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In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.
Assuntos
Inflamassomos , Análise de Célula Única , Animais , Apoptose , Caspase 8/metabolismo , Inflamassomos/metabolismo , Camundongos , Microscopia , PiroptoseRESUMO
Obesity is associated with an increase in adipose tissue, which is mediated by hyperplasia and hypertrophy. Therefore, inhibiting cell proliferation during mitotic clonal expansion (MCE) is one of the major strategies for preventing obesity. The antagonistic effects of Garcinia cambogia (G. cambogia) on obesity have been studied in animal experimental models. However, the effects of G. cambogia extract on MCE, and the underlying molecular mechanisms, are poorly understood. In this study, 3T3-L1 cells were used to investigate whether G. cambogia extract affected cell proliferation during MCE and to identify target molecules for any anti-adipogenic activity. G. cambogia extract suppressed isobutylmethylxanthine and dexamethasone-and-insulin (MDI)-induced adipogenesis at an early stage by attenuating MCE. In G. cambogia extract-treated preadipocytes, MDI-induced cell proliferation and cell cycle progression were inhibited by G0 /G1 arrest due to an increase in p21 and p27 expression, and inhibition of cyclin-dependent kinase 2, cyclin E1 expression, and retinoblastoma (Rb) phosphorylation. In addition, the MDI-induced phosphorylation and subsequent translocation into the nucleus of p90 ribosomal S6 kinase (p90RSK) and signal transducer and activator of transcription (Stat) 3 were suppressed. Specific inhibitors of p90RSK (FMK) and Stat3 (stattic) regulated cell proliferation and adipogenesis. In conclusion, this study demonstrated that G. cambogia extract inhibited MCE by regulating p90RSK, Stat3, and cell cycle proteins, leading to G0 /G1 arrest. These findings provide new insight into the mechanism by which G. cambogia suppresses adipocyte differentiation and show that p90RSK is critical for adipogenesis as a new molecular target.
Assuntos
Adipogenia , Garcinia cambogia/química , Mitose , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Clonais , Dexametasona/farmacologia , Insulina/farmacologia , Camundongos , Mitose/efeitos dos fármacos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
The secretion of platelet-derived growth factors (PDGFs) into vascular smooth muscle cells (VSMCs) induced by specific stimuli, such as oxidized low-density lipoprotein (LDL) cholesterol, initially increases the proliferation and migration of VSMCs, and continuous stimulation leads to VSMC apoptosis, resulting in the formation of atheroma. Autophagy suppresses VSMC apoptosis, and statins can activate autophagy. Thus, this study aimed to investigate the mechanism of the autophagy-mediated vasoprotective activity of rosuvastatin, one of the most potent statins, in VSMCs continuously stimulated with PDGF-BB, a PDGF isoform, at a high concentration (100 ng/ml) to induce phenotypic switching of VSMC. Rosuvastatin inhibited apoptosis in a concentration-dependent manner by reducing cleaved caspase-3 and interleukin-1ß (IL-1ß) levels and reduced intracellular reactive oxygen species (ROS) levels in PDGF-stimulated VSMCs. It also inhibited PDGF-induced p38 phosphorylation and increased the expression of microtubule-associated protein light chain 3 (LC3) and the conversion of LC3-I to LC3-II in PDGF-stimulated VSMCs. The ability of rosuvastatin to inhibit apoptosis and p38 phosphorylation was suppressed by treatment with 3-methyladenine (an autophagy inhibitor) but promoted by rapamycin (an autophagy activator) treatment. SB203580, a p38 inhibitor, reduced the PDGF-induced increase in intracellular ROS levels and inhibited the formation of cleaved caspase-3, indicating the suppression of apoptosis. In carotid ligation model mice, rosuvastatin decreased the thickness and area of the intima and increased the area of the lumen. In conclusion, our observations suggest that rosuvastatin inhibits p38 phosphorylation through autophagy and subsequently reduces intracellular ROS levels, leading to its vasoprotective activity. SIGNIFICANCE STATEMENT: This study shows the mechanism responsible for the vasoprotective activity of rosuvastatin in vascular smooth muscle cells under prolonged platelet-derived growth factor stimulation. Rosuvastatin inhibits p38 activation through autophagy, thereby suppressing intracellular reactive oxygen species levels, leading to the inhibition of apoptosis and reductions in the intima thickness and area. Overall, these results suggest that rosuvastatin can be used as a novel treatment to manage chronic vascular diseases such as atherosclerosis.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/toxicidade , Rosuvastatina Cálcica/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Propionate is a short-chain fatty acid (SCFA) mainly produced from carbohydrates by gut microbiota. Sodium propionate (SP) has shown to suppress the invasion in G protein-coupled receptor 41 (GPR41) and GPR43-overexpressing breast cancer cells. In this study we investigated the effects of SP on the proliferation, apoptosis, autophagy, and antioxidant production of breast cancer cells. We showed that SP (5-20 mM) dose-dependently inhibited proliferation and induced apoptosis in breast cancer cell lines JIMT-1 (ER-negative and HER2-expressing) and MCF7 (ER-positive type), and this effect was not affected by PTX, thus not mediated by the GPR41 or GPR43 SCFA receptors. Meanwhile, we demonstrated that SP treatment increased autophagic and antioxidant activity in JIMT-1 and MCF7 breast cancer cells, which might be a compensatory mechanism to overcome SP-induced apoptosis, but were not sufficient to overcome SP-mediated suppression of proliferation and induction of apoptosis. We revealed that the anticancer effect of SP was mediated by inhibiting JAK2/STAT3 signaling which led to cell-cycle arrest at G0/G1 phase, and increasing levels of ROS and phosphorylation of p38 MAPK which induced apoptosis. In nude mice bearing JIMT-1 and MCF7 cells xenograft, administration of SP (20 mg/mL in drinking water) significantly suppressed tumor growth by regulating STAT3 and p38 in tumor tissues. These results suggest that SP suppresses proliferation and induces apoptosis in breast cancer cells by inhibiting STAT3, increasing the ROS level and activating p38. Therefore, SP is a candidate therapeutic agent for breast cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Propionatos/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos Nus , Propionatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Mulberry is a Korean medicinal herb that shows effective prevention and treatment of obesity and diabetes. Bioconversion is the process of producing active ingredients from natural products using microorganisms or enzymes. METHODS: In this study, we prepared bioconverted mulberry leaf extract (BMLE) with Viscozyme L, which we tested in insulin-sensitive cells (i.e., skeletal muscle cells and adipocytes) and insulin-secreting pancreatic ß-cells, as well as obese diabetic mice induced by co-administration of streptozotocin (100 mg/kg, IP) and nicotinamide (240 mg/kg, IP) and feeding high-fat diet, as compared to unaltered mulberry leaf extract (MLE). RESULTS: BMLE increased the glucose uptake in C2C12 myotubes and 3 T3-L1 adipocytes and increased glucose-stimulated insulin secretion in HIT-T15 pancreatic ß-cells. The fasting blood glucose levels in diabetic mice treated with BMLE or MLE (300 and 600 mg/kg, PO, 7 weeks) were significantly lower than those of the vehicle-treated group. At the same concentration, BMLE-treated mice showed better glucose tolerance than MLE-treated mice. Moreover, the blood concentration of glycated hemoglobin (HbA1C) in mice treated with BMLE was lower than that in the MLE group at the same concentration. Plasma insulin levels in mice treated with BMLE or MLE tended to increase compared to the vehicle-treated group. Treatment with BMLE yielded significant improvements in insulin resistance and insulin sensitivity. CONCLUSION: These results indicate that in the management of diabetic condition, BMLE is superior to unaltered MLE due to at least, in part, high concentrations of maker compounds (trans-caffeic acid and syringaldehyde) in BMLE.
Assuntos
Glicemia/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Morus/química , Extratos Vegetais/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica , Hipoglicemiantes/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/metabolismo , Folhas de Planta/químicaRESUMO
Autophagy has been studied as a therapeutic strategy for cardiovascular diseases. However, insufficient studies have been reported concerning the influence of vascular smooth muscle cells (VSMCs) through autophagy regulation. The aim of the present study was to determine the effects of VSMCs on the regulation of autophagy under in vitro conditions similar to vascular status of the equipped microtubule target agent-eluting stent and increased release of platelet-derived growth factor-BB (PDGF-BB). Cell viability and proliferation were measured using MTT and cell counting assays. Immunofluorescence using an anti-α-tubulin antibody was performed to determine microtubule dynamic formation. Cell apoptosis was measured by cleavage of caspase-3 using western blot analysis, and by nuclear fragmentation using a fluorescence assay. Autophagy activity was assessed by microtubule-associated protein light chain 3-II (LC-II) using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured using H2DCFDA. The proliferation and viability of VSMCs were inhibited by microtubule regulation. Additionally, microtubule-regulated and PDGF-BB-stimulated VSMCs increased the cleavage of caspase-3 more than only the microtubule-regulated condition, similar to that of LC3-II, implying autophagy. Inhibitory autophagy of microtubule-regulated and PDGF-BB-stimulated VSMCs resulted in low viability. However, enhancement of autophagy maintained survival through the reduction of ROS. These results suggest that the apoptosis of conditioned VSMCs is decreased by the blocking generation of ROS via the promotion of autophagy, and proliferation is also inhibited. Thus, promoting autophagy as a therapeutic target for vascular restenosis and atherosclerosis may be a good strategy.
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Ascorbic acid is one of the most well-known nutritional supplement and antioxidant found in fruits and vegetables. Calcium ascorbate has been developed to mitigate the gastric irritation caused by the acidity of ascorbic acid. The aim of this study was to compare calcium ascorbate and ascorbic acid, focusing on their antioxidant activity and effects on gastric juice pH, total acid output, and pepsin secretion in an in vivo rat model, as well as pharmacokinetic parameters. Calcium ascorbate and ascorbic acid had similar antioxidant activity. However, the gastric fluid pH was increased by calcium ascorbate, whereas total acid output was increased by ascorbic acid. In the rat pylorus ligation-induced ulcer model, calcium ascorbate increased the gastric fluid pH without changing the total acid output. Administration of calcium ascorbate to rats given a single oral dose of 100 mg/kg as ascorbic acid resulted in higher plasma concentrations than that from ascorbic acid alone. The area under the curve (AUC) values of calcium ascorbate were 1.5-fold higher than those of ascorbic acid, and the Cmax value of calcium ascorbate (91.0 ng/ml) was higher than that of ascorbic acid (74.8 ng/ml). However, their Tmax values were similar. Thus, although calcium ascorbate showed equivalent antioxidant activity to ascorbic acid, it could attenuate the gastric high acidity caused by ascorbic acid, making it suitable for consideration of use to improve the side effects of ascorbic acid. Furthermore, calcium ascorbate could be an appropriate antioxidant substrate, with increased oral bioavailability, for patients with gastrointestinal disorders.
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BACKGROUND: Kiom-18 is a novel composition of Cinnamomum cassia, Pinus densiflora, Curcuma longa and Glycyrrhiza glabra. Curcuma longa and Glycyrrhiza glabra, which are traditional medicines in Asia, have been reported to demonstrate preventive effects against atherosclerosis; however, they have not yet been developed into functional atherosclerosis treatments. We therefore studied the anti-atherosclerotic effects and possible molecular mechanisms of Kiom-18 using vascular smooth muscle cells (VSMCs). METHODS: To assess the anti-proliferative effect of Kiom-18 in vitro, we performed thymidine incorporation, cell cycle progression, immunoblotting and immunofluorescence assays in VSMCs stimulated by platelet derived-growth factor (PDGF)-BB. In addition, we used LDLr knockout mice to identify the effects of Kiom-18 as a preliminary result in an atherosclerosis animal model. RESULTS: Kiom-18 inhibited platelet-derived growth factor (PDGF)-BB-stimulated-VSMC proliferation and DNA synthesis. Additionally, Kiom-18 arrested the cell cycle transition of G0/G1 stimulated by PDGF-BB and its cell cycle-related proteins. Correspondingly, the level of p27(kip1) expression was upregulated in the presence of the Kiom-18 extract. Moreover, in an atherosclerosis animal model of LDLr knockout mice, Kiom-18 extract showed a preventive effect for the formation of atherosclerotic plaque and suppressed body weight, fat weight, food treatment efficiency, neutrophil count, and triglyceride level. CONCLUSIONS: These results indicate that Kiom-18 exerts anti-atherosclerotic effects by inhibiting VSMC proliferation via G0/G1 arrest, which upregulates p27(Kip1) expression.
Assuntos
Aterosclerose/prevenção & controle , Cinnamomum aromaticum/química , Curcuma/química , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Glycyrrhiza/química , Pinus/química , Extratos Vegetais/farmacologia , Animais , Aorta , Aterosclerose/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Camundongos , Camundongos Knockout , Extratos Vegetais/química , Ratos , Regulação para Cima/efeitos dos fármacosRESUMO
Veratrum nigrum is recognized as a medicinal plant used for the treatment of hypertension, stroke, and excessive phlegm. Chemical investigation of the roots and rhizomes led to the isolation of five new steroidal alkaloids, jervine-3-yl formate (1), veramarine-3-yl formate (2), jerv-5,11-diene-3ß,13ß-diol (3), (1ß,3ß,5ß)-1,3-dihydroxyjervanin-12(13)-en-11-one (4), and veratramine-3-yl acetate (5). Compounds 1 and 5 exhibited potent inhibitory activity (11.3 and 4.7 µM, respectively) against protein tyrosine phosphatase 1B (PTP1B), which has emerged as a viable target for treatment of type 2 diabetes mellitus. On the basis of their PTP1B inhibitory activity, the compounds were evaluated for their potential to enhance glucose uptake in C2C12 skeletal muscle cells. The insulin-stimulated glucose uptake was enhanced upon treatment with compounds 1 and 5 (10 µM) by 49.9 ± 6.5% and 56.0 ± 9.7%, respectively, in a more potent manner than that with the positive control rosiglitazone (47.3 ± 3.4% at 30 µM). These results suggest that steroidal alkaloids serve as practical antidiabetes mellitus leads capable of enhancing glucose uptake.
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Glucose/metabolismo , Plantas Medicinais/química , Esteroides/isolamento & purificação , Esteroides/farmacologia , Veratrum/química , Alcaloides/química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Estrutura Molecular , Músculo Esquelético/metabolismo , Raízes de Plantas/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , República da Coreia , Rizoma/química , Rosiglitazona , Estereoisomerismo , Esteroides/química , Tiazolidinedionas/farmacologiaRESUMO
Glucose uptake into insulin-sensitive tissues is important for the regulation of blood glucose. This study has investigated whether the pentacyclic triterpenoids substituted with a carboxylic acid at the C-27 position isolated from Astilbe rivularis can enhance glucose uptake and subsequently to also examine their underlying molecular mechanisms. The structure of the new pentacyclic triterpenoid 1 was assigned by spectroscopic data interpretation. To evaluate the activity of compounds 1 and 2, glucose uptake and glucose transporter 4 (GLUT4) translocation were measured in C2C12 myotubes. The C-27-carboxylated triterpenoids 1 and 2 significantly increased basal and insulin-stimulated glucose uptake and GLUT4 translocation to plasma membrane. Both compounds stimulated the phosphorylation of insulin receptor substrate-1 (IRS-1), protein kinase B (Akt), and extracellular signal-regulated kinase 1/2 (Erk1/2). Pretreatment with the Akt inhibitor triciribine or the Erk1/2 inhibitor U0126 decreased the ability of both compounds to enhance basal- and insulin-stimulated glucose uptake and stimulate GLUT4 translocation. These results indicate that compounds 1 and 2 activated both the IRS-1/Akt and Erk1/2 pathways and subsequently stimulated GLUT4 translocation, leading to enhanced glucose uptake. Thus, these observations suggest that C-27-carboxylated-pentacyclic triterpenoids may serve as scaffolds for development as agents for the management of blood glucose levels in disease states such as diabetes.
Assuntos
Glucose/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Triterpenos Pentacíclicos/isolamento & purificação , Triterpenos Pentacíclicos/farmacologia , Saxifragaceae/química , Transporte Biológico , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Triterpenos Pentacíclicos/química , Fosforilação , Raízes de Plantas/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , VietnãRESUMO
"G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic ß-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with 30 µM linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus.
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The increased potential for vascular smooth muscle cell (VSMC) growth is a key abnormality in the development of atherosclerosis and post-angioplasty restenosis. Abnormally high activity of platelet-derived growth factor (PDGF) is believed to play a central role in the etiology of these pathophysiological situations. Here, we investigated the anti-proliferative effects and possible mechanism(s) of murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa Guillamin (Rutaceae), on PDGF-BB-stimulated VSMCs. Murrayafoline A inhibited the PDGF-BB-stimulated proliferation of VSMCs in a concentration-dependent manner, as measured using a non-radioactive colorimetric WST-1 assay and direct cell counting. Furthermore, murrayafoline A suppressed the PDGF-BB-stimulated progression through G0/G1 to S phase of the cell cycle, as measured by [(3)H]-thymidine incorporation assay and cell cycle progression analysis. This anti-proliferative action of murrayafoline A, arresting cell cycle progression at G0/G1 phase in PDGF-BB-stimulated VSMCs, was mediated via down-regulation of the expression of cyclin D1, cyclin E, cyclin-dependent kinase (CDK)2, CDK4, and proliferating cell nuclear antigen (PCNA), and the phosphorylation of retinoblastoma protein (pRb). These results indicate that murrayafoline A may be useful in preventing the progression of vascular complications such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis.
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G-protein-coupled receptors are a diverse class of cell surface receptors that orchestrate numerous physiological functions. The G-protein-coupled receptors, GPR41 and GPR43, sense short-chain fatty acids (SCFAs), which are metabolites of dietary fermentation by the host's intestinal bacteria. These receptors have gained attention as potential therapeutic targets against various diseases because of their SCFA-mediated beneficial effects on the host's intestinal health. Mounting evidence has associated the activity of these receptors with chronic metabolic diseases, including obesity, diabetes, inflammation, and cardiovascular disease. However, despite intensive research using various strategies, including gene knockout (KO) mouse models, evidence about the precise roles of GPR41 and GPR43 in disease treatment remains inconsistent. Here, we comprehensively review the latest findings from functional studies of the signaling mechanisms that underlie the activities of GPR41 and GPR43, as well as highlight their multifaceted roles in health and disease. We anticipate that this knowledge will guide future research priorities and the development of effective therapeutic interventions.
Assuntos
Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Humanos , Transdução de Sinais , Doenças Metabólicas/metabolismo , Ácidos Graxos Voláteis/metabolismoRESUMO
Regulated cell death (RCD) pathways, such as pyroptosis, apoptosis, and necroptosis, are essential for maintaining the body's balance, defending against pathogens, and eliminating abnormal cells that could lead to diseases like cancer. Although these pathways operate through distinct mechanisms, recent genetic and pharmacological studies have shown that they can interact and influence each other. The concept of "PANoptosis" has emerged, highlighting the interplay between pyroptosis, apoptosis, and necroptosis, especially during cellular responses to infections. This article provides a concise overview of PANoptosis and its molecular mechanisms, exploring its implications in various diseases. The review focuses on the extensive interactions among different RCD pathways, emphasizing the role of PANoptosis in infections, cytokine storms, inflammatory diseases, and cancer. Understanding PANoptosis is crucial for developing novel treatments for conditions involving infections, sterile inflammations, and cancer.
Assuntos
Inflamação , Necroptose , Neoplasias , Piroptose , Humanos , Inflamação/patologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Animais , Necroptose/efeitos dos fármacos , Necroptose/fisiologia , Piroptose/efeitos dos fármacos , Piroptose/fisiologia , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/fisiologia , Morte Celular Regulada/efeitos dos fármacos , Infecções/patologia , Infecções/imunologiaRESUMO
BACKGROUND: Because obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (-)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PURPOSE: This study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. METHODS: Preadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. RESULTS: Here, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. CONCLUSIONS: These findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.
Assuntos
Proliferação de Células , Citratos , Proteína Forkhead Box O1 , Obesidade , Proteínas Quinases S6 Ribossômicas 90-kDa , Animais , Camundongos , Células 3T3-L1/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Proliferação de Células/efeitos dos fármacos , Citratos/farmacologia , Citratos/uso terapêutico , Dieta Hiperlipídica/efeitos adversos , Proteína Forkhead Box O1/antagonistas & inibidores , Proteína Forkhead Box O1/metabolismo , Camundongos Endogâmicos C57BL , Mitose/efeitos dos fármacos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Innate immunity provides the first line of defense through multiple mechanisms, including pyrogen production and cell death. While elevated body temperature during infection is beneficial to clear pathogens, heat stress (HS) can lead to inflammation and pathology. Links between pathogen exposure, HS, cytokine release, and inflammation have been observed, but fundamental innate immune mechanisms driving pathology during pathogen exposure and HS remain unclear. Here, we use multiple genetic approaches to elucidate innate immune pathways in infection or LPS and HS models. Our results show that bacteria and LPS robustly increase inflammatory cell death during HS that is dependent on caspase-1, caspase-11, caspase-8, and RIPK3 through the PANoptosis pathway. Caspase-7 also contributes to PANoptosis in this context. Furthermore, NINJ1 is an important executioner of this cell death to release inflammatory molecules, independent of other pore-forming executioner proteins, gasdermin D, gasdermin E, and MLKL. In an in vivo HS model, mortality is reduced by deleting NINJ1 and fully rescued by deleting key PANoptosis molecules. Our findings suggest that therapeutic strategies blocking NINJ1 or its upstream regulators to prevent PANoptosis may reduce the release of inflammatory mediators and benefit patients.
Assuntos
Transtornos de Estresse por Calor , Lipopolissacarídeos , Humanos , Gasderminas , Morte Celular , Inflamação/genética , Caspases/genética , Resposta ao Choque Térmico/genética , Piroptose , Apoptose , Fatores de Crescimento Neural , Moléculas de Adesão Celular NeuronaisRESUMO
As the abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis and vascular restenosis, a candidate drug with antiproliferative properties is needed. We investigated the antiproliferative action and underlying mechanism of a newly synthesized naphthoquinone derivative, 5,8-dimethoxy-2-nonylamino-naphthalene-1,4-dione (2-nonylamino-DMNQ), using VSMCs treated with platelet-derived growth factor (PDGF). 2-Nonylamino-DMNQ inhibited proliferation and cell number of VSMCs induced by PDGF, but not epidermal growth factor (EGF), in a concentration-dependent manner without any cytotoxicity. This derivative suppressed PDGF-induced [(3)H]-thymidine incorporation, cell cycle progression from G0/G1 to S phase, and the phosphorylation of phosphor-retinoblastoma protein (pRb) as well as the expression of cyclin E/D, cyclin-dependent kinase (CDK) 2/4, and proliferating cell nuclear antigen (PCNA). Importantly, 2-nonylamino-DMNQ inhibited the phosphorylation of PDGF receptorß(PDGF-Rß) enhanced by PDGF at Tyr(579), Tyr(716), Tyr(751), and Tyr(1021) residues. Subsequently, 2-nonylamino-DMNQ inhibited PDGF-induced phosphorylation of STAT3, ERK1/2, Akt, and PLCγ1. Therefore, our results indicate that 2-nonylamino-DMNQ inhibits PDGF-induced VSMC proliferation by blocking PDGF-Rß autophosphorylation, and subsequently PDGF-Rß-mediated downstream signaling pathways.
RESUMO
Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum. This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including apoptosis, pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases, from other cell death pathways. Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.
Assuntos
Apoptose , Caspases , Humanos , Morte Celular/fisiologia , Apoptose/fisiologia , Caspases/metabolismo , Inflamação , Imunidade InataRESUMO
The overexpansion of adipose tissues leads to obesity and eventually results in metabolic disorders. Garcinia cambogia (G. cambogia) has been used as an antiobesity supplement. However, the molecular mechanisms underlying the effects of G. cambogia on cellular processes have yet to be fully understood. Here, we discovered that G. cambogia attenuated the expression of CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), an important adipogenic factor, suppressing its transcription in differentiated cells. In addition, G. cambogia inhibited macroautophagic/autophagic flux by decreasing autophagy-related gene expression and autophagosome formation. Notably, G. cambogia markedly elevated the expression of KLF3 (Kruppel-like factor 3 (basic)), a negative regulator of adipogenesis, by reducing SQSTM1/p62-mediated selective autophagic degradation. Furthermore, increased KLF3 induced by G. cambogia interacted with CTBP2 (C-terminal binding protein 2) to form a transcriptional repressor complex and inhibited Cebpa and Pparg transcription. Importantly, we found that RPS6KA1 and STAT3 were involved in the G. cambogia-mediated regulation of CEBPB and autophagic flux. In an obese animal model, G. cambogia reduced high-fat diet (HFD)-induced obesity by suppressing epididymal and inguinal subcutaneous white adipose tissue mass and adipocyte size, which were attributed to the regulation of targets that had been consistently identified in vitro. These findings provide new insight into the mechanism of G. cambogia-mediated regulation of adipogenesis and suggest molecular links to therapeutic targets for the treatment of obesity.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG: autophagy-related; Baf: bafilomycin A1; BECN1: beclin 1; CEBP: CCAAT/enhancer binding protein (C/EBP); CHX: cycloheximide; CREB: cAMP response element binding protein; CTBP: C-terminal binding protein; EGCG: (-)-epigallocatechin gallate; eWAT: epididymal white; G. cambogia: Garcinia cambogia; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HFD: high-fat diet; iWAT: inguinal subcutaneous white; KLF: Kruppel-like factor; LAP: liver-enriched transcriptional activating proteins; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; PPARG: peroxisome proliferator activated receptor gamma; qPCR: quantitative real-time PCR; RFP: red fluorescent protein; RPS6KA1: ribosomal protein S6 kinase A1; siRNA: small-interfering RNA; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TEM: transmission electron microscopy.
Assuntos
Adipogenia , Garcinia cambogia , Adipogenia/genética , Animais , Autofagia/fisiologia , Garcinia cambogia/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Obesidade , PPAR gama/metabolismo , Proteínas Serina-Treonina Quinases , Proteína Sequestossoma-1/metabolismoRESUMO
SCOPE: Garcinia cambogia (G. cambogia) is known to have antiobesity effects. In this study, the therapeutic effects of G. cambogia on glucose homeostasis in obesity-induced diabetes are explored and the underlying mechanisms are investigated. METHODS AND RESULTS: C2C12 myotubes are treated with G. cambogia; glucose uptake, intracellular Ca2+ levels, and related alterations in signaling pathways are examined. High-fat diet (HFD)-fed mice are administered G. cambogia for 8 weeks; oral glucose tolerance is evaluated, and the regulation of identified targets of signaling pathways in quadriceps skeletal muscle are examined in vivo. G. cambogia increases glucose uptake in C2C12 myotubes and induces the upregulation of AMPK, ACC, and p38 MAPK phosphorylation. Notably, G. cambogia markedly elevates both intracellular Ca2+ levels, activating CaMKII, a Ca2+ -sensing protein, and TBC1D4-mediated GLUT4 translocation, to facilitate glucose uptake. Furthermore, high-glucose-induced inhibition of glucose uptake and signal transduction is reverted by G. cambogia. In an HFD-induced diabetes mouse model, G. cambogia administration results in significant blood glucose-lowering effects, which are attributed to the regulation of targets that have been identified in vitro, in quadricep skeletal muscle. CONCLUSION: These findings provide new insights into the mechanism by which G. cambogia regulates glucose homeostasis in obesity-induced diabetes.