RESUMO
Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.
Assuntos
Complemento C3 , Mucosa Intestinal , Microbiota , Animais , Humanos , Camundongos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Neutrófilos , Complemento C3/metabolismo , Células Estromais/metabolismoRESUMO
The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.
Assuntos
Células T Matadoras Naturais , Serotonina , Serotonina/metabolismo , Lipídeos , Antígenos CD1d/metabolismoRESUMO
The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites1-3. Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine4. Gut commensal bacteria modify some unsaturated FAs-for example, linoleic acid (LA)-into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties5. However, little is known about how this diet-microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4+ intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4+CD8αα+ IELs in gnotobiotic mice. Restoration of CLAs increases CD4+CD8αα+ IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4+CD8αα+ IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4+ T cells that were CD4+CD8αα+.
Assuntos
Ácidos Graxos , Microbioma Gastrointestinal , Linfócitos Intraepiteliais , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Isomerismo , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Lipólise , Ácido Linoleico/metabolismo , Imunidade nas MucosasRESUMO
BACKGROUND: Enhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors. METHODS: We used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems. RESULTS: We showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment. CONCLUSIONS: Taken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.
RESUMO
Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21-based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non-canonical flexible ß-trefoil conformation affects the folding of ß2-ß3 hairpin and further overall protein stability. To modulate folding dynamics, we designed an FGF21-FGF19 chimera, FGF21SS . As expected, FGF21SS shows better thermostability without inducing hepatocyte proliferation. Functional characterization of FGF21SS shows its better insulin sensitivity, reduced inflammation in 3T3-L1 adipocytes, and lower blood glucose and insulin levels in ob/ob mice compared with wild type. Our dynamics-based rational design provides a promising approach for FGF21-based therapeutic development against T2DM.
Assuntos
Diabetes Mellitus Tipo 2 , Fatores de Crescimento de Fibroblastos , Resistência à Insulina , Animais , Glicemia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Fatores de Crescimento de Fibroblastos/genética , Resistência à Insulina/genética , CamundongosRESUMO
Marine collagen (MC) has recently attracted more attention in tissue engineering as a biomaterial substitute due to its significant role in cellular signaling mechanisms, especially in mesenchymal stem cells (MSCs). However, the actual signaling mechanism of MC in MSC growth, which is highly influenced by their molecular pattern, is poorly understood. Hence, we investigated the integrin receptors (α1ß1, α2ß1, α10ß1, and α11ß1) binding mechanism and proliferation of MCs (blacktip reef shark collagen (BSC) and blue shark collagen (SC)) compared to bovine collagen (BC) on MSCs behavior through functionalized collagen molecule probing for the first time. The results showed that BSC and SC had higher proliferation rates and accelerated scratch wound healing by increasing migratory rates of MSCs. Cell adhesion and spreading results demonstrated that MC had a better capacity to anchor MSCs and maintain cell morphology than controls. Living cell observations showed that BSC was gradually assembled by cells into the ECM network within 24 h. Interestingly, qRT-PCR and ELISA revealed that the proliferative effect of MC was triggered by interacting with specific integrin receptors such as α2ß1, α10ß1, and α11ß1 of MSCs. Accordingly, BSC accelerated MSCs' growth, adhesion, shape, and spreading by interacting with specific integrin subunits (α2 and ß1) and thereby triggering further signaling cascade mechanisms.
Assuntos
Células-Tronco Mesenquimais , Tubarões , Animais , Bovinos , Camundongos , Integrinas/metabolismo , Colágeno/metabolismo , Adesão Celular , Células-Tronco Mesenquimais/metabolismo , Tubarões/metabolismoRESUMO
Fungi fibrinolytic compound 1 (FGFC1) is a rare pyran-isoindolone derivative with fibrinolytic activity. The aim of this study was to further determine the effect of FGFC1 on fibrin clots lysis in vitro. We constructed a fibrinolytic system containing single-chain urokinase-type plasminogen activator (scu-PA) and plasminogen to measure the fibrinolytic activity of FGFC1 using the chromogenic substrate method. After FITC-fibrin was incubated with increasing concentrations of FGFC1, the changes in the fluorescence intensity and D-dimer in the lysate were measured using a fluorescence microplate reader. The fibrin clot structure induced by FGFC1 was observed and analyzed using a scanning electron microscope and laser confocal microscope. We found that the chromogenic reaction rate of the mixture system increased from (15.9 ± 1.51) × 10−3 min−1 in the control group to (29.7 ± 1.25) × 10−3 min−1 for 12.8 µM FGFC1(p < 0.01). FGFC1 also significantly increased the fluorescence intensity and d-dimer concentration in FITC fibrin lysate. Image analysis showed that FGFC1 significantly reduced the fiber density and increased the fiber diameter and the distance between protofibrils. These results show that FGFC1 can effectively promote fibrin lysis in vitro and may represent a novel candidate agent for thrombolytic therapy.
Assuntos
Trombose , Ativador de Plasminogênio Tipo Uroquinase , Fibrina , Fluoresceína-5-Isotiocianato , Humanos , Isoindóis , PiranosRESUMO
Fish collagen has been widely used in tissue engineering (TE) applications as an implant, which is generally transplanted into target tissue with stem cells for better regeneration ability. In this case, the success rate of this research depends on the fundamental components of fish collagen such as amino acid composition, structural and rheological properties. Therefore, researchers have been trying to find an innovative raw material from marine origins for tissue engineering applications. Based on this concept, collagens such as acid-soluble (ASC) and pepsin-soluble (PSC) were extracted from a new type of cartilaginous fish, the blacktip reef shark, for the first time, and were further investigated for physicochemical, protein pattern, microstructural and peptide mapping. The study results confirmed that the extracted collagens resemble the protein pattern of type-I collagen comprising the α1, α2, ß and γ chains. The hydrophobic amino acids were dominant in both collagens with glycine and hydroxyproline as major amino acids. From the FTIR spectra, α helix (27.72 and 26.32%), ß-sheet (22.24 and 23.35%), ß-turn (21.34 and 22.08%), triple helix (14.11 and 14.13%) and random coil (14.59 and 14.12%) structures of ASC and PSC were confirmed, respectively. Collagens retained their triple helical and secondary structure well. Both collagens had maximum solubility at 3% NaCl and pH 4, and had absorbance maxima at 234 nm, respectively. The peptide mapping was almost similar for ASC and PSC at pH 2, generating peptides ranging from 15 to 200 kDa, with 23 kDa as a major peptide fragment. The microstructural analysis confirmed the homogenous fibrillar nature of collagens with more interconnected networks. Overall, the preset study concluded that collagen can be extracted more efficiently without disturbing the secondary structure by pepsin treatment. Therefore, the blacktip reef shark skin could serve as a potential source for collagen extraction for the pharmaceutical and biomedical applications.
Assuntos
Pepsina A , Tubarões , Ácidos/química , Aminoácidos/química , Animais , Colágeno/química , Colágeno Tipo I/química , Peixes/metabolismo , Pepsina A/química , Tubarões/metabolismo , Pele/metabolismo , SolubilidadeRESUMO
A rapid, sensitive, and specific LC-MS/MS method was developed and fully validated for the detection of paeoniflorin only in rat plasma, and applied to pharmacokinetic studies, including intravenous, multi-dose oral and combined administrations with verapamil. In this study, tolbutamide was used as the internal standard, and the protein precipitation extraction method, using acetonitrile as the extraction agent, was used for the sample preparation. Subsequently, the supernatant samples were analyzed on a Phenomenex Gemini® NX-C18 column with a flow rate of 1.0 mL/min in a gradient elution procedure. In the extracted rat plasma, the method exhibited high sensitivity (LLOQ of 1.0 ng/mL) upon selecting ammonium adduct ions ([M+NH4]+) as the precursor ions and good linearity over the concentration range of 1.0−2000 ng/mL, with correlation coefficients >0.99. The intra- and inter-batch accuracy RE% values were within ±8.2%, and the precision RSD% values were ≤8.1% and ≤10.0%, respectively. The results show that the method can be successfully applied to quantitate paeoniflorin in biological samples. Additionally, paeoniflorin is subsequently confirmed to be the substrate of the P-gp transporter in vivo and in vitro for the first time, which would be necessary and beneficial to investigate the clinical safety and efficacy of PF with other drugs in the treatment of rheumatoid arthritis.
Assuntos
Espectrometria de Massas em Tandem , Verapamil , Ratos , Animais , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Monoterpenos/farmacocinética , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodosRESUMO
The discovery of cytokine tumor necrosis factor (TNF) in the 20th century revealed numerous secrets about organ development. In particular, the functions identified for the receptor activator of nuclear factor kappa-ß (NF-κß) ligand (also known as the RANKL/osteoprotegerin ligand (OPGL) or RANK ligand/TNFSF11) in the homeostasis of skeletal structure, function and regulation were not anticipated. Empirical evidence established the receptor-ligand interaction of RANKL with RANK in osteoclast formation. Reverse signaling of RANKL triggers NF-κß for the degradation of ß-catenin to inhibit bone formation. There is also evidence that RANKL modifies the behavior of other cells in the bone microenvironment, including osteoblasts, chondrocytes, endothelial cells and lymphocytes during normal (homeostatic) and diseased (osteoimmune) states. Two forms of RANKL, i.e., soluble and membrane-bound RANKL, are produced by bone cells. Even though soluble RANKL (sRANKL) and membrane-bound RANKL (mRANKL) both stimulate osteoclast formation in vitro, their biological roles are different. mRANKL triggers osteoclastogenesis by binding to RANK through cell-cell interaction; however, sRANKL released from osteogenic cells binds to RANK without cell-cell interaction. This review attempts to hypothesize how sRANKL functions biologically in bone and explore how this hypothesis might influence future research.
Assuntos
Osso e Ossos/metabolismo , Ligante RANK/metabolismo , Animais , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Células Endoteliais/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoclastos/metabolismoRESUMO
The inhibition of α-glucosidase activity is a prospective approach to attenuate postprandial hyperglycemia in the treatment of type 2 diabetes mellitus (T2DM). Herein, the inhibition of α-glucosidase by three compounds T1 -T3 of Akebia trifoliata stem, namely hederagenin (T1 ), 3-epiakebonoic acid (T2 ), and arjunolic acid (T3 ) were investigated using enzyme kinetics and molecular docking analysis. The three triterpenoids exhibited excellent inhibitory activities against α-glucosidase. T1 -T3 showed the strongest inhibition with IC50 values of 42.1±5.4, 19.6±3.2, and 11.2±2.3â µM, respectively, compared to the acarbose positive control (IC50 =106.3±8.2). Enzyme inhibition kinetics showed that triterpenoids T1 -T3 demonstrated competitive, mixed, and noncompetitive-type inhibition against α-glucosidase, respectively. The inhibition constant (Ki ) values were 21.21, 7.70, and 3.18â µM, respectively. Docking analysis determined that the interaction of ligands T1 -T3 and α-glucosidase was mainly forced by hydrogen bonds and hydrophobic interactions, which could result in improved binding to the active site of the target enzyme. The insulin resistant (IR)-HepG2â cell model used in this study (HepG2 cells exposed to 10-7 â M insulin for 24 h) and glucose uptake assays showed that compounds T1 -T3 had no cytotoxicity with concentrations ranging from 6.25 to 25â µM and displayed significant stimulation of glucose uptake in IR-HepG2 cells. Thus, triterpenoids T1 -T3 showed dual therapeutic effects of α-glucosidase inhibition and glucose uptake stimulation and could be used as potential medicinal resources to investigate new antidiabetic agents for the prevention or treatment of diabetes.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Inibidores de Glicosídeo Hidrolases/farmacologia , Hipoglicemiantes/farmacologia , Extratos Vegetais/farmacologia , Ranunculales/química , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Glucose/metabolismo , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Células Hep G2 , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Resistência à Insulina , Conformação Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Triterpenos/química , Triterpenos/isolamento & purificação , alfa-Glucosidases/metabolismoRESUMO
Fungi fibrinolytic compound 1 (FGFC1) is a rare marine-derived compound that can enhance fibrinolysis both in vitro and in vivo. The fibrinolytic activity characterization of FGFC1 mediated by plasminogen (Glu-/Lys-) and a single-chain urokinase-type plasminogen activator (pro-uPA) was further evaluated. The binding sites and mode of binding between FGFC1 and plasminogen were investigated by means of a combination of in vitro experiments and molecular docking. A 2.2-fold enhancement of fibrinolytic activity was achieved at 0.096 mM FGFC1, whereas the inhibition of fibrinolytic activity occurred when the FGFC1 concentration was above 0.24 mM. The inhibition of fibrinolytic activity of FGFC1 by 6-aminohexanoic acid (EACA) and tranexamic acid (TXA) together with the docking results revealed that the lysine-binding sites (LBSs) play a crucial role in the process of FGFC1 binding to plasminogen. The action mechanism of FGFC1 binding to plasminogen was inferred, and FGFC1 was able to induce plasminogen to exhibit an open conformation by binding through the LBSs. The molecular docking results showed that docking of ligands (EACA, FGFC1) with receptors (KR1-KR5) mainly occurred through hydrophilic and hydrophobic interactions. In addition, the binding affinity values of EACA to KR1-KR5 were -5.2, -4.3, -3.7, -4.5, and -4.3 kcal/moL, respectively, and those of FGFC1 to KR1-KR5 were -7.4, -9.0, -6.3, -8.3, and -6.7 kcal/moL, respectively. The findings demonstrate that both EACA and FGFC1 bound to KR1-KR5 with moderately high affinity. This study could provide a theoretical basis for the clinical pharmacology of FGFC1 and establish a foundation for practical applications of FGFC1.
Assuntos
Fibrinólise , Fibrinolíticos/química , Fungos/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , HumanosRESUMO
Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.
Assuntos
Candida albicans/patogenicidade , Candidíase/metabolismo , Estresse Oxidativo/fisiologia , Simportadores de Próton-Fosfato/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Transporte Biológico/fisiologia , Drosophila , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Fosfatos/metabolismo , Transdução de Sinais/fisiologia , VirulênciaRESUMO
Histone deacetylases (HDACs) have been found to be biomarkers of cancers and the corresponding inhibitors have attracted much attention these years. Herein we reported a near-infrared fluorescent HDAC inhibitor based on vorinostat (SAHA) and a NIR fluorophore. This newly designed inhibitor showed similar inhibitory activity to SAHA against three HDAC isoforms (HDAC1, 3, 6). The western blot assay showed significant difference in compared with the negative group. When used as probe for further kinematic imaging, Probe 1 showed enhanced retention in tumor cells and the potential of HDAC inhibitors in drug delivery was firstly brought out. The cytotoxicity assay showed Probe 1 had some anti-proliferation activities with corresponding IC50 values of 9.20 ± 0.96 µM on Hela cells and 5.91 ± 0.57 µM on MDA-MB-231 cells. These results indicated that Probe 1 could be used as a potential NIR fluorescent in the study of HDAC inhibitors and lead compound for the development of visible drugs.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HeLa , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Microscopia de Fluorescência , Vorinostat/químicaRESUMO
BACKGROUND: Cathepsin L and some other cathepsins have been implicated in the development of obesity in humans and mice. The functional inactivation of the proteases reduces fat accumulation during mammalian adipocyte differentiation. However, beyond degrading extracellular matrix protein fibronectin, the molecular mechanisms by which cathepsins control fat accumulation remain unclear. We now provide evidence from Caenorhabditis elegans and mouse models to suggest a conserved regulatory circuit in which peripheral cathepsin L inhibition lowers fat accumulation through promoting central serotonin synthesis. RESULTS: We established a C. elegans model of fat accumulation using dietary supplementation with glucose and palmitic acid. We found that nutrient supplementation elevated fat storage in C. elegans, and along with worm fat accumulation, an increase in the expression of cpl-1 was detected using real-time PCR and western blot. The functional inactivation of cpl-1 reduced fat storage in C. elegans through activating serotonin signaling. Further, knockdown of cpl-1 in the intestine and hypodermis promoted serotonin synthesis in worm ADF neurons and induced body fat loss in C. elegans via central serotonin signaling. We found a similar regulatory circuit in high-fat diet-fed mice. Cathepsin L knockout promoted fat loss and central serotonin synthesis. Intraperitoneal injection of the cathepsin L inhibitor CLIK195 similarly reduced body weight gain and white adipose tissue (WAT) adipogenesis, while elevating brain serotonin level and WAT lipolysis and fatty acid ß-oxidation. These effects of inhibiting cathepsin L were abolished by intracranial injection of p-chlorophenylalanine, inhibitor of a rate-limiting enzyme for serotonin synthesis. CONCLUSION: This study reveals a previously undescribed molecular mechanism by which peripheral CPL-1/cathepsin L inhibition induces fat loss in C. elegans and mice through promoting central serotonin signaling.
Assuntos
Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Obesidade/genética , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Collagen is widely used for dental therapy in several ways such as films, 3D matrix, and composites, besides traditional Chinese medicine (TCM), has been used in tissue regeneration and wound healing application for centuries. Hence, the present study was targeted for the first time to fabricate collagen film with TCM such as resveratrol and celastrol in order to investigate the human periodontal ligament fibroblasts (HPLF) growth and bone marrow macrophages (BMM) derived osteoclastogenesis. Further, the physicochemical, mechanical and biological activities of collagen-TCM films crosslinked by glycerol and EDC-NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysulfosuccinimide) were investigated. Collagen film characterization was significantly regulated by the nature of plasticizers like hydrophobic and degree of polarity. Interestingly, the collagen film's denaturation temperature was increased by EDC-NHS than glycerol. FT-IR data confirmed the functional group changes due to chemical interaction of collagen with TCM. Morphological changes of HPLF cells cultured in control and collagen films were observed by SEM. Importantly, the addition of resveratrol upregulated the proliferation of HPLF cells, while osteoclastogenesis of BMM cells treated with mCSF-RANKL was significantly downregulated by celastrol. Accordingly, the collagen-TCM film could be an interesting material for dental regeneration, and especially it is a therapeutic target to restrain the elevated bone resorption during osteoporosis.
Assuntos
Antioxidantes/farmacologia , Colágeno/farmacologia , Implantes Dentários , Triterpenos Pentacíclicos/farmacologia , Ligamento Periodontal/efeitos dos fármacos , Resveratrol/farmacologia , Antioxidantes/química , Compostos de Bifenilo/antagonistas & inibidores , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/química , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Estrutura Molecular , Osteogênese/efeitos dos fármacos , Triterpenos Pentacíclicos/química , Ligamento Periodontal/patologia , Picratos/antagonistas & inibidores , Resveratrol/química , Relação Estrutura-AtividadeRESUMO
Songorine isolated from Aconitum brachypodum Diels possesses prominent activity of inhibiting G protein-coupled receptors (GPCRs) in the early screening process. In this paper, a series of Songorine derivatives were synthesized and their inhibitory activities on GPCRs were also evaluated by using the Double Antibody Sandwich ELISA (DAS-ELISA) in vitro. Among them, three derivatives (3a, 4, 7) exhibited significant inhibitory activity against GPCRs with IC50 values of 0.08-0.29â¯nM. Moreover, the structure-activity relationships (SARs) of songorine derivatives were discussed in detail. They have great potentials as novel GPCRs antagonists in the future.
Assuntos
Alcaloides/química , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Aconitum/química , Aconitum/metabolismo , Alcaloides/metabolismo , Concentração Inibidora 50 , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-AtividadeRESUMO
Triple negative breast cancer (TNBC) is a more aggressive subtype of breast cancer and is characteristic of the absence of the expressions of estrogen receptor, progesterone receptor, and human epithelial growth factor receptor 2 in breast tumor tissues. This subtype of breast cancer has the poorest prognosis, compared to other subtypes of breast cancer. TNBC is heterogeneous by showing several different histo-pathological and molecular subtypes with different prognosis and is more commonly found in younger age of women, especially African-American and Hispanic women. Recent epidemiological data indicate that TNBC is highly associated with overweight/obesity. Due to the absence of the common tumor biomarkers of breast cancer, the current molecular target therapy is not effective. TNBC patients have a shorter survival rate and an increased tumor recurrence. The concept of cancer stem cells (CSC), also called tumor initiating cells (TIC) has been more and more accepted and considered to contribute to aggressive phenotypes of many tumors including breast cancer. Moreover, CSC/TIC has been identified in the tumor tissues of breast cancer including TNBC. These rare subpopulations of CSC/TIC cells might be one of the key contributors to the aggressive phenotypes of TNBC such as drug treatment resistance, metastasis, and tumor recurrence. Therefore, targeting these CSC/TIC cells will provide a new therapeutic strategy for the treatment of TNBC.
Assuntos
Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Feminino , Humanos , Terapia de Alvo Molecular , Recidiva Local de NeoplasiaRESUMO
A marine fibrinolytic compound was studied for use in thrombolytic therapy. Firstly, the absorption and transportation characteristics of 2,5-BHPA (2,5-BHPA:2,5-Bis-[8-(4,8-dimethyl-nona-3,7-dienyl)-5,7-dihydroxy-8-methyl-3-keto-1,2,7,8-tertahydro-6H-pyran[a]isoindol-2-yl]-pentanoic acid, a novel pyran-isoindolone derivative with bioactivity isolated from a rare marine microorganism in our laboratory) in the human Caco-2 cells monolayer model were investigated. We collected 2,5-BHPA in the cells to calculate the total recovery, and its concentration was analyzed by LC/MS/MS (Liquid Chromatography/ Mass Spectrum/ Mass Spectrum). The results showed that 2,5-BHPA has low permeability and low total recoveries in the Caco-2 cells membrane. Pharmacokinetics and tissue distribution of 2,5-BHPA were investigated in beagle dogs using HPLC (High Performance Liquid Chromatography) after intravenous administration of three different doses (7.5, 5.0, 2.5 mg·kg-1). Pharmacokinetic data indicated that 2,5-BHPA fitted well to a two-compartment model. Elimination half-lives (T1/2) were 49 ± 2, 48 ± 2, and 49 ± 2 min, respectively; the peak concentrations (Cmax) were 56.48 ± 6.23, 48.63 ± 5.53, and 13.64 ± 2.76 µg·mL-1, respectively; clearance rates (CL) were 0.0062 ± 0.0004, 0.0071 ± 0.0008, and 0.0092 ±0.0006 L·min-1·kg-1, respectively; mean retention times (MRT) were 28.17 ± 1.16, 26.23 ± 0.35, and 28.66 ± 0.84 min, respectively. The low penetrability of 2,5-BHPA indicated that the intravenous route of administration is more appropriate than the oral route. Meanwhile, 2,5-BHPA showed a good pharmacokinetic profile in beagle dogs. The tissue distribution showed that 2,5-BHPA could quickly distribute into the heart, intestines, liver, stomach, spleen, lungs, testicles, urine, intestine, kidneys, brain, and feces. The concentration of 2,5-BHPA was higher in the liver and bile. Interestingly, 2,5-BHPA was detected in the brain. Taken together, the above results suggested that our work might be beneficial in the development of agents for thrombolytic treatment.
Assuntos
Organismos Aquáticos/química , Fibrinolíticos/farmacocinética , Isoindóis/farmacocinética , Piranos/farmacocinética , Administração Oral , Animais , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Cães , Avaliação Pré-Clínica de Medicamentos , Fibrinolíticos/administração & dosagem , Fibrinolíticos/química , Humanos , Injeções Intravenosas , Isoindóis/administração & dosagem , Isoindóis/química , Masculino , Modelos Animais , Permeabilidade , Piranos/administração & dosagem , Piranos/química , Espectrometria de Massas em Tandem , Trombose/tratamento farmacológico , Distribuição TecidualRESUMO
The development of biomaterials with the potential to accelerate wound healing is a great challenge in biomedicine. In this study, four types of samples including pepsin soluble collagen sponge (PCS), acid soluble collagen sponge (ACS), bovine collagen electrospun I (BCE I) and bovine collagen electrospun II (BCE II) were used as wound dressing materials. We showed that the PCS, ACS, BCE I and BCE II treated rats increased the percentage of wound contraction, reduced the inflammatory infiltration, and accelerated the epithelization and healing. PCS, ACS, BCE I, and BCE II significantly enhanced the total protein and hydroxyproline level in rats. ACS could induce more fibroblasts proliferation and differentiation than PCS, however, both PCS and ACS had a lower effect than BCE I and BCE II. PCS, ACS, BCE I, and BCE II could regulate deposition of collagen, which led to excellent alignment in the wound healing process. There were similar effects on inducing the level of cytokines including EGF, FGF, and vascular endothelial marker CD31 among these four groups. Accordingly, this study disclosed that collagens (PCS and ACS) from tilapia skin and bovine collagen electrospun (BCE I and BCE II) have significant bioactivity and could accelerate wound healing rapidly and effectively in rat model.