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Rapid screening of botanical extracts for the discovery of bioactive natural products was performed using a fractionation approach in conjunction with flow-injection high-resolution mass spectrometry for obtaining chemical fingerprints of each fraction, enabling the correlation of the relative abundance of molecular features (representing individual phytochemicals) with the read-outs of bioassays. We applied this strategy for discovering and identifying constituents of Centella asiatica (C. asiatica) that protect against Aß cytotoxicity in vitro. C. asiatica has been associated with improving mental health and cognitive function, with potential use in Alzheimer's disease. Human neuroblastoma MC65 cells were exposed to subfractions of an aqueous extract of C. asiatica to evaluate the protective benefit derived from these subfractions against amyloid ß-cytotoxicity. The % viability score of the cells exposed to each subfraction was used in conjunction with the intensity of the molecular features in two computational models, namely Elastic Net and selectivity ratio, to determine the relationship of the peak intensity of molecular features with % viability. Finally, the correlation of mass spectral features with MC65 protection and their abundance in different sub-fractions were visualized using GNPS molecular networking. Both computational methods unequivocally identified dicaffeoylquinic acids as providing strong protection against Aß-toxicity in MC65 cells, in agreement with the protective effects observed for these compounds in previous preclinical model studies.
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Doença de Alzheimer , Centella , Ácido Quínico/análogos & derivados , Triterpenos , Humanos , Peptídeos beta-Amiloides/toxicidade , Doença de Alzheimer/tratamento farmacológico , Extratos Vegetais/farmacologia , Cognição , Centella/química , Triterpenos/análise , Bioensaio , Simulação por ComputadorRESUMO
Based on previous evidence that the non-steroidal estrogen receptor modulator STX mitigates the effects of neurotoxic Amyloid-ß (Aß) in vitro, we have evaluated its neuroprotective benefits in a mouse model of Alzheimer's disease. Cohorts of 5XFAD mice, which begin to accumulate cerebral Aß at two months of age, were treated with orally-administered STX starting at 6 months of age for two months. After behavioral testing to evaluate cognitive function, biochemical and immunohistochemical assays were used to analyze key markers of mitochondrial function and synaptic integrity. Oral STX treatment attenuated Aß-associated mitochondrial toxicity and synaptic toxicity in the brain, as previously documented in cultured neurons. STX also moderately improved spatial memory in 5XFAD mice. In addition, STX reduced markers for reactive astrocytosis and microgliosis surrounding amyloid plaques, and also unexpectedly reduced overall levels of cerebral Aß in the brain. The neuroprotective effects of STX were more robust in females than in males. These results suggest that STX may have therapeutic potential in Alzheimer's Disease.
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Doença de Alzheimer , Síndromes Neurotóxicas , Masculino , Feminino , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Camundongos Transgênicos , Peptídeos beta-Amiloides , Modelos Animais de Doenças , Placa Amiloide/tratamento farmacológicoRESUMO
Chronic or excess glucocorticoid exposure causes lipid disorders such as hypertriglyceridemia and hepatic steatosis. Angptl4 (angiopoietin-like 4), a primary target gene of the glucocorticoid receptor in hepatocytes and adipocytes, is required for hypertriglyceridemia and hepatic steatosis induced by the synthetic glucocorticoid dexamethasone. Angptl4 has also been shown to be required for dexamethasone-induced hepatic ceramide production. Here, we further examined the role of ceramide-mediated signaling in hepatic dyslipidemia caused by chronic glucocorticoid exposure. Using a stable isotope-labeling technique, we found that dexamethasone treatment induced the rate of hepatic de novo lipogenesis and triglyceride synthesis. These dexamethasone responses were compromised in Angptl4-null mice (Angptl4-/-). Treating mice with myriocin, an inhibitor of the rate-controlling enzyme of de novo ceramide synthesis, serine palmitoyltransferase long-chain base subunit 1 (SPTLC1)/SPTLC2, decreased dexamethasone-induced plasma and liver triglyceride levels in WT but not Angptl4-/- mice. We noted similar results in mice infected with adeno-associated virus-expressing small hairpin RNAs targeting Sptlc2. Protein phosphatase 2 phosphatase activator (PP2A) and protein kinase Cζ (PKCζ) are two known downstream effectors of ceramides. We found here that mice treated with an inhibitor of PKCζ, 2-acetyl-1,3-cyclopentanedione (ACPD), had lower levels of dexamethasone-induced triglyceride accumulation in plasma and liver. However, small hairpin RNA-mediated targeting of the catalytic PP2A subunit (Ppp2ca) had no effect on dexamethasone responses on plasma and liver triglyceride levels. Overall, our results indicate that chronic dexamethasone treatment induces an ANGPTL4-ceramide-PKCζ axis that activates hepatic de novo lipogenesis and triglyceride synthesis, resulting in lipid disorders.
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Proteína 4 Semelhante a Angiopoietina/metabolismo , Ceramidas/metabolismo , Dexametasona/toxicidade , Fígado/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteína 4 Semelhante a Angiopoietina/deficiência , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Hipertrigliceridemia/etiologia , Hipertrigliceridemia/metabolismo , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase C/antagonistas & inibidores , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
INTRODUCTION: The phytochemical composition of plant material governs the bioactivity and potential health benefits as well as the outcomes and reproducibility of laboratory studies and clinical trials. OBJECTIVE: The objective of this work was to develop an efficient method for the in-depth characterisation of plant extracts and quantification of marker compounds that can be potentially used for subsequent product integrity studies. Centella asiatica (L.) Urb., an Ayurvedic herb with potential applications in enhancing mental health and cognitive function, was used as a case study. METHODS: A quadrupole time-of-flight analyser in conjunction with an optimised high-performance liquid chromatography (HPLC) separation was used for in-depth untargeted fingerprinting and post-acquisition precursor ion quantification to determine levels of distinct phytochemicals in various C. asiatica extracts. RESULTS: We demonstrate the utility of this workflow for the characterisation of extracts of C. asiatica. This integrated workflow allowed the identification or tentative identification of 117 compounds, chemically interconnected based on Tanimoto chemical similarity, and the accurate quantification of 24 phytochemicals commonly found in C. asiatica extracts. CONCLUSION: We report a phytochemical analysis method combining liquid chromatography, high resolution mass spectral data acquisition, and post-acquisition interrogation that allows chemical fingerprints of botanicals to be obtained in conjunction with accurate quantification of distinct phytochemicals. The variability in the composition of specialised metabolites across different C. asiatica accessions was substantial, demonstrating that detailed characterisation of plant extracts is a prerequisite for reproducible use in laboratory studies, clinical trials and safe consumption. The methodological approach is generally applicable to other botanical products.
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Centella , Triterpenos , Cromatografia Líquida de Alta Pressão , Compostos Fitoquímicos , Extratos Vegetais , Reprodutibilidade dos Testes , Triterpenos/análiseRESUMO
Centella asiatica is a medicinal plant used to enhance memory. We have previously shown that a water extract of Centella asiatica (CAW) attenuates ß-amyloid (Aß)-induced spatial memory deficits in mice and improves neuronal health. Yet the effect of CAW on other cognitive domains remains unexplored as does its in vivo mechanism of improving Aß-related cognitive impairment. This study investigates the effects of CAW on learning, memory and executive function as well as mitochondrial function and antioxidant response in the 5xFAD model of Aß accumulation. Seven month old 5xFAD female mice were treated with CAW (2â¯mg/mL) in their drinking water for two weeks prior to behavioral testing. Learning, memory and executive function were assessed using the object location memory task (OLM), conditioned fear response (CFR) and odor discrimination reversal learning (ODRL) test. Mitochondrial function was profiled using the Seahorse XF platform in hippocampal mitochondria isolated from these animals and tissue was harvested for assessment of mitochondrial, antioxidant and synaptic proteins. CAW improved performance in all behavioral tests in the 5xFAD but had no effect on WT animals. Hippocampal mitochondrial function was improved and hippocampal and cortical expression of mitochondrial genes was increased in CAW-treated 5xFAD mice. Gene expression of the transcription factor NRF2, as well as its antioxidant target enzymes, was also increased with CAW treatment in both WT and 5xFAD mice. CAW treatment also decreased Aß-plaque burden in the hippocampus of treated 5xFAD mice but had no effect on plaques in the cortex. These data show that CAW can improve many facets of Aß-related cognitive impairment in 5xFAD mice. Oral treatment with CAW also attenuates hippocampal mitochondrial dysfunction in these animals. Because mitochondrial dysfunction and oxidative stress accompany cognitive impairment in many pathological conditions beyond Alzheimer's disease, this suggests potentially broad therapeutic utility of CAW.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Centella/metabolismo , Função Executiva/fisiologia , Memória/fisiologia , Mitocôndrias/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Hipocampo/metabolismo , Aprendizagem/fisiologia , Camundongos Transgênicos , Neurônios/metabolismo , Extratos Vegetais , Triterpenos/metabolismoRESUMO
Angptl4 (Angiopoietin-like 4) is a circulating protein secreted by white and brown adipose tissues and the liver. Structurally, Angptl4 contains an N-terminal coiled-coil domain (CCD) connected to a C-terminal fibrinogen-like domain (FLD) via a cleavable linker, and both full-length Angptl4 and its individual domains circulate in the bloodstream. Angptl4 inhibits extracellular lipoprotein lipase (LPL) activity and stimulates the lipolysis of triacylglycerol stored by adipocytes in the white adipose tissue (WAT). The former activity is furnished by the CCD, but the Angptl4 domain responsible for stimulating adipocyte lipolysis is unknown. We show here that the purified FLD of Angptl4 is sufficient to stimulate lipolysis in mouse primary adipocytes and that increasing circulating FLD levels in mice through adenovirus-mediated overexpression (Ad-FLD) not only induces WAT lipolysis in vivo but also reduces diet-induced obesity without affecting LPL activity. Intriguingly, reduced adiposity in Ad-FLD mice was associated with increased oxygen consumption, fat utilization, and the expression of thermogenic genes (Ucp1 and Ppargc1a) in subcutaneous WAT. Moreover, Ad-FLD mice exhibited increased glucose tolerance. Chronically enhancing WAT lipolysis could produce ectopic steatosis because of an overflow of lipids from the WAT to peripheral tissues; however, this did not occur when Ad-FLD mice were fed a high-fat diet. Rather, these mice had reductions in both circulating triacylglycerol levels and the mRNA levels of lipogenic genes in the liver and skeletal muscle. We conclude that separating the FLD from the CCD-mediated LPL-inhibitory activity of full-length Angptl4 reveals lipolytic and thermogenic properties with therapeutic relevance to obesity and diabetes.
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Gordura Abdominal/metabolismo , Angiopoietinas/metabolismo , Metabolismo Energético , Lipólise , Modelos Biológicos , Regulação para Cima , Gordura Abdominal/citologia , Gordura Abdominal/patologia , Tecido Adiposo Bege/citologia , Tecido Adiposo Bege/metabolismo , Tecido Adiposo Bege/patologia , Adiposidade , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/sangue , Angiopoietinas/química , Angiopoietinas/genética , Animais , Células Cultivadas , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Mutação , Obesidade/sangue , Obesidade/metabolismo , Obesidade/patologia , Obesidade/prevenção & controle , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismoRESUMO
This review describes in detail the phytochemistry and neurological effects of the medicinal herb Centella asiatica (L.) Urban. C. asiatica is a small perennial plant that grows in moist, tropical and sub-tropical regions throughout the world. Phytochemicals identified from C. asiatica to date include isoprenoids (sesquiterpenes, plant sterols, pentacyclic triterpenoids and saponins) and phenylpropanoid derivatives (eugenol derivatives, caffeoylquinic acids, and flavonoids). Contemporary methods for fingerprinting and characterization of compounds in C. asiatica extracts include liquid chromatography and/or ion mobility spectrometry in conjunction with high-resolution mass spectrometry. Multiple studies in rodent models, and a limited number of human studies support C. asiatica's traditional reputation as a cognitive enhancer, as well as its anxiolytic and anticonvulsant effects. Neuroprotective effects of C.asiatica are seen in several in vitro models, for example against beta amyloid toxicity, and appear to be associated with increased mitochondrial activity, improved antioxidant status, and/or inhibition of the pro-inflammatory enzyme, phospholipase A2. Neurotropic effects of C. asiatica include increased dendritic arborization and synaptogenesis, and may be due to modulations of signal transduction pathways such as ERK1/2 and Akt. Many of these neurotropic and neuroprotective properties of C.asiatica have been associated with the triterpene compounds asiatic acid, asiaticoside and madecassoside. More recently, caffeoylquinic acids are emerging as a second important group of active compounds in C. asiatica, with the potential of enhancing the Nrf2-antioxidant response pathway. The absorption, distribution, metabolism and excretion of the triterpenes, caffeoylquinic acids and flavonoids found in C. asiatica have been studied in humans and animal models, and the compounds or their metabolites found in the brain. This review highlights the remarkable potential for C. asiatica extracts and derivatives to be used in the treatment of neurological conditions, and considers the further research needed to actualize this possibility.
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Mitochondrial dysfunction is observed in brains of Alzheimer's Disease patients as well as many rodent model systems including those modeling mutations in preseinilin 1 (PSEN1). The aim of our study was to characterize mitochondrial function and number in fibroblasts from AD patients with PSEN1 mutations. We used biochemical assays, metabolic profiling and fluorescent labeling to assess mitochondrial number and function in fibroblasts from three AD patients compared to fibroblasts from three controls. The mutant AD fibroblasts had increased Aß42 relative to controls along with reduction in ATP, basal and maximal mitochondrial respiration as well as impaired spare mitochondrial respiratory capacity. Fluorescent staining and expression of genes encoding electron transport chain enzymes showed diminished mitochondrial content in the AD fibroblasts. This study demonstrates that mitochondrial dysfunction is observable in AD fibroblasts and provides evidence that this model system could be useful as a tool to screen disease-modifying compounds.
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Doença de Alzheimer/patologia , Fibroblastos/patologia , Fibroblastos/fisiologia , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Encéfalo/patologia , Encéfalo/fisiologia , Células Cultivadas , HumanosRESUMO
Oxidative stress and neuroinflammation are widespread in the Parkinson's disease (PD) brain and contribute to the synaptic degradation and dopaminergic cell loss that result in cognitive impairment and motor dysfunction. The polymethoxyflavone Gardenin A (GA) has been shown to activate the NRF2-regulated antioxidant pathway and inhibit the NFkB-dependent pro-inflammatory pathway in a Drosophila model of PD. Here, we evaluate the effects of GA on A53T alpha-synuclein overexpressing (A53TSyn) mice. A53TSyn mice were treated orally for 4 weeks with 0, 25, or 100â¯mg/kg GA. In the fourth week, mice underwent behavioral testing and tissue was harvested for immunohistochemical analysis of tyrosine hydroxylase (TH) and phosphorylated alpha synuclein (pSyn) expression, and quantification of synaptic, antioxidant and inflammatory gene expression. Results were compared to vehicle-treated C57BL6J mice. Treatment with 100â¯mg/kg GA improved associative memory and decreased abnormalities in mobility and gait in A53TSyn mice. GA treatment also reduced pSyn levels in both the cortex and hippocampus and attenuated the reduction in TH expression in the striatum seen in A53Tsyn mice. Additionally, GA increased cortical expression of NRF2-regulated antioxidant genes and decreased expression of NFkB-dependent pro-inflammatory genes. GA was readily detectable in the brains of treated mice and modulated the lipid profile in the deep gray brain tissue of those animals. While the beneficial effects of GA on cognitive deficits, motor dysfunction and PD pathology are promising, future studies are needed to further fully elucidate the mechanism of action of GA, optimizing dosing and confirm these effects in other PD models.
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Flavonas , Doença de Parkinson , Tirosina 3-Mono-Oxigenase , Camundongos , Animais , Tirosina 3-Mono-Oxigenase/metabolismo , Fator 2 Relacionado a NF-E2 , Antioxidantes/farmacologia , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Neurônios Dopaminérgicos/metabolismo , Cognição , Modelos Animais de DoençasRESUMO
Introduction: Centella asiatica (CA) is known in Ayurvedic medicine as a rejuvenating herb with particular benefits in the nervous system. Two groups of specialized metabolites found in CA and purported to contribute to its beneficial effects are triterpenes (TTs) and caffeoylquinic acids (CQAs). In order to evaluate the role and interactions of TTs and CQAs in the effects of CA, we examined the neurotrophic effects of a water extract of CA (CAW) and combinations of its TT and CQA components in mouse primary hippocampal neurons in vitro and in Drosophila melanogaster flies in vivo. Methods: Primary hippocampal neurons were isolated from mouse embryos and exposed in vitro for 5 days to CAW (50 µg/mL), mixtures of TTs, CQAs or TT + CQA components or to 4 TTs or 8 individual CQA compounds of CAW. Dendritic arborization was evaluated using Sholl analysis. Drosophila flies were aged to 28 days and treated for 2 weeks with CAW (10 mg/mL) in the food, mixtures of TTs, CQAs or TT + CQA and individual TT and CQA compounds. TTs and CQAs were tested at concentrations matching their levels in the CAW treatment used. After 2 weeks of treatment, Drosophila aged 42 days were evaluated for phototaxis responses. Results: In mouse primary hippocampal neurons, CAW (50 µg/mL), the TT mix, CQA mix, all individual TTs and most CQAs significantly increased dendritic arborization to greater than control levels. However, the TT + CQA combination significantly decreased dendritic arborization. In Drosophila, a marked age-related decline in fast phototaxis response was observed in both males and females over a 60 days period. However, resilience to this decline was afforded in both male and female flies by treatment from 28 days onwards with CAW (10 mg/mL), or equivalent concentrations of mixed TTs, mixed CQAs and a TT + CQA mix. Of all the individual compounds, only 1,5-diCQA slowed age-related decline in phototaxis in male and female flies. Discussion: This study confirmed the ability of CAW to increase mouse neuronal dendritic arborization, and to provide resilience to age-related neurological decline in Drosophila. The TT and CQA components both contribute to these effects but do not have a synergistic effect. While individual TTs and most individual CQAs increased dendritic arborization at CAW equivalent concentrations, in the Drosophila model, only 1,5-diCQA was able to slow down the age-related decline in phototaxis. This suggests that combinations (or potentially higher concentrations) of the other compounds are needed to provide resilience in this model.
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Introduction: A water extract of Centella asiatica (L.) Urban [Apiaceae] (CAW) has demonstrated cognitive-enhancing effects in mouse models of Alzheimer's disease and aging, the magnitude of which is influenced by whether CAW is delivered in the drinking water or the diet. These cognitive benefits are accompanied by improvements in oxidative stress and mitochondrial function in the brain, two pathways related to the neuroinflammatory response. The effect of CAW on neuroinflammation, however, has not been directly studied. Here, we investigated the effect of CAW on neuroinflammation in 5xFAD mice and compared plasma levels of CAW's active compounds following two modes of CAW administration. Methods: Eight-to-nine-month-old male and female 5xFAD mice and their wild-type littermates were administered CAW in their diet or drinking water (0 or 1,000 mg/kg/day) for five weeks. Immunohistochemistry was performed for ß-amyloid (Aß), glial fibrillary acidic protein (GFAP), and Griffonia simplicifolia lectin I (GSL I) in the cortex and hippocampus. Gene expression of inflammatory mediators (IL-6, TNFα, IL-1ß, TREM2, AIF1, CX3CR1, CX3CL1, CD36, C3AR1, RAGE, CCR6, CD3E) was measured in the deep grey matter. Results: CAW decreased cortical Aß plaque burden in female 5xFAD mice administered CAW in the drinking water but had no effect on Aß plaques in other treatment groups. CAW did not impact elevated levels of GFAP or GSL I in 5xFAD mice, regardless of sex, brain region, or mode of CAW administration. In the deep grey matter, CAW increased C3AR1 expression in 5xFAD females administered CAW in the drinking water and decreased IL-1ß expression in 5xFAD males administered CAW in the diet. CAW had no effect, however, on gene expression levels of any other inflammatory mediator in the deep grey, for either sex or mode of CAW administration. Mice administered CAW in the drinking water versus the diet had significantly higher plasma levels of CAW compounds. Discussion: CAW had little impact on the neuroinflammatory markers selected for evaluation in the present study, suggesting that the cognitive benefits of CAW may not be mediated by an anti-inflammatory effect or that additional molecular markers are needed to fully characterize the effect of CAW on neuroinflammation.
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We have previously reported that a water extract (CAW) of the Ayurvedic plant Centella asiatica administered in drinking water can improve cognitive deficits in mouse models of aging and neurodegenerative diseases. Here we compared the effects of CAW administered in drinking water or the diet on cognition, measures of anxiety and depression-like behavior in healthy aged mice. Three- and eighteen-month-old male and female C57BL6 mice were administered rodent AIN-93M diet containing CAW (0, 0.2, 0.5 or 1% w/w) to provide 0, 200 mg/kg/d, 500 mg/kg/d or 1000 mg/kg/d for a total of 5 weeks. An additional group of eighteen-month-old mice were treated with CAW (10 mg/mL) in their drinking water for a total of five weeks to deliver the same exposure of CAW as the highest dietary dose (1000 mg/kg/d). CAW doses delivered were calculated based on food and water consumption measured in previous experiments. In the fourth and fifth weeks, mice underwent behavioral testing of cognition, anxiety and depression (n=12 of each sex per treatment group in each test). Aged mice of both sexes showed cognitive deficits relative to young mice while only female aged mice showed increased anxiety compared to the young female mice and no differences in depression were observed between the different ages. CAW (1000 mg/kg/d) in the drinking water improved deficits in aged mice in learning, executive function and recognition memory in both sexes and attenuated the increased measures of anxiety observed in the aged female mice. However, CAW in the diet only improved executive function in aged mice at the highest dose (1000 mg/kg/d) in both sexes and did so less robustly than when given in the water. There were no effects of CAW on depression-like behavior in aged animals regardless of whether it was administered in the diet or the water. These results suggest that CAW can ameliorate age-related changes in measures of anxiety and cognition and that the mode of administration is important for the effects of CAW on resilience to these age-related changes.
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Background: A water extract (CAW) of the Ayurvedic plant Centella asiatica administered in drinking water has been shown to improve cognitive deficits in mouse models of aging and neurodegenerative diseases. Here the effects of CAW administered in drinking water or the diet on cognition, measures of anxiety and depression-like behavior in healthy aged mice are compared. Methods: Three- and eighteen-month-old male and female C57BL6 mice were administered rodent AIN-93M diet containing CAW (0, 0.2, 0.5 or 1% w/w) to provide 0, 200 mg/kg/d, 500 mg/kg/d or 1,000 mg/kg/d CAW for a total of 5 weeks. An additional group of eighteen-month-old mice were treated with CAW (10 mg/mL) in their drinking water CAW for a total of 5 weeks to deliver the same exposure of CAW as the highest dietary dose (1,000 mg/kg/d). CAW doses delivered were calculated based on food and water consumption measured in previous experiments. In the fourth and fifth weeks, mice underwent behavioral testing of cognition, anxiety and depression (n = 12 of each sex per treatment group in each test). Results: Aged mice of both sexes showed cognitive deficits relative to young mice while only female aged mice showed increased anxiety compared to the young female mice and no differences in depression were observed between the different ages. CAW (1,000 mg/kg/d) in the drinking water improved deficits in aged mice in learning, executive function and recognition memory in both sexes and attenuated the increased measures of anxiety observed in the aged female mice. However, CAW in the diet only improved executive function in aged mice at the highest dose (1,000 mg/kg/d) in both sexes and did so less robustly than when given in the water. There were no effects of CAW on depression-like behavior in aged animals regardless of whether it was administered in the diet or the water. Conclusions: These results suggest that CAW can ameliorate age-related changes in measures of anxiety and cognition and that the mode of administration is important for the effects of CAW on resilience to these age-related changes.
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Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice, and TG hydrolysis induced by fasts of 6 or 24 h was greatly reduced in mice lacking Angptl4 (Angptl4(-/-)). Glucocorticoid treatment mimicked the lipolytic effects of fasting, although with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this, cAMP levels and TG hydrolysis were reduced in primary Angptl4(-/-) murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4. Remarkably, human ANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4 modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance.
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Adipócitos Brancos/metabolismo , Tecido Adiposo Branco/metabolismo , Angiopoietinas/metabolismo , Jejum/fisiologia , Lipólise/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Angiopoietinas/genética , Angiopoietinas/farmacologia , Animais , AMP Cíclico/genética , AMP Cíclico/metabolismo , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Humanos , Resistência à Insulina/genética , Lipólise/efeitos dos fármacos , Camundongos , Camundongos Knockout , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
Oxidative stress and neuroinflammation are widespread in the Parkinson's disease (PD) brain and contribute to the synaptic degradation and dopaminergic cell loss that result in cognitive impairment and motor dysfunction. The polymethoxyflavone Gardenin A (GA) has been shown to activate the NRF2-regulated antioxidant pathway and inhibit the NFkB-dependent pro-inflammatory pathway in a Drosophila model of PD. Here, we evaluate the effects of GA on A53T alpha-synuclein overexpressing (A53TSyn) mice. A53TSyn mice were treated orally for 4 weeks with 0, 25, or 100 mg/kg GA. In the fourth week, mice underwent behavioral testing and tissue was harvested for immunohistochemical analysis of tyrosine hydroxylase (TH) and phosphorylated alpha synuclein (pSyn) expression, and quantification of synaptic, antioxidant and inflammatory gene expression. Results were compared to vehicle-treated C57BL6 mice. Treatment with 100 mg/kg GA improved associative memory and decreased abnormalities in mobility and gait in A53TSyn mice. GA treatment also reduced cortical and hippocampal levels of pSyn and attenuated the reduction in TH expression in the striatum. Additionally, GA increased cortical expression of NRF2-regulated antioxidant genes and decreased expression of NFkB-dependent pro-inflammatory genes. GA was readily detectable in the brains of treated mice and modulated the lipid profile in the deep gray brain tissue of those animals. While the beneficial effects of GA on cognitive deficits, motor dysfunction and PD pathology are promising, future studies are needed to further fully elucidate the mechanism of action of GA, optimizing dosing and confirm these effects in other PD models. Significance Statement: The polymethoxyflavone Gardenin A can improve cognitive and motor function and attenuate both increases in phosphorylated alpha synuclein and reductions in tyrosine hydroxylase expression in A53T alpha synuclein overexpressing mice. These effects may be related to activation of the NRF2-regulated antioxidant response and downregulation of NFkB-dependent inflammatory response by Gardenin A in treated animals. The study also showed excellent brain bioavailability of Gardenin A and modifications of the lipid profile, possibly through interactions between Gardenin A with the lipid bilayer, following oral administration. The study confirms neuroprotective activity of Gardenin A previously reported in toxin induced Drosophila model of Parkinson's disease.
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Centella asiatica (CA) is a culinary vegetable and well-known functional food that is widely used as a medicinal herb and dietary supplement. CA is rich in pentacyclic triterpenes (TTs), including asiaticoside (AS), madecassoside (MS) and the related aglycones asiatic acid (AA), madecassic acid (MA). Traditionally, TTs have been associated with the bioactivity and health promoting effect of CA. Recently, mono-caffeoylquinic acids (MonoCQAs) and di-caffeoylquinic acids (DiCQAs) have been found to contribute to the bioactivity of CA as well. This work reports an analytical strategy based on liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM-MS) for the simultaneous rapid and accurate quantification of 12 bioactive compounds in CA, namely AS, MS, AA, MA, 5-CQA, 4-CQA, 3-CQA, 1,3-DiCQA, 3,4-DiCQA, 1,5-DiCQA, 3,5-DiCQA, 4,5-DiCQA. Method selectivity, accuracy, precision, repeatability, robustness, linearity range, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The validated LC-MRM-MS method has been successfully applied to quantify the 12 bioactive compounds in CA aqueous extracts and two related formulations: a standardized CA product (CAP) used in a phase I clinical trial and formulated CA rodent diets used in preclinical studies. The validated method allows us to support the standardization of CA products used for clinical trials and conduct routine LC-MRM-MS analyses of formulated preclinical diets to confirm correct levels of CA phytochemical markers.
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Centella asiatica (Centella) is a traditional botanical medicine that shows promise in treating dementia based on behavioral alterations seen in animal models of aging and cognitive dysfunction. In order to determine if Centella could similarly improve cognitive function and reduce disease burden in multiple sclerosis (MS), we tested its effects in the neuroinflammatory experimental autoimmune encephalomyelitis (EAE) model of MS. In two independent experiments, C57BL/6J mice were treated following induction of EAE with either a standardized water extract of Centella (CAW) or placebo for 2 weeks. At the dosing schedule and concentrations tested, CAW did not improve behavioral performance, EAE motor disability, or degrees of demyelination. However, CAW-treated mice demonstrated increases in nuclear factor (erythroid-derived 2)-like 2 and other antioxidant response element genes, and increases in mitochondrial respiratory activity. Caw also decreased spinal cord inflammation. Our findings indicate that CAW can increase antioxidant gene expression and mitochondrial respiratory activity in mice with EAE, supporting investigation of the clinical effects of CAW in people with MS.
RESUMO
Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a redox-sensitive transcription factor that binds to the antioxidant response element consensus sequence, decreasing reactive oxygen species and regulating the transcription of a wide array of genes, including antioxidant and detoxifying enzymes, regulating genes involved in mitochondrial function and biogenesis. Moreover, NRF2 has been shown to directly regulate the expression of anti-inflammatory mediators reducing the expression of pro-inflammatory cytokines. In recent years, attention has turned to the role NRF2 plays in the brain in different diseases such Alzheimer's disease, Parkinson's disease, Huntington's disease and others. This review focused on the evidence, derived in vitro, in vivo and from clinical trials, supporting a role for NRF2 activation in maintaining and improving cognitive function and how its activation can be used to elicit neuroprotection and lead to cognitive enhancement. The review also brings a critical discussion concerning the possible prophylactic and/or therapeutic use of NRF2 activators in treating cognitive impairment-related conditions.
RESUMO
Due to an increase in the aging population, age-related diseases and age-related changes, such as diminished cognition and sleep disturbances, are an increasing health threat. It has been suggested that an increase in oxidative stress underlies many of these changes. Current treatments for these diseases and changes either have low efficacy or have deleterious side effects preventing long-time use. Therefore, alternative treatments that promote healthy aging and provide resilience against these health threats are needed. The herbs Withania somnifera and Centella asiatica may be two such alternatives because both have been connected with reducing oxidative stress and could therefore ameliorate age-related impairments. To test the effects of these herbs on behavioral phenotypes induced by oxidative stress, we used the Drosophila melanogaster sniffer mutant which has high levels of oxidative stress due to reduced carbonyl reductase activity. Effects on cognition and mobility were assessed using phototaxis assays and both, W. somnifera and C. asiatica water extracts improved phototaxis in sniffer mutants. In addition, W. somnifera improved nighttime sleep in male and female sniffer flies and promoted a less fragmented sleep pattern in male sniffer flies. This suggests that W. somnifera and C. asiatica can ameliorate oxidative stress-related changes in behavior and that by doing so they might promote healthy aging in humans.