RESUMEN
Crocetin is a main bioactive component with a carotenoid skeleton in Gardenia jasminoides, a typical traditional Chinese medicine with a long history in Southeast Asia. Crocetin is being commonly consumed as spices, dyes, and food colorants. Recent pharmacological studies had implied that crocetin may possess potent anti-inflammatory properties; however, the underlying molecular mechanism is not fully elucidated. In the present study, the regulatory effect of crocetin on redox balance was systematically investigated in lipopolysaccharide- (LPS-) stimulated RAW264.7 cells. The results showed that crocetin dose-dependently inhibited LPS-induced nitric oxide production and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells. Molecular data revealed that crocetin exerted its anti-inflammatory property by inhibiting the MEK1/JNK/NF-κB/iNOS pathway and activating the Nrf2/HO-1 pathway. The shRNA-knockdown (KD) of MEK1 and ERK1 confirmed that the activation of MEK1 and inhibition of JNK mediated the anti-inflammatory effect of crocetin. Moreover, the pull-down assay and computational molecule docking showed that crocetin could directly bind to MEK1 and JNK1/2. It is noticed that both KD and knockout (KO) of HO-1 gene blocked this action. More detailed data have shown that HO-1-KO blocked the inhibition of p-IκB-α by crocetin. These data indicated that crocetin exerted its anti-inflammatory property via modulating the crosstalk between the MEK1/JNK/NF-κB/iNOS pathway and the Nrf2/HO-1 pathway, highlighting HO-1 as a major player. Therefore, the present study reveals that crocetin can act as a potential candidate for redox-balancing modulation in charge of its anti-inflammatory and chemopreventive effect, which strengthens its potency in the subsequent clinic application in the near future.
Asunto(s)
Antiinflamatorios/farmacología , Carotenoides/farmacología , Transducción de Señal/efectos de los fármacos , Vitamina A/análogos & derivados , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Sitios de Unión , Carotenoides/química , Carotenoides/metabolismo , Hemo-Oxigenasa 1/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Factor 2 Relacionado con NF-E2/metabolismo , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Células RAW 264.7 , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Vitamina A/química , Vitamina A/metabolismo , Vitamina A/farmacologíaRESUMEN
Panaxynol (PAL) mainly comes from Umbelliferae plants, which has anti-inflammatory and neuroprotective activities. Lipopolysaccharide (LPS)-induced depression in mice was a classic model for studying the effects of drugs on depression in mice. The purpose of this study was to investigate the mechanism and effect of PAL on depression by LPS induced in mice. In the tail suspension test (TST) and forced swimming test (FST) results, PAL significantly reduced the immobility time of mice. In the result of the open field test (OFT) and the elevated plus maze test (EPM), improved their exploration ability. According to the results of ELISA, PAL could significantly reduce the tumor necrosis factor-α (TNF-α) and interleukin- 6 (IL-6) levels in serum. Increase the superoxide dismutase (SDO) level and decrease the malondialdehyde (MDA) level in hippocampus. According to Western blotting analysis results, PAL increased the protein expression of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB), decreased the nuclear transport of nuclear factor kappa-Bp65 (NF-κBp65) and phosphorylation of inhibitor of NF-κB (IκB-α). Meanwhile, PAL also inhibited the production of nitric oxide in BV-2 microglia and decreased the level of inflammatory factors. PAL also reduced levels of oxidative stress and inhibited protein expression in the NF-κB/IκB-α inflammatory pathway and increased the protein expression of BDNF/TrkB, thereby inhibiting the over-activation of BV-2 microglia. In conclusion, according to the results of the behavioral text, it is proved that PAL could effectively alleviate LPS induced depression behavior in mice. The mechanism may be that the anti-inflammatory and anti-oxidative stress effects of PAL reduce the release of inflammatory factors in the mouse brain. Meanwhile, PAL could improve brain neurotrophic factors, inhibit the excessive activation of BV-2 microglia, and further inhibit the depressive state of the mice.
Asunto(s)
Antidepresivos/farmacología , Diinos/farmacología , Alcoholes Grasos/farmacología , Microglía/efectos de los fármacos , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Extractos Vegetales/farmacología , Animales , Antidepresivos/uso terapéutico , Línea Celular , Depresión/tratamiento farmacológico , Depresión/metabolismo , Depresión/psicología , Diinos/uso terapéutico , Relación Dosis-Respuesta a Droga , Alcoholes Grasos/uso terapéutico , Inmovilización/métodos , Inmovilización/fisiología , Inmovilización/psicología , Masculino , Ratones , Ratones Endogámicos ICR , Microglía/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Extractos Vegetales/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Resultado del TratamientoRESUMEN
Lysozymes, which are secreted in many organisms, including invertebrates, mammals, plants, bacteria and fungus, exhibit antimicrobial, antiviral, antioxidant, and anti-inflammatory activities. Splys-i is an invertebrate-type (i-type) lysozyme isolated from Scylla paramamosain in 2017 and is involved in immune defense against bacteria. However, the antibacterial, antioxidant, and anti-inflammatory activities of Splys-i remain to be elucidated. In the current study, the expression parameters (including IPTG concentration, induction temperature, and induction duration) of Splys-i in Escherichia coli were optimized to achieve high-level yield through shake-flask cultivation with approximately 120 mg of Splys-i obtained from 1 L of LB medium. The purified Splys-i displayed low cytotoxicity to RAW264.7 macrophage cells and low hemolytic activity against erythrocytes of mouse, rat, and rabbit, respectively, and exhibited potent antibacterial activity against both Gram-positive and -negative bacteria with minimum concentrations ranging from 15 to 90 µg/mL. The antibacterial property of Splys-i was also unaffected when treated with various temperature, pHs, and salinity, respectively, and Splys-i showed resistance to proteinase digestion. Radical-scavenging rate assay (including ABTS+, DPPH, hydroyl free radical, and superoxide anion) indicated that Splys-i was an efficient antioxidant. Splys-i also exerted anti-inflammatory effect through the inhibition of IκBα and NF-κB(P65) phosphorylation, thereby reducing the secretion of pro-inflammatory cytokines. All these results suggested that Splys-i can be prepared from E. coli with potent biological property.
Asunto(s)
Antibacterianos/metabolismo , Antiinflamatorios/metabolismo , Antioxidantes/metabolismo , Proteínas de Artrópodos/genética , Braquiuros/química , Muramidasa/genética , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Proteínas de Artrópodos/metabolismo , Proteínas de Artrópodos/farmacología , Benzotiazoles/antagonistas & inhibidores , Benzotiazoles/química , Compuestos de Bifenilo/antagonistas & inhibidores , Compuestos de Bifenilo/química , Clonación Molecular , Eritrocitos/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Muramidasa/metabolismo , Muramidasa/farmacología , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Picratos/antagonistas & inhibidores , Picratos/química , Células RAW 264.7 , Conejos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Ácidos Sulfónicos/antagonistas & inhibidores , Ácidos Sulfónicos/química , Temperatura , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Due to unavailability of a specific drug/vaccine to attenuate severe acute respiratory syndrome coronavirus 2, the current strategy to combat the infection has been largely dependent upon the use of anti-inflammatory drugs to control cytokines storm responsible for respiratory depression. Thus, in this study, we discovered novel pyrazole analogs as a potent nuclear factor kappa B (NF-ĸB) inhibitor. The compounds were assessed for NF-ĸB transcriptional inhibitory activity in RAW264.7 cells after stimulation with lipopolysaccharides (LPS), revealing Compound 6c as the most potent analog among the tested series. The effect of Compound 6c was further investigated on the levels of interleukin-1ß, tumor necrosis factor-α, and interleukin-6 in LPS-stimulated RAW267.4 cells by enzyme immunoassay, where it causes a significant reduction in the level of these cytokines. In Western blot analysis, Compound 6c also causes the inhibition of inhibitor kappa B-α and NF-κB. It was found to be snugly fitted into the inner grove of the active site of NF-ĸB by forming H-bonds and a nonbonded interaction with Asn28 in a docking analysis.
Asunto(s)
Antiinflamatorios , Tratamiento Farmacológico de COVID-19 , COVID-19 , Simulación del Acoplamiento Molecular , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Pirazoles , SARS-CoV-2/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , COVID-19/metabolismo , COVID-19/patología , Citocinas/metabolismo , Ratones , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/química , Inhibidor NF-kappaB alfa/metabolismo , Pirazoles/química , Pirazoles/farmacología , Células RAW 264.7RESUMEN
BACKGROUND: Inflammatory bowel disease is a chronic immunoinflammatory disease of the gastrointestinal tract. Piperine, an alkaloid, has been reported to possess antioxidant, anti-inflammatory, antiapoptotic, and antiulcer potential. AIM: To elucidate the plausible mechanisms of action of piperine on experimental trinitrobenzenesufonic acid (TNBS)-induced colitis by assessing various biochemical, molecular, histological, and ultrastructural modifications. METHODS: Colitis was induced in male Sprague-Dawley rats via intrarectal instillation of TNBS. Then, the rats were treated with piperine (10, 20, and 40 mg/kg, p.o.) for 14 days. RESULTS: TNBS induced significant (p < 0.05) colonic damage, which was assessed by disease activity index, macroscopic score, and stool consistency. The administration of piperine (20 and 40 mg/kg) significantly inhibited (p < 0.05) these damages. Treatments with piperine (20 and 40 mg/kg) notably inhibited (p < 0.05) the TNBS-induced elevation of oxido-nitrosative stress (superoxide dismutase, glutathione, malondialdehyde, and nitric oxide), 5-hydroxytryptamine, and hydroxyproline content in the colon. Furthermore, colonic inducible nitric oxide synthase (iNOs), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, IL-6, interferon-gamma, and cyclooxygenase-2 (COX-2) messenger RNA (mRNA) expressions were upregulated after TNBS instillation and piperine (20 and 40 mg/kg) significantly attenuated (p < 0.05) these elevated mRNA expressions. TNBS decreased the expressions of tight junction (TJ) protein (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and increased the expressions of proapoptotic (caspase-1) protein. These expressions were markedly inhibited (p < 0.05) by piperine treatment. Histological and ultrastructural studies of transmission electron microscopy suggested that piperine significantly ameliorated (p < 0.05) TNBS-induced colonic aberrations. CONCLUSION: Piperine ameliorated the progression of TNBS-induced colitis by modulating the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha/nuclear factor-kappa B signaling pathway, thus inhibiting the overexpression of proinflammatory cytokines (TNF-α and IL's), COX-2, iNOs, oxido-nitrosative stress, and proapoptotic proteins (caspase-1) that may improve the expression of TJ protein (claudin-1, occludin, and ZO-1).
Asunto(s)
Alcaloides/farmacología , Benzodioxoles/farmacología , Colitis/prevención & control , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Proteínas de Uniones Estrechas/metabolismo , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Citocinas/genética , Modelos Animales de Enfermedad , Alimentos Funcionales , Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
Zinc finger and BTB domain containing 20 (ZBTB20), a sequence-specific transcriptional repressor, has been found to be involved in tumorigenesis. However, its role(s) in gastric cancer and the molecular mechanisms involved are poorly investigated. Here, our data demonstrated that ZBTB20 expression was markedly upregulated in gastric cancer cell lines infected with Helicobacter pylori (H. pylori) and in gastric cancer tumor samples. Loss- and gain-of-function studies showed that ZBTB20 promoted cell proliferation, invasion and migration of gastric cancer cell lines. Mechanistically, the phosphorylation of NF-κBp65 and expression and activity of MMP-2 and MMP-9 were increased, while IκBα expression was decreased by ZBTB20 in gastric cancer cells. We further revealed that IκBα overexpression significantly inhibited NF-κB signaling as well as cell migration, invasion and proliferation in gastric cancer cell lines induced by ZBTB20 overexpression. Therefore, our findings emphasize an important role for ZBTB20 in controlling gastric cancer development, which is helpful to identify potential therapeutic targets for its treatment.
Asunto(s)
Movimiento Celular , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Gástricas/patología , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Helicobacter pylori/fisiología , Humanos , Invasividad Neoplásica , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/microbiología , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The serine/threonine kinase IKBKE is frequently overexpressed or activated in a variety of human cancers. Ectopic expression of IKBKE induces malignant transformation, cell migration, invasion and chemoresistance. Thus, IKBKE is an attractive target for anti-cancer drug development. METHODS: By screening of NCI Diversity Set and Clinical Collection I and II compound libraries using cell-based assay, we identified several candidates of IKBKE inhibitors, which directly inhibited IKBKE kinase activity in vitro and in vivo. One of them, malachite green oxalate (MCCK1), was further characterized. The mechanism was examined by western blot, immunoprecipitation (IP) and Immunofluorescence. We also evaluated in a mouse xenograft model. In vitro kinase assay and luciferase reporter assay were also performed in our experiments. RESULTS: MCCK1 inhibits IKBKE kinase as well as its downstream targets such as IκBα, p65 and IRF3. MCCK1 is a selective inhibitor for IKBKE, with moderate effect on TBK1, but does not inhibit the activation of IKKα/ß, STAT3, Erk-1/2, p38 or JNK. The inhibition of IKBKE by MCCK1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor aberrant expression of IKBKE. Furthermore, MCCK1 inhibits tumor growth in nude mice of human cancer cells in which IKBKE is elevated but not of those cancer cells in which it is not. CONCLUSION: These data indicate that MCCK1 is an IKBKE inhibitor with anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients with tumors over expressing IKBKE.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Quinasa I-kappa B/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Colorantes de Rosanilina/farmacología , Células A549 , Animales , Apoptosis/efectos de los fármacos , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Humanos , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Neoplasias/patología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIMS: Although photodynamic therapy (PDT) can relieve esophageal obstruction and prolong survival time of patients with esophageal cancer, it can induce nuclear factor-kappa B (NF-κB) activation in many cancers, which plays a negative role in PDT. Dihydroartemisinin (DHA), the most potent artemisinin derivative, can enhance the effect of PDT on esophageal cancer cells. However, the mechanism is still unclear. METHODS: We generated stable cell lines expressing the super-repressor form of the NF-κB inhibitor IκBα and cell lines with lentivirus vector-mediated silencing of the HIF-1α gene. Esophageal xenograft tumors were created by subcutaneous injection of Eca109 cells into BALB/c nude mice. Four treatment groups were analyzed: a control group, photosensitizer alone group, light alone group, and PDT group. NF-κB expression was detected by an electrophoretic mobility shift assay, hypoxia-inducible factor α (HIF-1α) and vascular endothelial growth factor (VEGF) by real-time PCR, NF-κB, HIF-1α, and VEGF protein by western blot, and Ki-67, HIF-1α, VEGF, and NF-κB protein by immunohistochemistry. RESULTS: PDT increased NF-κB activity and the gene expression of HIF-1α and VEGF in vitro and in vivo. In contrast, the DHA groups, particularly the combined DHA and PDT treatment group, abolished the effect. The combined treatment significantly inhibited tumor growth in vitro and in vivo. NF-κB activity and HIF-1α expression were also reduced in the stable IκBα expression group, whereas the former showed no change in HIF-1α-silenced cells. CONCLUSION: DHA might increase the sensitivity of esophageal cancer cells to PDT by inhibiting the NF-κB/HIF-1α/VEGF pathway.
Asunto(s)
Artemisininas/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Crecimiento Endotelial Vascular/metabolismo , Ácido Aminolevulínico/uso terapéutico , Animales , Artemisininas/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Bigelovii A is a 30nortriterpenoid glycoside, isolated from Salicornia bigelovii Torr. Until now, the effect of Bigelovii A on breast cancer treatment was unknown. The present research indicated that Bigelovii A significantly inhibited the proliferation of human breast cancer cells (MCF7, MDAMB231 and MDAMB468) in a concentrationdependent manner. It was particularly effective in MCF7 cells, with an IC50 value of 4.10±1.19 µM. The antiproliferative effect of Bigelovii A was ascribed to the induction of apoptosis, which was characterized by chromatin condensation, externalization of phosphatidylserine on the plasma membrane, hypodiploid DNA, activation of caspases and poly (ADPribose) polymerase cleavage. Furthermore, Bigelovii A reduced B-cell lymphoma 2 (Bcl2) and Bcell lymphomaextra large (Bclxl) expression and caused disruption of mitochondrial membrane potential, which are indicative features of mitochondriadependent apoptotic signals. It was also identified that Bigelovii A downregulated the constitutive activation of nuclear factor (NF)κB, as indicated by the electrophoretic mobility gel shift assay and immunocytochemistry. Furthermore, Bigelovii A suppressed constitutive IκBα phosphorylation via inhibition of IκB kinase activity. In addition to the effects on Bcl2 and Bclxl, Bigelovii A also downregulated the expression of the NFκBregulated gene products, Cyclin D1 and cyclooxygenase2. This led to the induction of apoptosis and arrest of cells at the G1 phase of the cell cycle.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Chenopodiaceae/química , Regulación Neoplásica de la Expresión Génica , FN-kappa B/genética , Saponinas/farmacología , Triterpenos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/genética , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Células MCF-7 , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Saponinas/aislamiento & purificación , Transducción de Señal , Triterpenos/aislamiento & purificación , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Tanshinol, a water-soluble component isolated from Salvia miltiorrhiza Bunge, has a variety of biological activities involving anti-fibrotic effect. However, the exact role and the underlying mechanisms remain largely unclear. This study mainly focused on the anti-hepatic fibrotic activities and mechanisms of tanshinol on carbon tetrachloride (CCl4)-induced liver fibrosis in rats via anti-oxidative and anti-inflammation pathways. The rats were divided into 4 groups as follows: control, model, tanshinol 20 mg/kg, and tanshinol 40 mg/kg. Except for the control group, CCl4 was used to induce liver fibrosis processing for 8 weeks, meanwhile rats in tanshinol groups were intraperitoneally injected with additional tanshinol. Control group simultaneously received the same volumes of olive oil and saline. The potentially protective effect and mechanisms of tanshinol on liver fibrosis in rats were evaluated. The serum levels of alanine aminotransferase, aspartate aminotransferase, and total bilirubin were obviously lower in the tanshinol treatment groups related to model group. Compared with the model group, the levels of hyaluronic acid, type IV collagen, Laminin (LN), and procollagen III peptide (PIIIP) in serum were significantly decreased after tanshinol treatment. Furthermore, tanshinol could regulate Nrf2/HO-1 signaling pathway and increase the level of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and also decrease the level of malondialdehyde (MDA) to against damage induced by oxidative stress. Simultaneously tanshinol could regulate nuclear factor kappa B signaling pathway to inhibit expression of inflammation factors, including transforming growth factor-ß, tumor necrosis factor-α, Cox-2, interleukin-1ß, and interleukin-6. In summary, our research demonstrated that tanshinol has protective effect on CCl4-induced liver fibrosis via inhibiting oxidative stress and inflammation, which may be associated with the regulation of nuclear factor erythroid2-related factor 2/hemeoxygenase-a and nuclear factor kappa B/inhibitor of kappa B alpha signaling pathways.
Asunto(s)
Ácidos Cafeicos/farmacología , Hemo-Oxigenasa 1/antagonistas & inhibidores , Cirrosis Hepática/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Animales , Ácidos Cafeicos/administración & dosificación , Tetracloruro de Carbono , Hemo-Oxigenasa 1/metabolismo , Inyecciones Intraperitoneales , Cirrosis Hepática/inducido químicamente , Factor 2 Relacionado con NF-E2/metabolismo , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Conditional gene targeting at porcine IκBα may be a solution to delayed xenograft rejection, the main barrier to xenotransplantation. An oligonucleotidebased method was applied to construct the vector for conditional targeting of porcine IκBα. This method was free from PCR amplification during the assembling of the different vector elements, avoiding introduction of unwanted mutations. With the help of short doublestranded DNA fragments produced by annealing oligonucleotides, nondirectional cloning has also been avoided. By making the best of directional cloning, a highly complex targeting vector was built within 3 weeks. The present study also explained why the two recombinationbased methods (recombineering and gateway recombination), although having demonstrated to be highly efficient in constructing ordinary targeting vectors, were not appropriate in this context. The description in the present study of an additional method to efficiently construct targeting vectors is suggested to introduce more flexibility in the field therefore helping to meet the different needs of the researchers.
Asunto(s)
Ingeniería Genética/métodos , Vectores Genéticos , Inhibidor NF-kappaB alfa/genética , Oligonucleótidos/genética , Animales , Clonación Molecular , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Recombinación Genética , Porcinos , Trasplante Heterólogo/efectos adversosRESUMEN
Allantopyrone A is a fungal metabolite that uniquely possesses two α,ß-unsaturated carbonyl moieties. We recently reported that allantopyrone A inhibited the nuclear factor-κB (NF-κB) signaling pathway induced by tumor necrosis factor (TNF)-α in human lung carcinoma A549 cells. In the present study, the mechanism by which allantopyrone A inhibits the TNF-α-induced signaling pathway was investigated in more detail. Allantopyrone A blocked extensive modifications to receptor-interacting protein 1 (RIP1) in the TNF receptor 1 (TNF-R1) complex. Allantopyrone A augmented the high-MW bands of TNF-R1, TNF receptor-associated factor 2, RIP1, the NF-κB subunit RelA and inhibitor of NF-κB kinase ß in A549 cells, suggesting that it binds to and promotes the crosslinking of these proteins. The extracellular cysteine-rich domains of TNF-R1 were crosslinked by allantopyrone A more preferentially than its intracellular portion. The present results demonstrate that allantopyrone A interferes with multiple components of the TNF-R1 complex and blocks RIP1 modifications in the TNF-α-induced NF-κB signaling pathway.
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Antibióticos Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Pironas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Células A549 , Cisteína/química , Cisteína/metabolismo , Genes Reporteros/efectos de los fármacos , Células HEK293 , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Peso Molecular , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/química , Inhibidor NF-kappaB alfa/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/química , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factor 2 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 2 Asociado a Receptor de TNF/química , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
DNA vaccination is an attractive approach to elicit tumor-specific cytotoxic CD8+ T lymphocytes (CTL), which can mediate protective immunity against tumors. To initiate CTL responses, antigen-encoding plasmids employed for DNA vaccination need to activate dendritic cells (DC) through the stimulation of DNA-sensing innate immune receptors that converge in the activation of the master transcription factor NF-κB. To this end, NF-κB repressor IκBα needs to be degraded, allowing NF-κB to translocate to the nucleus and transcribe proinflammatory target genes, as well as its repressor IκBα. Therefore, NF-κB activation is self-limited by de novo synthesis of IκBa, which sequesters NF-κB in the cytosol. Hence, we tested whether co-delivering a shRNA-based adjuvant able to silence IκBα expression would further promote DNA-induced NFκB activation, DC activation and tumor-protective CTL responses induced by DNA vaccination in a preclinical model. First, an IκBα-targeting shRNA plasmid (shIκBα) was shown to reduce IκBα expression and promote NFκB-driven transcription in vitro, as well as up-regulate inflammatory target genes in vivo. Then, we showed that intradermal DNA electroporation induced the migration of skin migratory dendritic cells to draining lymph nodes and maturation of dermal dendritic cells (dDC). Interestingly, shIκBα further promoted the migration of mature skin migratory dendritic cells, in particular dDC, which are specialized in antigen cross-presentation and activation of CD8+ T cells. Consistently, mice vaccinated with a plasmid encoding the melanoma-associated antigen tyrosinase-related protein 2 (TRP2) in combination with shIκBα enhanced TRP2-specific CTL responses and reduced the number of lung melanoma foci in mice challenged with intravenous injection of B16F10 cells. Moreover, therapeutic vaccination with pTRP2 and shIκBα delayed the growth of B16F10 melanoma subcutaneous tumors. Our data suggest that adjuvants promoting NF-κB activation represent an attractive strategy to boost DC activation and promote the generation of tumor-protective CTL responses elicited by DNA vaccines.
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Vacunas contra el Cáncer/inmunología , Células de Langerhans/inmunología , Ganglios Linfáticos/inmunología , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/metabolismo , Animales , Vacunas contra el Cáncer/administración & dosificación , Movimiento Celular , Modelos Animales de Enfermedad , Células de Langerhans/fisiología , Pulmón/patología , Melanoma/patología , Melanoma/terapia , Ratones Endogámicos C57BL , Resultado del Tratamiento , Vacunación , Vacunas de ADN/administración & dosificaciónRESUMEN
Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high-intensity light-emitting diode array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.
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Ensayos Analíticos de Alto Rendimiento , Proteínas Luminiscentes/genética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Transcripción Genética/efectos de los fármacos , Bortezomib/farmacología , Línea Celular , Genes Reporteros , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Luminiscentes/antagonistas & inhibidores , Proteínas Luminiscentes/metabolismo , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We investigated the anti-inflammatory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid (HLEDA)-a lupane-type triterpene isolated from leaves of Acanthopanax gracilistylus W. W.Smith (AGS), as well as the underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results demonstrated that HLEDA concentration-dependently reduced the production of nitric oxide (NO), signiï¬cantly suppressed LPS-induced expression of TNF-α and IL-1ß at the mRNA and protein levels in RAW264.7 cells. Further analysis revealed that HLEDA could reduce the secretion of High Mobility Group Box 1 (HMGB1). Additionally, the results showed that HLEDA efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that HLEDA exerts anti-inflammatory properties in LPS-induced macrophages, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. These results warrant further studies that would concern candidate therapy for diseases, such as fulminant hepatitis and rheumatology of triterpenoids in AGS.
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Antiinflamatorios/farmacología , Medicamentos Herbarios Chinos/química , Proteína HMGB1/antagonistas & inhibidores , Interleucina-1beta/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Triterpenos/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antiinflamatorios/aislamiento & purificación , Línea Celular , Relación Dosis-Respuesta a Droga , Eleutherococcus , Expresión Génica , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamación , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Fosforilación/efectos de los fármacos , Triterpenos/aislamiento & purificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Modulators of the over-activation of myeloid dendritic cells (mDCs) by Toll-like receptors (TLRs) have an advantage in the treatment of systemic lupus erythematosus (SLE). This study was designed to evaluate the effects of FC-99, a novel benzenediamine derivative, on TLR-induced activation of mDCs, and to assess the efficacy of FC-99 in a murine model of SLE. In vitro, FC-99 inhibited the phenotypic (CD40 and MHC-II) and functional activation (IL-12 and CXCL10) of mDCs induced by TLR ligands. In vivo, MRLlpr/lpr mice displayed renal diseases associated with increased levels of proteinuria and immunoglobulin, which were ameliorated by FC-99. Enhanced accumulation and activation of mDCs in lymphoid organs was also impaired by FC-99. Additionally, FC-99 inhibited the activation of IκB-α and upregulated the expression of TNFα-induced protein 3 (TNFAIP3) in vitro and in vivo. These results indicate that FC-99 modulates TLR-induced activation of mDCs and ameliorates lupus-like syndrome in MRLlpr/lpr mice. This effect is closely associated with the inhibition of IκB-α and upregulation of TNFAIP3.
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Células Dendríticas/efectos de los fármacos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Células Mieloides/citología , Fenilendiaminas/química , Fenilendiaminas/farmacología , Receptores Toll-Like/metabolismo , Animales , Células Dendríticas/citología , Células Dendríticas/inmunología , Diseño de Fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inmunomodulación/efectos de los fármacos , Lupus Eritematoso Sistémico/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Fenilendiaminas/uso terapéutico , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have robust neuroprotective effects via its anti-inflammatory, anti-oxidative, and anti-apoptotic functions. In an effort to develop novel EP derivatives with greater protective potencies than EP, we generated four EP isosteres, among them the neuroprotective potency of N,N-diethyl-2-oxopropanamide (DEOPA), in which the ethoxy group of EP was replaced with diethylamine, was far greater than that of EP. When DEOPA was administered intravenously (5 mg/kg) to rat middle cerebral artery occlusion (MCAO) model at 6 hrs post-surgery, it suppressed infarct formation, ameliorated neurological and sensory/motor deficits, and inhibited microglial activation and neutrophil infiltrations in the postischemic brain more effectively than EP. In particular, DEOPA markedly suppressed LPS-induced nitrite production and cytokine/chemokine inductions in microglia, neutrophils, and endothelial cells and these effects are attributable to inhibition of the activity of NF-κB by suppressing IκB-α degradation and p65 to DNA binding. In addition, DEOPA suppressed NMDA-induced neuronal cell death in primary cortical neuron cultures by NAD replenishment and suppression of NF-κB activity. Together, these results indicate DEOPA has multi-modal protective effects against ischemic brain damage targeting numerous cell types in the brain and also against other inflammation-related diseases.
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Amidas/farmacología , Antiinflamatorios/farmacología , Encéfalo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Amidas/uso terapéutico , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Encéfalo/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/etiología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Inhibidor NF-kappaB alfa/antagonistas & inhibidores , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/uso terapéutico , Neutrófilos/citología , Neutrófilos/metabolismo , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismoRESUMEN
Post translational modifications, ubiquitination and its reversal by deubiquitination play an important role in regulating innate immune system. USP12 is a poorly studied deubiquitinase reported to regulate T-cell receptor signalling however the functional role of USP12 in macrophages, the principal architects of inflammation, is unknown. Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system. Here, we observed that the expression of USP12 was altered in time dependent manner in LPS stimulated RAW 264.7 macrophages at both mRNA and protein levels as revealed by qPCR and western blot analysis, respectively. Further analysis showed that LPS reduced the levels of Sp1 which enhanced the transcriptional levels of USP12. We observed that siRNA mediated ablation of USP12 expression in mouse macrophages suppressed the induction of LPS-induced iNOS and IL-6 expression but failed to alter IFN-ß synthesis, oxidative stress and phagocytic ability of macrophages. Mechanistic analysis suggest that USP12 may be required for the activation of NFκB pathway as knockdown of USP12 reduced the inhibitory phosphorylation of IκBα, a well characterized inhibitor of NFκB nuclear translocation. Further, USP12 was observed to be required for LPS elicited phosphorylation of ERK1/2 and p38. Collectively, our data suggest that USP12 may be a key mediator of LPS stimulated macrophage responses.