (MK2KO) experience significantly higher mortality from lipopolysaccharide (LPS), and have increased serum and tissue levels of pro-inflammatory cytokines such as IL-1β. IL-1β is a product of NLRP3 inflammasome priming and activation, which has been implicated in numerous inflammatory and immune-mediated diseases. Neutrophils are the first responders of the innate immune system and are responsible for bacterial antigen detection. We thus aimed to elucidate the role of KLF2 in the regulation of NLRP3 inflammasome activation and subsequent IL-1β release in the neutrophil compartment. Methods: 8-week old adult MK2KO ( Lyz2Cre/ Klf2fl/fl) and control ( Lyz2Cre) mice were used for obtaining neutrophils from the bone marrow. Neutrophils were plated in RPMI-1640 media and stimulated with 10 ng/mL of LPS for 1 hour (h) and 2 h, followed by nigericin for 30 minutes. LPS acts as a priming agent for NLRP3 inflammasome assembly, and nigericin is a potassium ionophore which enables the activation step and subsequent IL-1β release. Supernatant was used for IL-1β ELISA and cell lysate was used for protein extraction and Western blotting to quantify NLRP3 protein expression. GraphPad Prism was used for statistical analyses. Results: Neutrophils from both MK2KO and Cre mice have significantly increased production of IL-1β when stimulated with LPS followed by nigericin (Figure 1), and loss of KLF2 leads to significantly increased IL-1B release from neutrophils. MK2KO neutrophils also have significantly more NLRP3 protein expression as compared to Cre neutrophils at baseline, and in response to LPS and nigericin (Figure 2). This increase in NLRP3 protein expression was time dependent and dose-dependent (Figure 2). Conclusion: KLF2 negatively regulates the transcriptional expression of NLRP3 and loss of KLF2 leads to increased NLRP3 inflammasome priming, at baseline and in response to LPS. Loss of KLF2 also affects the second step of the NLRP3 inflammasome pathway - the activation step, leading to increased IL-1β release in neutrophils in adult mice. Ongoing studies are examining the mechanisms underlying KLF2 control of NLRP3 transcription and other aspects of inflammasome activation. Inflammasome activation has been implicated in sepsis-related mortality due to the pro-inflammatory cytokine storm, and thus modulating KLF2 might help in developing novel targets to reduce this burden of mortality.

L-ergothioneine reduces mitochondrial-driven NLRP3 activation in gestational diabetes mellitus

BackgroundMaternal hyperglycaemia has a significant impact on placental metabolism and mitochondrial function. The NLRP3 inflammasome is responsive to endogenous signals of mitochondrial dysfunction. We tested our hypothesis that mitochondrial dysfunction orchestrates activation of the NLRP3 inflammasome and contributes to inflammation in gestational diabetes mellitus (GDM). MethodsFasting blood, omental and placental tissue were collected on the day of caesarean section from nulliparous women with normal glucose tolerant (NGT) (n = 30) and GDM (n = 27) pregnancies. Cell-free mitochondrial DNA (cf-mtDNA) copy number was quantified by real-time PCR. M1-like (CD14+CD86+CD206-) and M2-like (CD14+CD86+CD206+) macrophage populations were characterized by flow cytometry. Immunoblotting for protein expression of NLRP3, ASC and caspase-1 was performed in maternal BMI and age-matched tissue samples. IL-1β and IL-18 were measured by multiplex ELISA. Placental explants from GDM participants were cultured for 24 h with 1 mM L-ergothioneine (antioxidant) and 1 µM MCC950 (NLRP3 inhibitor). ResultsCf-mtDNA copy numbers were significantly higher in GDM compared to NGT participants (p = 0.002). Placental populations of CD14+ (p = 0.02) and CD14+CD86+CD206- (p = 0.03) macrophages produced significantly increased levels of mitochondrial superoxide in GDM compared to NGT participants. Placental production of IL-18 (p = 0.04) was significantly increased in GDM. This increase in placental IL-18 was attenuated by treatment with 1 µM MCC950 (p = 0.0005), and 1 mM L-ergothioneine (p = 0.007). ConclusionPlacental inflammation is significantly increased in women with GDM. Furthermore, this increase may be initiated by elevated production of mitochondrial superoxide by macrophage subpopulations and orchestrated by the NLRP3 inflammasome. The mitochondrial antioxidant, L-ergothioneine, ameliorates NLRP3-induced placental inflammation in GDM, identifying a potential therapeutic role.

L-Carnitine augments probenecid anti-inflammatory effect in monoiodoacetate-induced knee osteoarthritis in rats: involvement of miRNA-373/P2X7/NLRP3/NF-κB milieu

Osteoarthritis (OA) is a degenerative joint disease, whereas the underlying molecular trails involved in its pathogenesis are not fully elucidated. Hence, the current study aimed to investigate the role of miRNA-373/P2X7/NLRP3/NF-κB trajectory in its pathogenesis as well as the possible anti-inflammatory effects of probenecid and l-carnitine in ameliorating osteoarthritis via modulating this pathway. In the current study, male Sprague Dawley rats were used and monoiodoacetate (MIA)-induced knee osteoarthritis model was adopted. Probenecid and/or L-carnitine treatments for 14 days succeeded in reducing OA knee size and reestablishing motor coordination and joint mobility assessed by rotarod testing. Moreover, different treatments suppressed the elevated serum levels of IL-1β, IL-18, IL-6, and TNF-α via tackling the miRNA-373/P2X7/NLRP3/NF-κB, witnessed as reductions in protein expressions of P2X7, NLRP3, cleaved caspase-1 and NF-κB. These were accompanied by increases in procaspase-1 and IκB protein expression and in miRNA-373 gene expression OA knee to various extents. In addition, different regimens reversed the abnormalities observed in the H and E as well as Safranin O-Fast green OA knees stained sections. Probenecid or l-carnitine solely showed comparable results on the aforementioned parameters, whereas the combination therapy had the most prominent effect on ameliorating the aforementioned parameters. In conclusion, l-carnitine augmented the probenecid’s anti-inflammatory effect to attenuate MIA-induced osteoarthritis in rats by provoking the miRNA-373 level and inhibiting the P2X7/NLRP3/NF-κB milieu, leading to the suppression of serum inflammatory cytokines: IL-1β, IL-18, IL-6, and TNF-α. These findings suggest the possibility of using probenecid and l-carnitine as a useful therapeutic option for treatment of osteoarthritis.Graphical abstract

Methotrexate associated with nanoparticles decreases NLRC4 inflammasome expression in infarcted rat hearts

Abstract Abstract: Following myocardial infarction (MI), molecules released from necrotic myocytes sensitize nucleotide-binding receptors (NLRs), activate inflammasome complex, and initiate acute inflammatory process and interleukin (IL)-1β production, essential for tissue damage resolution. However, excessive IL-1β production impairs cardiac remodeling. After MI, IL-1β is released by the NLRP3 inflammasome; involvement of the NLRC4 inflammasome in post-MI remodeling has not been established. Methotrexate (MTX) carried by lipid core nanoparticles in low density emulsion (MTX-LDE) reduces post-MI inflammatory process and improves cardiac function in rodents. Purpose To evaluate the influence of early treatment with MTX-LDE on myocardial expression of NLRP3 and NLRC4 inflammasomes in infarcted rats. Methods Male Wistar rats were divided into three groups: control (Sham, n=6); MI (n=6); and MI treated with MTX-LDE (MI-MTX, n=6). MI was induced by ligation of the left anterior descending coronary artery. Twenty-four hours later, the MI-MTX group was treated with 1 mg/kg of MTX-LDE, intraperitoneally. Euthanasia was performed 72 hours after surgery under anaesthesia with ketamine and xylazine. Infarct size was assessed by histology. Myocardial expression of NLRC4, NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1β and IL-1β was quantified by Western blotting. IL-1β concentration in serum and heart macerate supernatant was analyzed by ELISA. Statistical: ANOVA or Kruskal-Wallis; 5% significance level. Results Rats with MI lower than 30% of the left ventricle total area were excluded. Infarct size was larger in the MI-MTX group (43,8 ± 5,4) than MI (36,2 ± 6,22). NLRC4 and caspase-1 expression and IL-1β serum concentration were higher in MI than Sham and MI-MTX. Myocardial IL-1β concentration was higher in MI and MI-MTX than Sham and lower in MI-MTX than MI. NLRP3, ASC, pro-caspase-1, and IL-1β protein expression did not differ between groups. Conclusion Our study shows for the first time that NLRC4 inflammasome protein expression increases acutely after myocardial infarction in rats. Early treatment with nanoparticles-derived methotrexate reduces myocardial NLRC4 inflammasome expression and myocardial and serum IL-1β concentration.