RESUMEN
SCOPE: The disturbance of the hypothalamic-pituitary-gonadal (HPG) axis, gut microbiota (GM) community, and short-chain fatty acids (SCFAs) is a triggering factor for pubertal onset. The study investigates the effects of the long-term intake of aspartame on puberty and GM in animals and humans. METHODS AND RESULTS: Aspartame-fed female offspring rats result in vaginal opening time prolongation, serum estrogen reduction, and serum luteinizing hormone elevation. , 60 mg kg-1 aspartame treatment decreases the mRNA levels of gonadotropin-releasing hormone (GnRH), Kiss1, and G protein-coupled receptor 54 (GPR54), increases the mRNA level of RFamide-related peptide-3 (RFRP-3), and decreases the expression of GnRH neurons in the hypothalamus. Significant differences in relative bacterial abundance at the genus levels and decreased fecal SCFA levels are noted by 60 mg kg-1 aspartame treatment. Among which, Escherichia-Shigella is negatively correlated with several SCFAs. In girls, high-dose aspartame consumption decreases the risk of precocious puberty. CONCLUSIONS: Aspartame reduces the chance of puberty occurring earlier than usual in female offspring and girls. Particularly, 60 mg kg-1 aspartame-fed female offspring delays pubertal onset through the dysregulation of HPG axis and GM composition by inhibiting the Kiss1/GPR54 system and inducing the RFRP-3. An acceptable dose of aspartame should be recommended during childhood.
Asunto(s)
Kisspeptinas , Pubertad Tardía , Humanos , Ratas , Femenino , Animales , Kisspeptinas/metabolismo , Kisspeptinas/farmacología , Aspartame/efectos adversos , Aspartame/metabolismo , Pubertad Tardía/metabolismo , Ratas Sprague-Dawley , Maduración Sexual/fisiología , Hormona Liberadora de Gonadotropina/genética , Hipotálamo/metabolismo , Pubertad , ARN Mensajero/metabolismoRESUMEN
Mitochondrial dysfunction has been implicated in Parkinson's disease. Mic60 is a critical component of mitochondrial crista remodeling and participates in maintaining mitochondrial structure and function. This study investigated whether the carnosic acid (CA) of rosemary protects the mitochondria of SH-SY5Y cells against the neurotoxicity of 6-hydroxydopamine (6-OHDA) by regulating Mic60. Our results showed that CA pretreatment reversed the reduction in the Mic60 and citrate synthase proteins, as well as the protein induction of PKA caused by 6-OHDA. Moreover, Mic60 and PINK1 siRNAs blocked the ability of CA to lessen the release of mitochondrial cytochrome c by 6-OHDA. As shown by immunoprecipitation assay, in 6-OHDA-treated cells, the interaction of Mic60 with its phosphorylated threonine residue was decreased, but the interaction with its phosphorylated serine residue was increased. PINK1 siRNA and forskolin, a PKA activator, reversed these interactions. Moreover, forskolin pretreatment prevented CA from rescuing the interaction of PINK1 and Mic60 and the reduction in cytochrome c release and mitophagy impairment in 6-OHDA-treated cells. In conclusion, CA prevents 6-OHDA-induced cytochrome c release by regulating Mic60 phosphorylation by PINK1 through a downregulation of PKA. The regulation of Mic60 by CA can be considered as a protective mechanism for the prevention of Parkinson's disease.
Asunto(s)
Neuroblastoma , Enfermedad de Parkinson , Humanos , Oxidopamina/toxicidad , Citocromos c/metabolismo , Proteínas Mitocondriales/metabolismo , Enfermedad de Parkinson/metabolismo , Colforsina/metabolismo , Neuroblastoma/metabolismo , Mitocondrias/metabolismo , Línea Celular Tumoral , ARN Interferente Pequeño , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ApoptosisRESUMEN
BACKGROUND: Impairment of mitochondrial biogenesis is associated with the pathological progression of Parkinson's disease (PD). Parkin-interacting substrate (PARIS) can be ubiquitinated by parkin and prevents the repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α). PURPOSE: This study investigated whether the neuroprotective mechanism of carnosic acid (CA) from rosemary is mediated via the regulation of PARIS and PGC-1α by parkin. METHODS: The Western blotting and RT-PCR were used to determine protein and mRNA, respectively. To investigate the protein-protein interaction of between PARIS and ubiquitin, the immunoprecipitation assay (IP assay) was utilized. Silencing of endogenous parkin or PGC-1α was performed by using transient transfection of small interfering RNA (siRNA). RESULTS: SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) increased PARIS protein, decreased PGC-1α protein, and reduced protein and mRNA of mitochondrial biogenesis-related genes. CA pretreatment reversed the effects of 6-OHDA. By IP assay, the interaction of PARIS with ubiquitin protein caused by CA was stronger than that caused by 6-OHDA. Moreover, knockdown of parkin attenuated the ability of CA to reverse the 6-OHDA-induced increase in PARIS and decrease in PGC-1α expression. PGC-1α siRNA was used to investigate how CA influenced the effect of 6-OHDA on the modulation of mitochondrial biogenesis and apoptosis. In the presence of PGC-1α siRNA, CA could no longer significantly reverse the reduction of mitochondrial biogenesis or the induction of cleavage of apoptotic-related proteins by 6-OHDA. CONCLUSION: The cytoprotective of CA is related to the enhancement of mitochondrial biogenesis by inhibiting PARIS and inducing PGC-1α by parkin. The activation of PGC-1α-mediated mitochondrial biogenesis by CA prevents the degeneration of dopaminergic neurons, CA may have therapeutic application in PD.
Asunto(s)
Abietanos/farmacología , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Represoras/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Biogénesis de Organelos , Oxidopamina/toxicidad , Enfermedad de Parkinson/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , ARN Interferente Pequeño/farmacología , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
An imbalance in mitochondrial dynamics is strongly associated with Parkinson's disease. The fusion protein optic atrophy 1 (OPA1) is up-regulated through the activation of parkin-mediated IκB kinase γ (IKKγ)/p65 signaling. This study investigated whether the neuroprotection of carnosic acid (CA) from rosemary is involved in mitochondrial dynamics and OPA1 protein induction by parkin/IKKγ/p65 signaling. The neurotoxin 6-hydroxydopamine (6-OHDA) treated with SH-SY5Y cells decreased OPA1 and mitofusin 2 fusion proteins, but increased fission 1 and dynamin related protein 1 (DRP1) fission proteins. By immunofluorescence, 6-OHDA induced the fluorescence of green spots outside the mitochondria, indicating that cytochrome c was released to the cytoplasm. Except for the effects on DRP1 protein, CA pretreatment reversed these effects of 6-OHDA. Additionally, CA treatment increased the ubiquitination of IKKγ, nuclear p65 protein, OPA1-p65 DNA binding activity, and OPA1 protein. However, transfection of parkin small interfering RNA (siRNA) attenuated these effects of CA. Furthermore, transfection of OPA1 siRNA abolished the action of CA to reverse 6-OHDA-increased cytosolic cytochrome c protein, apoptotic-related protein cleavage, and cell death. In conclusion, the mechanism by which CA counteracts the toxicity of 6-OHDA is through modulation of mitochondrial dynamics and upregulation of OPA1 via activation of the parkin/IKKγ/p65 pathway.
Asunto(s)
Abietanos/farmacología , GTP Fosfohidrolasas/metabolismo , Quinasa I-kappa B/metabolismo , Fármacos Neuroprotectores/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Apoptosis/efectos de los fármacos , Secuencia de Bases , Línea Celular Tumoral , Humanos , Quinasa I-kappa B/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Neurotoxinas/toxicidad , Oxidopamina/toxicidad , Factor de Transcripción ReIA/metabolismo , Ubiquitinación/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Impairment in mitophagy contributes to the pathology of Parkinson's disease. This study investigated whether Phosphatase and tensin homologue (PTEN)-induced kinase 1 (PINK1)/parkin-mediated mitophagy is linked to the protective effect of carnosic acid (CA) from rosemary. Treatment of SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) disrupted the mitochondrial membrane potential, inhibited voltage-dependent anion channel 1 (VDAC1) protein, and induced cytosolic cytochrome c, but CA pretreatment reversed these findings. By immunofluorescence, CA pretreatment was shown to increase the co-localization of red fluorescence (parkin) and MitoTracker green FM fluorescence (mitochondria), indicating that CA promoted the translocation of parkin into mitochondria. Immunoprecipitation with VDAC1 antibody showed that 6-OHDA treatment decreased the interaction of ubiquitinated protein with VDAC1. However, CA pretreatment reversed this reduction in the interaction of ubiquitinated protein with VDAC1. Silencing of PINK1 and parkin by use of small interfering RNA (siRNA) attenuated the ability of CA to reverse 6-OHDA-inhibited autophagic vacuoles. Moreover, in PINK1 siRNA-transfected cells, CA no longer reversed these actions of 6-OHDA on the inhibition of mitophagy-related proteins (PINK1, parkin, VDAC1, and LC3-II) and anti-apoptotic Bcl-2 protein, as well as the induction of apoptotic-related proteins, and nuclear condensation. In conclusion, CA appears to counteract the neurotoxicity of 6-OHDA by activating PINK1/parkin-mediated mitophagy.
Asunto(s)
Abietanos/farmacología , Enfermedades Mitocondriales/prevención & control , Mitofagia/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Línea Celular Tumoral , Citocromos c/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Enfermedades Mitocondriales/inducido químicamente , Oxidopamina , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Induction of 4-hydroxynonenal (4-HNE), a major lipid peroxidation aldehyde, is observed in patients with obesity and type 2 diabetes mellitus. The lipolytic response by 4-HNE has been linked to insulin resistance. In this study, we investigated the effects of carnosic acid (CA) on 4-HNE-induced lipolysis and the inhibition of ß-oxidation in 3T3-L1 adipocytes. The results indicated that cells pretreated with CA reduced 4-HNE-mediated free fatty acid (FFA) release. Furthermore, CA reversed the inhibition of phosphorylation of Tyr632 of insulin receptor substrate-1 (IRS-1) and Akt and the phosphorylation of Ser307 of IRS-1. CA inhibited 4-HNE-induced phosphorylation of protein kinase A (PKA) and hormone-sensitive lipase (HSL), and reversed the suppression by 4-HNE of phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (pâ¯<â¯0.05). Pretreatment of cells with forskolin (a cAMP agonist) and compound C (an AMPK inhibitor) reversed these effects, respectively (pâ¯<â¯0.05). In human subcutaneous adipocytes, CA also attenuated 4-HNE-induced FFA release and the phosphorylation of PKA and HSL (pâ¯<â¯0.05). Moreover, CA increased the protein expression of glutathione S-transferase (GST) A and M. Pretreatment with ethacrynic acid, a GST inhibitor, prevented the 4-HNE-conjugated proteins suppression, the PKA and HSL phosphorylation reduction, and the FFA release inhibition by CA (pâ¯<â¯0.05). CONCLUSION: The attenuation by CA of the lipolytic response by 4-HNE is likely related to the induction of GST, which in turn reduced 4-HNE-conjugated proteins and decreased the activation of the PKA/HSL pathway. The observed effects may explain how CA improves 4-HNE-induced insulin resistance.
Asunto(s)
Abietanos/farmacología , Adipocitos/metabolismo , Aldehídos/farmacología , Ácidos Grasos no Esterificados/metabolismo , Glutatión Transferasa/metabolismo , Lipólisis/efectos de los fármacos , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Antioxidantes/farmacología , AMP Cíclico/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Humanos , Insulina/metabolismo , Ratones , Fosforilación , Transducción de SeñalRESUMEN
The mediation of apoptosis-related protein in the TGF-ß signaling pathway (ARTS) and X-liked inhibitor of apoptosis protein (XIAP) by parkin plays a critical role in preventing Parkinson's disease. We studied whether carnosic acid (CA) could prevent 6-hydroxydopamine (6-OHDA)-induced apoptosis by modulating ARTS and XIAP through parkin in SH-SY5Y cells. In cells treated with 6-OHDA, the protein expression of ARTS is increased and XIAP is decreased. Pretreatment of cells with CA reversed these effects. Moreover, CA attenuated the activation of caspase 9 and caspase 7 by 6-OHDA. By immunoprecipitation with ARTS antibody, we found that 6-OHDA increased the protein expression of XIAP. However, pretreatment of cells with CA reduced XIAP protein and increased the ubiquitination of ARTS. Silencing of parkin attenuated the ability of CA to reverse the induction of ARTS and apoptotic-related proteins and the reduction of XIAP and parkin protein by 6-OHDA. Similarly, reversal of 6-OHDA-induced nuclear condensation and apoptotic-related proteins by CA was inhibited in cells with XIAP silencing. In conclusion, CA induces parkin by enhancing the ubiquitination of ARTS, leading to induction of XIAP. This may be a novel strategy for preventing Parkinson's disease.
Asunto(s)
Abietanos/farmacología , Apoptosis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oxidopamina/farmacología , Septinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transducción de Señal/efectos de los fármacos , UbiquitinaciónRESUMEN
Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3) to regulate gene expression through epigenetic machinery. EZH2 functions as a double-facet molecule in regulation of gene expression via repression or activation mechanisms, depending on the different cellular contexts. EZH2 interacts with both histone and non-histone proteins to modulate diverse physiological functions including cancer progression and malignancy. In this review article, we focused on the updated information regarding microRNAs (miRNAs) and long non coding RNAs (lncRNAs) in regulation of EZH2, the oncogenic and tumor suppressive roles of EZH2 in cancer progression and malignancy, as well as current pre-clinical and clinical trials of EZH2 inhibitors.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias/genética , ARN Largo no Codificante/genética , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. CONCLUSION: CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway.
Asunto(s)
Abietanos/farmacología , Apoptosis/efectos de los fármacos , Gutatión-S-Transferasa pi/metabolismo , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Oxidopamina/toxicidad , Apoptosis/fisiología , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Gutatión-S-Transferasa pi/genética , Humanos , Isoquinolinas/farmacología , MAP Quinasa Quinasa 4/metabolismo , Fármacos Neuroprotectores/farmacología , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacologíaRESUMEN
Enhanced removal of abnormal protein aggregates or injured organelles through autophagy is related to neuroprotection in Parkinson's disease. In this study, we explored whether the induction of autophagy is associated with the neuroprotection of rosemary carnosic acid (CA) against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in SH-SY5Y cells. The results indicated that cells treated with CA had increased protein levels of parkin and autophagy-related markers, including phosphatidylinositol 3-kinase p100, Beclin1, autophagy-related gene 7, and microtubule-associated protein 1 light chain 3-II, as well as enhanced formation of autophagic vacuoles. Treatment of cells with 6-OHDA decreased the levels of parkin and the autophagy markers, but CA pretreatment reversed these effects. However, wortmannin (an autophagosome formation blocker) pretreatment attenuated the effect of CA. After CA pretreatment, the induction of cleaved caspase 3, cleaved poly-ADP ribose polymerase, and nuclear condensation by 6-OHDA were alleviated. Both wortmannin and bafilomycin A1 (an autophagosome-lysosome fusion blocker) inhibited the anti-apoptosis effects of CA. Additionally, we performed immunoprecipitation with anti-parkin antibody and found that the interaction of parkin and Beclin1 protein was reduced by 6-OHDA but that this effect was reversed in cells pretreated with CA. Moreover, transfection of parkin siRNA in cells inhibited the ability of CA to alleviate 6-OHDA-decreased autophagy-related markers and nuclear condensation. In conclusion, CA protects against 6-OHDA-induced apoptosis by inducing autophagy through the interaction of parkin and Beclin1. These results provide a future strategy for use of CA in the prevention of Parkinson's disease.
Asunto(s)
Abietanos/farmacología , Autofagia/efectos de los fármacos , Beclina-1/metabolismo , Neurotoxinas/toxicidad , Oxidopamina/toxicidad , Ubiquitina-Proteína Ligasas/metabolismo , Androstadienos/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Línea Celular Tumoral , Humanos , Macrólidos/farmacología , Fármacos Neuroprotectores/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , WortmaninaRESUMEN
Parkin is a Parkinson's disease (PD)-linked gene that plays an important role in the ubiquitin-proteasome system (UPS). This study explored whether carnosic acid (CA) from rosemary protects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via upregulation of parkin in vivo and in vitro. We found that the reduction in proteasomal activity by 6-OHDA was attenuated in SH-SY5Y cells pretreated with 1 µM CA. Immunoblots showed that CA reversed the induction of ubiquitinated protein and the reduction of PTEN-induced putative kinase 1 (PINK1) and parkin protein in 6-OHDA-treated SH-SY5Y cells and rats. Moreover, in a transgenic OW13 Caenorhabditis elegans model of PD that expresses human α-synuclein in muscle cells, CA reduced α-synuclein accumulation in a dose-dependent manner. In cells pretreated with the proteasome inhibitor MG132, CA no longer reversed the 6-OHDA-mediated induction of cleavage of caspase 3 and poly(ADP)-ribose polymerase and no longer reversed the suppression of proteasome activity. When parkin expression was silenced by use of small interfering RNA, the ability of CA to inhibit apoptosis and induce proteasomal activity was significantly reduced. The reduction in 6-OHDA-induced neurotoxicity by CA was associated with the induction of parkin, which in turn upregulated the UPS and then decreased cell death.
Asunto(s)
Abietanos/farmacología , Citoprotección/fisiología , Oxidopamina/toxicidad , Ubiquitina-Proteína Ligasas/biosíntesis , Regulación hacia Arriba/fisiología , Animales , Animales Modificados Genéticamente , Antioxidantes/farmacología , Caenorhabditis elegans , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Citoprotección/efectos de los fármacos , Humanos , Masculino , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , alfa-Sinucleína/biosíntesisRESUMEN
Induction of phase II enzymes is important in cancer chemoprevention. We compared the effect of rosemary diterpenes on the expression of the pi class of glutathione S-transferase (GSTP) in rat liver Clone 9 cells and the signaling pathways involved. Culturing cells with 1, 5, 10, or 20 µM carnosic acid (CA) or carnosol (CS) for 24 h in a dose-dependent manner increased the GSTP expression. CA was more potent than CS. The RNA level and the enzyme activity of GSTP were also enhanced by CA treatment. Treatment with 10 µM CA highly induced the reporter activity of the enhancer element GPEI. Furthermore, CA markedly increased the translocation of nuclear factor erythroid-2 related factor 2 (Nrf2) from the cytosol to the nucleus after 30 to 60 min. CA the stimulated the protein induction of p38, nuclear Nrf2, and GSTP was diminished in the presence of SB203580 (a p38 inhibitor). In addition, SB203580 pretreatment or silencing of Nrf2 by siRNA suppressed the CA-induced GPEI-DNA binding activity and GSTP protein expression. Knockdown of p38 or Nrf2 by siRNA abolished the activation of p38 and Nrf2 as well as the protein induction and enzyme activity of GSTP by CA. These results suggest that CA up-regulates the expression and enzyme activity of GSTP via the p38/Nrf2/GPEI pathway.
Asunto(s)
Abietanos , Anticarcinógenos , Gutatión-S-Transferasa pi , Hepatocitos , Glicoproteínas de Membrana , Transducción de Señal , Animales , Ratas , Abietanos/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Anticarcinógenos/química , Anticarcinógenos/farmacología , Células Clonales , Ensayo de Cambio de Movilidad Electroforética , Elementos de Facilitación Genéticos/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Reporteros/efectos de los fármacos , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Imidazoles/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Piridinas/farmacología , Proteínas Recombinantes/metabolismo , Interferencia de ARN , Rosmarinus/química , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
The neuroprotective effects of carnosic acid (CA), a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), have been widely investigated in recent years, however, its protection in in vivo still unclear. In this study, we investigated the behavioral activity and neuroprotective effects of CA in a rat model of Parkinson's disease (PD) induced by 6-hydroxydopamine (6-OHDA). Rats were treated with 20mg/kg body weight of CA for 3 weeks before 6-OHDA exposure. Results indicated that CA improved the locomotor activity and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Significant protection against lipid peroxidation and GSH reduction was observed in the 6-OHDA rats pretreated with CA. Pretreatment with CA increased the protein expression of γ-glutamate-cysteine ligase catalytic subunit, γ-glutamate-cysteine ligase modifier subunit, superoxide dismutase, and glutathione reductase compared with 6-OHDA-stimulated rats and SH-SY5Y cells. Immunoblots showed that the reduction of the Bcl-2/Bax ratio, the induction of caspase 3 cleavage, and the induction of poly(ADP-ribose) polymerase (PARP) cleavage by 6-OHDA was reversed in the presence of SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) in SH-SY5Y cells. Rats treated with CA reversed the 6-OHDA-mediated the activation of c-Jun NH2-terminal kinase and p38, the down-regulation of the Bcl-2/Bax ratio, the up-regulation of cleaved caspase 3/caspase 3 and cleaved PARP/PARP ratio, and the down-regulation of tyrosine hydroxylase protein. However, BAM7, an activator of Bax, attenuated the effect of CA on apoptosis in SH-SY5Y cells. These results suggest that CA protected against 6-OHDA-induced neurotoxicity is attributable to its anti-apoptotic and anti-oxidative action. The present findings may help to clarify the possible mechanisms of rosemary in the neuroprotection of PD.
Asunto(s)
Abietanos/farmacología , Antioxidantes/farmacología , Enfermedad de Parkinson/metabolismo , Extractos Vegetales/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Modelos Animales de Enfermedad , Glutamato-Cisteína Ligasa/análisis , Glutamato-Cisteína Ligasa/metabolismo , Glutatión Reductasa/análisis , Glutatión Reductasa/metabolismo , Humanos , Masculino , Actividad Motora/fisiología , Oxidopamina/administración & dosificación , Enfermedad de Parkinson/enzimología , Poli(ADP-Ribosa) Polimerasas/análisis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Proteína X Asociada a bcl-2/análisis , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Carnosic acid (CA), a diterpene found in the rosemary (Rosmarinus officinalis), has been reported to have a neuroprotective effect. Glutathione S-transferase (GST) P (GSTP) is a phase II detoxifying enzyme that provides a neuroprotective effect. The aim of this study was to explore whether the neuroprotective effect of CA is via an upregulation of GSTP expression and the possible signaling pathways involved. SH-SY5Y cells were pretreated with 1 µM CA followed by treatment with 100 µM 6-hydroxydopamine (6-OHDA). Both immunoblotting and enzyme activity results show that CA also induced protein expression and enzyme activity of GSTP. Moreover, CA significantly increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt, the nuclear translocation of p65, but not mitogen-activated protein kinases (p < 0.05). Pretreatment with LY294002 (a PI3K/Akt inhibitor) suppressed the CA-induced phosphorylation of IκB kinase (IKK) and IκBα, p65 nuclear translocation, and nuclear factor-kappa B (NF-κB)-DNA binding activity as well as GSTP protein expression. Furthermore, CA attenuated 6-OHDA-induced caspase 3 activation, and cell death was reversed by GSTP siRNA or LY294002 treatment. Additionally, male Wistar rats with lesions induced by 6-OHDA treatment in the right striatum responded to treatment with CA, which significantly reversed the reduction in GSTP protein expression that resulted from lesioning. We suggest that CA prevents 6-OHDA-induced apoptosis through an increase in GSTP expression via activation of the PI3K/Akt/NF-κB pathway. Therefore, CA may be a promising candidate for use in the prevention of Parkinson's disease.
Asunto(s)
Abietanos/farmacología , Gutatión-S-Transferasa pi/biosíntesis , FN-kappa B/metabolismo , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/prevención & control , Fosfatidilinositol 3-Quinasas/metabolismo , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Abietanos/uso terapéutico , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Inducción Enzimática , Gutatión-S-Transferasa pi/genética , Humanos , Masculino , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/enzimología , Síndromes de Neurotoxicidad/inmunología , Oxidopamina/farmacología , Extractos Vegetales/uso terapéutico , Ratas Wistar , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Understanding the neuroprotective effects of the rosemary phenolic diterpene carnosic acid (CA) has attracted increasing attention. We explored the mechanism by which CA modulates the neurotoxic effects of 6-hydroxydopamine (6-OHDA) in SH-SY5Y cells. Cells were pretreated with CA for 12 h followed by treatment with 100 µM 6-OHDA for 12 or 24 h. Cell viability determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide (MTT) assay indicated that 0.1 to 1 µM CA dose-dependently attenuated the cell death induced by 6-OHDA, whereas the effect of 3-5 µM CA was weaker. CA at 1 µM suppressed the 6-OHDA-induced nuclear condensation, reactive oxygen species generation, and cleavage of caspase 3 and PARP. Immunoblots showed that the phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 by 6-OHDA was reduced in the presence of CA. Incubation of cells with CA resulted in significant increases in the total glutathione (GSH) level and the protein expression of the γ-glutamylcysteine ligase catalytic subunit and modifier subunit. L-Buthionine-sulfoximine, an inhibitor of GSH synthesis, attenuated the effect of CA on cell death and apoptosis. Treatment with CA also led to an increase in nuclear factor erythroid-2 related factor 2 (Nrf2) activation, antioxidant response element (ARE)-luciferase reporter activity, and DNA binding to the ARE. Silencing of Nrf2 expression alleviated the reversal of p38 and JNK1/2 activation by CA. These results suggest that the attenuation of 6-OHDA-induced apoptosis by CA is associated with the Nrf2-driven synthesis of GSH, which in turn down-regulates the JNK and p38 signaling pathways. The CA compound may be a promising candidate for neuroprotection in Parkinson's disease.
Asunto(s)
Abietanos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Glutatión/metabolismo , Oxidopamina/toxicidad , Extractos Vegetales/farmacología , Abietanos/química , Antioxidantes/química , Butionina Sulfoximina/química , Butionina Sulfoximina/farmacología , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Glutamato-Cisteína Ligasa/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Oxidopamina/química , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Sales de Tetrazolio/química , Sales de Tetrazolio/metabolismo , Tiazoles/química , Tiazoles/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The anticarcinogenic effect of rosemary has been partly attributed to the modulation of the activity and expression of phase II detoxification enzymes. Here we compared the effects of phenolic diterpenes from rosemary on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Cells were treated with 1-20 µmol/L of carnosic acid (CA) or carnosol (CS) for 24 h. Both CA and CS dose dependently increased NQO1 enzyme activity and protein expression, and the induction potency of CA was stronger than that of CS. The increase in NQO1 enzyme activity in cells treated with 10 µmol/L CA and CS was 4.1- and 1.9-fold, respectively (P < 0.05). RT-PCR showed that CA and CS induced NQO1 mRNA in a dose-dependent manner. Furthermore, CA dose dependently induced transcription of nuclear factor erythroid-2 related factor 2 (Nrf2) and antioxidant response element (ARE)-luciferase reporter activity. Silencing of Nrf2 expression alleviated NQO1 protein expression and ARE-luciferase activity by CA. Moreover, the phosphorylation of p38 was mainly stimulated in the presence of CA. Pretreatment with SB203580 or silencing of p38 expression inhibited Nrf2 activation and NQO1 induction. These results suggest that the increased NQO1 expression by CA is likely related to the p38-Nrf2 pathway and help to clarify the possible molecular mechanism of action of rosemary phenolic compounds in drug metabolism and cancer prevention.
Asunto(s)
Abietanos/farmacología , Anticarcinógenos/administración & dosificación , NAD(P)H Deshidrogenasa (Quinona)/genética , Factor 2 Relacionado con NF-E2/genética , Extractos Vegetales/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Antioxidantes , Western Blotting , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Silenciador del Gen , Hígado/enzimología , Hígado/metabolismo , Luciferasas , NAD(P)H Deshidrogenasa (Quinona)/efectos de los fármacos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Fosforilación/efectos de los fármacos , Plásmidos , ARN Mensajero , Ratas , Elementos de Respuesta/efectos de los fármacos , Transducción de Señal , Transcripción Genética , Transfección , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Carnosic acid (CA), a rosemary phenolic compound, has been shown to display anti-cancer activity. We examined the apoptotic effect of CA in human neuroblastoma IMR-32 cells and elucidated the role of the reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) associated with carcinogenesis. The result indicated that CA decreased the cell viability in a dose-dependent manner. Further investigation in IMR-32 cells revealed that cell apoptosis following CA treatment is the mechanism as confirmed by flow cytometry, hoechst 33258, and caspase-3/-9 and poly(ADP-ribose) polymerase (PARP) activation. Immunoblotting suggested a down-regulation of anti-apoptotic Bcl-2 protein in the CA-treated cells. In flow cytometric analysis, CA caused the generation of reactive oxygen species (ROS); however, pretreatment with the antioxidant N-acetylcysteine (NAC) attenuated the CA-induced generation of ROS and apoptosis. This effect was accompanied by increased activation of p38 and by decreased activation of extracellular signal-regulated kinase (ERK) as well as activation of c-Jun NH(2)-terminal kinase (JNK). Moreover, NAC attenuated the CA-induced phosphorylation of p38. Silencing of p38 by siRNA gene knockdown reduced the CA-induced activation of caspase-3. In conclusion, ROS-mediated p38 MAPK activation plays a critical role in CA-induced apoptosis in IMR-32 cells.