RESUMO
Periodontitis is a chronic inflammatory disease involving plaque biofilm as a pathogenic factor. Potassium ion plays an important role in cellular homeostasis; a large outflow of potassium may lead to local inflammation progression. In this work, the multifunctional short peptide molecule BmKTX-33 was designed by modifying the BmKTX, a Kv1.3 potassium channel inhibitor. This was to explore its antibacterial properties, capability of maintaining cell ion homeostasis, and bone-forming capacity. The results showed that BmKTX-33 had inhibitory effects on S. gordonii, F. nucleatum, and P. gingivalis. Moreover, BmKTX-33 also inhibited excessive potassium outflow in inflammatory environments. Finally, BmKTX-33 promoted MC3T3-E1 early osteogenesis while suppressing the NLRP3 inflammasome's production. In conclusion, BmKTX-33 not only has antibacterial properties, but also can inhibit the expression of NLRP3 inflammasome and play an anti-inflammatory role.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR , Periodontite , Animais , Periodontite/tratamento farmacológico , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/uso terapêutico , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/uso terapêutico , Porphyromonas gingivalis/efeitos dos fármacos , Potássio/metabolismo , Linhagem Celular , Osteogênese/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Biofilmes/efeitos dos fármacosRESUMO
Acute pancreatitis (AP) is a sudden-onset disease of the digestive system caused by abnormal activation of pancreatic enzymes. Dual oxidase 2 (DUOX2) has been found to be elevated in the progression of a variety of inflammatory diseases. Therefore, we analyzed the specific roles of DUOX2 in AP development. Blood samples were collected from of AP patients and healthy people, and the caerulein- stimulated human pancreatic duct cells (H6C7) were utilized to establish an AP cell model. Cell growth and apoptosis were measured using an MTT assay and TUNEL staining. Additionally, RT-qPCR and western blot assays were conducted to assess the RNA and protein expressions of the cells. ELISA kits were used to determine TNF-α, IL-6, IL-8, and IL-1ß levels. The interaction between DUOX2 and miR-605-3p was predicted using the Targetscan database and confirmed by dual-luciferase report assay. We found that DUOX2 increased while miR-605-3p decreased in the blood of AP patients and caerulein-stimulated H6C7 cells. DUOX2 was targeted by miR-605-3p. Furthermore, DUOX2 knockdown or miR-605-3p overexpression promoted cell viability, decreased the TNF-α, IL-6, IL-8, and IL-1ß levels, and inhibited apoptosis rate in caerulein-stimulated H6C7 cells. DUOX2 knockdown or miR-605-3p overexpression also increased the Bcl-2 protein levels and down-regulated Bax, cleaved-caspase-1, NLRP3 and p-p65. Interestingly, DUOX2 overexpression reversed the miR-605-3p mimic function in the caerulein-treated H6C7 cells. In conclusion, our research demonstrated that DUOX2 knockdown relieved the injury and inflammation in caerulein-stimulated H6C7 cells.
Assuntos
Ceruletídeo , Oxidases Duais , MicroRNAs , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pancreatite , Piroptose , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Oxidases Duais/metabolismo , Oxidases Duais/genética , Pancreatite/patologia , Pancreatite/metabolismo , Pancreatite/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , NF-kappa B/metabolismo , Transdução de Sinais , Masculino , Linhagem Celular , Ductos Pancreáticos/patologia , Ductos Pancreáticos/metabolismo , Apoptose , Feminino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: The drug resistance of multiple myeloma (MM) cells is one of the main causes of relapse, refractory and progression of MM. METHODS: First, Western blot analysis was used to detect the expression levels of NLRP3, ASC, pro-IL-1ß and cleaved IL-1ß, and RT-qPCR was used to detect the mRNA expression levels of them. The expression levels of IL-1ß and IL-18 in the supernatant were detected by ELISA, and the expression levels of these factors in the activated group and the control group were compared to verify the activation of BMMCs and KM3. RESULT: 1. The protein expression of NLRP3 and cleavd-IL-1ß in the BMMCs cells was significantly higher than that of the control group (P < 0.05). The mRNA expression levels of caspase-1 and IL-1ß were higher than those of the control group (P = 0.03, P = 0.02). 2. The protein expression levels of NLRP3 and cleaved-IL-1ß in the KM3 cells were significantly higher than those of the control group (P < 0.05). The expressions of caspase-1 mRNA(P = 0.016) and IL-1ß mRNA(P = 0.037) were significantly increased compared with the control group. 3. The early apoptosis results of BMMCs showed that the apoptosis rate of the LPS+ATP+Dex group was lower than that of the Dex group (P = 0.017). The early apoptosis rate of the LPS+ATP+Dex+Vel group was decreased compared with the Dex+Vel group (P = 0.045). 4. The early apoptosis rate of KM3 in the LPS+ATP+Dex group was lower than that in the Dex group (P = 0.03). CONCLUSION: 1. LPS+ATP can activate NLRP3 inflammasome in multiple myeloma cells. 2. Activation of NLRP3 inflammasome inhibits the early apoptosis of myeloma cells induced by dexamethasone and bortezomib.
Assuntos
Inflamassomos , Mieloma Múltiplo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Linhagem Celular Tumoral , Masculino , Feminino , Pessoa de Meia-IdadeRESUMO
Minocycline (Min), as an antibiotic, possesses various beneficial properties such as anti-inflammatory, antioxidant, and anti-apoptotic effects. Despite these known qualities, the precise cardioprotective effect and mechanism of Min in protecting against sepsis-induced cardiotoxicity (SIC) remain unspecified. To address this, our study sought to assess the protective effects of Min on the heart. Lipopolysaccharide (LPS) was utilized to establish a cardiotoxicity model both in vivo and in vitro. Min was pretreated in the models. In the in vivo setting, evaluation of heart tissue histopathological injury was performed using hematoxylin and eosin (H&E) staining and TUNEL. Immunohistochemistry (IHC) was employed to evaluate the expression levels of NLRP3 and Caspase-1 in the heart tissue of mice. During in vitro experiments, the viability of H9c2 cells was gauged utilizing the CCK8 assay kit. Intracellular ROS levels in H9c2 cells were quantified using a ROS assay kit. Both in vitro and in vivo settings were subjected to measurement of oxidative stress indexes, encompassing glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. Additionglly, myocardial injury markers like lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) activity were quantified using appropriate assay kits. Western blotting (WB) analysis was conducted to detect the expression levels of NOD-like receptor protein-3 (NLRP3), caspase-1, IL-18, and IL-1ß, alongside apoptosis-related proteins such as Bcl-2 and Bax, and antioxidant proteins including superoxide dismutase-1 (SOD-1) and antioxidant proteins including superoxide dismutase-1 (SOD-2), both in H9c2 cells and mouse heart tissues. In vivo, Min was effective in reducing LPS-induced inflammation in cardiac tissue, preventing cell damage and apoptosis in cardiomyocytes. The levels of LDH and CK-MB were significantly reduced with Min treatment. In vitro studies showed that Min improved the viability of H9C2 cells, reduced apoptosis, and decreased ROS levels in these cells. Further analysis indicated that Min decreased the protein levels of NLRP3, Caspase-1, IL-18, and IL-1ß, while increasing the levels of SOD-1 and SOD-2 both in vivo and in vitro. Min alleviates LPS-induced SIC by suppressing the NLRP3/Caspase-1 signalling pathway in vivo and in vitro.
Assuntos
Cardiotoxicidade , Caspase 1 , Lipopolissacarídeos , Minociclina , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Caspase 1/metabolismo , Cardiotoxicidade/metabolismo , Cardiotoxicidade/tratamento farmacológico , Camundongos , Minociclina/farmacologia , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , RatosRESUMO
Purpose: Ischemic stroke is a refractory disease wherein the reperfusion injury caused by sudden restoration of blood supply is the main cause of increased mortality and disability. However, current therapeutic strategies for the inflammatory response induced by cerebral ischemia-reperfusion (I/R) injury are unsatisfactory. This study aimed to develop a functional nanoparticle (MM/ANPs) comprising apelin-13 (APNs) encapsulated in macrophage membranes (MM) modified with distearoyl phosphatidylethanolamine-polyethylene glycol-RVG29 (DSPE-PEG-RVG29) to achieve targeted therapy against ischemic stroke. Methods: MM were extracted from RAW264.7. PLGA was dissolved in dichloromethane, while Apelin-13 was dissolved in water, and CY5.5 was dissolved in dichloromethane. The precipitate was washed twice with ultrapure water and then resuspended in 10 mL to obtain an aqueous solution of PLGA nanoparticles. Subsequently, the cell membrane was evenly dispersed homogeneously and mixed with PLGA-COOH at a mass ratio of 1:1 for the hybrid ultrasound. DSPE-PEG-RVG29 was added and incubated for 1 h to obtain MM/ANPs. Results: In this study, we developed a functional nanoparticle delivery system (MM/ANPs) that utilizes macrophage membranes coated with DSPE-PEG-RVG29 peptide to efficiently deliver Apelin-13 to inflammatory areas using ischemic stroke therapy. MM/ANPs effectively cross the blood-brain barrier and selectively accumulate in ischemic and inflamed areas. In a mouse I/R injury model, these nanoparticles significantly improved neurological scores and reduced infarct volume. Apelin-13 is gradually released from the MM/ANPs, inhibiting NLRP3 inflammasome assembly by enhancing sirtuin 3 (SIRT3) activity, which suppresses the inflammatory response and pyroptosis. The positive regulation of SIRT3 further inhibits the NLRP3-mediated inflammation, showing the clinical potential of these nanoparticles for ischemic stroke treatment. The biocompatibility and safety of MM/ANPs were confirmed through in vitro cytotoxicity tests, blood-brain barrier permeability tests, biosafety evaluations, and blood compatibility studies. Conclusion: MM/ANPs offer a highly promising approach to achieve ischemic stroke-targeted therapy inhibiting NLRP3 inflammasome-mediated pyroptosis.
Assuntos
Inflamassomos , AVC Isquêmico , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanopartículas , Piroptose , Animais , Camundongos , AVC Isquêmico/tratamento farmacológico , Células RAW 264.7 , Piroptose/efeitos dos fármacos , Nanopartículas/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Masculino , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/química , Polietilenoglicóis/química , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/tratamento farmacológico , Fosfatidiletanolaminas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismoRESUMO
NLRP3 is an inflammasome seeding pattern recognition receptor activated in response to multiple danger signals which perturb intracellular homeostasis. Electrostatic interactions between the NLRP3 polybasic (PB) region and negatively charged lipids on the trans-Golgi network (TGN) have been proposed to recruit NLRP3 to the TGN. In this study, we demonstrate that membrane association of NLRP3 is critically dependant on S-acylation of a highly conserved cysteine residue (Cys-130), which traps NLRP3 in a dynamic S-acylation cycle at the Golgi, and a series of hydrophobic residues preceding Cys-130 which act in conjunction with the PB region to facilitate Cys-130 dependent Golgi enrichment. Due to segregation from Golgi localised thioesterase enzymes caused by a nigericin induced breakdown in Golgi organisation and function, NLRP3 becomes immobilised on the Golgi through reduced de-acylation of its Cys-130 lipid anchor, suggesting that disruptions in Golgi homeostasis are conveyed to NLRP3 through its acylation state. Thus, our work defines a nigericin sensitive S-acylation cycle that gates access of NLRP3 to the Golgi.
Assuntos
Complexo de Golgi , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nigericina , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Complexo de Golgi/metabolismo , Humanos , Acilação , Nigericina/farmacologia , Animais , Inflamassomos/metabolismo , Células HEK293RESUMO
The activation of the NLRP3 inflammasome has been linked to several inflammatory and autoinflammatory diseases. Despite cases of potential hearing improvement in immune-mediated diseases, direct evidence of the efficacy of targeting this mechanism in the inner ear is still lacking. Previously, we discovered that macrophages are associated with Sensorineural Hearing loss (SNHL) in Chronic Suppurative Otitis Media (CSOM), the leading cause of this permanent hearing loss in the developing world and incurring costs of $4 to $11 billion dollars in the United States. However, the underlying mechanism remained unknown. Here, we investigate how macrophages drive permanent hearing loss in CSOM. We first confirmed the occurrence of NLRP3 inflammasome activation in cochlear macrophages in CSOM. We then revealed that Outer Hair Cells (OHCs) were protected in CSOM by macrophage depletion and subsequently confirmed the same protection in the NLRP3 knockout condition. Furthermore, we showed that therapeutic inhibition of NLRP3 inflammasome activation and downstream inhibition of IL-1ß protects OHCs in CSOM. Collectively, our data demonstrates that the main driver for hearing loss in CSOM is NLRP3 inflammasome activation in cochlear macrophages and this is therapeutically targetable, leading the way for the development of interventions to prevent the leading cause of permanent hearing loss and a costly disease in the developed world.
Assuntos
Cóclea , Inflamassomos , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Otite Média Supurativa , Animais , Feminino , Humanos , Masculino , Camundongos , Doença Crônica , Cóclea/metabolismo , Cóclea/patologia , Modelos Animais de Doenças , Perda Auditiva/etiologia , Perda Auditiva/prevenção & controle , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidoresRESUMO
Excessive proinflammatory cytokine release induced by pyroptosis plays a vital role in intestinal mucosal inflammation in ulcerative colitis (UC). Several pyroptosis-related factors are regulated by the centrosome. Pericentriolar material 1 (PCM1) is a primary component of centriolar satellites that is present as cytoplasmic granules around the centrosome. Our previous study revealed that PCM1 was highly expressed in UC patients, but the role of PCM1 in UC remains unknown. This study aimed to elucidate the role of PCM1 in the development of UC, especially the mechanism in pyroptosis process of UC. Clinical mucosal sample and dextran sulfate sodium (DSS)-induced colitis mouse were used to reveal the association between PCM1 and intestinal inflammation. Intestinal epithelial cell-specific PCM1-knockout mice were constructed to determine the role of PCM1 in colitis. Finally, PCM1 RNA interference and overexpression assays in THP1 cells were employed to study the molecular mechanisms of PCM1 in inflammatory responses and pyroptosis. We found that PCM1 expression was upregulated in the colonic mucosa of UC patients and positively correlated with inflammatory indicators. PCM1 expression was elevated in DSS-induced colitis mice and was reduced after methylprednisolone treatment. In the DSS colitis model, intestinal-specific PCM1-knockout mice exhibited milder intestinal inflammation and lower pyroptosis levels than wild-type mice. In cell level, PCM1 exerted a proinflammatory effect by activating the NLRP3 inflammasome and triggering subsequent gasdermin D-mediated pyroptosis to release IL-1ß and IL-18. In conclusion, PCM1 mediates activation of the NLRP3 inflammasome and gasdermin D-dependent pyroptosis, ultimately accelerating intestinal inflammation in UC. These findings revealed a previously unknown role of PCM1 in initiating intestinal mucosal inflammation and pyroptosis in UC, and this factor is expected to be a regulator in the complex inflammatory network of UC.
Assuntos
Colite Ulcerativa , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas de Ligação a Fosfato , Piroptose , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/fisiologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Camundongos , Humanos , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Feminino , Sulfato de Dextrana/toxicidade , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , GasderminasRESUMO
BACKGROUND: Radiation-induced skin injury is a significant adverse reaction to radiotherapy. However, there is a lack of effective prevention and treatment methods for this complication. Ferulic acid (FA) has been identified as an effective anti-radiation agent. Conventional administrations of FA limit the reaching of it on skin. We aimed to develop a novel FA hydrogel to facilitate the use of FA in radiation-induced skin injury. METHODS: We cross-linked carbomer 940, a commonly used adjuvant, with FA at concentrations of 5%, 10%, and 15%. Sweep source optical coherence tomography system, a novel skin structure evaluation method, was applied to investigate the influence of FA on radiation-induced skin injury. Calcein-AM/PI staining, CCK8 assay, hemolysis test and scratch test were performed to investigate the biocompatibility of FA hydrogel. The reducibility of DPPH and ABTS radicals by FA hydrogel was also performed. HE staining, Masson staining, laser Doppler blood flow monitor, and OCT imaging system are used to evaluate the degree of skin tissue damage. Potential differentially expressed genes were screened via transcriptome analysis. RESULTS: Good biocompatibility and in vitro antioxidant ability of the FA hydrogels were observed. 10% FA hydrogel presented a better mechanical stability than 5% and 15% FA hydrogel. All three concentrations of FA remarkably promoted the recovery of radiation-induced skin injury by reducing inflammation, oxidative conidiation, skin blood flow, and accelerating skin tissue reconstruction, collagen deposition. FA hydrogel greatly inhibiting the levels of NLRP3, caspase-1, IL-18, pro-IL-1ß and IL-1ß in vivo and vitro levels through restraining the activation of NLRP3 inflammasome. Transcriptome analysis indicated that FA might regulate wound healing via targeting immune response, inflammatory response, cell migration, angiogenesis, hypoxia response, and cell matrix adhesion. CONCLUSIONS: These findings suggest that the novel FA hydrogel is a promising therapeutic method for the prevention and treatment of radiation-induced skin injury patients.
Assuntos
Ácidos Cumáricos , Hidrogéis , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pele , Cicatrização , Ácidos Cumáricos/farmacologia , Ácidos Cumáricos/química , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Cicatrização/efeitos dos fármacos , Inflamassomos/metabolismo , Camundongos , Hidrogéis/química , Hidrogéis/farmacologia , Pele/efeitos dos fármacos , Masculino , Humanos , Antioxidantes/farmacologia , Antioxidantes/química , Lesões por Radiação/tratamento farmacológico , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Radiodermatitis (RD) is an inflammatory lesion of skin mucosa caused by radiation, which causes itching and pain in patients' skin. Hypericum sampsonii has an anti-inflammatory effect. This study aims to explore the potential effect and mechanism of H. sampsonii on RD. MATERIALS AND METHODS: The RD model was established using X-ray irradiation of mice and the pain response of mice under different treatment methods. Serum levels of IL-1ß, IL-6, and TNF-α were measured by ELSA. The RD cell model was constructed by RAW264.7 cell, H. sampsonii intervention was conducted, and the changes of the NLRP3 inflammasome in the cells were detected by qRT-PCR. The cells were stimulated with LPS and the protein changes of TLR4/NF-κB were investigated by Western Blotting. RESULTS: H. sampsonii can better improve the skin status of RD mice, relieve pain, and reduce the secretion of serum inflammatory factors IL-1ß, IL-6, and TNF-α. H. sampsonii significantly down-regulated the expression of NLRP3, Caspase-1, pro IL-1ß, and IL-1ß. Lps-induced activation of the TLR4/NF-κB pathway promotes the expression of NLRP3 and pro-IL-1ß, and H. sampsonii can inhibit this promotion. CONCLUSION: H. sampsonii may inhibit NLRP3 inflammatory vesicle activation via interfering with TLR4/NF-κB signaling to reduce the inflammatory response in macrophages and thus play a role in the treatment of RD.
Assuntos
Hypericum , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Radiodermite , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Camundongos , Inflamassomos/metabolismo , Hypericum/química , Radiodermite/patologia , Células RAW 264.7 , Modelos Animais de Doenças , Extratos Vegetais/farmacologia , Masculino , Anti-Inflamatórios/farmacologiaRESUMO
No single treatment significantly reduces the mortality rate and improves neurological outcomes after intracerebral haemorrhage (ICH). New evidence suggests that pyroptosis-specific proteins are highly expressed in the perihaematomal tissues of patients with ICH and that the disulfiram (DSF) inhibits pyroptosis. An ICH model was established in C57BL/6 mice by intracranial injection of collagenase, after which DSF was used to treat the mice. Cell model of ICH was constructed, and DSF was used to treat the cells. HE, TUNEL, Nissl, FJC and IF staining were performed to evaluate the morphology of brain tissues; Western blotting and ELISA were performed to measure the protein expression of NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin D (GSDMD) classical pyroptosis pathway and Toll-likereceptor4 (TLR4)/nuclear factor-kappaB (NF-κB) inflammatory signaling pathway and bloodâbrain barrier-associated factoes, and the wet/dry weight method was used to determine the brain water content. The expression of proteins related to the NLRP3/Caspase-1/GSDMD pathway and the TLR4/NF-κB pathway was upregulated in tissues surrounding the haematoma compared with that in control tissues; Moreover, the expression of the blood-brain barrier structural proteins occludin and zonula occludens-1 (ZO-1) was downregulated, and the expression of Aquaporin Protein-4 (AQP4) and matrix metalloprotein 9 (MMP-9) was upregulated. DSF significantly inhibited these changes, reduced the haematoma volume, decreased the brain water content, reduced neuronal death and degeneration and improved neurological function after ICH. ICH activated the classical pyroptosis pathway and TLR4/NF-κB inflammatory pathway, disruped the expression of blood-brain barrier structural proteins, and exacerbated brain injury and neurological dysfunction. DSF inhibited these changes and exerted the therapeutic effects on pathological changes and dysfunction caused by ICH.
Assuntos
Barreira Hematoencefálica , Dissulfiram , Camundongos Endogâmicos C57BL , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Piroptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Dissulfiram/farmacologia , Transdução de Sinais/efeitos dos fármacos , Masculino , Receptor 4 Toll-Like/metabolismo , NF-kappa B/metabolismo , Modelos Animais de Doenças , Caspase 1/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/metabolismo , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Humanos , GasderminasRESUMO
This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. The cell counting kit-8(CCK-8) assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells. Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-α in macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose. Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α, interleukin(IL)-18, and IL-1ß secreted by macrophages. The expression level of nuclear factor-kappa B(NF-κB) p65 in macrophages exposed to high glucose was detected by immunofluorescence, and the level of intracellular reactive oxygen species(ROS) was detected by the DCFH-DA fluorescent probe. The mRNA levels of NLRP3, TNF-α, and IL-18 in macrophages were determined by qRT-PCR. The results showed that treatment with 30 mmol·L~(-1) glucose for 48 h was the best condition for the modeling of macrophage injury. Compared with the blank group, the model group showed improved polarization of macrophages, increased secretion of TNF-α, IL-18, and IL-1ß, elevated ROS level, and up-regulated expression of NF-κB p65. In addition, the modeling up-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 and the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1ß, cleaved IL-1ß, and pro-IL-18. Compared with the model group, daidzein(10, 20, and 40 µmol·L~(-1)) lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 as well as the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1ß, cleaved IL-1ß, and pro-IL-18. In addition, daidzein reduced intracellular ROS. According to the available reports and the experimental results, high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines. Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway, thereby reducing the inflammation of macrophages exposed to high glucose.
Assuntos
Glucose , Inflamassomos , Isoflavonas , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Animais , Camundongos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Glucose/efeitos adversos , Isoflavonas/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Células RAW 264.7 , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-18/imunologiaRESUMO
Bovine clinical mastitis is characterized by inflammation and immune responses, with apoptosis of mammary epithelial cells as a cellular reaction to infection. PIEZO1, identified as a mechanotransduction effector channel in nonruminant animals and sensitive to both mechanical stimuli or inflammatory signals like lipopolysaccharide (LPS). However, its role in inflammatory processes in cattle has not been well-documented. The aim of this study was to elucidate the in situ expression of PIEZO1 in bovine mammary gland and its potential involvement in clinical mastitis. We observed widespread distribution and upregulation of PIEZO1 in mammary epithelial cells in clinical mastitis cows and LPS-induced mouse models, indicating a conserved role across species. In vitro studies using mammary epithelial cells (MAC-T) revealed that LPS upregulates PIEZO1. Notably, the effects of PIEZO1 artificial activator Yoda1 increased apoptosis and NLRP3 expression, effects mitigated by PIEZO1 silencing or NLRP3 inhibition. In conclusion, the activation of the PIEZO1-NLRP3 pathway induces abnormal apoptosis in mammary epithelial cells, potentially serving as a regulatory mechanism to combat inflammatory responses to abnormal stimuli.
Assuntos
Apoptose , Células Epiteliais , Canais Iônicos , Lipopolissacarídeos , Mastite Bovina , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais , Animais , Feminino , Apoptose/efeitos dos fármacos , Camundongos , Lipopolissacarídeos/farmacologia , Bovinos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Mastite Bovina/genética , Mastite Bovina/metabolismo , Mastite Bovina/imunologia , Transdução de Sinais/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/genética , Inflamação/imunologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/citologia , Mastite/imunologia , Mastite/genética , Mastite/metabolismoRESUMO
Acute kidney injury (AKI) is characterized by a sudden decline in renal function. The inflammatory response is the fundamental pathologic alteration throughout AKI, regardless of the various causal factors. Macrophages are the main immune cells involved in the inflammatory microenvironment in AKI. Consequently, targeting macrophages might become a novel strategy for the treatment of AKI. In this study, we demonstrated that pseudoginsenoside-F11 (PF11), a distinctive component of Panax quinquefolius L., regulated macrophage function and protected renal tubular epithelial cells TCMK-1 from lipopolysaccharide (LPS) in vitro. PF11 also alleviated renal injuries in an LPS-induced AKI mouse model, decreased the levels of inflammatory cytokines, reduced macrophage inflammatory infiltration, and promoted the polarization of M1 macrophages to M2c macrophages with suppression of the nuclear factor-κB/NOD-like receptor thermal protein domain-associated protein 3/interleukin-1ß (NF-κB/NLRP3/IL-1ß) signaling pathway. To further investigate whether this nephroprotective effect of PF11 is mediated by macrophages, we performed macrophage depletion by injection of clodronate liposomes in mice. Macrophage depletion abolished PF11's ability to protect against LPS-induced kidney damage with downregulating the NF-κB/NLRP3/IL-1ß signaling pathway. In summary, this is the first study providing data on the efficacy and mechanism of PF11 in the treatment of AKI by regulating macrophage function.
Assuntos
Injúria Renal Aguda , Ginsenosídeos , Lipopolissacarídeos , Macrófagos , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/metabolismo , Ginsenosídeos/farmacologia , Ginsenosídeos/administração & dosagem , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Lipopolissacarídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Panax/química , Transdução de Sinais/efeitos dos fármacosRESUMO
The higher prevalence of ulcerative colitis (UC) and the side effects of its therapeutic agents contribute to finding novel treatments. This study aimed to investigate whether kynurenine (KYN), a tryptophan metabolite, has the possibility of alleviating UC and further clarifying the underlying mechanism. The effect of KYN on treating UC was evaluated by intestinal pathology, inflammatory cytokines, and tight-junction proteins in colitis mice and LPS-stimulated Caco-2 cells. Our results revealed that KYN relieved pathological symptoms of UC, improved intestinal barrier function, enhanced AhR expression, and inhibited NF-κB signaling pathway activation in the colon of colitis mice. Moreover, the improved intestinal barrier function, the decreased inflammasome production, and the inhibited activation of the NF-κB signaling pathway by KYN were dependent on AhR in Caco-2 cells. KYN could trigger AhR activation, inactivate the NF-κB signaling pathway, and inhibit NLRP3 inflammasome production, thus alleviating intestinal epithelial barrier dysfunction and reducing intestinal inflammation. In conclusion, the present study reveals that KYN ameliorates UC by improving the intestinal epithelial barrier and activating the AhR-NF-κB-NLRP3 signaling pathway, and it can be a promising therapeutic agent and dietary supplement for alleviating UC.
Assuntos
Colite Ulcerativa , Cinurenina , Camundongos Endogâmicos C57BL , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores de Hidrocarboneto Arílico , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Animais , Cinurenina/metabolismo , Humanos , Camundongos , Células CACO-2 , NF-kappa B/genética , NF-kappa B/metabolismo , NF-kappa B/imunologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Transdução de Sinais/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamassomos/genética , Inflamassomos/efeitos dos fármacosRESUMO
Obesity-related metabolic diseases are associated with a chronic inflammatory state. Calenduloside E (CE) is a triterpene saponin from sugar beet. In mouse models, CE reduced pro-inflammatory cytokines in white adipose tissue (WAT) and decreased macrophage infiltration of WAT. And CE inhibited pyroptosis in J774A.1 cells and WAT by inhibiting the activation of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome. Moreover, CE could trigger the activation of Sirtuin 2 (SIRT2), leading to a decrease in the acetylation of NLRP3, particularly at the K24 site. In addition, it has been shown that CE can reduce inflammation in adipocytes that have been induced by macrophage-conditioned medium. However, the selective SIRT2 inhibitor AGK2 hindered the beneficial effects of CE. In summary, CE has the capacity to impede NLRP3-mediated pyroptosis by triggering SIRT2 activity, thus positioning CE as a promising therapeutic avenue for combating obesity-related metabolic disorders.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Sirtuína 2 , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Animais , Camundongos , Sirtuína 2/metabolismo , Sirtuína 2/genética , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/imunologia , Saponinas/farmacologia , Saponinas/química , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/imunologiaRESUMO
BACKGROUND: Renal tubular injury induced by free fatty acid bound to albumin is the key pathological basis for the progression of diabetic kidney disease. However, effective interventions are limited. Astragaloside IV, as a major bioactive component purified from Astragalus membranaceus (Fisch.) Bunge, possesses pharmacological properties of lowering blood glucose and proteinuria, and renal tubular protection in diabetic kidney disease. Further work is needed to understand the underlying molecular mechanisms. PURPOSE: This study was designed to investigate the mechanism of renal tubular protection by astragaloside IV in diabetic kidney disease. METHODS: Rats receiving high-fat diet combined with streptozotocin (30 mg/kg, i.p.) were gavaged with astragaloside IV (10 mg/kg/d or 20 mg/kg/d) or empagliflozin (1.72 mg/kg/d) for 8 weeks. In vitro, the NRK-52E cells were treated with free fatty acid-deleted BSA or palmitic acid-bound BSA in the presence or absence of astragaloside IV (5 µM, 10 µM, 20 µM) or 5 µM of mcc950. The effects of astragaloside IV on mitochondrial function, NLRP3/ASC/IL-18/IL-1ß inflammatory cascade, and renal tubular injury were detected by pathological staining, immunoblotting, MitoSOX Red staining. Next, to investigate the mechanism of renal tubular protection by astragaloside IV, we transfected Fatp2 siRNA into BSA-PA-treated NRK-52E cells and injected lipofermata (a FATP2 inhibitor) intraperitoneally into free fatty acid-bound BSA overloaded rats with concomitant astragaloside IV treatment. RESULTS: Treatment with astragaloside IV for 8 weeks dose-dependently attenuated the blood glucose, ratio of urinary albumin to creatinine, disorder of lipid metabolism, and pathological injury in diabetic kidney disease rats. In addition, astragaloside IV dose-dependently attenuated mitochondrial-derived reactive oxygen species and subsequent inhibiting NLRP3-mediated inflammatory cascade in diabetic kidney disease rats and palmitic acid-bound BSA-treated NRK-52E cells, thereby exerting renal tubular protection. More importantly, the effects of astragaloside IV on restoration of mitochondrial function, inhibition of inflammatory response and amelioration of renal tubular injury in vivo and in vitro were further enhanced when used in combination with Fatp2 siRNA or lipofermata. CONCLUSION: Astragaloside IV exerts antioxidant and anti-inflammatory effects in diabetic kidney disease by inhibiting FATP2-mediated fatty acid transport, thereby attenuating renal tubular injury.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Túbulos Renais , Saponinas , Triterpenos , Animais , Masculino , Ratos , Astragalus propinquus/química , Linhagem Celular , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos , Interleucina-1beta/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Saponinas/farmacologia , Transportador 2 de Glucose-Sódio , Triterpenos/farmacologiaRESUMO
BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease complicated by diabetes mellitus (DM) is the leading cause of death in diabetic patients, and it is strongly associated with macrophages and inflammasomes. It has been found that activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is closely associated with phosphatidylinositol 4-phosphate (PI4P) on the trans-Golgi. However, how PI4P and NLRP3 regulate macrophage function and its role in diabetic atherosclerotic plaques is unclear. METHODS: The expression of Pi4p and Nlrp3-inflammasome-related proteins in atherosclerosis in apolipoprotein E-deficient (Apoe-/-) and Apoe-/- DM mice was investigated. Then, Pi4p levels were affected by shRNA-Pi4kb or cDNA-Sac1 plasmid to investigate the effects of changes in Pi4p-related metabolic enzymes on macrophage function. Finally, genetically modified macrophages were injected into diabetic Apoe-/- mice to explore the effects on atherosclerosis. RESULTS: DM promoted plaque progression in atherosclerotic mice and increased expression of Pi4p and Nlrp3 in plaques. In addition, impaired macrophage function induced by high glucose was reversed by transfected shRNA-Pi4kb or cDNA-Sac1 plasmid. Furthermore, decreased levels of Pi4p reduced plaque area in diabetic Apoe-/- mice. CONCLUSIONS: Our data suggests that Pi4p/Nlrp3 in macrophages play an important role in the exacerbation of atherosclerosis in diabetic mice. Pi4p-related metabolizing enzymes (PI4KB and SAC1) may be a potential therapeutic strategy for diabetic atherosclerosis, and macrophage therapy is also a potential treatment.
Assuntos
Aterosclerose , Diabetes Mellitus Experimental , Progressão da Doença , Macrófagos , Placa Aterosclerótica , Transdução de Sinais , Animais , Masculino , Camundongos , Apolipoproteínas E/genética , Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Diabetes Mellitus Experimental/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genéticaRESUMO
The liver has a distinctive capacity to regenerate, yet severe acute injury can be life-threatening if not treated appropriately. Inflammation and oxidative stress are central processes implicated in the pathophysiology of acute livery injury. NOX isoforms are important enzymes for ROS generation, NF-κB and NLRP3 activation, its inhibition could be vital in alleviating acute liver injury (ALI). Here in our study, we used apocynin, a natural occurring potent NOX inhibitor, to exploreits potential protective effect against thioacetamide (TAA)-induced ALI through modulating crucial oxidative and inflammatory pathways. Rats were injected once with TAA (500 mg/kg/i.p) and treated with apocynin (10 mg/kg/i.p) twice before TAA challenge. Sera and hepatic tissues were collected for biochemical, mRNA expression, western blot analysis and histopathological assessments. Pretreatment with apocynin improved liver dysfunction evidenced by decreased levels of aminotransferases, ALP, GGT and bilirubin. Apocynin reduced mRNA expression of NOX1 and NOX4 which in turn alleviated oxidative stress, as shown by reduction in MDA and NOx levels, and elevation in GSH levels andcatalase and SOD activities. Moreover, apocynin significantly reduced MPO gene expression. We also demonstrate that apocynin ameliorated inflammation through activating IκBα and suppressing IKKα, IKKß, NF-κBp65 and p-NF-κBp65, IL-6 andTNF-α. Additionally, apocynin potentiated the gene expression of anti-inflammatory IL-10 and reduced levels of hepatic NLRP3, Caspase-1 and IL-1ß. These results suggest that apocynin protects against ALI in association with the inhibition of NOX1 and NOX4 and regulating oxidative and inflammatory pathways.
Assuntos
Acetofenonas , Fígado , NADPH Oxidase 1 , NADPH Oxidase 4 , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estresse Oxidativo , Transdução de Sinais , Tioacetamida , Animais , Acetofenonas/farmacologia , NADPH Oxidase 4/metabolismo , NADPH Oxidase 1/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , NF-kappa B/metabolismo , Masculino , Ratos , Transdução de Sinais/efeitos dos fármacos , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Estresse Oxidativo/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Ratos Sprague-Dawley , Inflamação/metabolismo , Inflamação/tratamento farmacológicoRESUMO
OBJECTIVE: Traumatic brain injury (TBI)-induced anxiety is a common but under-investigated disorder, for which neuroinflammation is a significant contributor. Here we aim to investigate the protective effects of genistein, a plant-derived anti-inflammatory drug, against TBI-induced anxiety, and the underlying mechanisms. METHODS: A rat model of TBI was constructed using the lateral fluid percussion injury method. Genistein at the doses of 5, 10, and 20 mg/kg were used to treat rats at 30 min, 12 h, 24 h, 48 h, and 72 h up to 14 days after TBI. The evaluation of neurological deficit was performed preoperatively, on days 1, 3, 7, and 14 after TBI. The elevated plus maze test was carried out to assess anxiety and explorative behaviours, and the open field test was performed to assess locomotive activities. Brain injury was assessed by measuring brain water content and TdT-mediated dUTP Nick-End Labeling staining. Inflammatory responses were examined using enzyme-linked immunosorbent assay. The mRNA and protein expression were analysed using real-time polymerase chain reaction and Western blot, respectively. RESULTS: In the behavioural level, genistein treatment alleviated TBI-induced anxiety behaviours and neurological deficit in rats. In the meanwhile, brain oedema was also reduced by genistein treatment, showing alleviating effects of genistein at the pathological level. TUNEL staining also showed reduced apoptosis in rats treated with genistein. Genistein also inhibited Nlrp3/caspase-1 signalling, unveiling the effects of genistein in altering molecular pathways in brains with TBI. CONCLUSION: Genistein alleviates anxiety-like behaviours in TBI rats, which may be mediated via inhibiting Nlrp/caspase-1 signalling pathway.