Estudo do efeito neuroprotetor e imunomodulador de flavonoides em modelos in vitro da doen??a de Parkinson

Submitted by P??s Imunologia ([email protected]) on 2017-03-14T18:18:14Z No. of bitstreams: 1 Cleonice Creuza.pdf: 2460625 bytes, checksum: 893afe600ef298af83303b91d5023108 (MD5)Approved for entry into archive by Delba Rosa ([email protected]) on 2017-04-24T12:58:29Z (GMT) No. of bitstreams: 1 Cleonice Creuza.pdf: 2460625 bytes, checksum: 893afe600ef298af83303b91d5023108 (MD5)Made available in DSpace on 2017-04-24T12:58:29Z (GMT). No. of bitstreams: 1 Cleonice Creuza.pdf: 2460625 bytes, checksum: 893afe600ef298af83303b91d5023108 (MD5)CapesA doen??a de Parkinson (DP) ?? caracterizada pela perda de neur??nios dopamin??rgicos no mesenc??falo. No entanto, mecanismos molecular respons??vel pelo processo degenerativo no sistema dopamin??rgico nigroestriatal durante a doen??a de Parkinson permanecem desconhecidos. Atualmente, concorda-se que disfun????o mitocondrial, agrega????o de ??- sinucle??na, estresse oxidativo, neuroinflama????o e degrada????o prot??ica s??o causas envolvidas. Estudos farmacol??gicos t??m focado no uso de metabolitos secund??rios de plantas e, entre estes, os flavonoides t??m mostrado atividade biol??gica com efeitos neuroprotetores, antiinflamat??rios e antioxidantes. Neste sentido, o objetivo deste trabalho foi estudar os efeitos neuroprotetores e imunomodulat??rios de flavonoides bioativos usando modelos in vitro de DP. Foram usados diferentes modelos de culturas neuronais, avaliadas pelo testes de MTT e an??lises morfol??gicas para avaliar os efeitos citot??xicos ou neuroprotetores da rutina e quercetina. Em seguida, foram usados diferentes modelos de cultura prim??ria de c??lulas gliais e neuronais e testes de MTT, exclus??o ao azul de tripan, Fluoro-Jade B, Western Blot ou imunocitoqu??mica para ??-III-Tubulina, Western Blot para tirosina hidroxilase ou para GFAP para investigar o efeito citot??xico induzido por aminocromo e/ou o efeito neuroprotetor induzido por rutina. Tamb??m investigamos a resposta imunoinflamat??ria ou imunomodulat??ria dosando citocinas (IL6, IL10, TNF-alpha) usando o m??todo de ELISA. Nossos resultados demonstraram que rutina e quercetina (10-50??M) n??o induziram decr??scimo no metabolismo mitocondrial em linhagens celulares SHSY-5Y ou co-cultura prim??ria de neur??nios/c??lulas gliais, contudo estes aumentaram o metabolismo mitocondrial nas concentra????es de 50 e 100 ??M em c??lulas da linhagem PC12. Al??m disso, rutina (10??M) induziu altera????es morfol??gicas em c??lulas PC12. Tamb??m foi visto que aminocromo (250- 500??M) induziu uma redu????o na viabilidade celular em co-cultura de mesenc??falo e em cultura de microglia, cultura de c??lulas gliais e cultura de neur??nios corticais. Ainda, a exposi????o ao aminocromo reduziu a express??o de ??-III-tubulina, tirosina hidroxilase e GFAP em co-culturas de mesenc??falo e cultura de c??lulas gliais. Nossos resultados demonstram que a rutina protege c??lulas neurais frente a dano induzido por aminocromo, aumenta a express??o de ??-III-tubulina e protege a rede de neuritos. Al??m do mais, aminocromo induziu redu????o de n??veis de citocinas IL-6, IL-10 e TNF-alfa que foram reguladas a n??veis basais quando a cocultura de mesenc??falo foi tratada com rutina. Conclu??mos ent??o que a rutina protege c??lulas PC12 contra danos celulares induzidos por MPTP, co-culturas de mesenc??falo e c??lulas gliais contra danos induzidos por aminocromo. Podemos sugerir que a redu????o no n??vel de citocinas ?? conseq????ncia da perda de c??lulas gliais induzidas por aminocromo e que o retorno a n??veis basais podem ser resultado de glioprote????o e neuroprote????o induzida pela rutina.Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the midbrain. The molecular mechanism responsible for degenerative process in the nigrostriatal dopaminergic system in PD remains unknown. Currently, there is a general agreement that mitochondrial dysfunction, ??-synuclein aggregation, oxidative stress, neuroinflammation and impaired protein degradation are involved. Pharmacological studies have been focusing in using plant secondary metabolites and, among these, flavonoids have shown biological activity such neuroprotective, anti-inflammatory and antioxidant. In this sense, the objective of this work was to study the neuroprotective and immunomodulatory effects of a bioactive flavonoid using in vitro models of PD. We used different models of neuronal culture and MTT test and morphological analysis to make a screening of cytotoxic and/or neuroprotection by rutin and quercetin. After we used different models of primary culture of neuronal and glial cells and MTT test, Tripan Blue, Fluoro-Jade B, western blot or immunocytochemistry of ??-III-Tubulin, tyrosine hydroxylase or GFAP in order to investigate the cytotoxic effect induced by aminochrome and/or neuroprotection induced by rutin. We also investigated immunoinflamatory response or immunomodulation by measuring cytokines (IL6, IL10, TNF-alpha) using ELISA. Our results demonstrated that rutin and quercetin (10-50??M) did not induce decrease in mitochondrial metabolism on SHSY-5Y cells line, or primary coculture of neuron/glial cells, however it increased the mitochondrial metabolism at concentrations 50 and 100??M on PC12 cells line. Moreover rutin (10??M) induce alterations on PC12 cells morphology. We also saw that aminocrhome (250-500??M) induced a decrease on cell viability at midbrain co-culture and microglia culture, glial cells culture, cortical neurons culture. In addition, the exposure to aminocrhome decreased expression of ??-IIItubulin, tyrosine hydroxylase and GFAP on midbrain co-cultures and glial cells culture. Our results demonstrate that rutin protects neural cells from damage induced by aminocrhome, increases ??-III-tubulin expression and protects neurite network. Furthermore, aminocrhome induced reduction of cytokines IL-6, IL-10 and TNF-alpha levels that were regulated to basal levels when the midbrain co-culture was treated with rutin. We concluded that rutin protects against PC12 cells cell against death induced by MPTP and neuronal and glial cells against death induced by aminocrhome. We can suggest that the reduction in cytokine levels are consequence of the loss of glial cells induced by aminocrhome and that the return to baseline levels may result from glioprotection and neuroprotection induced by rutin

Impact of Plant-Derived Flavonoids on Neurodegenerative Diseases

© 2016, Springer Science+Business Media New York.Neurodegenerative disorders have a common characteristic that is the involvement of different cell types, typically the reactivity of astrocytes and microglia, characterizing gliosis, which in turn contributes to the neuronal dysfunction and or death. Flavonoids are secondary metabolites of plant origin widely investigated at present and represent one of the most important and diversified among natural products phenolic groups. Several biological activities are attributed to this class of polyphenols, such as antitumor activity, antioxidant, antiviral, and anti-inflammatory, among others, which give significant pharmacological importance. Our group have observed that flavonoids derived from Brazilian plants Dimorphandra mollis Bent., Croton betulaster Müll. Arg., e Poincianella pyramidalis Tul., botanical synonymous Caesalpinia pyramidalis Tul. also elicit a broad spectrum of responses in astrocytes and neurons in culture as activation

Amburana cearensis seed extracts protect PC-12 cells against toxicity induced by glutamate

Amburana cearensis (Allemão) A.C. Sm., Fabaceae, has been widely studied for its medicinal activities. Many neurodegenerative disorders are caused by oxidative stress, mitochondrial dysfunction, excitotoxicity induced by glutamate and ultimately cell death. This study describes the chemical profile of the ethanolic, hexane, dichloromethane, and ethyl acetate extracts obtained from seeds of A. cearensis. The objective of this study was to investigate the chemical profile of extracts obtained from seeds of A. cearensis, as well as their cytotoxicity and neuroprotective effects in cultures of neural PC12 cells. Metabolite profile was performed by GC–MS. PC12 cells were treated with increasing concentrations of the extracts (0.01–2000 g/ml) and the cell viability was analyzed after 24 and 72 h using an MTT test. For the excitotoxicity assay, PC12 cells were pre-treated with glutamate (1 mM) for 6 h and treated with increasing concentrations (0.1–1000 g/ml) of the extracts. The chromatographic analysis of the extracts detected various compounds with antioxidant properties, with the majority of peaks corresponding to the isoflavone coumarin. Only the hexane extract showed toxicity after 72 h exposure at the highest concentration (1000 g/ml). By contrast, all extracts increased the cellular viability of PC12 cells against the toxicity caused by glutamate. Therefore,the extracts from the seeds of A. cearensis showed no toxicity and have neuroprotective potential against neuronal damage induced by glutamate, which may be related to their antioxidant properties. © 2016 Sociedade Brasilei

The Phytochemical Agathisflavone Modulates miR146a and miR155 in Activated Microglia Involving STAT3 Signaling

MicroRNAs (miRs) act as important post-transcriptional regulators of gene expression in glial cells and have been shown to be involved in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD). Here, we investigated the effects of agathisflavone, a biflavonoid purified from the leaves of Cenostigma pyramidale (Tul.), on modulating the expression of miRs and inflammatory mediators in activated microglia. C20 human microglia were exposed to oligomers of the β-amyloid peptide (Aβ, 500 nM) for 4 h or to lipopolysaccharide (LPS, 1 µg/mL) for 24 h and then treated or not with agathisflavone (1 µM) for 24 h. We observed that β-amyloid and LPS activated microglia to an inflammatory state, with increased expression of miR-146a, miR-155, IL1-β, IL-6, and NOS2. Treatment with agathisflavone resulted in a significant reduction in miR146a and miR-155 induced by LPS or Aβ, as well as inflammatory cytokines IL1-β, IL-6, and NOS2. In cells stimulated with Aβ, there was an increase in p-STAT3 expression that was reduced by agathisflavone treatment. These data identify a role for miRs in the anti-inflammatory effect of agathisflavone on microglia in models of neuroinflammation and AD

Structural Design, Synthesis and Antioxidant, Antileishmania, Anti-Inflammatory and Anticancer Activities of a Novel Quercetin Acetylated Derivative

Quercetin (Q) is a bioflavonoid with biological potential; however, poor solubility in water, extensive enzymatic metabolism and a reduced bioavailability limit its biopharmacological use. The aim of this study was to perform structural modification in Q by acetylation, thus, obtaining the quercetin pentaacetate (Q5) analogue, in order to investigate the biological potentials (antioxidant, antileishmania, anti-inflammatory and cytotoxicity activities) in cell cultures. Q5 was characterized by FTIR, 1H and 13C NMR spectra. The antioxidant potential was evaluated against the radical ABTS•+. The anti-inflammatory potential was evaluated by measuring the pro-inflammatory cytokine tumor necrosis factor (TNF) and the production of nitric oxide (NO) in peritoneal macrophages from BALB/c mice. Cytotoxicity tests were performed using the AlamarBlue method in cancer cells HepG2 (human hepatocarcinoma), HL-60 (promyelocytic leukemia) and MCR-5 (healthy human lung fibroblasts) as well as the MTT method for C6 cell cultures (rat glioma). Q and Q5 showed antioxidant activity of 29% and 18%, respectively, which is justified by the replacement of hydroxyls by acetyl groups. Q and Q5 showed concentration-dependent reductions in NO and TNF production (p 40µM when compared to dexamethasone (20 µM). For the HL-60 lineage, Q5 demonstrated selectivity, inducing death in cancer cells, when compared to the healthy cell line MRC-5 (IC50 > 80 µM). Finally, the cytotoxic superiority of Q5 was verified (IC50 = 11 µM), which, at 50 µM for 24 h, induced changes in the morphology of C6 glioma cells characterized by a round body shape (not yet reported in the literature). The analogue Q5 had potential biological effects and may be promising for further investigations against other cell cultures, particularly neural ones

Structural Design, Synthesis and Antioxidant, Antileishmania, Anti-Inflammatory and Anticancer Activities of a Novel Quercetin Acetylated Derivative

Quercetin (Q) is a bioflavonoid with biological potential; however, poor solubility in water, extensive enzymatic metabolism and a reduced bioavailability limit its biopharmacological use. The aim of this study was to perform structural modification in Q by acetylation, thus, obtaining the quercetin pentaacetate (Q5) analogue, in order to investigate the biological potentials (antioxidant, antileishmania, anti-inflammatory and cytotoxicity activities) in cell cultures. Q5 was characterized by FTIR, 1H and 13C NMR spectra. The antioxidant potential was evaluated against the radical ABTS•+. The anti-inflammatory potential was evaluated by measuring the pro-inflammatory cytokine tumor necrosis factor (TNF) and the production of nitric oxide (NO) in peritoneal macrophages from BALB/c mice. Cytotoxicity tests were performed using the AlamarBlue method in cancer cells HepG2 (human hepatocarcinoma), HL-60 (promyelocytic leukemia) and MCR-5 (healthy human lung fibroblasts) as well as the MTT method for C6 cell cultures (rat glioma). Q and Q5 showed antioxidant activity of 29% and 18%, respectively, which is justified by the replacement of hydroxyls by acetyl groups. Q and Q5 showed concentration-dependent reductions in NO and TNF production (p < 0.05); Q and Q5 showed higher activity at concentrations > 40µM when compared to dexamethasone (20 µM). For the HL-60 lineage, Q5 demonstrated selectivity, inducing death in cancer cells, when compared to the healthy cell line MRC-5 (IC50 > 80 µM). Finally, the cytotoxic superiority of Q5 was verified (IC50 = 11 µM), which, at 50 µM for 24 h, induced changes in the morphology of C6 glioma cells characterized by a round body shape (not yet reported in the literature). The analogue Q5 had potential biological effects and may be promising for further investigations against other cell cultures, particularly neural ones