RESUMEN
Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory tract infections. There are no convenient small-animal infection models. Here, we show viral replication in the upper and lower airways of AG129 mice (double IFNα/ß and IFNγ receptor knockout mice) upon intranasal inoculation. By multiplex fluorescence RNAscope and immunohistochemistry followed by confocal microscopy, we demonstrate viral tropism to ciliated cells and club cells of the bronchiolar epithelium. HPIV-3 causes a marked lung pathology. No virus transmission of the virus was observed by cohousing HPIV-3-infected AG129 mice with other mice. Oral treatment with GS-441524, the parent nucleoside of remdesivir, reduced infectious virus titers in the lung, with a relatively normal histology. Intranasal treatment also affords an antiviral effect. Thus, AG129 mice serve as a robust preclinical model for developing therapeutic and prophylactic strategies against HPIV-3. We suggest further investigation of GS-441524 and its prodrug forms to treat HPIV-3 infection in humans.
Asunto(s)
Antivirales , Modelos Animales de Enfermedad , Pulmón , Ratones Noqueados , Virus de la Parainfluenza 3 Humana , Infecciones por Respirovirus , Animales , Pulmón/virología , Pulmón/patología , Pulmón/efectos de los fármacos , Ratones , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Virus de la Parainfluenza 3 Humana/fisiología , Antivirales/farmacología , Infecciones por Respirovirus/tratamiento farmacológico , Infecciones por Respirovirus/virología , Humanos , Replicación Viral/efectos de los fármacos , Femenino , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Receptor de Interferón alfa y beta/deficiencia , Adenosina/análogos & derivados , Adenosina/farmacología , Tropismo Viral , Benzamidas , FtalimidasRESUMEN
Recently a novel genetically modified mouse strain with serum carboxylesterase knocked-out and the human acetylcholinesterase gene knocked-in (KIKO) was created to simulate human responses to nerve agent (NA) exposure and its standard medical treatment. A1 adenosine receptor (A1AR) agonist N-bicyclo-(2.2.1)-hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA) alone is a potent anticonvulsant and neuroprotectant (A/N) in both rat and KIKO mouse soman (GD) seizure models. In this study we utilized the KIKO mouse to evaluate further the basic pharmacologic A/N effects of ENBA as an adjunct to standard NA medical treatments (i.e., atropine sulfate, pralidoxime chloride [2-PAM], and midazolam). Male mice, implanted with cortical electroencephalographic (EEG) electrodes, were pretreated with asoxime (HI-6) and exposed to an epileptogenic dose of GD (33 µg/kg, s.c.) or saline (sham exposure) and then treated 15 min after seizure onset with ENBA at 15 mg/kg, i.p. (a minimum efficacy dose in suppressing NA-induced seizure) alone or as an adjunct to standard medical treatments. We collected EEG activity, seizure suppression outcomes, daily body temperature and weight, heart rate, toxic signs, neuropathology, and lethality data for up to 14 days. Without ENBA, death from NA exposure was 45%, while with ENBA, either alone or in combination with midazolam, the survival improved to 80% and 90%, respectively. Additionally, seizure was suppressed quickly and permanently, toxic signs, hypothermia, and bradycardia recovered by 48 h, and no neuropathology was evident. Our findings confirmed that ENBA is a potent A/N adjunct for delayed medical treatments of NA exposure.
Asunto(s)
Acetilcolinesterasa , Agonistas del Receptor de Adenosina A1 , Modelos Animales de Enfermedad , Convulsiones , Soman , Animales , Soman/toxicidad , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Masculino , Agonistas del Receptor de Adenosina A1/farmacología , Humanos , Ratones , Acetilcolinesterasa/metabolismo , Electroencefalografía , Adenosina/análogos & derivados , Adenosina/farmacología , Ratones Noqueados , Anticonvulsivantes/farmacología , Anticonvulsivantes/toxicidadRESUMEN
Liver transplantation remains the only definitive treatment for end-stage liver diseases. However, the increasing prevalence of fatty liver disease among potential donors exacerbates the shortage of suitable organs. This study evaluates the efficacy of the preservation solution Institut Georges Lopez-2 (IGL-2) compared to Histidine-Tryptophan-Ketoglutarate (HTK) and University of Wisconsin (UW) preservation solutions in mitigating ischemia-reperfusion injury (IRI) in steatotic livers. Using Zucker Obese rat livers, we assessed the impact of 24-h static cold storage (SCS) with each solution on transaminase release, glutathione redox balance, antioxidant enzyme activity, lipoperoxidation, and inflammation markers. IGL-2 and UW solutions demonstrated reduced transaminase and lactate levels compared to HTK, indicating better preservation of liver integrity. IGL-2 maintained a higher reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, suggesting more effective management of oxidative stress. Antioxidant enzyme activities catalase, superoxide dismutase, and glutathione peroxidase (CAT, SOD, GPX) were higher in IGL-2 preserved livers, contributing to decreased oxidative damage. Lipid peroxidation markers and inflammatory markers were lower in IGL-2 than in HTK, indicating reduced oxidative stress and inflammation. Additionally, improved mitochondrial function was observed in the IGL-2 group, correlating with reduced reactive oxygen species (ROS) production and lipid peroxidation. These findings suggest that IGL-2 offers superior preservation of liver viability, reduces oxidative stress, and minimizes inflammation compared to HTK and UW solutions. By maintaining a higher ratio of reduced glutathione and antioxidant enzyme activity, IGL-2 effectively mitigates the harmful effects of ischemia-reperfusion injury. The reduced lipid peroxidation and inflammation in the IGL-2 group further underscore its potential in improving liver transplant outcomes. These results highlight the importance of optimizing preservation solutions to enhance the viability and functionality of donor organs, potentially expanding the donor pool and improving the success rates of liver transplantation. Future research should focus on refining preservation techniques and exploring additional protective agents to further improve organ preservation and transplant outcomes.
Asunto(s)
Adenosina , Alopurinol , Antioxidantes , Hígado Graso , Insulina , Hígado , Soluciones Preservantes de Órganos , Procaína , Rafinosa , Ratas Zucker , Daño por Reperfusión , Animales , Soluciones Preservantes de Órganos/farmacología , Ratas , Rafinosa/farmacología , Insulina/metabolismo , Adenosina/metabolismo , Adenosina/farmacología , Hígado Graso/metabolismo , Hígado Graso/tratamiento farmacológico , Hígado Graso/patología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/prevención & control , Antioxidantes/farmacología , Antioxidantes/metabolismo , Hígado/metabolismo , Hígado/efectos de los fármacos , Hígado/patología , Alopurinol/farmacología , Masculino , Procaína/farmacología , Inflamación/metabolismo , Inflamación/patología , Inflamación/tratamiento farmacológico , Glucosa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Glutatión/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Manitol/farmacología , Isquemia Fría/efectos adversos , Cloruro de Potasio/farmacología , Preservación de Órganos/métodos , Trasplante de Hígado/métodosRESUMEN
The skeleton is a metabolically active organ undergoing continuous remodeling initiated by bone marrow stem cells (BMSCs). Recent research has demonstrated that BMSCs adapt the metabolic pathways to drive the osteogenic differentiation and bone formation, but the mechanism involved remains largely elusive. Here, using a comprehensive targeted metabolome and transcriptome profiling, we revealed that one-carbon metabolism was promoted following osteogenic induction of BMSCs. Methotrexate (MTX), an inhibitor of one-carbon metabolism that blocks S-adenosylmethionine (SAM) generation, led to decreased N6-methyladenosine (m6A) methylation level and inhibited osteogenic capacity. Increasing intracellular SAM generation through betaine addition rescued the suppressed m6A content and osteogenesis in MTX-treated cells. Using S-adenosylhomocysteine (SAH) to inhibit the m6A level, the osteogenic activity of BMSCs was consequently impeded. We also demonstrated that the pro-osteogenic effect of m6A methylation mediated by one-carbon metabolism could be attributed to HIF-1α and glycolysis pathway. This was supported by the findings that dimethyloxalyl glycine rescued the osteogenic potential in MTX-treated and SAH-treated cells by upregulating HIF-1α and key glycolytic enzymes expression. Importantly, betaine supplementation attenuated MTX-induced m6A methylation decrease and bone loss via promoting the abundance of SAM in rat. Collectively, these results revealed that one-carbon metabolite SAM was a potential promoter in BMSC osteogenesis via the augmentation of m6A methylation, and the cross talk between metabolic reprogramming, epigenetic modification, and transcriptional regulation of BMSCs might provide strategies for bone regeneration.
The bone is a self-renewing tissue that continues to reshape throughout life. Bone marrow mesenchymal stem cells (BMSCs) are essential for bone homeostasis as they are capable of osteogenic differentiation. Recent evidence suggests that BMSCs drive the osteogenic differentiation through metabolic reprogramming, but the mechanism remains unclear. In this paper, we explored the metabolic alteration following osteogenic induction of BMSCs and found that one-carbon metabolism was obviously promoted in this process. The underlining mechanisms of the osteogenic potential driven by one-carbon metabolism seem to be its contribution on N6-methyladenosine (m6A) methylation and consequent glycolysis level by providing methyl donor. We demonstrated that one-carbon metabolism-mediated m6A methylation was a potential promoter in BMSC osteogenesis, and metabolic-epigenetic coupling might provide novel therapeutic targets for bone regeneration.
Asunto(s)
Adenosina , Carbono , Osteogénesis , Ratas Sprague-Dawley , S-Adenosilmetionina , Animales , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacología , Osteogénesis/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/metabolismo , Metilación/efectos de los fármacos , Carbono/metabolismo , Carbono/farmacología , Ratas , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Metotrexato/farmacología , Glucólisis/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacosRESUMEN
Hepatocellular carcinoma (HCC) is an aggressive cancer that has an effect on human health. As a first-line drug for HCC, despite its excellent efficacy, lenvatinib (Len) is prone to developing drug resistance in HCC patients. The N6-methyladenosine (m6A) modification is not only related to the development of HCC but also shows great potential in overcoming HCC resistance. Using Dot Blot, our group first screened a small molecule m6A regulator, lobeline (Lob), from a library of 390 compounds (mostly natural products). In vitro experiments demonstrated that Lob could significantly enhance the sensitivity to Len of Len-resistant HCC (HCC/Len) and inhibit migration of resistant cells. In Len-resistant cell-derived and patient-derived xenograft models, Lob could reverse the resistant phenotype, with reductions in tumor volume of 68% and 60%, respectively. Furthermore, MeRIP-m6A sequencing results indicated that the underlying molecular mechanism of Lob reversal of HCC drug resistance was related to UBE3B. Taken together, this study highlighted that Lob, a plant derived natural product, could reverse the resistance of HCC to Len by regulating the m6A levels. It is hoped that this will provide a pharmacological research basis for the clinical treatment of HCC patients.
Asunto(s)
Carcinoma Hepatocelular , Resistencia a Antineoplásicos , Neoplasias Hepáticas , Compuestos de Fenilurea , Quinolinas , Animales , Humanos , Ratones , Adenosina/análogos & derivados , Adenosina/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Productos Biológicos/farmacología , Productos Biológicos/química , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Estructura Molecular , Compuestos de Fenilurea/farmacología , Quinolinas/farmacología , Quinolinas/química , LobelinaRESUMEN
The currently available immune checkpoint therapy shows a disappointing therapeutic efficacy for glioblastoma multiforme (GBM), and it is of great importance to discover better immune checkpoints and develop innovative targeting strategies. The discovered metabolic immune checkpoint ecto-5-nucleotidase (CD73) in a tumor contributes to its immune evasion due to the dysregulation of extracellular adenosine (ADO), which significantly inhibits the function of antitumor T cells and increases the activity of immunosuppressive cells. Herein, we drastically inhibit the expression of CD73 to reduce the production of ADO by using versatile Au@Cu2-xSe nanoparticles (ACS NPs). ACS NPs can decrease the expression of CD73 by alleviating the tumor hypoxia through their Fenton-like reaction to weaken the ADO-driven immunosuppression for enhancing antitumor T cell infiltration and activity of GBM. The copper ions (Cu2+) released from ACS NPs can chelate with disulfide, leading to the formation of cytotoxic bis(N,N-diethyldithiocarbamate)-copper complex (CuET), which can be combined with radiotherapy to recruit more antitumor T cells to infiltrate into the tumor site. Based on the inhibition of CD73 to promote the infiltration and activity of antitumor T cells, a cascade of enhancing GBM immunotherapy effects can be achieved. The significant increase in CD8+ T and CD4+ T cells within the tumor and the memory T cells in the spleen effectively reduces tumor size by 92%, which demonstrates the excellent efficacy of immunotherapy achieved by a combination of metabolic immune checkpoint CD73 inhibition with chemoradiotherapy. This work demonstrates that modulation of CD73-mediated tumor immunosuppression is an important strategy of improving the outcome of GBM immunotherapy.
Asunto(s)
5'-Nucleotidasa , Glioblastoma , Inmunoterapia , Glioblastoma/terapia , Glioblastoma/inmunología , Glioblastoma/patología , Glioblastoma/tratamiento farmacológico , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/antagonistas & inhibidores , Animales , Humanos , Ratones , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Cobre/química , Cobre/farmacología , Oro/química , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Nanopartículas del Metal/química , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/antagonistas & inhibidores , Adenosina/química , Adenosina/farmacologíaRESUMEN
Circular RNAs (circRNAs) are stable non-coding RNAs characterized by the absence of the conventional 5' cap and 3' polyadenylated tail structure. Its involvement in various aspects of cancers underscores its significance in oncology. Elevated expression of circ_0000620 was observed in both lung adenocarcinoma (LUAD) tissues and cell lines. In vitro, experiments demonstrated that the downregulation of circ_0000620 increased cisplatin sensitivity and promoted cell apoptosis while suppressing malignant characteristics such as cell migration and proliferation. Further investigation into the mechanism underlying the increased expression of circ_0000620 revealed that Methyltransferase 3, N6-Adenosine-Methyltransferase Complex Catalytic Subunit (METTL3) mediates the m6A methylation modification of circ_0000620, thereby promoting its stability and expression. Furthermore, circ_0000620 modulates the miR-216b-5p/KRAS axis to influence apoptosis and cisplatin sensitivity in both A549 and H1299 cell lines. These findings were corroborated by in vivo nude mouse experiments, which showed that knockdown of circ_0000620 inhibited tumor growth and proliferation. In summary, METTL3 plays a role in regulating the stability of circ_0000620 expression, and circ_0000620 exerts its effects on LUAD apoptosis and cisplatin sensitivity through the miR-216b-5p/KRAS signaling pathway.
Asunto(s)
Adenocarcinoma del Pulmón , Apoptosis , Cisplatino , Neoplasias Pulmonares , Metiltransferasas , Ratones Desnudos , MicroARNs , ARN Circular , Humanos , Cisplatino/farmacología , Cisplatino/uso terapéutico , MicroARNs/metabolismo , MicroARNs/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , ARN Circular/metabolismo , ARN Circular/genética , Apoptosis/efectos de los fármacos , Animales , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Ratones , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Ratones Endogámicos BALB C , Células A549 , Femenino , MasculinoRESUMEN
Medium chain fatty acids are commonly consumed as part of diets for endurance sports and as medical treatment in ketogenic diets where these diets regulate energy metabolism and increase adenosine levels. However, the role of the equilibrative nucleoside transporter 1 (ENT1), which is responsible for adenosine transport across membranes in this process, is not well understood. Here, we investigate ENT1 activity in controlling the effects of two dietary medium chain fatty acids (decanoic and octanoic acid), employing the tractable model system Dictyostelium. We show that genetic ablation of three ENT1 orthologues unexpectedly improves cell proliferation specifically following decanoic acid treatment. This effect is not caused by increased adenosine levels triggered by both fatty acids in the presence of ENT1 activity. Instead, we show that decanoic acid increases expression of energy-related genes relevant for fatty acid ß-oxidation, and that pharmacological inhibition of ENT1 activity leads to an enhanced effect of decanoic acid to increase expression of tricarboxylicacid cycle and oxidative phosphorylation components. Importantly, similar transcriptional changes have been shown in the rat hippocampus during ketogenic diet treatment. We validated these changes by showing enhanced mitochondria load and reduced lipid droplets. Thus, our data show that ENT1 regulates the medium chain fatty acid-induced increase in cellular adenosine levels and the decanoic acid-induced expression of important metabolic enzymes in energy provision, identifying a key role for ENT1 proteins in metabolic effects of medium chain fatty acids.
Asunto(s)
Metabolismo Energético , Tranportador Equilibrativo 1 de Nucleósido , Adenosina/metabolismo , Adenosina/farmacología , Caprilatos/farmacología , Proliferación Celular/efectos de los fármacos , Dictyostelium/metabolismo , Dictyostelium/genética , Dictyostelium/efectos de los fármacos , Dieta Cetogénica , Grasas de la Dieta/farmacología , Grasas de la Dieta/metabolismo , Metabolismo Energético/efectos de los fármacos , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/genética , Regulación de la Expresión Génica/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacosRESUMEN
AIMS: It is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called "extracellular cAMP-adenosine pathway". Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. MAIN METHODS: The effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. KEY FINDINGS: While 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5'-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SIGNIFICANCE: Our data show that activation of ß-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.
Asunto(s)
5'-Nucleotidasa , AMP Cíclico , Contracción Muscular , Ratas Wistar , Receptor de Adenosina A1 , Conducto Deferente , Masculino , Animales , Conducto Deferente/efectos de los fármacos , Conducto Deferente/metabolismo , AMP Cíclico/metabolismo , 5'-Nucleotidasa/metabolismo , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A1/efectos de los fármacos , Ratas , Contracción Muscular/efectos de los fármacos , Adenosina/farmacología , Adenosina/análogos & derivados , Adenosina/metabolismo , Isoproterenol/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Colforsina/farmacologíaRESUMEN
Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch - a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.
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Adenosina , Proliferación Celular , Cricetulus , Células CHO , Animales , Adenosina/metabolismo , Adenosina/farmacología , Proliferación Celular/efectos de los fármacos , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/metabolismo , Técnicas de Cultivo Celular por Lotes , Cricetinae , Reactores BiológicosRESUMEN
Background and objectives: The main problem of vascular preservation is the maintenance of vessel graft quality and function following extended storage. Conventional preservation solutions such as histidine-tryptophan-ketoglutarate (HTK) solution, Phosphate-Buffer Solution (PBS), or sodium chloride 0.9% has been shown to be inadequate in preserving vascular physiological function after 3 days of cold storage. This study aimed to evaluate whether adenosine and lidocaine (AL) in a modified Krebs-Henseleit (KH) solution can preserve the function and histological structure of rat aortic rings after 6 days. Materials and Methods: Thirty-five aortic rings from male Wistar rats (200-300 g) were harvested and immediately immersed in one of the assigned cold preservation solutions: standard KH, modified KH (mod KH) with lower calcium (Ca2+) and higher magnesium content (Mg2+) with or without adenosine and lidocaine (mod KH-AL), and modified KH with AL, insulin, and melatonin (Mod KH-ALMI). The contraction and relaxation function of the aortic rings were examined using an isometric force transducer after 6 days of cold preservation. Hematoxylin and eosin staining were used to analyze the rings' histological structure. Results: Vascular contraction and relaxation functions were severely affected after a 6-day cold storage period in standard KH. Modifying the KH solution by reducing the Ca2+ and increasing the Mg2+ levels greatly recovered the vessel functions. The addition of AL or ALMI to the modified KH did not further recover vascular contractility. However, only the addition of AL to the modified KH increased the ACh-induced relaxation at 6 days when compared to the conventional KH, suggesting that endothelium preservation is improved. From histological analysis, it was found that the addition of AL but not ALMI further improved the endothelial lining and the structure of the elastic membrane layers of the preserved vessels after 6 days of cold preservation. Conclusions: The addition of AL to low calcium-high magnesium KH solution significantly enhanced endothelial preservation and improved endothelial-induced relaxation of preserved vessels after 6 days of cold storage.
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Adenosina , Calcio , Lidocaína , Magnesio , Soluciones Preservantes de Órganos , Ratas Wistar , Animales , Lidocaína/farmacología , Adenosina/farmacología , Ratas , Masculino , Calcio/análisis , Magnesio/farmacología , Soluciones Preservantes de Órganos/farmacología , Aorta/efectos de los fármacos , Aorta/fisiología , Glucosa/farmacología , Cloruro de Potasio/farmacología , TrometaminaRESUMEN
INTRODUCTION: Non-compressible torso hemorrhagic (NCTH) shock is the leading cause of potentially survivable trauma on the battlefield. New hypotensive drug therapies are urgently required to resuscitate and protect the heart and brain following NCTH. Our aim was to examine the strengths and limitations of permissive hypotension and discuss the development of small-volume adenosine, lidocaine, and Mg2+ (ALM) fluid resuscitation in rats and pigs. MATERIALS AND METHODS: For review of permissive hypotension, a literature search was performed from inception up to November 2023 using PubMed, Cochrane, and Embase databases, with inclusion of animal studies, clinical trials and reviews with military and clinical relevance. For the preclinical study, adult female pigs underwent laparoscopic liver resection. After 30 minutes of bleeding, animals were resuscitated with 4 mL/kg 3% NaCl ± ALM bolus followed 60 minutes later with 4 h 3 mL/kg/h 0.9% NaCl ± ALM drip (n = 10 per group), then blood transfusion. Mean arterial pressure (MAP) and cardiac output (CO) were continuously measured via a left ventricular pressure catheter and pulmonary artery catheter, respectively. Systemic vascular resistance (SVR) was calculated using the formula: 80 × (MAP - CVP)/CI. Oxygen delivery was calculated as the product of CO and arterial oxygen content. RESULTS: Targeting a MAP of â¼50 mmHg can be harmful or beneficial, depending on how CO and SVR are regulated. A theoretical example shows that for the same MAP of 50 mmHg, a higher CO and lower SVR can lead to a nearly 2-fold increase in O2 supply. We further show that in animal models of NCTH, 3% NaCl ALM bolus and 0.9% NaCl ALM drip induce a hypotensive, high flow, vasodilatory state with maintained tissue O2 supply and neuroprotection. ALM therapy increases survival by resuscitating the heart, reducing internal bleeding by correcting coagulopathy, and decreasing secondary injury. CONCLUSIONS: In rat and pig models of NCTH, small-volume ALM therapy resuscitates at hypotensive pressures by increasing CO and reducing SVR. This strategy is associated with heart and brain protection and maintained tissue O2 delivery. Translational studies are required to determine reproducibility and optimal component dosing. ALM therapy may find wide utility in prehospital and far-forward military environments.
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Adenosina , Hipotensión , Resucitación , Animales , Porcinos , Resucitación/métodos , Ratas , Hipotensión/etiología , Hipotensión/fisiopatología , Adenosina/administración & dosificación , Adenosina/farmacología , Lidocaína/farmacología , Lidocaína/uso terapéutico , Lidocaína/administración & dosificación , Femenino , Choque Hemorrágico/terapia , Choque Hemorrágico/complicaciones , Choque Hemorrágico/fisiopatologíaRESUMEN
Knee osteoarthritis (KOA) is a chronic joint disease that significantly affects the health of the elderly. As an herbal remedy, Gubi decoction (GBD) has been traditionally used for the treatment of osteoarthritis-related syndromes. However, the anti-KOA efficacy and mechanism of GBD remain unclear. This study aimed to experimentally investigate the anti-KOA efficacy and the underlying mechanism of GBD. The medial meniscus (DMM) mice model and IL-1ß-stimulated chondrocytes were, respectively, constructed as in vivo and in vitro models of KOA to evaluate the osteoprotective effect and molecular mechanism of GBD. The UPLC-MS/MS analysis showed that GBD mainly contained pinoresinol diglucoside, rehmannioside D, hesperidin, liquiritin, baohuoside I, glycyrrhizic acid, kaempferol and tangeretin. Animal experiment showed that GBD could alleviate articular cartilage destruction and recover histopathological alterations in DMM mice. In addition, GBD inhibited chondrocyte apoptosis and restored DMM-induced dysregulated autophagy evidenced by the upregulation of ATG7 and LC3 II/LC3 I but decreased P62 level. Mechanistically, METTL3-mediated m6A modification decreased the expression of ATG7 in DMM mice, as it could be significantly attenuated by GBD. METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy. Further research showed that GBD promoted proteasome-mediated ubiquitination degradation of METLL3. Our findings suggest that GBD could act as a protective agent against KOA. The protective effect of GBD may result from its promotion on chondrocyte autophagy by suppressing METTL3-dependent ATG7 m6A methylation.
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Proteína 7 Relacionada con la Autofagia , Autofagia , Condrocitos , Metiltransferasas , Osteoartritis de la Rodilla , Animales , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Autofagia/efectos de los fármacos , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patología , Osteoartritis de la Rodilla/tratamiento farmacológico , Ratones , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , Metiltransferasas/metabolismo , Metilación/efectos de los fármacos , Masculino , Medicamentos Herbarios Chinos/farmacología , Modelos Animales de Enfermedad , Apoptosis/efectos de los fármacos , Ratones Endogámicos C57BL , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/metabolismo , Humanos , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patologíaRESUMEN
BACKGROUND: Ferroptosis is caused by iron-dependent peroxidation of membrane phospholipids and chondrocyte ferroptosis contributes to osteoarthritis (OA) progression. Glutathione peroxidase 4 (GPX4) plays a master role in blocking ferroptosis. N6-methyladenosine (m6A) is an epigenetic modification among mRNA post-transcriptional modifications. This study investigated the effect of methyltransferase-like 14 (METTL14), the key component of the m6A methyltransferase, on chondrocyte ferroptosis via m6A modification. METHODS: An OA rat model was established through an intra-articular injection of monosodium iodoacetate in the right knee. OA cartilages in rat models were used for gene expression analysis. Primary mouse chondrocytes or ADTC5 cells were stimulated with IL-1ß or erastin. The m6A RNA methylation quantification kit was used to measure m6A level. The effect of METTL14 and GPX4 on ECM degradation and ferroptosis was investigated through western blotting, fluorescence immunostaining, propidium iodide staining, and commercially available kits. The mechanism of METTL14 action was explored through MeRIP-qPCR assays. RESULTS: METTL14 and m6A expression was upregulated in osteoarthritic cartilages and IL-1ß-induced chondrocytes. METTL14 depletion repressed the IL-1ß or erastin-stimulated ECM degradation and ferroptosis in mouse chondrocytes. METTL14 inhibited GPX4 gene through m6A methylation modification. GPX4 knockdown reversed the si-METTL14-mediated protection in IL-1ß-induced chondrocytes. CONCLUSION: METTL14 depletion inhibits ferroptosis and ECM degradation by suppressing GPX4 mRNA m6A modification in injured chondrocytes.
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Condrocitos , Ferroptosis , Metiltransferasas , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Animales , Condrocitos/efectos de los fármacos , Condrocitos/patología , Condrocitos/metabolismo , Condrocitos/enzimología , Ferroptosis/efectos de los fármacos , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones , Masculino , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacología , Osteoartritis/patología , Osteoartritis/metabolismo , Osteoartritis/enzimología , Osteoartritis/genética , Osteoartritis/inducido químicamente , Cartílago Articular/patología , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Ratas , Humanos , Ratas Sprague-DawleyRESUMEN
The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.
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Adenosina Monofosfato , Adenosina , Alanina , Antivirales , COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Antivirales/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Humanos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Alanina/química , COVID-19/inmunología , COVID-19/virología , Animales , Agentes Inmunomoduladores/farmacología , Agentes Inmunomoduladores/química , Antagonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/química , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Pandemias , Tratamiento Farmacológico de COVID-19 , Chlorocebus aethiops , Replicación Viral/efectos de los fármacos , Células Vero , Betacoronavirus/efectos de los fármacos , Betacoronavirus/inmunología , Receptor de Adenosina A2A/metabolismo , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virologíaRESUMEN
N6-clyclohexyladenosine (CHA) is an adenosine A1 receptor agonist that inhibits thermogenesis. Cardiovascular side effects however, limit use of CHA as a therapeutic. We and others have shown that this can be reversed by administering 8-p-(sulfophenyl)theophylline (8-SPT), a nonspecific antagonist that does not cross the BBB. Other evidence shows that CNS actions of CHA may contribute to bradycardia through enhanced vagal tone and other mechanisms. Here we test the hypothesis that 8-SPT pretreatment alone is sufficient to prevent hypotension caused by CHA. To test this hypothesis, we pretreated rats with 8-SPT alone, and in combination with other antagonists to test the hypothesis that direct action of CHA on the heart is the primary mechanism by which CHA induces bradycardia and hypotension. Results show that pretreatment with 8-SPT alone is not sufficient to prevent CHA-induced hypotension. Pretreatment with 8-SPT or atropine alone did not prevent the fall in mean arterial pressure (MAP) and heart rate (HR), however, pretreatment with 8-SPT (25 mg/kg) and atropine (1 mg/kg) 15 min before CHA (1 mg/kg) preserves MAP and HR baseline values after CHA administration. We next asked if blood pressure was managed during the transition into a hypometabolic state, would prolong CHA-mediated inhibition of metabolism after cardiac arrest improve outcome better than anti-shivering medications meperidine and buspirone. We found that CHA-mediated hypotension can be mitigated by pretreatment with atropine and 8-SPT. This combination administered after cardiac arrest facilitated temperature management and metabolic suppression better than meperidine and buspirone, however, did not improve survival.
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Adenosina , Buspirona , Paro Cardíaco , Meperidina , Ratas Sprague-Dawley , Animales , Ratas , Adenosina/análogos & derivados , Adenosina/farmacología , Buspirona/farmacología , Buspirona/uso terapéutico , Masculino , Paro Cardíaco/tratamiento farmacológico , Meperidina/farmacología , Meperidina/análogos & derivados , Meperidina/uso terapéutico , Frecuencia Cardíaca/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Teofilina/análogos & derivados , Teofilina/farmacología , Teofilina/uso terapéutico , Resultado del TratamientoRESUMEN
BACKGROUND: Facet joint osteoarthritis (FJOA) is a common lumbar osteoarthritis characterized by degeneration of small joint cartilage. Bushen Huoxue decotion (BSHXD) has good therapeutic effects on OA. Our work aimed to further probe the pharmacological effects of BSHXD-containing serum (BSHXD-CS) on FJOA and define underlying the mechanisms invovled. METHODS: To establish a FJOA cell model, primary rat chondrocytes were treated with LPS. The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. The secretion levels of pro-inflammatory cytokines were measured by ELISA. Cell viability was determined by CCK8 assay. The global m6A level was detected by the kit, and NLRP3 mRNA m6A level was determined by Me-RIP assay. The molecular interactions were analyzed by RIP and RNA pull-down assays. RESULTS: BSHXD-CS treatment relieved LPS-induced cell injury, inflammation, NLRP3 inflammasome and pyroptosis in chondrocytes (all p < 0.05). LPS-induced NLRP3 upregulation in chondrocytes was related to its high m6A modification level (p < 0.05). It was also observed that BSHXD-CS reduced LPS-induced m6A modification in chondrocytes via repressing STAT3 (all p < 0.05), suggesting BSHXD-CS could repress NLRP3 expression via m6A-dependent manner. Moreover, DAA, a m6A specific inhibitor, was proved to strengthen the protectively roles of BSHXD-CS on LPS-challenged pytoptosis (all p < 0.05). CONCLUSION: BSHXD-CS inhibited NLRP3 inflammasome activation and pyroptosis in chondrocytes to repress OA progression by reducing RNA m6A modification.
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Condrocitos , Medicamentos Herbarios Chinos , Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , Articulación Cigapofisaria , Animales , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Condrocitos/patología , Piroptosis/efectos de los fármacos , Ratas , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Articulación Cigapofisaria/patología , Células Cultivadas , Ratas Sprague-Dawley , Osteoartritis/patología , Masculino , Modelos Animales de Enfermedad , Suero , Inflamasomas/metabolismo , Humanos , Adenosina/análogos & derivados , Adenosina/farmacologíaRESUMEN
Collagen hydrogel has been shown promise as an inducer for chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), contributing to the repair of cartilage defects. However, the precise molecular mechanism underlying this phenomenon remains poorly elucidated. Here, we induced chondrogenic differentiation of BMSCs using collagen hydrogel and identified 4451 differentially expressed genes (DEGs) through transcriptomic sequencing. Our analysis revealed that DEGs were enriched in the focal adhesion pathway, with a notable decrease in expression levels in the collagen hydrogel group compared to the control group. Protein-protein interaction network analysis suggested that actinin alpha 1 (ACTN1) and actinin alpha 4 (ACTN4), two proteins also involved in cytoskeletal recombination, may be crucial in collagen hydrogel-induced chondrogenic differentiation of BMSCs. Additionally, we found that N6-methyladenosine RNA methylation (m6A) modification was involved in collagen hydrogel-mediated chondrogenic differentiation, with fat mass and obesity-associated protein (FTO) implicated in regulating the expression of ACTN1 and ACTN4. These findings suggest that collagen hydrogel might regulate focal adhesion and actin cytoskeletal signaling pathways through down-regulation of ACTN1 and ACTN4 mRNA via FTO-mediated m6A modification, ultimately driving chondrogenic differentiation of BMSCs. In conclusion, our study provides valuable insights into the molecular mechanisms of collagen hydrogel-induced chondrogenic differentiation of BMSCs, which may aid in developing more effective strategies for cartilage regeneration.
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Diferenciación Celular , Condrogénesis , Colágeno , Perfilación de la Expresión Génica , Hidrogeles , Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , Diferenciación Celular/efectos de los fármacos , Hidrogeles/química , Colágeno/química , Animales , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Transcriptoma/efectos de los fármacos , Actinina/metabolismo , Actinina/genética , Células Cultivadas , Metilación , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , RatasRESUMEN
DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-l-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-l-homocysteine (SAH) into L-homocysteine. As a result, the SAM-to-SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts, but there has yet to be a study looking at the potential downstream impact of DZNep treatment on proteins other than histones. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small-molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize the protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in the SAM/SAH levels. This study systematically evaluates DZNep's impact on the transcriptome, the proteome, and the thermal stability of proteins.
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Adenosina , Estabilidad Proteica , Humanos , Células HEK293 , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Estabilidad Proteica/efectos de los fármacos , Metilación , Adenosilhomocisteinasa/antagonistas & inhibidores , Adenosilhomocisteinasa/metabolismo , TemperaturaRESUMEN
N6-methyladenosine (m6A) is one of the most prevalent and conserved RNA modifications. It controls several biological processes, including the biogenesis and function of circular RNAs (circRNAs), which are a class of covalently closed-single stranded RNAs. Several studies have revealed that proteotoxic stress response induction could be a relevant anticancer therapy in Acute Myeloid Leukemia (AML). Furthermore, a strong molecular interaction between the m6A mRNA modification factors and the suppression of the proteotoxic stress response has emerged. Since the proteasome inhibition leading to the imbalance in protein homeostasis is strictly linked to the stress response induction, we investigated the role of Bortezomib (Btz) on m6A regulation and in particular its impact on the modulation of m6A-modified circRNAs expression. Here, we show that treating AML cells with Btz downregulated the expression of the m6A regulator WTAP at translational level, mainly because of increased oxidative stress. Indeed, Btz treatment promoted oxidative stress, with ROS generation and HMOX-1 activation and administration of the reducing agent N-acetylcysteine restored WTAP expression. Additionally, we identified m6A-modified circRNAs modulated by Btz treatment, including circHIPK3, which is implicated in protein folding and oxidative stress regulation. These results highlight the intricate molecular networks involved in oxidative and ER stress induction in AML cells following proteotoxic stress response, laying the groundwork for future therapeutic strategies targeting these pathways.