RESUMO
BACKGROUND: Ginsenosides have received increased amounts of attention due to their ability to modulate the intestinal flora, which may subsequently alleviate alcoholic liver disease (ALD). The effects of ginseng fermentation solution (GFS) on the gut microbiota and metabolism in ALD patients have not been explored. PURPOSE: This research aimed to explore the regulatory effect of GFS on ALD both in vitro and in vivo. METHOD: This study assessed the anti-ALD efficacy of GFS using an LO2 cell model and a zebrafish model. Untargeted metabolomics was used for differentially abundant metabolite analysis, and high-throughput 16S rRNA sequencing was used to examine the effect of GFS on ALD. RESULTS: The LO2 cell line experiments demonstrated that GFS effectively mitigated alcohol-induced oxidative stress and reduced apoptosis by upregulating PI3K and Bcl-2 expression and decreasing the levels of malondialdehyde, total cholesterol, and triglycerides. In zebrafish, GFS improved morphological and physiological parameters and diminished oxidative stress-induced ALD. Meanwhile, the results from Western blotting indicated that GFS enhanced the expression of PI3K, Akt, and Bcl-2 proteins while reducing Bax protein expression, thereby ameliorating the ALD model in zebrafish. Metabolomics data revealed significant changes in a total of 46 potential biomarkers. Among them, metabolites such as prostaglandin F2 alpha belong to arachidonic acid metabolism. In addition, GFS also partly reversed the imbalance of gut microbiota composition caused by alcohol. At the genus level, alcohol consumption elevated the presence of Flectobacillus, Curvibacter, among others, and diminished Elizabethkingia within the intestinal microbes of zebrafish. Conversely, GFS reversed these effects, notably enhancing the abundance of Proteobacteria and Archaea. Correlation analyses further indicated a significant negative correlation between prostaglandin F2 alpha, 11,14,15-THETA, Taurocholic acid and Curvibacter. CONCLUSION: This study highlights a novel mechanism by which GFS modulates anti-ALD activity through the PI3K/Akt signalling pathway by influencing the intestinal flora-metabolite axis. These results indicate the potential of GFS as a functional food for ALD treatment via modulation of the gut flora.
Assuntos
Microbioma Gastrointestinal , Hepatopatias Alcoólicas , Panax , Animais , Humanos , Apoptose/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Fermentação , Microbioma Gastrointestinal/efeitos dos fármacos , Ginsenosídeos/farmacologia , Hepatopatias Alcoólicas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Panax/química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Peixe-ZebraRESUMO
RATIONALE: Spleen-qi deficiency syndrome, a common weakness syndrome in traditional Chinese medicine, results from insufficient spleen-qi levels. For centuries, ginseng has been relied upon as a traditional Chinese medicine to treat spleen-qi deficiency syndrome. Until now, the mechanism feature of ginseng in treating temper deficiency through intestinal bacteria and short-chain fatty acid (SCFA) metabolites has not been fully elucidated. METHODS: This study established a rat model of spleen-qi deficiency via multi-factor compound modeling that involved fatigue injury and a controlled diet. The content of SCFAs between different treatment groups was determined by gas chromatography-mass spectrometry. And the 16s rRNA sequencing technology was applied to reveal the effects of ginseng on the intestinal microecological environment of the rats. RESULTS: It was found that the ginseng treatment group exhibited the most remarkable regulatory effect on propionic acid, surpassing all other administration groups. Ginseng increased the relative abundance of beneficial bacteria and decreased that of harmful bacteria at the genus level in rats with spleen-qi deficiency syndrome. And propionic acid is significantly positively correlated with Lactobacillus level and significantly negatively correlated with uncultured_bacterium_f_Muribaculaceae (p < 0.05). n-Butyric acid is negatively correlated with the Faecalibaculum level (p < 0.01). n-Valeric acid is significantly negatively correlated with the Romboutsia level (p < 0.01). CONCLUSION: The mechanism of ginseng treatment for spleen-qi deficiency is elucidated from the perspective of gut microbiota and its metabolite SCFAs. It provides a new way for further development and utilization of ginseng and a theoretical basis.
Assuntos
Medicamentos de Ervas Chinesas , Microbioma Gastrointestinal , Panax , Ratos , Animais , Baço , RNA Ribossômico 16S/genética , Qi , Cromatografia Gasosa-Espectrometria de Massas , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Panax/química , Ácidos Graxos VoláteisRESUMO
Patients with a spleen-qi deficiency often exhibit dysfunction in the metabolic system. Metabolites are considered the most direct reflection of individual physiological and pathological conditions and represent attractive candidates to provide deep insights into disease phenotypes. This study examines the potential therapeutic mechanism of wild ginseng on spleen-qi deficiency through the analysis of serum and urine metabolomics using rapid-resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. The reasons for the superiority of wild ginseng treatment over cultivated ginseng were also analyzed in depth. After wild ginseng intervention, anandamide, urobilinogen, aldosterone, and testosterone glucuronide were significantly reduced in serum. Meanwhile, argininosuccinic acid, L-cysteine, and seven other metabolites were significantly elevated in serum. Nine metabolites, including L-acetylcarnitine, and citrulline were elevated in the urine, and trimethylamine N-oxide, adrenic acid, and 10 other metabolites were reduced. Arginine biosynthesis, pantothenate and CoA biosynthesis, thiamin metabolism, taurine, and tryptophan metabolism pathways were mainly improved. Further analysis was conducted on the relationship between Lactobacillus and Akkermansia bacteria with metabolites, and it was found that they are mainly related to amino acid metabolites. This study provides strong theoretical support and direction for further explanation of the immune mechanism of wild ginseng and lays the foundation for future studies.
Assuntos
Panax , Baço , Ratos , Animais , Qi , Panax/química , Cromatografia Líquida , Metabolômica/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão/métodos , BiomarcadoresRESUMO
Pseudomonas aeruginosa is an important opportunistic pathogen that is lethal to cystic fibrosis (CF) patients. Glycerol generated during the degradation of phosphatidylcholine, the major lung surfactant in CF patients, could be utilized by P. aeruginosa. Previous studies have indicated that metabolism of glycerol by this bacterium contributes to its adaptation to and persistence in the CF lung environment. Here, we investigated the metabolic mechanisms of glycerol and its important metabolic intermediate glycerol 3-phosphate (G3P) in P. aeruginosa PAO1. We found that G3P homeostasis plays an important role in the growth and virulence factor production of P. aeruginosa PAO1. The G3P accumulation caused by the mutation of G3P dehydrogenase (GlpD) and exogenous glycerol led to impaired growth and reductions in pyocyanin synthesis, motilities, tolerance to oxidative stress, and resistance to kanamycin. Transcriptomic analysis indicates that the growth retardation caused by G3P stress is associated with reduced glycolysis and adenosine triphosphate (ATP) generation. Furthermore, two haloacid dehalogenase-like phosphatases (PA0562 and PA3172) that play roles in the dephosphorylation of G3P in strain PAO1 were identified, and their enzymatic properties were characterized. Our findings reveal the importance of G3P homeostasis and indicate that GlpD, the key enzyme for G3P catabolism, is a potential therapeutic target for the prevention and treatment of infections by this pathogen. IMPORTANCE In view of the intrinsic resistance of Pseudomonas aeruginosa to antibiotics and its potential to acquire resistance to current antibiotics, there is an urgent need to develop novel therapeutic options for the treatment of infections caused by this bacterium. Bacterial metabolic pathways have recently become a focus of interest as potential targets for the development of new antibiotics. In this study, we describe the mechanism of glycerol utilization in P. aeruginosa PAO1, which is an available carbon source in the lung environment. Our results reveal that the homeostasis of glycerol 3-phosphate (G3P), a pivotal intermediate in glycerol catabolism, is important for the growth and virulence factor production of P. aeruginosa PAO1. The mutation of G3P dehydrogenase (GlpD) and the addition of glycerol were found to reduce the tolerance of P. aeruginosa PAO1 to oxidative stress and to kanamycin. The findings highlight the importance of G3P homeostasis and suggest that GlpD is a potential drug target for the treatment of P. aeruginosa infections.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Humanos , Pseudomonas aeruginosa/genética , Glicerol/metabolismo , Fatores de Virulência/genética , Fibrose Cística/microbiologia , Antibacterianos/metabolismo , Canamicina/metabolismo , Oxirredutases/metabolismo , Infecções por Pseudomonas/microbiologiaRESUMO
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
Assuntos
Antimaláricos , Malária Falciparum , Terapia de Alvo Molecular , Plasmodium falciparum , Biossíntese de Proteínas , Proteínas de Protozoários , Tirosina-tRNA Ligase , Adenosina/análogos & derivados , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cristalografia por Raios X , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Ácidos Sulfônicos/química , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/metabolismoRESUMO
Root radial transport is important for cadmium (Cd) absorption and root-shoot translocation. However, the relationship between root structural characteristics and radial transport of Cd in wheat is still unclear. Six wheat cultivars with different Cd tolerance and accumulation characteristics were used to investigate the roles of root phenotype, microstructure, and apoplastic and symplastic pathways in Cd uptake and root-shoot transport in pot culture. Longer root length, smaller root diameter, and more numerous root tips were more conducive to Cd absorption, while thicker roots were able to retain more Cd, thus reducing root-shoot transport and improving Cd tolerance of shoots. Cd stress can induce the deposition of apoplastic barriers in wheat roots, and the deposition of the apoplastic barrier increases under greater stress. The formation of apoplastic barriers can reduce Cd absorption and transfer to the shoot, and the presence of passage cells can weaken this effect. The cell wall thickening induced by Cd stress enhanced Cd adsorption capacity in wheat roots, but there was no significant correlation between Cd content and polysaccharide content in the cell wall. The up-regulated expression of TaHMA3 and TaVP1, which encode proteins related to Cd compartmentalization, was associated with increased Cd tolerance in wheat and decreased Cd translocation to aboveground parts. The morphology and anatomy of roots appear to play critical roles in Cd tolerance, uptake, and translocation in wheat. The present study provides useful information for the selection of wheat cultivars with low Cd accumulation.
Assuntos
Cádmio , Poluentes do Solo , Adsorção , Transporte Biológico , Cádmio/análise , Cádmio/toxicidade , Raízes de Plantas/química , Poluentes do Solo/análise , Triticum/genéticaRESUMO
Stimulator of Interferon Genes (STING) plays an important role in innate immunity by inducing type I interferon production upon infection with intracellular pathogens. STING activation can promote increased T-cell activation and inflammation in the tumor microenvironment, resulting in antitumor immunity. Natural and synthetic cyclic dinucleotides (CDNs) are known to activate STING, and several synthetic CDN molecules are being investigated in the clinic using an intratumoral administration route. Here, we describe the identification of STING agonist 15a, a cyclic dinucleotide structurally diversified from natural ligands with optimized properties for systemic intravenous (iv) administration. Our studies have shown that STING activation by 15a leads to an acute innate immune response as measured by cytokine secretion and adaptive immune response via activation of CD8+ cytotoxic T-cells, which ultimately provides robust antitumor efficacy.
Assuntos
Proteínas de Membrana/agonistas , Nucleotídeos Cíclicos/química , Pirimidinas/química , Administração Intravenosa , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Meia-Vida , Humanos , Imunoterapia , Proteínas de Membrana/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Neoplasias/patologia , Neoplasias/terapia , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/uso terapêutico , Fosfatos/química , Ratos , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.
Assuntos
Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Descoberta de Drogas , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ácidos Sulfônicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/químicaRESUMO
ß-Cell dysfunction and reduction in ß-cell mass are hallmark events of diabetes mellitus. Here we show that ß-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in ß-cells. Kindlin-2 loss activates GSK-3ß and downregulates ß-catenin, leading to reduced ß-cell proliferation and mass. Kindlin-2 loss reduces the percentage of ß-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of ß-catenin in ß-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of ß-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Proteínas Musculares/metabolismo , beta Catenina/metabolismo , Animais , Proliferação de Células , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Insulina/genética , Resistência à Insulina , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Estabilidade Proteica , beta Catenina/genéticaRESUMO
Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tetra-Hidronaftalenos/química , Animais , Linhagem Celular Tumoral , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Mutagênese , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/farmacocinética , Tetra-Hidronaftalenos/uso terapêutico , Transplante HeterólogoRESUMO
Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.
Assuntos
Antineoplásicos/síntese química , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Tetra-Hidronaftalenos/síntese química , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Cristalografia por Raios X , Desenho de Fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Modelos Moleculares , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Estereoisomerismo , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/química , Tetra-Hidronaftalenos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.