41,924 research outputs found

    Reduction of lipopolysaccharide content and detoxification in Bordetella pertussis and Bordetella bronchiseptica

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    O g√©nero Bordetella √© composto por m√ļltiplas bact√©rias que causam doen√ßas a n√≠vel respirat√≥rio. Pert√ļssis, ou tosse convulsa, √© causada pelo patog√©nico humano Bordetella pertussis, enquanto Bordetella bronchiseptica afeta uma ampla variedade de mam√≠feros, mas raramente humanos. Embora raramente sejam infetados, a transmiss√£o ocasional em humanos ocorre geralmente em indiv√≠duos imunocomprometidos. Estirpes de B. bronchiseptica isoladas de diferentes esp√©cies hospedeiras possuem caracter√≠sticas gen√©ticas e fenot√≠picas √ļnicas. Infe√ß√£o por esta bact√©ria predisp√Ķe o animal √† coloniza√ß√£o por outros patog√©nicos. Ambas as esp√©cies demonstram uma associa√ß√£o clara com a idade. Na ind√ļstria su√≠na, B. bronchiseptica afeta amplamente as popula√ß√Ķes de jovens leit√Ķes enquanto nos humanos a morbidade e mortalidade s√£o mais graves em crian√ßas infetadas com B. pertussis. Antes da introdu√ß√£o das primeiras vacinas contra a tosse convulsa, esta doen√ßa era uma das principais causas de morte. A primeira gera√ß√£o de vacinas desenvolvida na d√©cada de 1950 √© composta de c√©lulas n√£o vi√°veis do patog√©nico. As severas rea√ß√Ķes adversas provocadas pela administra√ß√£o destas vacinas levaram ao desenvolvimento das vacinas de segunda gera√ß√£o, ou acelulares. Integram estas vacinas componentes bacterianos purificados. Indiv√≠duos vacinados com vacinas acelulares apresentam r√°pido decl√≠nio da imunidade e podem ser portadores assintom√°ticos. A imuniza√ß√£o contra B. bronchiseptica, tanto de c√©lulas n√£o vi√°veis quanto acelulares, foi considerada parcialmente ineficaz ou segura para os animais. O lipopolissacar√≠deo (LPS) ou lipoglicano √© o principal respons√°vel pelas rea√ß√Ķes adversas das vacinas de primeira gera√ß√£o. O LPS √© um dos principais componentes da membrana externa das bact√©rias Gram-negativas e constitui a camada externa da membrana externa. O LPS contribui para a assimetria lip√≠dica na membrana externa, oferecendo uma barreira de permeabilidade eficiente a antimicrobianos e outros fatores de stress. A toxicidade do LPS deriva da sua parte lip√≠dica, denominado l√≠pido A. A bioss√≠ntese do l√≠pido A, ou via de Raetz, inclui nove etapas conservadas catalisadas por enzimas. Esta via tem in√≠cio com a acila√ß√£o de UDP-N-acetilglucosamina (UDPGlcNAc) pela enzima LpxA e subsequente desacetila√ß√£o pela enzima LpxC. A LpxC √© uma enzima dependente de zinco que se correlaciona diretamente com a quantidade de LPS presente na membrana externa. Nas esp√©cies de Bordetella a estrutura do l√≠pido A √© completada ap√≥s a introdu√ß√£o de cadeias acil secund√°rias catalisadas pelas aciltransferases LpxL1 e LpxL2 na cadeia acil prim√°ria. O n√ļmero de cadeias acil √© determinante na atividade endot√≥xica da bact√©ria. A enzima LpxL1 n√£o √© normalmente expressa em B. pertussis, resultando numa estrutura com cinco cadeias acil. Em B. bronchiseptica a express√£o de ambas as enzimas resultam em seis cadeias acil no l√≠pido A distribu√≠das de forma irregular e produzindo uma estrutura assim√©trica. O LPS √© transportado atrav√©s da membrana interna pelo transportador ABC (cassete de liga√ß√£o de ATP) MsbA e posteriormente para a camada externa da membrana externa pela maquinaria de transporte do LPS (Lpt). O transporte mediado pelo MsbA √© espec√≠fico para a acila√ß√£o do l√≠pido A e a sua deple√ß√£o leva √† acumula√ß√£o de LPS e glicerofosfol√≠pidos na membrana interna da bact√©ria. A superexpress√£o deste gene essencial mostrou compensar a inativa√ß√£o do gene lpxL no crescimento de E. coli e Burkholderia cenocepacia. M√ļltiplos mecanismos foram identificados em bact√©rias Gram-negativas para preservar a assimetria lip√≠dica na membrana externa. Perturba√ß√Ķes da membrana ou rutura da bioss√≠ntese do LPS ou do seu sistema de transporte causam uma invers√£o compensat√≥ria de fosfol√≠pidos para a camada externa da membrana externa de forma a acomodar a redu√ß√£o nos n√≠veis de LPS. A fosfolipase PldA atua como um sensor de assimetria lip√≠dica na membrana externa, degradando fosfol√≠pidos que destabilizam a homeostasia. Os √°cidos gordos produzidos pela hidr√≥lise dos fosfol√≠pidos sinalizam para a inibi√ß√£o da degrada√ß√£o da enzima LpxC e consequente aumento do LPS. O sistema de transporte ABC, Mla, facilita o transporte retr√≥grado de fosfol√≠pidos de volta para a membrana interna. Esta via √© regulada positivamente ap√≥s a perda de LPS. Muta√ß√Ķes que resultam na perda de fun√ß√£o de qualquer um dos genes que codificam o transportador Mla afetam toda a via, levando √† rutura do equil√≠brio lip√≠dico na membrana externa e √† acumula√ß√£o de fosfol√≠pidos na superf√≠cie celular. Ap√≥s a interrup√ß√£o dos n√≠veis de LPS, a remo√ß√£o destes dois sistemas permite que a membrana externa recupere a homeostasia entre as camadas da bicamada lip√≠dica. Exemplos de bact√©rias Gramnegativas capazes de sobreviver na aus√™ncia de l√≠pido A s√£o Acinetobacter baumanni, Neisseria meningitidis e Moraxella catarrhalis. Existe a necessidade de vacinas mais seguras e eficientes para B. bronchiseptica e B. pertussis. As vacinas acelulares s√£o menos reativas do que as vacinas de c√©lulas n√£o vi√°veis devido √† quantidade m√≠nima ou inexistente de LPS. O objetivo deste estudo foi aplicar o mesmo princ√≠pio numa vacina com c√©lulas n√£o vi√°veis. Reduzir a quantidade de LPS e a sua endotoxicidade em ambas as esp√©cies envolveu a interfer√™ncia na bioss√≠ntese e estrutura do l√≠pido A, com a suposi√ß√£o de que menos LPS e l√≠pido A com menos cadeias acil traduzem em bact√©rias menos endot√≥xicas. A produ√ß√£o das enzimas-chave da s√≠ntese, LpxA, LpxC, LpxL1 e LpxL2 foi interrompida ou regulada com promotores induz√≠veis. A interfer√™ncia com as duas primeiras enzimas afeta a quantidade de l√≠pido A produzido. A inativa√ß√£o do gene lpxA por meio da introdu√ß√£o de um marcador antibi√≥tico foi combinada com um plasm√≠deo de express√£o contendo o gene expresso a partir de um promotor control√°vel. A constru√ß√£o do vetor contendo o gene lpxA interrompido pela sequ√™ncia de resist√™ncia ao antibi√≥tico cloranfenicol falhou pois n√£o foi poss√≠vel introduzir a sequ√™ncia de resist√™ncia ao antibi√≥tico no vetor. O principal desafio foi conseguir transformantes resistentes a cloranfenicol. A mesma estrat√©gia foi usada para os genes lpxL1 e lpxL2 em simult√Ęneo (lpxL1-2) e lpxL2 em diferentes estirpes de Bordetella. A inativa√ß√£o de lpxL1-2 foi tamb√©m tentada na estirpe deficiente B. pertussis őĒPdlA-MlaF usando um vetor suicida para recombina√ß√£o hom√≥loga e troca al√©lica. A estirpe mutante permite que a c√©lula atinja a homeostasia quando a estrutura de LPS √© deficiente em cadeias acil. A inativa√ß√£o de lpxL1-2 no cromossoma foi efetuada com a colabora√ß√£o de uma c√≥pia dos genes sob um promotor induz√≠vel num plasm√≠deo fora do cromossoma. O mutante n√£o apresentou diferen√ßas de crescimento em compara√ß√£o com a estirpe selvagem (WT). A estrutura do LPS divergiu do controlo WT apresentando uma cadeia acil suplementar. Numa abordagem diferente, a express√£o dos genes lpxC e lpxL2 foi regulada por meio de um promotor control√°vel inserido no cromossoma, todavia a inser√ß√£o cromoss√≥mica foi inexequ√≠vel.Bordetella species cause respiratory diseases in several animals. Pertussis, or whooping cough, is caused by the human pathogen Bordetella pertussis, while Bordetella bronchiseptica affects a broad range of mammals, but rarely humans. Severe morbidity and mortality in young children caused by B. pertussis led to the development of wholecell vaccines. The reactogenicity of the vaccine produced several side effects that later led to development of second-generation acellular vaccines. Individuals vaccinated with acellular vaccines present a rapid waning of immunity and can be asymptomatic carriers. Immunization against B. bronchiseptica, either whole-cell or acellular, has been regarded as not fully effective or safe for the animals. The lipopolysaccharide (LPS) is the main reason for the high reactogenicity of the firstgeneration vaccines. LPS is one of the main components of the outer membrane of Gramnegative bacteria. LPS contributes to the lipid asymmetry in the outer membrane offering an efficient permeability barrier to antimicrobials and other stressors. LPS endotoxicity derives from its lipid A moiety. This study aimed to reduce the LPS amount and endotoxicity in the bacterial cell through reduction of lipid A biosynthesis or by modifying its structure, with the assumption that less LPS and less-acylated lipid A translates in cells with reduced endotoxicity. Interference with lipid A biosynthesis and structure involved the inactivation of the lpxA, lpxL2 and lpxL1 and lpxL2 genes simultaneously and chromosomal regulation of the expression of the lpxC and lpxL2 genes. Constructs were created for the insertion of an inducible promoter that would allow for the regulation of lpxC and lpxL2 expression. However, the chromosomal insertion was unfeasible. Chromosomal knock-out with a copy under an inducible promoter on a plasmid did not present fitness differences compared to the wild-type (WT) strain. LPS structure diverged from the WT control and presented hexa-acylated lipid A.Project under the BacVactory program - A Technology Center for Bacterial Vaccines ‚Äď on Whole-cell and outer-membrane-vesicle vaccines under supervision of Prof. dr. J.P.M. Tommassen at Utrecht University, Faculty of Science, Biology Department, Utrecht, The Netherlands. Financial support for mobility granted by Erasmus

    Efecto de rosuvastatina sobre la expresión in vitro de pecam-1 en células endoteliales humanas estimuladas con los de porphyromonas gingivalis

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    La presencia de bacterias periodonto-patógenas como Pophyromonas gingivalis en lesiones ateroscleróticas y procesos de endotoxemias en pacientes con enfermedad periodontal pueden ser factores que desencadenarían a nivel endotelial una respuesta inflamatoria pro-aterosclerótica. La expresión de moléculas de adhesión tras la activación endotelial como PECAM-1 toma gran importancia en la adhesión y migración de monocitos durante el proceso inflamatorio pro-aterosclerótico. El manejo en la reducción de estos marcadores inflamatorios ha motivado a buscar minuciosamente posibles efectos anti-inflamatorios en medicamentos convencionalmente formulados para prevenir el riesgo de enfermedad vascular siendo actualmente la Rosuvastatina un fármaco con potencial antiinflamatorio, por lo que se evaluó sobre la expresión in vitro de PECAM-1 en células HCAEC, estimuladas con LPS de P. gingivalis 33277 mediante la técnica Cell ELISA Fluorométrica, de igual modo se evaluó el efecto del fármaco sobre la viabilidad celular utilizando la prueba espectrofotométrica de resazurina. Los resultados obtenidos demostraron que Rusuvastatina disminuye la expresión de PECAM-1 en células HCAEC de manera concentración dependiente (p<0.05) y no inducen citotoxicidad sobre las células, a concentraciones de 100 uM- 6,5uM durante un periodo de exposición de 24 horas. Los hallazgos de este estudio sugieren que Rosuvastatina posee un perfil farmacológico promisorio dado que posee efectos moduladores en la adhesión, vislumbrando propiedades anti-inflamatorias y un alto rango de seguridad toxicológica.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias1308-519-28960Inducción de disfunción endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibición de la inflamación por resolvina (rvd1) y estatina (rosuvastatina)n

    Lipopolysaccharides isolated from Eikenella corrodens but not from Porphyromonas gingivalis W83 induce proatherosclerotic inflammatory responses in human coronary artery endothelial cells

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    Eikenella corrodens and Porphyromonas gingivalis are oral microorganisms associated with the periodontal disease and have been identified in atherosclerotic lesions. The pro-atherosclerotic potential of a periodontopathic species depends on the ability of the strain to infect the endothelium. Lipopolysaccharide (LPS) from atherosclerosis-associated bacteria causes innate inflammatory responses in the pathogenic processes induced by microorganisms. The purpose of this study was to compare the pro-inflammatory responses of human coronary artery endothelial cells (HCAECs) to LPS isolated from E. corrodens 23834 and P. gingivalis W83.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias1308-519-28960Inducción de disfunción endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibición de la inflamación por resolvina (rvd1) y estatina (rosuvastatina)n

    NOTCH-1 signalling and other molecular mechanisms in osteoarthritis: potential therapeutic role of olive-derived polyphenols

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    Osteoarthritis (OA) is the most common joint disease and the primary cause of disability in the elderly. Chondrocytes, the only cell type in articular cartilage, are primarily involved in the progression of the disease. The molecular mechanisms underlying OA modifications are not entirely known. Current research in the field is focused on finding new targets and novel therapeutic approaches. Among these, nutraceuticals (including food-derived bioactive compounds) present an interesting option. Our study investigated the molecular mechanisms involved in the onset and progression of OA and, in particular, the role of NOTCH-1 signalling, a novel target of interest in the disease. NOTCH-1 is a major regulator of chondrocyte homeostasis in mature cartilage, but it is deregulated in OA therefore suggesting a potential role in the pathogenesis. We used primary chondrocytes derived from patients with OA undergoing total knee arthroplasty and C28/I2 cells, a human chondrocyte cell line. Our results indicate that this signalling pathway exerts a pivotal role in the progression of OA and especially in chondrocyte differentiation and matrix remodelling by the regulation of key pathways in OA, such as MMP-13, RUNX-2, VEGFA, NFKB1 and its regulator IKKőĪ. NOTCH-1 silencing also prevented cartilage calcification and reduced cell death. Furthermore, we investigated the potential chondroprotective role of two olive-derived nutraceuticals, oleuropein (OE) and hydroxytyrosol (HT), in vitro. Our data showed an antioxidant effect of both compounds against LPS-induced ROS production in C28/I2 cells. In addition to that, OE and HT were effective in decreasing the expression of inflammation markers, as well as of NOTCH-1 and its downstream targets. In conclusion, our research proved that NOTCH-1 signalling pathway is a potential target of OA. Furthermore, olive-derived polyphenols are able to modulate this pathway suggesting a promising strategy in OA management

    Purification and characterization of lipopolysaccharide from Eikenella corrodens 23834 and Porphyromonas gingivalis W83

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    La purificaci√≥n de lipopolisac√°ridos (LPS) o endotoxinas y su caracterizaci√≥n es un aspecto esencial para estudios que buscan aclarar el papel de estas biomol√©culas de bacterias Gram negativas presentes en la cavidad oral y su relaci√≥n con enfermedades locales periodontales y sist√©micas. Este estudio implementa una metodolog√≠a para la extracci√≥n, purificaci√≥n y caracterizaci√≥n de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83, utilizando t√©cnicas previamente descritas. La extracci√≥n cruda de LPS se realiz√≥ con fenol-agua caliente; la purificaci√≥n se realiz√≥ con tratamiento enzim√°tico con nucleasas y proteasa, seguido de cromatograf√≠a de exclusi√≥n por tama√Īo (Sephacryl S-200 HR) con deoxicolato de sodio como fase m√≥vil. La caracterizaci√≥n de los extractos purificados se realiz√≥ por barrido espectrofotom√©trico, pruebas bioqu√≠micas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromog√©nica de LAL. Como control para la identificaci√≥n y caracterizaci√≥n de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis. La metodolog√≠a implementada permiti√≥ la obtenci√≥n de LPS de elevada pureza con la identificaci√≥n de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83. Ambos LPS purificados mostraron capacidad endot√≥xica a bajas concentraciones. La metodolog√≠a implementada en este estudio para la purificaci√≥n y caracterizaci√≥n de LPS a partir de bacteria completa fue eficiente al compararla con los LPS comerciales.Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarify the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed by using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli, Salmonella typhimurium, P. gingivalis, and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low valoraconcentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when it was compared with commercial LPS.Departamento Administrativo de Ciencia, Tecnolog√≠a e Innovaci√≥n [CO] Colciencias1308-519-28960Inducci√≥n de disfunci√≥n endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibici√≥n de la inflamaci√≥n por resolvina (rvd1) y estatina (rosuvastatina)n

    Rosuvastatin inhibits IL-8 and IL-6 production in human coronary artery endothelial cells stimulated with Aggregatibacter actinomycetemcomitans

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    Aggregatibacter actinomycetemcomitans (A.a) is a Gram negative periodontopathogen that has been highly associated with endocarditis and atherosclerosis. However, the potential mechanisms by which A.a could be contributing to atherosclerosis remain unclear. The purpose of this study was to determine the effect of purified LPS from A.a (Aa-LPS) on the expression of pro-inflammatory molecules (i.e., adhesion molecules, Toll-like receptors and cytokines/chemokines) associated with the pathogenesis of atherosclerosis in human coronary artery endothelial cells (HCAECs), as well as evaluating the potential of Rosuvastatin (RSV) for inhibiting the A.a-induced endothelial responses. HCAECs were stimulated with purified A. a-LPS and cytokine expression levels determined by qPCR and flow cytometry, and TLR2 and TLR4 expression evaluated by ELISA fluorometric assay. The effect of RSV in Aa-LPS-induced pro-inflammatory responses was also studied using similar experimental approaches. A. a-LPS increased the expression of IL-6, IL-8, and TLR2 in HCAECs. No effects in the expression of adhesion molecules were observed. Aa-induced IL-6 and IL-8 production was inhibited by RSV particularly at higher doses. These results suggest that Aa-LPS plays a role in pro-inflammatory endothelial responses that could be contributing to the atherosclerotic process, and the use of statins (i.e., RSV) could be reducing the likelihood for Aa-induced pro-atherosclerotic endothelial responses.Departamento Administrativo de Ciencia, Tecnología e Innovación [CO] Colciencias1308-519-28960Inducción de disfunción endotelial in vitro por lipopolisacarido de bacterias periodontopaticas e inhibición de la inflamación por resolvina (rvd1) y estatina (rosuvastatina)n

    Bioprospecção de lípidos da alga vermelha Palmaria palmata como fonte de compostos bioactivos

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    Palmaria palmata is an edible red seaweed rich in the omega-3 eicosapentaenoic acid (EPA), mostly esterified with polar lipids (phospholipids and glycolipids). EPA is known for its positive effects in health, namely in the prevention of non-communicable diseases. However, the polar lipid content of P. palmata is still undervalued and poorly explored. Therefore, this work aimed to (a) bioprospect the polar lipids from P. palmata as a source of add-value compounds with potential health benefits, focusing on their anti-inflammatory and antioxidant properties, and (b) correlate the bioactive potential with the lipid composition, through an untargeted lipidomic approach, using HILIC-ESI-MS/MS to identify and characterize the polar lipid profile. The total lipid extract as well as the glycolipid- and phospholipid-enriched extracts were shown to be sources of compounds with antioxidant potential, scavenging the radicals ABTS‚óŹ+ and DPPH‚óŹ in chemico, and reducing the intracellular oxidative stress in vitro, in RAW macrophages treated with lipopolysaccharide (LPS). The lipid extracts also showed an anti-inflammatory potential, inhibiting the COX-2 activity in chemico, and reducing the production of nitric oxide in vitro and the transcription of pro-inflammatory genes such as Nos2, IL1b, IL6, Tnf e Ptgs2, triggered by LPS in RAW macrophages. These results show that both the phospholipid- and glycolipid-enriched extracts seem to have a relevant role in the bioactivities demonstrated by the total lipid extract. The characterization of the lipid extracts by HILIC-ESI-MS/MS highlighted that the biomass of the seaweed P. palmata farmed in an integrated multitrophic aquaculture (IMTA) is a source of compounds with high nutritional value ‚Äď namely omega-3 PUFAs ‚Äď and bioactive compounds, mainly EPA-rich polar lipids, thus contributing to its valorisation as a healthy and functional food. Future studies are necessary to evaluate the bioaccessibility and bioavailability of these bioactive lipids, in order to ensure that the properties observed in chemico and in vitro will show in their in vivo utilization.A Palmaria palmata √© uma macroalga vermelha comest√≠vel com um elevado teor de √°cido √≥mega-3 eicosapentan√≥ico (EPA) maioritariamente esterificado na forma de l√≠pidos polares (fosfol√≠pidos e glicol√≠pidos). O EPA √© conhecido pelos seus efeitos positivos na sa√ļde, nomeadamente na preven√ß√£o de doen√ßas n√£o comunic√°veis. No entanto, o conte√ļdo de l√≠pidos polares da P. palmata continua desvalorizado e pouco explorado. Assim, este trabalho teve como objetivos (a) a bioprospec√ß√£o dos l√≠pidos polares da P. palmata como fonte de compostos de valor acrescentado e potencialmente ben√©ficos para a sa√ļde, com √™nfase nas suas propriedades anti-inflamat√≥rias e antioxidantes, e (b) relacionar o potencial bioativo com a composi√ß√£o de l√≠pidos polares, atrav√©s duma abordagem lipid√≥mica, usando HILIC-ESI-MS/MS para a identifica√ß√£o e carateriza√ß√£o do perfil lip√≠dico. Quer o extrato lip√≠dico total quer as fra√ß√Ķes enriquecidas em fosfol√≠pidos e glicol√≠pidos revelaram-se fontes de compostos com potencial antioxidante, reduzindo os radicais ABTS‚óŹ+ e DPPH‚óŹ in chemico e diminuindo o stress oxidativo intracelular in vitro, em macr√≥fagos RAW tratados com lipopolissacar√≠deo (LPS). Os extratos lip√≠dicos tamb√©m demonstraram ter potencial anti-inflamat√≥rio, inibindo a atividade da COX-2 in chemico, e diminuindo a produ√ß√£o de √≥xido n√≠trico in vitro e a transcri√ß√£o de genes pro-inflamat√≥rios tais como Nos2, IL1b, IL6, Tnf e Ptgs2, desencadeada por LPS em macr√≥fagos RAW. Estes resultados mostram que as fra√ß√Ķes de fosfol√≠pidos e glicol√≠pidos parecem ter um papel relevante nas bioatividades demonstradas pelo extrato lip√≠dico total. A caracteriza√ß√£o dos extratos lip√≠dicos por HILIC-ESI-MS/MS evidenciou a sua riqueza em esp√©cies moleculares contendo EPA, tendo este perfil sido conservado ap√≥s o fracionamento em fosfol√≠pidos e glicolipidos. Este trabalho demonstrou que a biomassa da macroalga P. palmata cultivada em aquacultura multitr√≥fica integrada (IMTA) √© uma fonte de compostos de elevado valor nutricional - nomeadamente √≥mega-3 PUFAs - e de compostos bioactivos, nomeadamente l√≠pidos polares ricos em EPA, contribuindo assim para a sua valoriza√ß√£o enquanto alimento saud√°vel e funcional. Estudos futuros s√£o necess√°rios para avaliar a bioacessibilidade e biodisponibilidade destes l√≠pidos bioativos, de forma a garantir que as propriedades observadas in chemico e in vitro tenham reflexo na sua utiliza√ß√£o in vivo.Mestrado em Bioqu√≠mic

    Apigenin Inhibits the Progression of Osteoarthritis by Mediating Macrophage Polarization

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    Objective: The overall purpose of this study was to investigate the mechanism of macrophage polarization on chondrocyte injury in osteoarthritis and the protective effect of apigenin on chondrocytes in osteoarthritis. Method: Primary chondrocytes were isolated from the knee cartilage of three-day-old mice, and cells positive for Alsine blue staining and type II collagen immunocytochemical staining were identified and used in followup experiments. Transwell coculture was performed. Chondrocytes were inoculated in the inferior compartment, and macrophages were inoculated in the upper compartment. The experimental groups were the N group, LPS group, and LPS+ apigenin group. The effect of macrophage polarization on chondrocyte inflammation and the protective effect of apigenin on chondrocytes were verified by the drug administration. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of RNA and protein. Experimental OA was induced by modified Hulth surgery in mice. Modified Hulth surgery was performed on the mouse‚Äôs right knee to induce experimental osteoarthritis in mice, with the nonoperative right knee serving as an ipsilateral control. The mice were randomly assigned to three groups (six mice per group): the sham group, the modified Hulth group, and the modified Hulth + apigenin group. Animals were given gavage for four weeks. The protective effect of apigenin on articular cartilage was verified by histological staining and immunohistochemical analysis. Results: Histological staining showed that apigenin had a protective effect on cartilage degeneration induced by modified Hulth surgery. The PCR results showed that apigenin significantly reduced the expression levels of IL-1, IL-6, MMP3, and MMP13 in the articular cartilage of OA mice, and it had a protective effect on articular cartilage. Apigenin reduced the levels of IL-1, IL-6, TNF-őĪ, and IL-12 in macrophages and increased the levels of MG-L1, MG-L2, ARG-1, and IL-10, which can inhibit the M1 polarization of macrophages and promote M2 polarization. In the coculture system, apigenin decreased the protein levels of TRPM7, P-mTOR, BAX, and c-caspase3 in macrophages, while significantly increasing the protein levels of Bcl2. The levels of IL-1, IL-6, MMP13, TNF-őĪ, P38, JNK, and ERK phosphorylation were reduced in chondrocytes. Conclusion: Apigenin alleviates cartilage injury in OA mice induced by modified Hulth. Apigenin inhibits chondrocyte inflammation through the MAPK pathway. Apigenin alleviates macrophage-polarization-induced inflammatory response and chondrocyte apoptosis in the macrophage‚Äďchondrocyte coculture system through the TRPM7-mTOR pathway

    Extraction optimization, structure features, and bioactivities of two polysaccharides from Corydalis decumbens

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    Two polysaccharides (CPS1 and CPW2) from Corydalis decumbens were obtained to develop insights into natural medical resources. Optimal extraction conditions of total sugars were researched using the method of response surface methodology, polysaccharides were purified using a combination of ethanol precipitation and anion-exchange chromatography, and structure features were analyzed by scanning electron microscopy, transmission electron microscopy, and Congo-red assay. The bioactivities were estimated in terms of antioxidant and anti-inflammatory effects. Total sugars were extracted with an experimental yield of 32.74% under optimum conditions. CPS1 and CPW2 were purified with yields of 12.01% and 8.23%, respectively. CPS1 was a unique polysaccharide with a molecular weight (Mw) of 360 kDa and consisted of glucose, galactose, mannose, and arabinose in a ratio of 4.9:2.0:1:1.9, and CPW2 was composed of glucose with the Mw of 550 kDa. CPS1 possessed a four-helix conformation, and CPW2 was identified as a linear molecule without branched and entangled chains. The mRNA expressions of TNF-őĪ (71.80%), IL-1ő≤ (56.55%), IL-6 (43.98%), and COX-2 (91.88%) in LPS-stimulated RAW 264.7 cells were significantly inhibited by 75 őľg/mL CPS1 (P < 0.0001), while CPW2 showed lower inhibitory effects than CPS1. Compared with CPW2, CPS1 showed stronger scavenging abilities for hydroxyl (EC50 = 520.46 őľg/mL), ABTS (EC50 = 533.99 őľg/mL), and superoxide (EC50 = 1512.06 őľg/mL) radicals. CPS1 with four-helix conformation exhibited more outstanding bioactivities than CPW2 without entangled chains

    Mushroom ő≤-glucan and polyphenol formulations as natural immunity boosters and balancers: nature of the application

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    Mushrooms are experiencing a kind of renaissance as a part of the contemporary human diet. These valuable organisms are more than food, they fi t in perfectly as a novel market group known as nutra-mycoceuticals. Immune-balancing mushroom dietary fibers and secondary metabolites such as polyphenols are the main focus of the healthcare industry. Wellness and cosmetic companies are increasingly using mushroom extracts rich in these ingredients. This review considers the basic molecular immunomodulatory mechanisms of action of the most commonly used mushroom dietary fibers, ő≤-glucans. The literature data on their bioavailability, metabolic transformations, preclinical and human clinical research, and safety are discussed. Immunomodulatory mechanisms of polyphenol ingredients are also considered. These molecules present great potential in the design of the new immunity balancer formulations according to their widespread structural diversity. Finally, we draw attention to the perspectives of modern trends in mushroom nutraceutical and cosmeceutical formulations to strengthen and balance immunity
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