RESUMO
Glutamine:fructose-6-phosphate aminotransferases (GFATs) and phosphofructokinase (PFKs) are the principal rate-limiting enzymes involved in hexosamine biosynthesis pathway (HBP) and glycolysis pathway, respectively. In this study, the NlGFAT and NlPFK were knocked down through RNA interference (RNAi) in Nilaparvata lugens, the notorious brown planthopper (BPH), and the changes in energy metabolism were determined. Knockdown of either NlGFAT or NlPFK substantially reduced gene expression related to trehalose, glucose, and glycogen metabolism pathways. Moreover, trehalose content rose significantly at 72 h after dsGFAT injection, and glycogen content increased significantly at 48 h after injection. Glucose content remained unchanged throughout the experiment. Conversely, dsPFK injection did not significantly alter trehalose, but caused an extreme increase in glucose and glycogen content at 72 h after injection. The Knockdown of NlGFAT or NlPFK significantly downregulated the genes in the glycolytic pathway, as well as caused a considerable and significant decrease in pyruvate kinase (PK) activity after 48 h and 72 h of inhibition. After dsGFAT injection, most of genes in TCA cycle pathway were upregulated, but after dsNlPFK injection, they were downregulated. Correspondingly, ATP content substantially increased at 48 h after NlGFAT knockdown but decreased to an extreme extent by 72 h. In contrast, ATP content decreased significantly after NlPFK was knocked down and returned. The results have suggested the knockdown of either NlGFAT or NlPFK resulted in metabolism disorders in BPHs, highlighting the difference in the impact of those two enzyme genes on energy metabolism. Given their influence on BPHs energy metabolism, developing enzyme inhibitors or activators may provide a biological control for BPHs.
RESUMO
Glutamine:fructose-6-phosphate aminotransferase (GFAT) and phosphofructokinase (PFK) are enzymes related to chitin metabolism. RNA interference (RNAi) technology was used to explore the role of these two enzyme genes in chitin metabolism. In this study, we found that GFAT and PFK were highly expressed in the wing bud of Nilaparvata lugens and were increased significantly during molting. RNAi of GFAT and PFK both caused severe malformation rates and mortality rates in N. lugens. GFAT inhibition also downregulated GFAT, GNPNA, PGM1, PGM2, UAP, CHS1, CHS1a, CHS1b, Cht1-10, and ENGase. PFK inhibition significantly downregulated GFAT; upregulated GNPNA, PGM2, UAP, Cht2-4, Cht6-7 at 48 h and then downregulated them at 72 h; upregulated Cht5, Cht8, Cht10, and ENGase; downregulated Cht9 at 48 h and then upregulated it at 72 h; and upregulated CHS1, CHS1a, and CHS1b. In conclusion, GFAT and PFK regulated chitin degradation and remodeling by regulating the expression of genes related to the chitin metabolism and exert opposite effects on these genes. These results may be beneficial to develop new chitin synthesis inhibitors for pest control.
Assuntos
Quitina/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Hemípteros/genética , Fosfofrutoquinases/genética , Animais , Quitina/metabolismo , Quitina Sintase/genética , Regulação da Expressão Gênica/genética , Proteínas de Insetos/genética , Interferência de RNARESUMO
The brown planthopper, Nilaparvata lugens is one of the most serious pests of rice, and there is so far no effective way to manage this pest. However, RNA interference not only can be used to study gene function, but also provide potential opportunities for novel pest management. The development of wing plays a key role in insect physiological activities and mainly involves chitin. Hence, the regulating role of trehalase (TRE) genes on wing bud formation has been studied by RNAi. In this paper, the activity levels of TRE and the contents of the two sugars trehalose and glucose were negatively correlated indicating the potential role of TRE in the molting process. In addition, NlTRE1-1 and NlTRE2 were expressed at higher levels in wing bud tissue than in other tissues, and abnormal molting and wing deformity or curling were noted 48 h after the insect was injected with any double-stranded TRE (dsTRE), even though different TREs have compensatory functions. The expression levels of NlCHS1b, NlCht1, NlCht2, NlCht6, NlCht7, NlCht8, NlCht10, NlIDGF, and NlENGase decreased significantly 48 h after the insect was injected with a mixture of three kinds of dsTREs. Similarly, the TRE inhibitor validamycin can inhibit NlCHS1 and NlCht gene expression. However, the wing deformity was the result of the NlIDGF, NlENGase, NlAP, and NlTSH genes being inhibited when a single dsTRE was injected. These results demonstrate that silencing of TRE gene expression can lead to wing deformities due to the down-regulation of the AP and TSH genes involved in wing development and that the TRE inhibitor validamycin can co-regulate chitin metabolism and the expression of wing development-related genes in wing bud tissue. The results provide a new approach for the prevention and management of N. lugens.
RESUMO
Anion exchanger 3 (AE3) is known to serve crucial roles in maintaining intracellular chloride homeostasis by facilitating the reversible electroneutral exchange of Cl for HCO3 across the plasma membrane. Our previous studies reported that sasanquasaponin (SQS) can inhibit hypoxia/reoxygenation (H/R)induced elevation of intracellular Cl concentration ([Cl]i) and elicit cardioprotection by favoring Cl/HCO3 exchange of AE3. However, the molecular basis for SQSinduced increase of Cl/HCO3 exchange of AE3 remains unclear. The present study demonstrated that SQS activates protein kinase Cε (PKCε) and stimulates the phosphorylation of AE3 in H9c2 cells. Notably, SQSinduced AE3 phosphorylation was blocked by the PKCε selective inhibitor εV12, and a S67A mutation of AE3, indicating that SQS could promote phosphorylation of Ser67 of AE3 via a PKCεdependent regulatory signaling pathway. Additionally, both inhibition of PKCε by εV12 and S67A mutation of AE3 eradicated the SQSinduced increase of AE3 activity, reversed the inhibitory effect of SQS on H/Rinduced elevation of [Cl]i, Ca2+ overload and generation of reactive oxygen species, and eliminated SQSinduced cardioprotection. In conclusion, PKCεdependent phosphorylation of serine 67 on AE3 may be responsible for the increase of Cl/HCO3 exchange of AE3 and intracellular chloride efflux by SQS, and contributes to the cardioprotection of SQS against H/R in H9c2 cells.
Assuntos
Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Saponinas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Cardiotônicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Creatina Quinase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteína Quinase C-épsilon/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl-]i). Given that the increased [Cl-]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10µM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl-]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl--free condition created by replacing Cl- with equimolar gluconate resulted in a decrease in [Cl-]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl-]i.
Assuntos
Cardiotônicos/farmacologia , Cloretos/química , Citoplasma/química , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Saponinas/farmacologia , Animais , Atractilosídeo/farmacologia , Linhagem Celular , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/fisiopatologiaRESUMO
DJ1 protein, as a multifunctional intracellular protein, has been demonstrated to serve a critical role in regulating cell survival and oxidative stress. To provide in vivo evidence that DJ1 is involved in the delayed cardioprotection induced by ischemic preconditioning (IPC) against oxidative stress caused by ischemia/reperfusion (I/R), the present study subjected male SpragueDawley rats to IPC (3 cycles of 5min coronary occlusion/5min reperfusion) 24 h prior to I/R (30min coronary occlusion/120min reperfusion). A lentiviral vector containing short hairpin RNA was injected into the left ventricle three weeks prior to IPC, to knockdown DJ1 in situ. Lactate dehydrogenase (LDH) and creatine kinaseMB (CKMB) release, infarct size, cardiac function, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, malondialdehyde (MDA), intracellular reactive oxygen species (ROS), and DJ1 protein expression levels were assessed. IPC caused a significant increase in the expression levels of DJ1 protein. In addition, IPC reduced LDH and CKMB release, attenuated myocardial infarct size, improved cardiac function following I/R, and inhibited the elevation of ROS and MDA and the decrease in activities of the antioxidant enzymes SOD, CAT and GPx. However, in situ knockdown of DJ1 attenuated the IPCinduced delayed cardioprotection, and reversed the inhibitory effect of IPC on I/Rinduced oxidative stress. The present study therefore provided novel evidence that DJ1 is involved in the delayed cardioprotection of IPC against I/R injury in vivo. Notably, DJ1 is required for IPC to inhibit I/Rinduced oxidative stress.
Assuntos
Precondicionamento Isquêmico Miocárdico , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Estresse Oxidativo , Proteína Desglicase DJ-1/genética , Animais , Precondicionamento Isquêmico Miocárdico/métodos , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Proteína Desglicase DJ-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Sasanquasaponin (SQS) is an active component of Camellia oleifera Abel. A recent study by our group demonstrated that SQS was able to inhibit ischemia/reperfusioninduced elevation of the intracellular chloride ion concentration ([Cl]i) and exerted cardioprotective effects; however, the underlying intracellular signal transduction mechanisms have yet to be elucidated. As protein kinase C ε (PKCε) is able to mediate Cl homeostasis, the present study investigated its possible involvement in the effects of SQS on cardiomyocytes subjected to ischemia/reperfusion injury. Cardiomyocytes were pretreated with or without SQS or SQS plus εV12, a selective PKCε inhibitor, followed by simulated ischemia/reperfusion (sI/R). The effects on cell viability, PKCε phosphorylation levels, [Cl]i, mitochondrial membrane potential and reactive oxygen species (ROS) production were assessed using an MTS assay, western blot analysis, colorimetric assays and flow cytometry. The results revealed that treatment with SQS prior to sI/R increased the viability of cardiomyocytes, and efficiently attenuated lactate dehydrogenase and creatine phosphokinase release induced by sI/R. In addition, SQS promoted PKCε phosphorylation and inhibited sI/Rinduced elevation of [Cl]i, paralleled by the attenuation of mitochondrial membrane potential loss and ROS generation. However, when the cardiomyocytes were treated with εV12 prior to SQS preconditioning, the cardioprotection induced by SQS was reduced and the inhibitory effects of SQS on sI/Rinduced elevation of [Cl]i, production of ROS and loss of mitochondrial membrane potential were also attenuated. These findings indicated that SQS may inhibit sI/Rinduced elevation of [Cl]i through the PKCε signaling pathway to elicit cardioprotection in cultured cardiomyocytes.
Assuntos
Cardiotônicos/farmacologia , Cloretos/metabolismo , Proteína Quinase C-épsilon/metabolismo , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Hipóxia Celular , Células Cultivadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
DJ-1 protein, as a multifunctional intracellular protein, has an important role in transcriptional regulation and anti-oxidant stress. A recent study by our group showed that DJ-1 can regulate the expression of certain antioxidant enzymes and attenuate hypoxia/reoxygenation (H/R)induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2like 2 (Nrf2) is an essential transcription factor that regulates the expression of several antioxidant genes via binding to the antioxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of antioxidative enzymes by DJ1 and contributes to the protective functions of DJ1 against H/Rinduced oxidative stress in H9c2 cells. The results demonstrated that DJ1overexpressing H9c2 cells exhibited antioxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/Rinduced oxidative stress compared with native cells, whereas DJ1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ1mediated antioxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, AREbinding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ1mediated induction of antioxidant enzymes and cytoprotection against oxidative stress induced by H/R. In conclusion, these findings indicated that activation of the Nrf2 pathway is a critical mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2 cells.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/metabolismo , Animais , Catalase/genética , Catalase/metabolismo , Hipóxia Celular , Linhagem Celular , Indução Enzimática , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteína Desglicase DJ-1 , Ratos , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
We have recently shown that DJ-1 is implicated in the delayed cardioprotective effect of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R) injury as an endogenous protective protein. This study aims to further investigate the underlying mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress. Using a well-characterized cellular model of HPC from rat heart-derived H9c2 cells, we found that HPC promoted nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein-1 (Keap1) dissociation and resulted in increased nuclear translocation, antioxidant response element-binding, and transcriptional activity of Nrf2 24 hours after HPC, with subsequent upregulation of manganese superoxide dismutase (MnSOD) and heme oxygenase-1 (HO-1), which provided delayed protection against H/R-induced oxidative stress in normal H9c2 cells. However, the aforementioned effects of HPC were abolished in DJ-1-knockdown H9c2 cells, which were restored by restoration of DJ-1 expression. Importantly, we showed that inhibition of the Nrf2 pathway in H9c2 cells mimicked the effects of DJ-1 knockdown and abolished HPC-derived induction of antioxidative enzymes (MnSOD and HO-1) and the delayed cardioprotection. In addition, inhibition of Nrf2 also reversed the effects of restored DJ-1 expression on induction of antioxidative enzymes and delayed cardioprotection by HPC in DJ-1-knockdown H9c2 cells. Taken together, this work revealed that activation of Nrf2 pathway and subsequent upregulation of antioxidative enzymes could be a critical mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress in H9c2 cells.