RESUMO
Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.
Assuntos
Artrite Reumatoide , Vitamina B 12 , Camundongos , Animais , Desiminases de Arginina em Proteínas/metabolismo , Hidrolases/metabolismo , Proteína-Arginina Desiminase do Tipo 4 , Citrulina/metabolismo , Anticorpos , ColágenoRESUMO
BACKGROUND: The role of nerve growth factor (NGF)/tyrosine kinase A receptor (TrKA) signaling, which is activated in a variety of pain states, in regulating membrane-associated δ-opioid receptor (mDOR) expression is poorly understood. The hypothesis was that elevated NGF in bone cancer tumors could upregulate mDOR expression in spinal cord neurons and that mDOR agonism might alleviate bone cancer pain. METHODS: Bone cancer pain (BCP) was induced by inoculating Lewis lung carcinoma cells into the femoral marrow cavity of adult C57BL/6J mice of both sexes. Nociceptive behaviors were evaluated by the von Frey and Hargreaves tests. Protein expression in the spinal dorsal horn of animals was measured by biochemical analyses, and excitatory synaptic transmission was recorded in miniature excitatory synaptic currents. RESULTS: The authors found that mDOR expression was increased in BCP mice (BCP vs. sham, mean ± SD: 0.18 ± 0.01 g vs. mean ± SD: 0.13 ± 0.01 g, n = 4, P < 0.001) and that administration of the DOR agonist deltorphin 2 (Del2) increased nociceptive thresholds (Del2 vs. vehicle, median [25th, 75th percentiles]: 1.00 [0.60, 1.40] g vs. median [25th, 75th percentiles]: 0.40 [0.16, 0.45] g, n = 10, P = 0.001) and reduced miniature excitatory synaptic current frequency in lamina II outer neurons (Del2 vs. baseline, mean ± SD: 2.21 ± 0.81 Hz vs. mean ± SD: 2.43 ± 0.90 Hz, n = 12, P < 0.001). Additionally, NGF expression was increased in BCP mice (BCP vs. sham, mean ± SD: 0.36 ± 0.03 vs. mean ± SD: 0.16 ± 0.02, n = 4, P < 0.001), and elevated NGF was associated with enhanced mDOR expression via TrKA signaling. CONCLUSIONS: Activation of mDOR produces analgesia that is dependent on the upregulation of the NGF/TrKA pathway by increasing mDOR levels under conditions of BCP in mice.
Assuntos
Analgesia , Neoplasias Ósseas , Dor do Câncer , Ratos , Masculino , Feminino , Camundongos , Animais , Dor do Câncer/tratamento farmacológico , Receptores Proteína Tirosina Quinases , Ratos Sprague-Dawley , Fator de Crescimento Neural/metabolismo , Camundongos Endogâmicos C57BL , Dor , Neoplasias Ósseas/complicações , Corno Dorsal da Medula Espinal , Receptores OpioidesRESUMO
OBJECTIVE: To evaluate the early and mid-term outcomes of open repair in patients with thoracoabdominal aortic aneurysm (TAAA) after thoracic endovascular aortic repair (TEVAR). METHODS: This was a retrospective single center study. Data were retrospectively collected and analyzed for consecutive patients undergoing open TAAA repair (TAAAR) after TEVAR from November 2016 to June 2021. Indications for TAAAR included aneurysm progression due to endoleak, persisted false lumen perfusion, proximal/distal disease progression, and aorta rupture. The risk factor of operative mortality was analyzed by multivariable logistic regression model and the survival was evaluated by Kaplan-Meier. RESULTS: Sixty-three patients who met the inclusion criteria for the study were identified. The mean age at TAAAR was 41 ± 12 years and 43 (68.3%) were male. Marfan syndrome (MFS) was presented in 39 patients (61.9%). 60 (95.2%) patients presented with post-dissection aneurysm and 3 (4.8%) patients with degenerative aneurysm. The extent of TAAA was Crawford I in 9 (14.3%), II in 22 (34.9%), III in 23 (36.5%), and IV in 9 (14.3%). Emergent TAAAR was done in 10 (15.9%) patients, and deep hypothermic circulatory arrest was used in 22 (34.6%). Endograft was explanted in 31 (49.2%). Operative mortality was 11 (17.5%). Stroke, paraplegia, and acute kidney failure occurred in 5 (7.9%), 7 (11.1%), and 6 (9.5%) patients, respectively. Pulmonary complications occurred in 19 (30.2%) patients. The estimated survival was 74.8 ± 4.9% at 5 years. Late reoperations were performed in 2 patients at 2.5 years and 1.3 years, respectively. CONCLUSIONS: In this series of TAAA after TEVAR, TAAAR was related with a high risk of operative mortality and morbidity and the midterm outcomes represented a durable treatment and were respectable.
Assuntos
Aneurisma da Aorta Torácica , Aneurisma da Aorta Toracoabdominal , Implante de Prótese Vascular , Procedimentos Endovasculares , Humanos , Masculino , Feminino , Correção Endovascular de Aneurisma , Prótese Vascular/efeitos adversos , Estudos Retrospectivos , Implante de Prótese Vascular/efeitos adversos , Resultado do Tratamento , Aneurisma da Aorta Torácica/cirurgia , Fatores de Risco , Procedimentos Endovasculares/efeitos adversos , Complicações Pós-OperatóriasRESUMO
The purpose of this study is to analyze the clinical characteristics of patients with Van der Woude syndrome (VWS) and to detect variations in each patient. Finally, the combination of genotype and phenotype can make a clear diagnosis of VWS patients with different phenotype penetrance.Five Chinese VWS pedigree were enrolled. Whole exome sequencing of the proband was performed, and the potential pathogenic variation was further verified by Sanger sequencing in the patient and their parents. The human mutant IRF6 coding sequence was generated from the human full-length IRF6 plasmid by site-directed mutagenesis and cloned into the GV658 vector, RT-qPCR and Western blot were used to detect the expression of IRF6.We found one de novo nonsense variation (p. Gln118Ter) and three novel missense variations (p. Gly301Glu, p. Gly267Ala, and p. Glu404Gly) co-segregated with VWS. RT-qPCR analysis revealed that p. Glu404Gly significantly reduced the expression level of IRF6 mRNA. Western blot of cell lysates confirmed that IRF6 p. Glu404Gly abundance levels were lower than those for IRF6 wild type.This discovery of the novel variation (IRF6 p. Glu404Gly) expands the spectrum of known variations in VWS in Chinese humans. Genetic results combined with clinical phenotypes and differential diagnosis points from other diseases can make a definitive diagnosis and provide genetic counseling for families.
RESUMO
OBJECTIVE: Paired box 7 (PAX7) has been considered as a candidate gene for non-syndromic cleft lip with or without palate (NSCL/P). However, there is no research for the XXX, and previous studies concentrated on limited variants. This study aimed to conduct sufficiently dense and powerful scans of variants at PAX7 and explored the roles of variants at PAX7 in NSCL/P among the XXX. DESIGN: Targeted region sequencing was performed to thoroughly screen variations, followed by a two-phase association analysis. 159 NSCL/P cases and 542 controls were analyzed in phase 1. Then in phase 2, the validation study was performed using 1626 cases and 2255 controls. We also explored the roles of variants at PAX7 gene in NSCL/P subtypes. Additionally, indirect associations were found by calculating LD and haplotypes. SETTING: The study was conducted in XXX. PATIENTS, PARTICIPANTS: 159 NSCL/P cases and 542 controls were analyzed in phase 1. Then in phase 2, the validation study was performed using 1626 cases and 2255 controls. INTERVENTIONS: Blood samples were collected. MAIN OUTCOME MEASURES: To explore the association analysis between variants at PAX7 and NSCL/P in XXX. RESULTS: The results showed that rs2236810, rs114882979 and rs2236804 were significantly associated with NSCL/P, which were predicted to have regulatory functions. Besides, variants at PAX7 function differently in the NSCL/P subtypes. We also discovered a PAX7 missense variant, NM_001135254 p.A369â V (NM_002584.2:c.1106C > T). CONCLUSIONS: In summary, we confirmed 3 SNPs at PAX7 were significantly associated with NSCL/P in XXX and identified a missense variant, NM_001135254 p.A369â V (NM_002584.2:c.1106C > T).
RESUMO
BACKGROUND: People with human immunodeficiency virus (PWH) have an increased risk of developing chronic obstructive pulmonary disease (COPD). METHODS: We phenotyped lung macrophages in 4 subgroups-M1 (CD40+CD163-), M2 (CD40-CD163+), double positives (CD40+CD163+), and double negatives and (CD40-CD163-)-and we determined their phagocytic capacity in PWH with and without COPD. RESULTS: People with human immunodeficiency virus with COPD have more double-negative macrophages (84.1%) versus PWH without (54.3%) versus controls (23.9%) (P=.004) and reduced phagocytosis (P=.012). Double-negative macrophages had the worst phagocytic capacity (P<.001). CONCLUSIONS: People with human immunodeficiency virus with COPD have an abundance of nonpolarized macrophages, which have poor phagocytic capacity and therefore predispose PWH to increased risk of disease progression.
Assuntos
Macrófagos Alveolares , Doença Pulmonar Obstrutiva Crônica , HIV , Humanos , Pulmão , Macrófagos , FagocitoseRESUMO
Rationale: Inhaled corticosteroids (ICS) are commonly prescribed with long-acting ß2-agonists (LABA) in chronic obstructive pulmonary disease (COPD). To date, the effects of ICS therapy on the airway microbiome in COPD are unknown. Objectives: To determine the effects of ICS/LABA on the airway microbiome of patients with COPD. Methods: Clinically stable patients with COPD were enrolled into a 4-week run-in period during which ICS was discontinued and all participants were placed on formoterol (Form) 12 µg twice daily (BID). The participants were then randomized to budesonide/formoterol (Bud + Form; 400/12 µg BID), fluticasone/salmeterol (Flu + Salm; 250/50 µg BID), or formoterol only (12 µg BID) for 12 weeks. Participants underwent bronchoscopy before and after the 12-week treatment period. The primary endpoint was the comparison of changes in the airway microbiome over the trial period between the ICS/LABA and LABA-only groups. Measurements and Main Results: Sixty-three participants underwent randomization: Bud + Form (n = 20), Flu + Salm (n = 22), and Form (n = 21) groups; 56 subjects completed all visits. After the treatment period, changes in α-diversity were significantly different across groups, especially between Flu + Salm and Form groups (Δrichness: P = 0.02; ΔShannon index: P = 0.03). Longitudinal differential abundance analyses revealed more pronounced microbial shifts from baseline in the fluticasone (vs. budesonide or formoterol only) group. Conclusions: Fluticasone-based ICS/LABA therapy modifies the airway microbiome in COPD, leading to a relative reduction in α-diversity and a greater number of bacterial taxa changes. These data may have implications in patients who develop pneumonia on ICS. Clinical trial registered with www.clinicaltrials.gov (NCT02833480).
Assuntos
Corticosteroides/uso terapêutico , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Combinação de Medicamentos , Microbiota/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/uso terapêutico , Administração por Inalação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Anthocyanins are water-soluble pigments in plants known for their photoprotective role against photoinhibitory and photooxidative damage under high light (HL). However, it remains unclear whether light-shielding or antioxidant activity plays a major role in the photoprotection exerted by anthocyanins under HL stress. To shed light on this question, we analyzed the physiological and biochemical responses to HL of three Arabidopsis thaliana lines (Col, chi, ans) with different light absorption and antioxidant characteristics. Under HL, ans had the highest antioxidant capacity, followed by Col, and finally chi; Col had the strongest light attenuation capacity, followed by chi, and finally ans. The line ans had weaker physiological activity of chloroplasts and more severe oxidative damage than chi after HL treatment. Col with highest photoprotection of light absorption capacity had highest resistance to HL among the three lines. The line ans with high antioxidant capacity could not compensate for its disadvantages in HL caused by the absence of the light-shielding function of anthocyanins. In addition, the expression level of the Anthocyanin Synthase (ANS) gene was most upregulated after HL treatment, suggesting that the conversion of colorless into colored anthocyanin precursors was necessary under HL. The contribution of anthocyanins to flavonoids, phenols, and antioxidant capacity increased in the late period of HL, suggesting that plants prefer to synthesize red anthocyanins (a group of colored antioxidants) over other colorless antioxidants to cope with HL. These experimental observations indicate that the light attenuation role of anthocyanins is more important than their antioxidant role in photoprotection.
Assuntos
Adaptação Ocular/fisiologia , Antocianinas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Proteção Radiológica , Luz Solar/efeitos adversos , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Mutação , Estresse Oxidativo/fisiologia , Fenótipo , Fotossíntese/fisiologiaRESUMO
As part of the immune response, leukocytes can directly transmigrate through the body of endothelial cells or through the gap between adjacent endothelial cells. These are known, respectively, as the transcellular and paracellular route of diapedesis. What determines the usage of one route over the other is unclear. A recently proposed tenertaxis hypothesis claims that leukocytes choose the path with less mechanical resistance against leukocyte protrusions. We examined this hypothesis using numerical simulation of the mechanical resistance during paracellular and transcellular protrusions. By using parameters based on human lung endothelium, our results show that the required force to breach the endothelium through the transcellular route is greater than paracellular route, in agreement with experiments. Moreover, experiments have demonstrated that manipulation of the relative strength between the two routes can make the transcellular route preferable. Our simulations have demonstrated this reversal and thus tentatively confirmed the hypothesis of tenertaxis.
Assuntos
Células Endoteliais , Migração Transendotelial e Transepitelial , Movimento Celular , Humanos , Leucócitos , Testes MecânicosRESUMO
Interruption of the Warburg effect - the observation that un-stimulated macrophages reprogram their core metabolism from oxidative phosphorylation toward aerobic glycolysis to become pro-inflammatory M1 macrophages upon stimulation - is an emerging strategy for the treatment of cancer and anti-inflammatory diseases such as rheumatoid arthritis. We studied this process with view to the discovery of novel therapeutics, and found that tylophorine-based compounds targeted a ribonucleoprotein complex containing caprin-1 and mRNAs of c-Myc and HIF-1α in LPS/IFN-γ stimulated Raw264.7 cells, diminished the protein levels of c-Myc and HIF-1α, and consequently downregulated their targeted genes that are associated with the Warburg effect, as well as the pro-inflammatory iNOS and COX2. The tylophorine-based compound DBQ 33b significantly meliorated the severity and incidence of type II collagen-monoclonal antibody-induced rheumatoid arthritis and diminished gene expressions of c-Myc, HIF-1α, iNOS, COX2, TNFα, and IL-17A in vivo. Moreover, pharmacological inhibition of either c-Myc or HIF-1α exhibited similar effects as the tylophorine-based compound DBQ 33b, even though inhibition of c-Myc reversed the induction of iNOS and COX2 in LPS/IFN-γ stimulated Raw264.7 cells to a lesser degree. Therefore, simultaneous inhibition of both c-Myc and HIF-1α is efficacious for anti-inflammation in vitro and in vivo and merits further study.
Assuntos
Alcaloides/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Indolizinas/uso terapêutico , Fenantrenos/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Alcaloides/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Proteínas de Ciclo Celular , Ciclo-Oxigenase 2/genética , Edema/tratamento farmacológico , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indolizinas/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/genética , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genéticaRESUMO
Cardenolides are plant-derived toxic substances. Their cytotoxicity and the underlying mechanistic signaling axes have been extensively documented, but only a few anti-viral activities of cardenolides and the associated signaling pathways have been reported. Previously, we reported that a variety of cardenolides impart anti-transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells, through targeting of the cell membrane sodium/potassium pump, Na+/K+-ATPase. Herein, we further explore the potential signaling cascades associated with this anti-TGEV activity in ST cells. Ouabain, a representative cardenolide, was found to potently diminish TGEV titers and inhibit the TGEV-induced production of IL-6 in a dose dependent manner, with 50% inhibitory concentrations of 37â¯nM and 23â¯nM respectively. By pharmacological inhibition and gene silencing, we demonstrated that PI3K_PDK1_RSK2 signaling was induced in TGEV-infected ST cells, and ouabain imparted a degree of anti-TGEV activity via further augmentation of this existing PI3K_PDK1 axis signaling, in a manner dependent upon its association with the Na+/K+-ATPase. Finally, inhibition of PI3K by LY294002 or PDK1 by BX795 antagonized the anti-viral activity of ouabain and restored the TGEV virus titer and yields. This finding is the first report of a PI3K_PDK1 signaling axis further induced by ouabain and implicated in the suppression of TGEV activity and replication; greatly illuminates the underlying mechanism of cardenolide toxicity; and is expected to result in one or more anti-viral applications for the cardenolides in the future.
Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Ouabaína/farmacologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/biossíntese , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Cromonas/farmacologia , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inativação Gênica , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Camundongos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/genética , Pirimidinas/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil , Tiofenos/farmacologiaRESUMO
In surface-based biosensors, the nonspecific or undesired adsorption of the probe is an important characteristic that is typically difficult to measure and therefore to control or eliminate. A methodology for measuring and then minimizing or eliminating this problem on gold surfaces, readily applicable to many common surface modifications is presented. Combining electrochemical perturbation and fluorescence microscopy, we show that the potential at which the adsorbed species is removed can be used as an estimate of the strength of the adsorbate-surface interaction. This desorption potential can be easily measured through an increase in fluorescence intensity as the potential is manipulated. Furthermore, this method can be used to evaluate strategies for preventing or removing nonspecific adsorption. This is demonstrated for a wide variety of surface modifications, from strongly chemisorbed monolayers such as thiol self-assembled monolayers (SAMs) to physisorbed monolayers as well as for complex surface structures like peptide and DNA mixed-component SAMs. The use of a coadsorption strategy or small magnitude potential-step cycles was shown to significantly decrease the amount of nonspecifically or noncovalently bound probe, creating better defined surfaces.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Ouro/química , Ácidos Nucleicos/análise , Peptídeos/análise , Adsorção , Microscopia de Fluorescência , Propriedades de SuperfícieRESUMO
A series of naturally occurring cardenolides that exhibit potent anti-transmissible gastroenteritis virus (TGEV) activity in swine testicular (ST) cells has been identified. In an immunofluorescence assay, these cardenolides were found to diminish the expressions of TGEV nucleocapsid and spike protein, which was used as an indication for viral replication; block TGEV infection induced apoptosis and cytopathic effects; and impart the same trend of inhibitory activity against Na+/K+-ATPase as for anti-TGEV activity. The viral titer inhibition was found to take place in a dose-dependent manner. Knocking down expression of Na+/K+-ATPase, the cellular receptor of cardenolides, in ST cells was found to significantly impair the susceptibility of ST cells to TGEV infectivity. Thus, we have identified Na+/K+-ATPase as an anti-viral drug target and its antagonists, cardenolides, a novel class of anti- TGEV agents.
Assuntos
Antivirais/farmacologia , Cardenolídeos/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Vírus da Gastroenterite Transmissível/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Inativação Gênica , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , Vírus da Gastroenterite Transmissível/fisiologia , Replicação ViralAssuntos
Infecções por Coronavirus/epidemiologia , Pandemias/estatística & dados numéricos , Peptidil Dipeptidase A/sangue , Pneumonia Viral/epidemiologia , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Fumar/epidemiologia , Idoso , Manuseio das Vias Aéreas/métodos , Enzima de Conversão de Angiotensina 2 , Biomarcadores/sangue , COVID-19 , Comorbidade , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/terapia , Feminino , Saúde Global , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/diagnóstico , Pneumonia Viral/terapia , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Medição de Risco , Índice de Gravidade de Doença , Fumar/efeitos adversos , Populações VulneráveisRESUMO
Hypertension is the most prevalent non-communicable disease, affecting billions of people worldwide. Discovery and development of natural antihypertensive lead compounds or drugs are important to resolve the limitations of existing antihypertensive drug safety and resistance. This investigation verified that carnosic acid (CA), an important active ingredient of rosemary, an edible spice plant, indicates a significant anti-hypertensive activity in spontaneous hypertension rats by targeting AT1R. Moreover, our research indicated that CA shared a comparable antagonistic mechanism with established synthetic angiotensin II receptor blockers (ARBs), as it occupies the binding sites of Angiotensin II (AngII) at His6 and Pro7 within the AT1R's ligand-binding pocket. Notably, CA exerted better anti-hypertensive activity since it could not break the Asn1113.35-Asn2957.46 hydrogen bond to stabilize the AT1R inactive state. As the first potent AT1R antagonist identified in a natural food source, CA is poised to become a novel anti-hypertensive lead compound, distinguished by its unique skeleton structure different from conventional ARBs. This research lays a valuable theoretical groundwork for the future exploration of CA and rosemary extract in both fundamental studies and clinical applications.
RESUMO
Tylophorine, a phenanthroindolizidine alkaloid, is the major medicinal constituent of herb Tylophora indica. Tylophorine treatment increased the accumulation of c-Jun protein, a component of activator protein 1 (AP1), in carcinoma cells. An in vitro kinase assay revealed that the resultant c-Jun phosphorylation was primarily mediated via activated c-Jun N-terminal protein kinase (JNK). Moreover, flow cytometry indicated that ectopically overexpressed c-Jun in conjunction with tylophorine significantly increased the number of carcinoma cells that were arrested at the G1 phase. The tylophorine-mediated downregulation of cyclin A2 protein levels is known to be involved in the primary G1 arrest. Chromatin immunoprecipitation and reporter assays revealed that tylophorine enhanced the c-Jun downregulation of the cyclin A2 promoter activity upon increased binding of c-Jun to the deregulation AP1 site and decreased binding to the upregulation activating transcription factor (ATF) site in the cyclin A2 promoter, thereby reducing cyclin A2 expression. Further, biochemical studies using pharmacological inhibitors and RNA silencing approaches demonstrated that tylophorine-mediated elevation of the c-Jun protein level occurs primarily via two discrete prolonged signaling pathways: (i) the NF-κB/PKCδ_(MKK4)_JNK cascade, which phosphorylates c-Jun and increases its stability by slowing its ubiquitination, and (ii) the PI3K_PDK1_PP2A_eEF2 cascade, which sustains eukaryotic elongation factor 2 (eEF2) activity and thus c-Jun protein translation. To the best of our knowledge, this report is the first to demonstrate the involvement of c-Jun in the anticancer activity of tylophorine and the release of c-Jun translation from a global translational blockade via the PI3K_PDK1_eEF2 signaling cascade.
Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Carcinoma/tratamento farmacológico , Indolizinas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator 1 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Ciclina A2/biossíntese , Ciclina A2/genética , Regulação para Baixo , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Células Hep G2 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Fenantrolinas , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína Quinase C-delta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição AP-1/metabolismo , TylophoraRESUMO
Fucoidans are a class of long chain sulfated polysaccharides and have multiple biological functions. Herein, four natural fucoidans extracted from Fucus vesiculosus, F. serratus, Laminaria japonica and Undaria pinnatifida, were tested for their HCoV-OC43 inhibition and found to demonstrate EC50 values ranging from 0.15 to 0.61 µg/mL. That from U. pinnatifida exhibited the most potent anti-HCoV-OC43 activity with an EC50 value of 0.15 ± 0.02 µg/mL, a potency largely independent of its sulfate content. Comparison of the gene expression profiles of fucoidan-treated and untreated cells infected with HCoV-OC43 revealed that fucoidan treatment effectively diminished HCoV-OC43 gene expressions associated with induced chemokines, cytokines and viral activities. Further studies using a highly fucoidan-resistant HCoV-OC43 determined that fucoidan inhibited HCoV-OC43 infection via interfering with viral entry and led to the identification of the specific site on the N-terminal region of spike protein, that located adjacent to the host cell receptor binding domain, targeted by the virus. Furthermore, in a SARS-CoV-2 pseudovirus neutralization assay, fucoidan also blocked SARS-CoV-2 entry. In vitro and in vivo, fucoidan decreased SARS-CoV-2 viral loads and inhibited viral infection in Calu-3 or Vero E6 cells and SARS-CoV-2 infected hamsters, respectively. Fucoidan was also found to inhibit furin activity, and reported furin inhibitors were found to inhibit viral infection by wild type HCoV-OC43 or SARS-CoV-2. Accordingly, we conclude that fucoidans inhibit coronaviral infection by targeting viral spike protein and host cell furin to interfere with viral entry.
Assuntos
COVID-19 , Coronavirus Humano OC43 , Animais , Cricetinae , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Furina/metabolismoRESUMO
In the original publication [...].