Aumento da resist?ncia ao estresse oxidativo induzido pelo extrato hidroalco?lico de carqueja (Baccharis trimera) no Caenorhabditis elegans : seria atrav?s de um mecanismo SKN-1/p38 MAPK e DAF-16 dependente?

O alto consumo de comidas e bebidas ricas em flavon?ides tem sido associado a um menor risco de doen?as degenerativas cr?nicas como aterosclerose e c?ncer, devido tanto ?s suas atividades antioxidantes diretas de remo??o de radicais livres, quanto ? sua capacidade de atuar como antioxidante indireto ativando diferentes vias de sinaliza??o em resposta ao estresse oxidativo. Baccharis trimera, conhecida como carqueja, ? uma planta subarbustiva cujos extratos aquoso e hidroalc?olico s?o ricos em compostos antioxidantes tais como os ?steres do ?cido qu?nico e os flavon?ides nepetina, isoquercetina e quercetina. Recentemente, diversos estudos tem destacado as propriedades antioxidantes, anti-inflamat?ria, anti-proteol?tica e anti-hemorr?gica tanto em ensaios in vitro quanto in vivo. Neste projeto, n?s investigamos o potencial antioxidante e pr?-longevidade do extrato hidroalc?olico de carqueja (EHC) no organismo modelo Caenorhabiditis elegans. Para testar se o tratamento com o extrato hidroalc?olico de carqueja (EHC) aumenta a resist?ncia ao estresse oxidativo, animais tipo selvagem foram tratados por 48 horas com 0,5, 5 e 50 mg/mL de EHC por 48 horas e depois submetidos ao estresse provocado por hidroper?xido de terc-butila (t-BOOH). O tratamento com 5 mg/ml aumentou significativamente a sobreviv?ncia dos animais submetidos a 5 mM de t-BOOH. Embora o tratamento com 5 mg/ml por 48 horas ter aumentado a resist?ncia ao estresse oxidativo, este mesmo tratamento n?o aumentou a resist?ncia ao estresse t?rmico a 35 o C. O tratamento cont?nuo com 5 mg/ml de extrato de carqueja tamb?m n?o aumentou a longevidade dos animais tipo selvagem em condi??es normais. Apesar de apresentarem uma maior resist?ncia ao estresse, os animais tratados com 5mg/ml do extrato de carqueja por 48 horas n?o apresentaram uma diferen?a significativa quanto aos marcadores bioqu?micos (atividade da catalase) e indicadores de resist?ncia (n?veis de prote?na carbonilada e sulfidrila totais) analisados em rela??o aos animais do grupo controle. Para averiguar se o aumento da resist?ncia ao estresse oxidativo promovido pelo tratamento com extrato de carqueja depende da ativa??o dos fatores de transcri??o SKN-1 e DAF-16, os ensaios de resist?ncia ao estresse oxidativo foram repetidos usando mutantes de dele??o ou ?knockdown? para estes dois fatores. O tratamento com carqueja n?o aumentou a resist?ncia ao estresse oxidativo dos mutantes skn-1(RNAi), sek-1 e daf-16. Por outro lado, tamb?m n?o foi observada a indu??o da localiza??o nuclear nos animais tratados contendo gene rep?rter SKN-1::GFP e DAF-16::GFP. O presente trabalho sugere que EHC aumenta a resist?ncia ao estresse oxidativo de uma maneira SKN/p38 MAPK e DAF-16 dependente. Por?m, as an?lises de genes rep?rteres realizadas at? o momento n?o corroboraram com estas observa??es.High consumption of foods and beverages rich in flavonoids have been associated with reduced risk of chronic degenerative diseases such as atherosclerosis and cancer due to both their direct antioxidant activities by neutralizing free radicals, and indirect antioxidant activity by activating different pathways signaling against oxidative stress. Baccharis trimera, known as carqueja, undergrowth is a plant whose aqueous and hydroalcoholic are rich in antioxidant compounds such as esters of quinic acid and flavonoids nepetin, isoquercetina and quercetin. Recently, several studies have highlighted the carqueja antioxidant, anti-inflammatory, anti-proteolytic and antihemorrhagic properties both in vitro and in vivo. In this project, we investigated the antioxidant potential and pro-longevity of the hydroalcoholic extract of carqueja in the model organism Caenorhabditis elegans. To test whether treatment EHC increases resistance to oxidative stress, wild-type animals were treated for 48 hours with 0.5, 5 and 50 mg / mL of EHC for 48 hours and then subjected the stress caused by tert-butyl hydroperoxide (t-BOOH). Treatment with 5 mg / ml significantly increased the survival of animals submitted to 5 mM t-BOOH. Despite the 5 mg/mL of EHC treatment for 48 hours have increased resistance to oxidative stress, this same treatment did not increase the resistance to heat stress at 35 o C. Continuous treatment with 5 mg/mL of EHC did not increase the longevity of wild-type animals under normal conditions. Although providing a higher resistance to stress, the animals treated with 5 mg/mL of EHC for 48 hours did not show a significant difference regarding biochemical markers (catalase activity) and indicators of resistance (total sulfhydryl and protein carbonyl levels) analyzed compared to control animals. In order to investigate whether the increased oxidative stress resistance induced by EHC treatment depends on the activation of transcription factors SKN-1 and DAF-16, the oxidative stress resistance assays were repeated using mutants or "knockdown" for these two factors. Treatment with EHC did not increase oxidative stress resistance in the mutants skn-1(RNAi), sek-1 and daf-16. However, treatment with EHC did not SKN-1:: GFP and DAF-16:: GFP nuclear localization. In conclusion, EHC treatment increases oxidative stress resistance in a way SKN-1/p38 MAPK and DAF-16 dependent. But the analysis of reporters genes performed to date does not corroborate these observation

Carqueja (Baccharis trimera) Protects against Oxidative Stress and -Amyloid-Induced Toxicity in Caenorhabditis elegans

Carqueja (Baccharis trimera) is a native plant found throughout South America. Several studies have shown that Carqueja has antioxidant activity in vitro, as well as anti-inflammatory, antidiabetic, analgesic, antihepatotoxic, and antimutagenic properties. However, studies regarding its antioxidant potential in vivo are limited. In this study, we used Caenorhabditis elegans as a model to examine the antioxidant effects of a Carqueja hydroalcoholic extract (CHE) on stress resistance and lifespan and to investigate whether CHE has a protective effect in a C. elegans model for Alzheimer's disease. Here, we show for the first time, using in vivo assays, that CHE treatment improved oxidative stress resistance by increasing survival rate and by reducing ROS levels under oxidative stress conditions independently of the stress-related signaling pathways (p38, JNK, and ERK) and transcription factors (SKN-1/Nrf and DAF-16/Foxo) tested here. CHE treatment also increased the defenses against -amyloid toxicity in C. elegans, in part by increasing proteasome activity and the expression of two heat shock protein genes. Our findings suggest a potential neuroprotective use for Carqueja, supporting the idea that dietary antioxidants are a promising approach to boost the defensive systems against stress and neurodegeneration

Chronic treatment with ivabradine does not affect cardiovascular autonomic control in rats.

A low resting heart rate (HR) would be of great benefit in cardiovascular diseases. Ivabradine-a novel selective inhibitor of hyperpolarization-activated cyclic nucleotide gated (HCN) channels- has emerged as a promising HR lowering drug. Its effects on the autonomic HR control are little known. This study assessed the effects of chronic treatment with ivabradine on the modulatory, reflex and tonic cardiovascular autonomic control and on the renal sympathetic nerve activity (RSNA). Male Wistar rats were divided in 2 groups, receiving intraperitoneal injections of vehicle (VEH) or ivabradine (IVA) during 7 or 8 consecutive days. Rats were submitted to vessels cannulation to perform arterial blood pressure (AP) and HR recordings in freely moving rats. Time series of resting pulse interval and systolic AP were used to measure cardiovascular variability parameters. We also assessed the baroreflex, chemoreflex and the Bezold-Jarish reflex sensitivities. To better evaluate the effects of ivabradine on the autonomic control of the heart, we performed sympathetic and vagal autonomic blockade. As expected, ivabradine-treated rats showed a lower resting (VEH: 362 ? 16 bpm vs. IVA: 260 ? 14 bpm, p = 0.0005) and intrinsic HR (VEH: 369 ? 9 bpm vs. IVA: 326 ? 11 bpm, p = 0.0146). However, the chronic treatment with ivabradine did not change normalized HR spectral parameters LF (nu) (VEH: 24.2 ? 4.6 vs. IVA: 29.8 ? 6.4; p > 0.05); HF (nu) (VEH: 75.1 ? 3.7 vs. IVA: 69.2 ? 5.8; p > 0.05), any cardiovascular reflexes, neither the tonic autonomic control of the HR (tonic sympathovagal index; VEH: 0.91? 0.02 vs. IVA: 0.88 ? 0.03, p = 0.3494). We performed the AP, HR and RSNA recordings in urethane-anesthetized rats. The chronic treatment with ivabradine reduced the resting HR (VEH: 364 ? 12 bpm vs. IVA: 207 ? 11 bpm, p < 0.0001), without affecting RSNA (VEH: 117 ? 16 vs. IVA: 120 ? 9 spikes/s, p = 0.9100) and mean arterial pressure (VEH: 70 ? 4 vs. IVA: 77 ? 6 mmHg, p = 0.3293). Our results suggest that, in health rats, the long-term treatment with ivabradine directly reduces the HR without changing the RSNA modulation and the reflex and tonic autonomic control of the heart

Baccharis trimera protege contra o estresse oxidativo e toxicidade induzida pelo peptídeo β-amilóide no Caenorhabditis elegans.

Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto.Uma planta muito utilizada na medicina popular é a Baccharis trimera (B. trimera), conhecida como Carqueja, planta nativa encontrada em toda a América do Sul, rica em antioxidantes tais como os ésteres do ácido quínico e os flavonóides nepetina, quercetina e isoquercetina. Vários estudos têm mostrado que a B. trimera tem atividade antioxidante in vitro, no entanto, os efeitos antioxidantes desta planta ainda foram pouco explorados in vivo. Neste projeto, nós usamos o organismo modelo Caenorhabiditis elegans para investigar o potencial antioxidante in vivo do Extrato Hidroalcoólico de B. trimera (EHB) e seus possíveis mecanismos subjacentes, além de investigar se o EHB possui efeito protetor em um modelo C. elegans para a doença de Alzheimer. A suplementação com 50 mg/mL de EHB não alterou a longevidade, a reprodução, o desenvolvimento ou o comportamento alimentar do C. elegans. O EHB inibiu o crescimento microbiano, mas esta propriedade bacteriostática não influenciou no aumento da resistência ao estresse oxidativo promovido pelo EHB. Os efeitos benéficos do EHB foram independentes das vias de sinalização testadas sugerindo que o aumento observado na resistência ao estresse oxidativo possa ser através de um mecanismo de ação direto, já que o EHB foi capaz de diminuir a produção de ROS e a expressão de sod-3 e gcs-1. O EHB aliviou a paralisia induzida pelo β-amilóide em um modelo de C. elegans para doença de Alzheimer. Nossos resultados sugerem que o possível efeito neuroprotetor do EHB contra o peptídeo Aβ1-42 é, em parte pela ação antioxidante do EHB como também um resultado de um aumento da homeostase protéica, já que o EHB aumentou a atividade do proteassoma e expressão gênica de duas proteínas de choque térmico. Em conjunto, todos os resultados desse trabalho sugerem um uso potencial neuroprotetor para B. trimera, apoiando a ideia de que o uso de antioxidantes na dieta é uma abordagem promissora para aumentar os sistemas de defesa contra o estresse e neurodegeneração.A plant widely used in folk medicine is Baccharis trimera (B. trimera), known like Carqueja, native plant found throughout South America, rich in antioxidants such as acid quinic esters and nepetin, quercetin and isoquercetin flavonoids. Several studies have shown that B. trimera has antioxidant activity in vitro, however, the antioxidant effects of this plant still been little explored in vivo. In this project, we used the Caenorhabiditis elegans to investigate the antioxidant potential of B. trimera Hydroalcoholic Extract (EHB) in vivo and its possible underlying mechanisms, and investigate whether EHB has protective effect in C. elegans model for Alzheimer's disease. Supplementation with EHB 50 mg/mL did not affect the longevity, reproduction, development feeding behavior of C. elegans. EHB inhibited microbial growth, but this bacteriostatic property did not affect the increased resistance to oxidative stress caused by the EHB. The EHB beneficial effects were independent of the signaling pathways here tested suggesting that the increase in oxidative stress resistance may be through a direct mechanism of action, since the EHB was capable of reducing ROS production and sod- 3 and gcs-1 expression. EHB delayed paralysis induced by β-amyloid in a C. elegans model for Alzheimer's disease. Our results suggest that the possible neuroprotective effect of EHB against Aβ1-42-peptide is partly antioxidant activity of the EHB as well as a result of an increase in protein homeostasis, since the EHB increased proteasome activity and two heat shock proteins gene expression. Together, all the results of this study suggest a potential neuroprotective use for B. trimera, supporting the idea that the use of antioxidants in the diet is a promising approach to increase the defense mechanisms against stress and neurodegeneration

Guarana (Paullinia cupana) Extract Protects Caenorhabditis elegans Models for Alzheimer Disease and Huntington Disease through Activation of Antioxidant and Protein Degradation Pathways

Guarana (Paullinia cupana) is largely consumed in Brazil in high energy drinks and dietary supplements because of its stimulant activity on the central nervous system. Although previous studies have indicated that guarana has some protective effects in Parkinson’s (PD), Alzheimer’s (AD), and Huntington’s (HD) disease models, the underlying mechanisms are unknown. Here, we investigated the protective effects of guarana hydroalcoholic extract (GHE) in Caenorhabditis elegans models of HD and AD. GHE reduced polyglutamine (polyQ) protein aggregation in the muscle and also reduced polyQ-mediated neuronal death in ASH sensory neurons and delayed β-amyloid-induced paralysis in a caffeine-independent manner. Moreover, GHE’s protective effects were not mediated by caloric restriction, antimicrobial effects, or development and reproduction impairment. Inactivation of the transcription factors SKN-1 and DAF-16 by RNAi partially blocked the protective effects of GHE treatment in the AD model. We show that the protective effect of GHE is associated with antioxidant activity and modulation of proteostasis, since it increased the lifespan and proteasome activity, reduced intracellular ROS and the accumulation of autophagosomes, and increased the expression of SOD-3 and HSP-16.2. Our findings suggest that GHE has therapeutic potential in combating age-related diseases associated with protein misfolding and accumulation

Carqueja (Baccharis trimera) Protects against Oxidative Stress and β-Amyloid-Induced Toxicity in Caenorhabditis elegans

Carqueja (Baccharis trimera) is a native plant found throughout South America. Several studies have shown that Carqueja has antioxidant activity in vitro, as well as anti-inflammatory, antidiabetic, analgesic, antihepatotoxic, and antimutagenic properties. However, studies regarding its antioxidant potential in vivo are limited. In this study, we used Caenorhabditis elegans as a model to examine the antioxidant effects of a Carqueja hydroalcoholic extract (CHE) on stress resistance and lifespan and to investigate whether CHE has a protective effect in a C. elegans model for Alzheimer's disease. Here, we show for the first time, using in vivo assays, that CHE treatment improved oxidative stress resistance by increasing survival rate and by reducing ROS levels under oxidative stress conditions independently of the stress-related signaling pathways (p38, JNK, and ERK) and transcription factors (SKN-1/Nrf and DAF-16/Foxo) tested here. CHE treatment also increased the defenses against β-amyloid toxicity in C. elegans, in part by increasing proteasome activity and the expression of two heat shock protein genes. Our findings suggest a potential neuroprotective use for Carqueja, supporting the idea that dietary antioxidants are a promising approach to boost the defensive systems against stress and neurodegeneration

Effect of açaí aqueous extract (AAE) on redox status in wild-type <i>C. elegans</i> and HUVECs.

<p><b>A</b>) <i>C. elegans</i> was treated with control solution (S basal) or 100 mg/mL AAE for 48 h and then submitted to the presence or absence of 1 mM H₂O₂ for 2 h. The results are expressed as H₂DCFDA fluorescence relative to the untreated control. <b>B</b>) HUVECs were treated with or without 2.5 mg/mL AAE for 16 h and then incubated in 0.25 mM H₂O₂ for 1 h. The fluorescence was measured by flow cytometry. The results are expressed as H₂DCFDA fluorescence relative to the untreated control. Different letters indicate significant differences by one-way ANOVA followed by Tukey's post-test. <b>C</b>) To measure the levels of total SH groups, animals were treated with control solution (S basal) or 100 mg/mL AAE from L1 until L4 and then incubated with or without 5 mM t-BOOH for 1 h. *p values were determined by a two-tailed Student's <i>t-</i>test, and groups were significantly different when p<0.05 in <i>C. elegans</i>. Transgenic worms containing reporter genes were treated with control solution (S basal) or 100 mg/mL AAE for 48 h beginning at L1 and then with or without the oxidative stress condition. After a 1-h hour recovery period, photographs were taken on a fluorescence microscope. For (<b>D</b>) <i>gcs-1::GFP</i> and (<b>E</b>) <i>gst-4::GFP</i> animals, GFP fluorescence signals were measured using NIH Image J software. Different letters correspond to significant differences by the Kruskal-Wallis test followed by Dunn's post-test.</p

Effect of açaí aqueous extract (AAE) on <i>C. elegans</i> grown under normal and stress conditions.

<p><b>A</b>) <i>fem-1(hc17)</i> mutants were treated at 25°C with control solution (S basal) or 100 mg/mL AAE beginning at L1. Surviving and dead animals were counted daily until all nematodes had died. Log-rank (Mantel-Cox) analysis showed no significant difference between the curves. <b>B</b>) Animals were treated with control solution (S basal) or 100 mg/mL AAE from L1 until L4 and then submitted to 7.5 mM t-BOOH in M9. The survival was measured at 6, 9 and 12 h. The survival curves show that AAE treatment increased <i>C. elegans</i> oxidative stress resistance. ***p<0.001 by the Log-rank (Mantel-Cox) test. <b>C</b>) Animals were treated with control solution (S basal) or 100 mg/mL AAE beginning at L1. After five days at 20°C, the animals were incubated at 35°C and survival was monitored at 6, 9 and 12 h. There was no significant difference between curves by the Log-rank (Mantel-Cox) test. <b>D</b>) Animals were treated with control solution (S basal) or 100 mg/mL AAE for 68 h beginning at L1 and then transferred to new plates containing 500 mM NaCl. The percentage of worms that moved outside a 7-mm circle was monitored at 15, 30 and 60 min. *p<0.05 by a two-tailed Student's <i>t-</i>test.</p

Effect of açaí aqueous extract (AAE) on the bacterial growth and oxidative stress resistance of wild-type <i>C. elegans</i> grown on dead bacteria.

<p><b>A</b>) <i>E. coli</i> OP50 growth was evaluated over 4 h in the presence of 100 mg/mL AAE. The OD of the control group at time zero was used to normalize all other OD readings. * Treatment of 100 mg/mL AAE decreased bacteria growth at all times analyzed with p<0.05, determined by a two-tailed Student's <i>t-</i>test. <b>B</b>) Animals were treated with or without 100 mg/mL AAE, mixed with either <i>E. coli</i> OP50 or <i>E. coli</i> OP50 treated with 10 mM Kanamycin (KAN), from L1 until L4 and then submitted to 7.5 mM t-BOOH in M9. The survival was measured at 6, 9 and 12 h. The survival curves show that AAE treatment increased <i>C. elegans</i> oxidative stress resistance independent of its antibacterial effect. ***p<0.001 related to the respective control by the Log-rank (Mantel-Cox) test.</p