RESUMEN
The purpose of this study was to reveal the antibacterial activity and mechanism of Polygonatum sibiricum extract (PSE) against Bacillus cereus and further analyze the application of PSE in pasteurized milk (PM). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values and growth curve analysis were used to evaluate the antibacterial activity of PSE against B. cereus. The changes in contents of intracellular adenosine 5'-triphosphate (ATP) and reactive oxygen species (ROS), activities of ß-galactosidase, adenosine triphosphatase (ATPase) and alkaline phosphatase (AKP), cell membrane potential, protein and nucleic acid leakage, and cell morphology were used to reveal the antibacterial mechanism. The effects of PSE on viable count and sensory evaluation of PM during storage were analyzed. The results showed that the MIC and MBC values of PSE against B. cereus were 2 and 4 mg/mL, respectively. Growth curve analysis showed that PSE with a concentration of 2 MIC could completely inhibit the growth of B. cereus. After treatments with PSE, the levels of intracellular ATP and ROS, and activities of ß-galactosidase, ATPase and AKP of B. cereus were significantly reduced (p < 0.05). Cell membrane was depolarized, amounts of protein and nucleic acid leakage were significantly increased (p < 0.05), and cell morphology was destroyed. Furthermore, PSE significantly reduced the viable count of B. cereus in PM and improved the sensory quality of PM during storage (p < 0.05). Together, our findings suggested that PSE had the desired effect as a natural preservative applied in PM.
Asunto(s)
Ácidos Nucleicos , Polygonatum , Animales , Leche/microbiología , Bacillus cereus , Especies Reactivas de Oxígeno/farmacología , Antibacterianos/farmacología , beta-Galactosidasa/farmacología , Extractos Vegetales/farmacología , Adenosina Trifosfatasas/farmacología , Adenosina TrifosfatoRESUMEN
OBJECTIVE: Skin ageing is linked to the accumulation of senescent cells and a "senescence-associated secretory phenotype" (SASP). SASP factors include chemokines, cytokines, and small extracellular vesicles (EVs) containing miRNAs. We characterized SASP profile markers in normal human dermal fibroblasts (HDFs) and evaluated the effect of Haritaki fruit extract on these senescence markers. METHODS: Senescence was induced in HDFs by ionizing radiation (X ray), followed by 14 days of culture. Parallel incubations included fibroblasts treated for 12 days with 10 or 100 µg/mL Haritaki (a standardized extract of Terminalia chebula fruit). Senescence was assessed on Day 14 according to cell morphology, ß-galactosidase activity, RT-qPCR measurement of SASP genes, as well as semi-quantitative (RT-qPCR) expression of miRNAs contained in EVs isolated from the medium. The size and distribution of EVs were measured by Nanoparticle Tracking Analysis. RESULTS: Human dermal fibroblasts exhibited a senescent phenotype 14 days after ionizing-radiation, demonstrated by a flattened and irregular shape, increased ß-galactosidase activity and over-expression of SASP genes. CSF3, CXCL1, IL1ß, IL6 and IL8 genes were increased by 1492%, 1041%, 343%, 478%, 2960% and 293%, respectively. The cell cycle inhibitor, CDKN1A, was increased by 357%, while COL1A1, was decreased by 56% and MMP1 was increased by 293%. NTA analysis of the EVs size distribution indicated a mix of exosomes (45-100 nm) and microvesicles (100-405 nm). miRNA expression in EVs was increased in senescent fibroblasts. miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p and miR 186-5p were increased in senescent HDF by 4.17-, 2.43-, 1.17-, 2.01, 12.5-fold, respectively. Incubation of senescent fibroblasts with Haritaki extract strongly decreased SASP mRNA levels and miRNA expression in EVs. CONCLUSION: Haritaki strongly reduced SASP expression and EV-shuttled miRNAs in senescent fibroblasts. These results indicate that Haritaki has strong senomorphic properties and may be a promising ingredient for the development of new anti-ageing dermo-cosmetic products by inhibiting deleterious effects of senescent cells.
OBJECTIF: Le vieillissement cutané est lié à l'accumulation de cellules sénescentes et à un « phénotype sécrétoire associé à la sénescence ¼ (SASP). Le SASP est constitué de chimiokines, cytokines et de petites vésicules extracellulaires (VE) contenant des miARN. Nous avons caractérisé les marqueurs du SASP dans des fibroblastes dermiques humains normaux (HDF) et évalué l'effet d'un extrait de fruit d'Haritaki sur ces marqueurs de la sénescence. MÉTHODES: La sénescence a été induite dans les HDF par des rayonnements ionisants (rayons X), suivis de 14 jours de culture. Parallèlement, des HDF ont été traités pendant 12 jours avec 10 ou 100 µg/mL d'Haritaki (un extrait standardisé de fruit de Terminalia chebula). La sénescence a été évaluée au jour 14 en fonction de la morphologie cellulaire, de l'activité ß-galactosidase, de la mesure des gènes du SASP par RT-PCR, ainsi que de l'expression semi-quantitative (RT-qPCR) des miARN contenus dans les VE isolées du milieu. La taille et la distribution des VE ont été mesurées par Nanoparticle Tracking Analysis (NTA). RÉSULTATS: Les HDF ont présenté un phénotype sénescent 14 jours après le rayonnement ionisant, en effet, ils avaient une forme aplatie et irrégulière, une activité ß-galactosidase accrue et une surexpression des gènes du SASP. Les ARNm de CSF3, CXCL1, IL1ß, IL6 et IL8 ont été augmentés de 1492%, 1041%, 343%, 478%, 2960% et 293%, respectivement. L'inhibiteur du cycle cellulaire, CDKN1A, a été augmenté de 357%, tandis que le COL1A1 a diminué de 56% et la MMP1 a augmenté de 293%. L'analyse NTA de la distribution de taille des VE a montré un mélange d'exosomes (45-100 nm) et de microvésicules (100-405 nm). L'expression des miARN dans les VE a augmenté dans les fibroblastes sénescents. Les miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p et miR 186-5p ont été augmentés dans le HDF sénescent de, respectivement, 4,17-, 2,43-, 1,17-, 2,01 et 12,5- fois. L'incubation de fibroblastes sénescents avec l'extrait de Haritaki a fortement diminué les niveaux d'ARNm du SASP et l'expression de miARN dans les VE. CONCLUSION: L'extrait d'Haritaki a fortement réduit l'expression du SASP et de miARN contenus dans les VE des fibroblastes sénescents. Ces résultats indiquent que Haritaki possède de fortes propriétés sénomorphiques et pourrait être un ingrédient prometteur pour le développement de nouveaux produits dermo-cosmétiques anti-âge en inhibant les effets délétères des cellules sénescentes.
Asunto(s)
Exosomas , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Exosomas/metabolismo , Senescencia Celular , Frutas/metabolismo , Fenotipo , Fibroblastos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
INTRODUCTION: Vancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown. OBJECTIVES: This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect. METHODS: The influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, ß-galactosidase assay, and electrophoresis mobility shift assay (EMSA). RESULTS: Compared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled ß-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation. CONCLUSION: The results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Resistencia a la Vancomicina , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Coagulasa/genética , Coagulasa/farmacología , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/metabolismo , Ratones , Pruebas de Sensibilidad Microbiana , Mutación , Staphylococcus aureus/genética , Resistencia a la Vancomicina/genética , Staphylococcus aureus Resistente a Vancomicina , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/farmacología , beta-Galactosidasa/genética , beta-Galactosidasa/farmacologíaRESUMEN
Cardiac fibroblasts (CFs) undergo senescence in reaction to different stressors, leading to a poor prognosis of cardiac disease. Doxorubicin (Doxo) is an antineoplastic drug with strong cardiotoxic effects, which induces IL-1ß secretion and thus, triggers a potent pro-inflammatory response. Doxo induces CFs senescence; however, the mechanisms are not fully understood. Different pharmacological strategies have been used to eliminate senescent cells by inducing their apoptosis or modifying their secretome. However, Resolvin E1 (RvE1), a lipid derivative resolutive mediator with potent anti-inflammatory effects has not been used before to prevent CFs senescence. CFs were isolated from adult male C57BL/6J mice and subsequently stimulated with Doxo, in the presence or absence of RvE1. Senescence-associated ß-galactosidase activity (SA-ß-gal), γ-H2A.X, p53, p21, and senescence-associated secretory phenotype (SASP) were evaluated. The involvement of the NLRP3 inflammasome/interleukin-1 receptor (IL-1R) signaling pathway on CFs senescence was studied using an NLRP3 inhibitor (MCC950) and an endogenous IL-1R antagonist (IR1A). Doxo is able to trigger CFs senescence, as evidenced by an increase of γ-H2A.X, p53, p21, and SA-ß-gal, and changes in the SASP profile. These Doxo effects were prevented by RvE1. Doxo triggers IL-1ß secretion, which was dependent on NLRP3 activation. Doxo-induced CFs senescence was partially blocked by MCC950 and IR1A. In addition, IL-1ß also triggered CFs senescence, as evidenced by the increase of γ-H2A.X, p53, p21, SA-ß-gal activity, and SASP. All these effects were also prevented by RvE1 treatment. CONCLUSION: These data show the anti-senescent role of RvE1 in Doxo-induced CFs senescence, which could be mediated by reducing IL-1ß secretion.
Asunto(s)
Inflamasomas , Interleucina-1beta/metabolismo , Animales , Antiinflamatorios/farmacología , Senescencia Celular , Doxorrubicina/farmacología , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacología , Fibroblastos/metabolismo , Furanos , Indenos , Inflamasomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptores de Interleucina-1/metabolismo , Sulfonamidas , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
Natural mineral waters (NMWs) emerge from the earth as springs and their beneficial therapeutic effect has been empirically recognized in different countries. Portugal has diverse NMW resources that are sought for the relief of different afflictions including dermatological complications. However, there is a lack of scientific validation supporting this empiric knowledge. In this study, we aimed to screen the in vitro bioactivity of Portuguese NMWs with different chemical profiles, namely sulfurous/bicarbonate/sodic (SBS), bicarbonate/magnesium, sulfated/calcic, sulfurous/chlorinated/sodic, sulfurous/bicarbonate/fluoridated/sodic, and chlorinated/sodic, focusing on aging-related skin alterations. Mouse skin fibroblasts and macrophages were exposed to culture medium prepared in different NMWs. Cellular viability was evaluated by MTT assay and etoposide-induced senescence was analyzed through the beta-galactosidase staining kit. Wound healing was investigated by the scratch assay, and phototoxicity/photoprotection after UVA irradiation was evaluated using a neutral red solution. ROS production was quantified using the 2'7'-dichlorofluorescin diacetate dye, and the activity of superoxide dismutase (SOD) was analyzed by a commercial kit after lipopolysaccharide exposure. NMWs within the SBS profile demonstrated anti-senescence activity in skin fibroblasts, along with a variable effect on cellular viability. Among the tested NMWs, two decreased cellular senescence and preserved cell viability and were therefore selected for subsequent studies, together with a SBS NMW with therapeutic indications for dermatologic diseases. Overall, the selected NMW promoted wound healing in skin fibroblasts and activated SOD in macrophages, thus suggesting an anti-oxidant effect. None of the NMWs prevented phototoxicity after UV irradiation. Our results shed a light on the anti-aging potential of Portuguese NMW, supporting their putative application in cosmetic or medical products.
Asunto(s)
Aguas Minerales , Envejecimiento de la Piel , Animales , Antioxidantes/farmacología , Bicarbonatos , Células Cultivadas , Etopósido/farmacología , Lipopolisacáridos/farmacología , Magnesio , Ratones , Rojo Neutro/farmacología , Portugal , Especies Reactivas de Oxígeno , Piel , Superóxido Dismutasa , Rayos Ultravioleta , beta-Galactosidasa/farmacologíaRESUMEN
Astragaloside IV (AS-IV), as one of the main active components of Astragalus membranaceus, has been reported to have cardiovascular protective effects. However, the role and molecular mechanism of AS-IV in vascular senescence have not been clearly stated. The in vitro aging model was constructed using bleomycin (BLM) in vascular smooth muscle cells (VSMCs). Cell senescence were assessed through Western blotting analysis of aging markers, flow cytometry, and the ß-galactosidase (SA-ß-Gal) kit. Mitophagy was determined through transmission electron microscopy, TMRM staining, and Western blotting analysis of p62. A model of aging blood vessels was induced by D-gal. The vascular wall thickness of mice was also evaluated by H&E staining. Our data proved that AS-IV plays an anti-senescent role in vitro and in vivo. Results showed that AS-IV effectively improved mitochondrial injury, raised MMP, and mediated mitophagy in BLM-induced senescent VSMCs and D-gal induced aging mice. Parkin expression strengthened AS-IV's anti-senescent function. In conclusions, AS-IV attenuated BLM-induced VSMC senescence via Parkin to regulate mitophagy. Therefore, AS-IV-mediated Parkin might be a latent therapeutic agent and target for VSMC senescence.
Asunto(s)
Mitofagia , Músculo Liso Vascular , Animales , Bleomicina/metabolismo , Bleomicina/farmacología , Senescencia Celular , Ratones , Músculo Liso Vascular/metabolismo , Saponinas , Triterpenos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , beta-Galactosidasa/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
PURPOSE: The NAD+-dependent deacetylase, sirtuin 1 (SIRT1), plays an important role in vascular calcification induced by high glucose and/or high phosphate levels. However, the mechanism by which SIRT1 regulates this process is still not fully understood. Thus, this study aimed to determine the role of high glucose and phosphate in vascular calcification and the molecular mechanisms underlying SIRT1 regulation. METHODS: Vascular smooth muscle cells (VSMCs) were cultured under normal, high phosphate, and/or high-glucose conditions for 9 days. Alizarin red staining and calcification content analyses were used to determine calcium deposition. VSMC senescence was detected by ß-galactosidase (SA-ß-Gal) staining and p21 expression. RESULTS: Mouse VSMCs exposed to high phosphate and high glucose in vitro showed increased calcification, which was correlated with the induction of cell senescence, as confirmed by the increased SA-ß-galactosidase activity and p21 expression. SRT1720, an activator of SIRT1, inhibits p65 acetylation, the nuclear factor-κ-gene binding (NF-κB) pathway, and VSMC transdifferentiation, prevents senescence and reactive oxygen species (ROS) production, and reduces vascular calcification. In contrast, sirtinol, an inhibitor of SIRT1, increases p65 acetylation, activates the NF-κB pathway, induces vascular smooth muscle cell transdifferentiation and senescence, and promotes vascular calcification. CONCLUSIONS: High glucose and high phosphate levels induce senescence and vascular calcification in VSMCs, and the combined effect of high glucose and phosphate can inhibit SIRT1 expression. SIRT1 inhibits vascular smooth muscle cell senescence and osteogenic differentiation by inhibiting NF-κB activity, thereby inhibiting vascular calcification.
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Músculo Liso Vascular , Calcificación Vascular , Animales , Células Cultivadas , Glucosa/metabolismo , Glucosa/farmacología , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Osteogénesis/fisiología , Fosfatos , Sirtuina 1/metabolismo , Calcificación Vascular/metabolismo , beta-Galactosidasa/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
Diabetes mellitus contributes to intervertebral disc degeneration. Nucleus pulposus cell senescence plays an important role in intervertebral disc degeneration. However, the effects of hyperglycemia on human nucleus pulposus cells and the underlying process remains poorly understood. In the current study, we evaluated the effects of high glucose levels on human nucleus pulposus cell senescence in vitro and the effects of hyperglycemia on rat nucleus pulposus aging in vivo. Human nucleus pulposus cells were cultured in high-glucose medium (200 mM glucose) for 48 h. Senescence-associated ß-galactosidase staining, western blot analysis, and enzyme-linked immunosorbent assays were performed to evaluate human nucleus pulposus cell senescence. Flow cytometry and enzyme-linked immunosorbent assays were used to evaluate reactive oxygen species and advanced glycation end-product levels. Transcriptome sequencing followed by bioinformatics analysis was used to understand the abnormal biological processes of nucleus pulposus cells cultured in high-glucose medium. Diabetes mellitus rat models were established and histopathological and immunohistochemical analysis was conducted to examine nucleus pulposus tissue senescence in vivo. Exposure to a high glucose concentration promoted human nucleus pulposus cell senescence and increased the senescence-related secretion phenotype in human nucleus pulposus cells in vitro and in rat nucleus pulposus tissue in vivo. Bioinformatics analysis showed that hub genes were involved in nucleus pulposus cell cycle activities and cell senescence. The results suggest that appropriate blood glucose control may be key to preventing intervertebral disc degeneration in diabetic patients.
Asunto(s)
Diabetes Mellitus , Hiperglucemia , Degeneración del Disco Intervertebral , Núcleo Pulposo , Animales , Senescencia Celular , Diabetes Mellitus/metabolismo , Glucosa/metabolismo , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Hiperglucemia/patología , Degeneración del Disco Intervertebral/patología , Núcleo Pulposo/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , beta-Galactosidasa/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.
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Biocatálisis , Carbono/química , Carbono/farmacología , Nitrógeno/química , Nitrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Corona de Proteínas/metabolismo , Puntos Cuánticos/química , Puntos Cuánticos/metabolismo , Células A549 , Animales , Apirasa/química , Apirasa/farmacología , Catalasa/química , Catalasa/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/farmacología , Células HeLa , Humanos , Ratas , Especies Reactivas de Oxígeno/metabolismo , beta-Galactosidasa/química , beta-Galactosidasa/farmacologíaRESUMEN
In prior research it has been demonstrated that methylated ß-cyclodextrins-threaded acid-labile polyrotaxanes (Me-PRXs) exhibit a lower critical solution temperature (LCST) and form coacervate droplets above their LCST. In this study, the encapsulation of proteins in coacervate droplets and the pH-responsive release of proteins, through the acid-induced dissociation of the Me-PRX, were investigated. The coacervate droplets encapsulate various proteins, such as bovine serum albumin (BSA), lysozyme, and ß-galactosidase, at pH 7.4, into their hydrophobic inner phase. Concomitant with the pH-dependent dissociation of the Me-PRXs, the coacervates degraded below pH 6.5 and released encapsulated proteins, with the intrinsic activity of proteins maintained. Additionally, the subcutaneous injection of coacervate droplets encapsulating BSA in mice revealed that the retention time of the BSA at the injection site was prolonged compared to that of free BSA. Altogether, the coacervate droplets of the Me-PRX would be utilized as a new class of pH-responsive and injectable carrier for the controlled release of therapeutic proteins.
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Portadores de Fármacos , Muramidasa , Rotaxanos , Albúmina Sérica Bovina , beta-Galactosidasa , Animales , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacología , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos BALB C , Muramidasa/química , Muramidasa/farmacocinética , Muramidasa/farmacología , Rotaxanos/química , Rotaxanos/farmacocinética , Rotaxanos/farmacología , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/farmacocinética , Albúmina Sérica Bovina/farmacología , beta-Galactosidasa/química , beta-Galactosidasa/farmacocinética , beta-Galactosidasa/farmacologíaRESUMEN
The fabrication and performance of mechanically responsive multilayer superhydrophobic composites are reported. The application of tensile strain triggers the release of small molecules and proteins from these composites, with different tensile strain magnitudes and coating thickness influencing agent release. These mechanoresponsive composites consist of an absorbent drug core surrounded by an electrosprayed superhydrophobic protective coating that limits drug release in the absence of tensile strain. Coating thickness and applied tensile strain control release of chemotherapeutic cisplatin and enzyme ß-galactosidase, as measured by atomic absorption and UV-vis spectrophotometry, respectively, with preserved in vitro activity. Such mechanically responsive drug delivery devices, when coupled to existing dynamic mechanical forces in the body or integrated with mechanical medical devices, such as stents, will provide local controlled dosing.
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Cisplatino , Sistemas de Liberación de Medicamentos/métodos , Interacciones Hidrofóbicas e Hidrofílicas , beta-Galactosidasa , Línea Celular Tumoral , Cisplatino/química , Cisplatino/farmacocinética , Cisplatino/farmacología , Humanos , beta-Galactosidasa/química , beta-Galactosidasa/farmacocinética , beta-Galactosidasa/farmacologíaRESUMEN
AIM: Polymersomes are created to deliver an enzyme-based therapy to the brain in lysosomal storage disease patients. MATERIALS & METHODS: Polymersomes are formed via the injection method using poly(ethylene glycol)-b-poly(lactic acid) (PEGPLA) and bound to apolipoprotein E, to create a brain-targeted delivery vehicle. RESULTS: Polymersomes have a smallest average diameter of 145 ± 21 nm and encapsulate ß-galactosidase at 72.0 ± 12.2% efficiency. PEGPLA polymersomes demonstrate limited release at physiologic pH (7.4), with a burst release at the acidic pH (4.8) of the lysosome. PEGPLA polymersomes facilitate delivery of active ß-galactosidase to an in vitro model of GM1 gangliosidosis. CONCLUSION: The foundation has been laid for testing of PEGPLA polymersomes to deliver enzymatic treatments to the brain in lysosomal storage disorders for the first time.
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Portadores de Fármacos/química , Terapia de Reemplazo Enzimático/métodos , Lactatos/química , Polietilenglicoles/química , beta-Galactosidasa/farmacología , Encéfalo/metabolismo , Línea Celular , Liberación de Fármacos , Gangliosidosis GM1/tratamiento farmacológico , Humanos , Concentración de Iones de Hidrógeno , Nanopartículas/química , Tamaño de la Partícula , Permeabilidad , Propiedades de SuperficieRESUMEN
ß-galactosidase was successfully encapsulated within an amino-functionalised silica matrix using a "fish-in-net" approach and molecular imprinting technique followed by covalent binding of lysozyme via a glutaraldehyde-based method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy were used to characterise the silica matrix hosting the two enzymes. Both encapsulated ß-galactosidase and bound lysozyme exhibited high enzymatic activities and outstanding operational stability in model reactions. Moreover, enzyme activities of the co-immobilised enzymes did not obviously change relative to enzymes immobilised separately. In antibacterial tests, bound lysozyme exhibited 95.5% and 89.6% growth inhibition of Staphylococcus aureus ATCC (American type culture collection) 653 and Escherichia coli ATCC 1122, respectively. In milk treated with co-immobilised enzymes, favourable results were obtained regarding reduction of cell viability and high lactose hydrolysis rate. In addition, when both co-immobilised enzymes were employed to treat milk, high operational and storage stabilities were observed. The results demonstrate that the use of co-immobilised enzymes holds promise as an industrial strategy for producing low lactose milk to benefit people with lactose intolerance.
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Antiinfecciosos/química , Lactosa/química , Muramidasa/química , Dióxido de Silicio/química , beta-Galactosidasa/química , Animales , Antiinfecciosos/farmacología , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Microscopía Electrónica de Rastreo , Leche/química , Impresión Molecular , Muramidasa/farmacología , Espectroscopía Infrarroja por Transformada de Fourier , Staphylococcus aureus/efectos de los fármacos , Difracción de Rayos X , beta-Galactosidasa/farmacologíaRESUMEN
We have developed cationic polyrotaxanes composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (α-CDs) threaded along a poly(ethylene glycol) (PEG) chain capped with a terminal bulky stopper (DMAE-PRX) for the intracellular delivery of proteins through the polyelectrolyte complexation. Herein, to ascertain the effect of supramolecular backbone structure of cationic polyrotaxanes, the physicochemical properties and biological activity of polyelectrolyte complex with anionic ß-galactosidase (ß-gal) were investigated in comparison to a cationic linear polymer, poly[2-(N,N-dimethylaminoethyl) methacrylate] (PDMAEMA). In the cellular experiments, the DMAE-PRX/ß-gal complexes exhibited higher intracellular uptake of ß-gal and sustainable enzymatic activity of delivered ß-gal than the PDMAEMA/ß-gal complexes. It is considered that the cationic polyrotaxanes are promising supramolecular backbone structure for the intracellular protein delivery.
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Sistemas de Liberación de Medicamentos , Metacrilatos/química , Nylons/química , Rotaxanos/química , Ciclodextrinas/química , Ciclodextrinas/farmacología , Células HeLa , Humanos , Metacrilatos/farmacología , Nylons/farmacología , Poliaminas/química , Poliaminas/farmacología , Polielectrolitos , Polietilenglicoles/química , Polietilenglicoles/farmacología , Rotaxanos/farmacología , beta-Galactosidasa/química , beta-Galactosidasa/farmacologíaRESUMEN
Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol abundant in red wine, is known to extend the life span of diverse species. On the contrary, it was reported that angiotensin (Ang) II enhances senescence of vascular smooth muscle cells (VSMCs). We, therefore, examined whether resveratrol attenuates Ang II-induced senescence of VSMC. Senescence-associated ß-galactosidase (SA ß-gal) assay showed that Ang II induced senescence of VSMC. The Ang II-induced senescence was inhibited by losartan, an Ang II type 1 receptor (AT1R) antagonist but not by PD123319, Ang II type 2 receptor antagonist, indicating that AT1R is responsible for the induction of senescence. Resveratrol suppressed Ang II-induced senescence of VSMC in a dose-dependent manner. In addition, resveratrol suppressed Ang II-induced induction of p53 and its downstream target gene p21, both of which play an important role in the induction of senescence. Resveratrol suppressed senescence of VSMC possibly through inhibition of AT1R-dependent induction of p53/p21. Suppression of p53 induction may be involved in the longevity by resveratrol.
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Angiotensina II/efectos adversos , Senescencia Celular/efectos de los fármacos , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Estilbenos/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Western Blotting , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Pruebas de Enzimas , Imidazoles/farmacología , Losartán/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Receptor de Angiotensina Tipo 1/metabolismo , Resveratrol , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.
Asunto(s)
ADN/farmacología , Portadores de Fármacos/síntesis química , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Técnicas de Transferencia de Gen , Metacrilatos/síntesis química , beta-Galactosidasa/farmacología , Acrilatos/química , Animales , Cationes/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Microscopía por Crioelectrón , ADN/genética , ADN/metabolismo , Portadores de Fármacos/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Haplorrinos , Humanos , Metacrilatos/metabolismo , Micelas , Plásmidos , Polietilenglicoles/química , Polímeros/química , Poliestirenos/química , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
G(M1) -gangliosidosis is a fatal neurodegenerative disorder caused by deficiency of lysosomal acid ß-galactosidase (ß-gal). Accumulation of its substrate ganglioside G(M1) (G(M1) ) in lysosomes and other parts of the cell leads to progressive neurodegeneration, but underlying mechanisms remain unclear. Previous studies demonstrated an essential role for interaction of G(M1) with tropomyosin receptor kinase (Trk) receptors in neuronal growth, survival and differentiation. In this study we demonstrate accumulation of G(M1) in the cell-surface rafts and lysosomes of the ß-gal knockout (ß-gal-/-) mouse brain association with accumulation of Trk receptors and enhancement of its downstream signaling. Immunofluorescence and subcellular fractionation analysis revealed accumulation of Trk receptors in the late endosomes/lysosomes of the ß-gal-/- mouse brain and their association with ubiquitin and p62. Administration of a chemical chaperone to ß-gal-/- mouse expressing human mutant R201C protein resulted in a marked reduction of intracellular storage of G(M1) and phosphorylated Trk. These findings indicate that G(M1) accumulation in rafts causes activation of Trk signaling, which may participate in the pathogenesis of G(M1) -gangliosidosis.
Asunto(s)
Química Encefálica/fisiología , Gangliosidosis GM1/metabolismo , Hexosaminas/farmacología , Lisosomas/metabolismo , Chaperonas Moleculares/farmacología , Receptor trkA/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Encéfalo/patología , Química Encefálica/efectos de los fármacos , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Microdominios de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/fisiología , Fosforilación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , beta-Galactosidasa/genética , beta-Galactosidasa/farmacologíaRESUMEN
An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide bonds in the main chain, by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA), histamine (HIS) to CBA, and ABOL to bis(acryloyl)piperazine (BAP). These water-soluble PAAs efficiently condense ß-galactosidase by self-assembly into nanoscaled and positively-charged complexes. Stable under neutral extracellular conditions, the disulfide-containing nanocomplexes rapidly destabilized in a reductive intracellular environment. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially non-toxic. ß-Galactosidase was successfully internalized into cells, with up to 94% of the cells showing ß-galactosidase activity, whereas the enzyme alone was not taken up by the cells. The results indicate that these poly(amidoamine)s have excellent properties as highly potent and non-toxic intracellular protein carriers, which should create opportunities for novel applications in protein delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Poliaminas/química , Proteínas/administración & dosificación , Concentración de Iones de Hidrógeno , Nanopartículas , Poliaminas/administración & dosificación , beta-Galactosidasa/administración & dosificación , beta-Galactosidasa/química , beta-Galactosidasa/farmacocinética , beta-Galactosidasa/farmacologíaRESUMEN
Homocysteine plays a key role in endothelial cell senescence associated with atherosclerosis-based cardiovascular diseases. Selaginellin, a component extracted from Selaginella pulvinata (Hook. et Grev.) Maximo, was assessed for its ability to protect human umbilical vein endothelial cells against homocysteine-induced senescence. The endothelial cells were pretreated with various concentrations (10(-7), 3 x 10(-7), or 10(-6) M) of selaginellin for 1 hour before exposure to homocysteine. Selaginellin was shown to protect endothelial cells against homocysteine-induced senescence, as determined by senescence-associated beta-galactosidase activity, telomerase activity, and cell cycle distribution. In addition, the increase in levels of intracellular reactive oxygen species and downregulation of SIRT1 gene expression induced by homocysteine were significantly reversed by selaginellin. Our study suggests that selaginellin has a protective effect against homocysteine-induced senescence through mechanisms related to antioxidation via scavenging reactive oxygen species and upregulating the expression of SIRT1 gene.
Asunto(s)
Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Envejecimiento/efectos de los fármacos , Antioxidantes/metabolismo , Antioxidantes/farmacología , Ciclo Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio/metabolismo , Homocisteína/metabolismo , Homocisteína/farmacología , Homocisteína/fisiología , Humanos , Óxido Nítrico Sintasa de Tipo III , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Venas Umbilicales/metabolismo , Regulación hacia Arriba/efectos de los fármacos , beta-Galactosidasa/metabolismo , beta-Galactosidasa/farmacologíaRESUMEN
Two synthesis methods of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez AN 119) nanoparticles (NP) (used for oral vaccination) were compared. Wheat germ agglutinin (WGA) was used as ligand to enhance the bioadhesive properties of NP and beta-galactosidase as antigen. The first method encapsulated beta-galactosidase in NP by co-precipitation in an acetone/water mixture containing 44% acetone. In the second method, antigen addition occurred in 100% acetone. To improve stability, NP were crosslinked with 1,3-diaminopropane. The stability of WGA-conjugated NP with encapsulated antigen diminished at lower pH and when decreasing the amount of crosslinker. The binding type between WGA and polymer depended on the synthesis method: predominantly ionic bonds were formed using the 44% acetone method, whereas synthesis via the 100% acetone method resulted in covalent bonds. The biological activity of the WGA coating, evaluated via a pig gastric mucin binding test, was lower in NP prepared via the 100% acetone method. No release of native antigen was detected after hydrolysis of NP, due to the covalent antigen binding during antigen encapsulation and the high reactivity of the polymer. Moreover, the mucosal irritation capacity was evaluated upon nanoparticle hydrolysis using a slug mucosal irritation assay. Herein, hydrolysed NP of the 44% acetone method were classified as mild irritative.