RESUMO
Redox impairment, inflammation, and increased rates of cell death are central players during neurodegeneration. In that context, activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been viewed as an interesting strategy in order to reduce the impact of redox dysfunction and neuroinflammation on cell fate. There is evidence indicating that the benefits caused by natural products in the brain may be due to the ability of these agents in upregulating Nrf2. Gastrodin (GAS) induces anti-oxidant, anti-inflammatory, and anti-apoptotic actions in brain cells. Nonetheless, the mechanisms underlying such effects are not clear yet. Therefore, we investigated here whether GAS would affect apoptosis and inflammation in the human neuroblastoma cell line (SH-SY5Y) exposed to hydrogen peroxide (H2O2). GAS at 1-25 µM was administrated to the cells during 30 min before a challenge with H2O2 at 300 µM for additional 24 h. GAS prevented the activation of the intrinsic apoptotic pathway by modulating the levels of Bcl-2 and Bax, causing a decrease in the release of cytochrome c to the cytosol. GAS also prevented the activation of the pro-apoptotic enzymes caspase-9 and caspase-3. Consequently, GAS abrogated poly (ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation in the H2O2-treated SH-SY5Y cells. Moreover, GAS reduced the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) and the activity of nuclear factor-κB in H2O2-treated cells. Silencing of Nrf2 by small interfering RNA (siRNA) suppressed the GAS-induced cytoprotection. Thus, GAS elicited anti-apoptotic and anti-inflammatory effects by a mechanism involving Nrf2 in SH-SY5Y cells.
Assuntos
Apoptose , Álcoois Benzílicos/farmacologia , Glucosídeos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/farmacologia , Linhagem Celular Tumoral , Fragmentação do DNA , Humanos , Peróxido de Hidrogênio/farmacologia , Interleucina-1beta/metabolismo , NF-kappa B/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mitochondria are double-membrane organelles involved in the transduction of energy from different metabolic substrates into adenosine triphosphate (ATP) in mammalian cells. The oxidative phosphorylation system is comprised by the activity of the respiratory chain and the complex V (ATP synthase/ATPase). This system is dependent on oxygen gas (O2) in order to maintain a flux of electrons in the respiratory chain, since O2 is the final acceptor of these electrons. Electron leakage from this complex system leads to the continuous generation of reactive species in the cells. The mammalian cells exhibit certain mechanisms to attenuate the consequences originated from the constant exposure to these reactive species. In this context, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and one of the enzymes whose expression is modulated by Nrf2, heme oxygenase-1 (HO-1), take a central role in inducing cytoprotection in humans. Mitochondrial abnormalities are observed during intoxication and in certain diseases, including neurodegeneration. Mitochondrial protection promoted by natural compounds has attracted the attention of researchers due to the promising effects these agents induce experimentally. In this regard, we examined here whether and how gastrodin (GAS), a phenolic glucoside, would prevent the paraquat (PQ)-induced mitochondrial impairment in the SH-SY5Y cells. The cells were exposed to GAS (25 µM) for 4 h prior to the challenge with PQ at 100 µM for additional 24 h. The silencing of Nrf2 by siRNA or the inhibition of HO-1 by ZnPP IX suppressed the GAS-elicited cytoprotection. Therefore, GAS promoted mitochondrial protection by an Nrf2/HO-1-dependent manner.
Assuntos
Álcoois Benzílicos/farmacologia , Glucosídeos/farmacologia , Heme Oxigenase-1/metabolismo , Herbicidas/farmacologia , Mitocôndrias/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Paraquat/farmacologia , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nuclear factor erythroid-related factor 2 (Nrf2) has been implicated in several detoxifying and antioxidant defense processes. Nrf2-mediated heme oxygenase-1 (HO-1) expression was demonstrated to play a key role against oxidative stress. Gastrodin (GSTD) is a well-known active compound isolated from the roots of Rhizoma gastrodiae, a plant used in ancient Chinese traditional medicine. The aim of this work was to investigate whether GSTD could alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells (LSECs). In LSECs exposed to 1 mM H2O2, treatment with GSTD (1, 10, or 50 µM) resulted in higher cell viability than the untreated control. Treated cells maintained a higher Bcl2/Bax ratio and suppressed caspase-9 expression compared with untreated cells, reducing cell apoptosis. GSTD was protective for H2O2-induced oxidative injury by reducing the generation of intracellular reactive oxygen species and malondialdehyde. HO-1 and Nrf2 expressions were synergistically upregulated by GSTD. Inhibition of HO-1 by 10 µM zinc protoporphyrin resulted in less protective effects on cell viability and malondialdehyde reduction by GSTD treatment in H2O2-exposed LSECs. Additionally, phosphorylated p38 in LSECs exposed to H2O2 was elevated by GSTD. Inhibition of p38 phosphorylation by SB203580 did not induce Nrf2 and HO-1 expression after 1 or 10 µM GSTD treatment and the protective effect on cell viability and malondialdehyde reduction in H2O2-exposed LSECs was reduced. The data conclusively demonstrated that GSTD-induced HO-1 and Nrf2 expression is involved in protection of LSECs from H2O2-induced oxidative injury, which may be regulated by p38 phosphorylation.
Assuntos
Álcoois Benzílicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Glucosídeos/farmacologia , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Malondialdeído/metabolismo , Camundongos , Modelos Teóricos , Estresse Oxidativo/fisiologia , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/sangueRESUMO
Mitochondrion is the main site of ATP production in animal cells and also orchestrates signaling pathways associated with cell survival and death. Mitochondrial dysfunction has been linked to bioenergetics and redox impairment in human diseases, such as neurodegeneration and cardiovascular disease. Protective agents able to attenuate mitochondrial impairment are of pharmacological interest. Gastrodin (GAS; 4-hydroxybenzyl alcohol 4-O-beta-D-glucoside) is a phenolic glucoside obtained from the Chinese herbal medicine Gastrodia elata Blume and exhibits antioxidant, anti-inflammatory, and antiapoptotic effects in several cell types. GAS is able to cross the blood-brain barrier, reducing the impact of different stressors on the cognition of experimental animals. In the present work, we investigated whether GAS would protect mitochondria of human SH-SY5Y neuroblastoma cells against an exposure to a pro-oxidant agent. The cells were treated with GAS at 25 µM for 30 min before the administration of hydrogen peroxide (H2O2) at 300 µM for an additional 3 or 24 h, depending on the assay. We evaluated both mitochondrial redox state and function parameters and analyzed the mechanism by which GAS protected mitochondria in this experimental model. Silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor suppressed the GAS-induced mitochondrial protection seen here. Moreover, Nrf2 knockdown abrogated the effects of GAS on cell viability, indicating a potential role for Nrf2 in both mitochondrial and cellular protection promoted by GAS. Further research would be necessary to investigate whether GAS would be able to induce similar effects in in vivo experimental models.
Assuntos
Antioxidantes/farmacologia , Álcoois Benzílicos/farmacologia , Glucosídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , OxirreduçãoRESUMO
Nuclear factor erythroid-related factor 2 (Nrf2) has been implicated in several detoxifying and antioxidant defense processes. Nrf2-mediated heme oxygenase-1 (HO-1) expression was demonstrated to play a key role against oxidative stress. Gastrodin (GSTD) is a well-known active compound isolated from the roots of Rhizoma gastrodiae, a plant used in ancient Chinese traditional medicine. The aim of this work was to investigate whether GSTD could alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells (LSECs). In LSECs exposed to 1 mM H2O2, treatment with GSTD (1, 10, or 50 µM) resulted in higher cell viability than the untreated control. Treated cells maintained a higher Bcl2/Bax ratio and suppressed caspase-9 expression compared with untreated cells, reducing cell apoptosis. GSTD was protective for H2O2-induced oxidative injury by reducing the generation of intracellular reactive oxygen species and malondialdehyde. HO-1 and Nrf2 expressions were synergistically upregulated by GSTD. Inhibition of HO-1 by 10 µM zinc protoporphyrin resulted in less protective effects on cell viability and malondialdehyde reduction by GSTD treatment in H2O2-exposed LSECs. Additionally, phosphorylated p38 in LSECs exposed to H2O2 was elevated by GSTD. Inhibition of p38 phosphorylation by SB203580 did not induce Nrf2 and HO-1 expression after 1 or 10 µM GSTD treatment and the protective effect on cell viability and malondialdehyde reduction in H2O2-exposed LSECs was reduced. The data conclusively demonstrated that GSTD-induced HO-1 and Nrf2 expression is involved in protection of LSECs from H2O2-induced oxidative injury, which may be regulated by p38 phosphorylation.
Assuntos
Animais , Coelhos , Álcoois Benzílicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Heme Oxigenase-1/metabolismo , Glucosídeos/farmacologia , Peróxido de Hidrogênio/farmacologia , Regulação para Cima/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Malondialdeído/metabolismo , Modelos TeóricosRESUMO
BACKGROUND: The aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit has demonstrated hypoglycemic effect, which may be attributed to some components in the extract. However, the major secondary metabolites in this fruit have not yet been identified and little is known about its extra-pancreatic action, in particular, on liver carbohydrate metabolism. Therefore, in addition to the isolation and structural elucidation of the principal components in the aqueous extract of C. ficifolia, the aim of this study was to determine whether or not the hypoglycemic effect of the aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit is due to accumulation of liver glycogen in diabetic mice. MATERIALS AND METHODS: The aqueous extract from fruit of C. ficifolia was fractionated and its main secondary metabolites were purified and chemically characterized (NMR and GC-MS). Alloxan-induced diabetic mice received daily by gavage the aqueous extract (30 days). The liver glycogen content was quantified by spectroscopic method and by PAS stain; ALT and AST by spectrometric method; glycogen synthase, glycogen phosphorylase and GLUT2 by Western blot; the mRNA expression of GLUT2 and glucagon-receptor by RT-PCR; while serum insulin was quantified by ELISA method. A liver histological analysis was also performed by H&E stain. RESULTS: Chemical fingerprint showed five majoritarian compounds in the aqueous extract of C. ficifolia: p-coumaric acid, p-hydroxybenzoic acid, salicin, stigmast-7,2,2-dien-3-ol and stigmast-7-en-3-ol. The histological analysis showed accumulation of liver glycogen. Also, increased glycogen synthase and decreased glycogen phosphorylase were observed. Interestingly, the histological architecture evidenced a liver-protective effect due the extract. CONCLUSION: Five compounds were identified in C. ficifolia aqueous extract. The hypoglycemic effect of this extract may be partially explained by liver glycogen accumulation. The bioactive compound responsible for the hypoglycemic effect of this extract will be elucidated in subsequent studies.
Assuntos
Cucurbita/química , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/química , Glicogênio Hepático/metabolismo , Compostos Fitoquímicos/análise , Fitoterapia/métodos , Extratos Vegetais/química , Aloxano , Animais , Álcoois Benzílicos/análise , Álcoois Benzílicos/farmacologia , Ácidos Cumáricos/análise , Ácidos Cumáricos/farmacologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Glucosídeos/análise , Glucosídeos/farmacologia , Hidroxibenzoatos/análise , Hidroxibenzoatos/farmacologia , Hipoglicemiantes/farmacologia , Insulina/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Propionatos , Sitosteroides/análise , Sitosteroides/farmacologiaAssuntos
Ansiolíticos , Broncodilatadores , Aprovação de Drogas , Ansiolíticos/análise , Ansiolíticos/farmacologia , Ansiolíticos/uso terapêutico , Álcoois Benzílicos/análise , Álcoois Benzílicos/farmacologia , Álcoois Benzílicos/uso terapêutico , Broncodilatadores/análise , Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Clorobenzenos , Maitansina/análogos & derivados , Maitansina/uso terapêutico , Piperazinas/análise , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Pirazóis/análise , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas , Sulfetos , Sulfonamidas , VortioxetinaRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a serious health concern due to the lack of effective vaccines or satisfactory treatment. In the search for new compounds against this neglected disease, we have previously demonstrated that the compound 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile) (MBHA3), derived from the Morita-Baylis-Hillman reaction, effectively caused a loss of viability in both the epimastigote and trypomastigote forms. However, the mechanisms of parasite death elicited by MBHA3 remain unknown. The aim of this study was to better understand the morphophysiological changes and the mechanism of cell death induced by MBHA3 treatment on T. cruzi. To perform this analysis, we used confocal microscopy and flow cytometry to monitor the fluorescent probes such as annexin-V/propidium iodide (AV/PI), calcein-AM/ethidium homodimer (CA/EH), acridine orange (AO) and rhodamine 123 (Rho 123). Lower concentrations of MBHA3 led to alterations in the mitochondrial membrane potential and AO labeling, but did not decrease the viability of the epimastiogote forms, as determined by the CA/EH and AV/PI assays. Conversely, treatment with higher concentrations of MBHA3 led to extensive plasma membrane damage, loss of mitochondrion membrane potential, DNA fragmentation and acidification of the cytoplasm. Our findings suggest that at higher concentrations, MBHA3 induces T. cruzi epimastigote death by necrosis in a mitochondrion-dependent manner.
Assuntos
Acrilonitrila/análogos & derivados , Álcoois Benzílicos/farmacologia , Morte Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Trypanosoma cruzi/efeitos dos fármacos , Acrilonitrila/farmacologia , Acrilonitrila/uso terapêutico , Álcoois Benzílicos/uso terapêutico , Membrana Celular/efeitos dos fármacos , Doença de Chagas/parasitologia , Citoplasma/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , NitrilasRESUMO
OBJECTIVES: Gastrodia elata (GE) Blume (Orchidaceae) has been previously known for its therapeutic benefits against neurodegenerative diseases. Microglial activation and death have been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease. In this study, GE and its pure components, gastrodin and 4-hydroxybenzyl alcohol (4HBA), were applied to ß-amyloid-induced BV2 mouse microglial cells. MATERIALS AND METHODS: Cell viability was assessed by the MTT assay and Western blotting was also performed. RESULTS: ß-amyloid-induced cell death was shown to be induced time- and dose-dependently. To examine the cell death mechanism, we confirmed the involvement of ER stress signaling. C/EBP homologous protein (CHOP), a pro-apoptotic ER stress protein, was expressed at high levels but glucose-regulated protein 78 (GRP78), an anti-apoptotic ER stress protein with chaperone activity, was only slightly affected by treatment with ß-amyloid. However, pretreatment with GE and its components inhibited the expression of CHOP but increased that of GRP78 in ß-amyloid-treated cells. This study also showed that a single treatment with GE extracts, gastrodin, or 4HBA induced the expression of GRP78, a marker for enhanced protein folding machinery, suggesting a protective mechanism for GE against ß-amyloid. CONCLUSIONS: This study reveals the protective effects of GE against ß-amyloid-induced cell death, possibly through the enhancement of protein folding machinery of a representative protein, GRP78, and the regulation of CHOP in BV2 mouse microglial cells.
Assuntos
Amiloide/farmacologia , Álcoois Benzílicos/farmacologia , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Gastrodia/química , Glucosídeos/farmacologia , Microglia/efeitos dos fármacos , Animais , Álcoois Benzílicos/isolamento & purificação , Chaperona BiP do Retículo Endoplasmático , Glucosídeos/isolamento & purificação , CamundongosRESUMO
Objectives: Gastrodia elata (GE) Blume (Orchidaceae) has been previously known for its therapeutic benefits against neurodegenerative diseases. Microglial activation and death have been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease. In this study, GE and its pure components, gastrodin and 4-hydroxybenzyl alcohol (4HBA), were applied to β-amyloid-induced BV2 mouse microglial cells. Materials and Methods Cell viability was assessed by the MTT assay and Western blotting was also performed. Results: β-amyloid-induced cell death was shown to be induced time- and dose-dependently. To examine the cell death mechanism, we confirmed the involvement of ER stress signaling. C/EBP homologous protein (CHOP), a pro-apoptotic ER stress protein, was expressed at high levels but glucose-regulated protein 78 (GRP78), an anti-apoptotic ER stress protein with chaperone activity, was only slightly affected by treatment with β-amyloid. However, pretreatment with GE and its components inhibited the expression of CHOP but increased that of GRP78 in β-amyloid-treated cells. This study also showed that a single treatment with GE extracts, gastrodin, or 4HBA induced the expression of GRP78, a marker for enhanced protein folding machinery, suggesting a protective mechanism for GE against β-amyloid. Conclusions: This study reveals the protective effects of GE against β-amyloid-induced cell death, possibly through the enhancement of protein folding machinery of a representative protein, GRP78, and the regulation of CHOP in BV2 mouse microglial cells.