Reactive Oxygen Species In PC12 Cells Research Articles

A phlorotannin isolated from Ecklonia radiata, Dibenzodioxin-fucodiphloroethol, inhibits neurotoxicity and aggregation of β-amyloid

BackgroundThe polyphenolic phlorotannins derived from brown macroalgae consist of complex polymers of phloroglucinol residues with diverse structures ascribed pleiotropic bioactivity. This study aimed to isolate and purify the phlorotannins from Ecklonia radiata and to investigate their neuroprotective activity in PC-12 cells. MethodsThis study used high performance counter-current chromatography (HPCCC) combined with size exclusion chromatography to isolate and purify a phlorotannin from the marine brown algae Ecklonia radiata. Nuclear magnetic resonance (NMR) including 1H and 13C and 2D COZY, HSQC, HMBC (broad and band selective), and NOESY along with mass spectroscopy enabled us to identify the phlorotannin as dibenzodioxin-fucodiphloroethol (DFD). The compound was subsequently investigated for protective bioactivity against the neurotoxic β-amyloid protein Aβ1–42, and intracellular reactive oxygen species (ROS) scavenging activity in PC-12 cells along with in silico studies. ResultsDFD was nontoxic up to 50 µM in the neuronal PC-12 cell line and significantly prevented loss of cell viability in response to Aβ1–42 (0–1.5 µM). Furthermore, it significantly reduced the aggregation of Aβ1–42 as evidenced by transmission electron microscopy, while molecular docking studies revealed DFD binding to key Aβ1–42 residues associated with fibrillisation propensity. Additionally, DFD demonstrated moderate cholinesterase inhibitory activity (IC50 value of 41 µM), sharing similar interacting binding residues to donepezil in the crystallized structure, and was also able to significantly scavenge ROS in PC-12 cells. ConclusionsCollectively, DFD possesses neuroprotective actions mitigating several mechanisms ascribed to amyloid β neurotoxicity, making this macroalgal phlorotannin an excellent candidate for further studies targeting neurodegenerative pathways in Alzheimer's disease.

Analogs of imine resveratrol alleviate oxidative stress-induced neurotoxicity in PC12 cells via activation of Nrf2.

Oxidative stress is closely associated with neurodegenerative, cardiovascular and metabolic diseases. Resveratrol and related compounds have shown great potential as antioxidants via either direct scavenging of abundant reactive oxygen species (ROS) or activation of the Kelch‐like ECH‐associated protein 1‐nuclear factor (erythroid‐derived 2)‐like 2‐antioxidant response elements pathway. In the present study, we evaluated imine resveratrol analogs (IRAs) for their neuroprotective effects against ROS in PC12 cells, which are a commonly employed model system for studies of neuronal development and function. We identified that IRA‐3 (4‐[[(4‐hydroxyphenyl)methylene]amino]‐phenol) was more potent than resveratrol at rescuing PC12 cells from H2O2‐induced oxidative damage, exhibiting a recovery percentage of 60.4% at 50 μm. Our findings suggest that the neuroprotective effect of IRA‐3 was achieved via multiple routes, including direct scavenging of ROS, rescue of endogenous antioxidants and activation of the Kelch‐like ECH‐associated protein 1‐nuclear factor (erythroid‐derived 2)‐like 2‐antioxidant response elements pathway. Our results suggest that IRA‐3 may have potential for development into a possible treatment for neurodegenerative diseases.

Oleanolic Acid Mitigates 6-Hydroxydopamine Neurotoxicity by Attenuating Intracellular ROS in PC12 Cells and Striatal Microglial Activation in Rat Brains.

Oleanolic acid (OA), a biologically active pentacyclic triterpenoid compound, has been implicated in a number of clinical benefits including antioxidant, and anti-inflammatory properties. OA has been previously shown to ameliorate the toxic effects of 6-hydroxydopamine (6-OHDA), however, the mechanism by which this effect is exhibited is not clearly understood. In the present study, we investigated the role of OA in attenuation of microglial activation in 6-OHDA induced Parkinsonian rat model. We also explored the ability of OA to attenuate 6-OHDA-induced intracellular reactive oxygen species (ROS), and thus prevent cell death in PC12 cells. We accessed the utility of immunohistochemistry to assess striatal microglial activation, where shape descriptors such as area, perimeter, Feret’s diameter, aspect ratio and solidity were determined using the Fiji ImageJ software. Intracellular ROS and cell viability were assessed in PC12 cells using the OxiSelectTM Intracellular ROS Assay Kit and MTT assay, respectively. We found that microglial activation was decreased in rats pre-treated with OA prior to 6-OHDA insult as well as in rats treated with OA 1 day post 6-OHDA exposure when compared to untreated rats, as determined by shape descriptors. This finding was in correlation with significantly improved motor symptoms and increased striatal dopamine in treated rats as compared to non-treated rats. Flow cytometry assessment of PC12 cells revealed a decreased amount of intracellular ROS in cells pre-treated with OA 6 h prior to 6-OHDA exposure and cells treated with OA 1 h post 6-OHDA exposure, suggesting that OA provides neuroprotection in PC12 cells by removing intracellular ROS, thereby reducing oxidative stress. Our finding suggest that OA exhibits its neuroprotective effect by attenuating striatal microglial activation, which results in neuroinflammation that is implicated in Parkinson’s disease pathology. Further studies detailing the mechanism by which OA interacts with microglia may be useful in understanding the role of OA in attenuating neuroinflammation.

Celastrol ameliorates Cd-induced neuronal apoptosis by targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway.

Celastrol, a plant-derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species (ROS). Recently, we have reported that cadmium (Cd) activates c-Jun N-terminal kinase (JNK) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 (PP5), and celastrol prevents Cd-activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS-dependent PP5-JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd-induced expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22phox , p40phox , p47phox , p67phox , and Rac1), as well as the generation of ROS in PC12 cells and primary neurons. Also, N-acetyl-l-cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd-evoked NOX2-derived ROS. Further research revealed that celastrol was involved in the regulation of PP5 inactivation and JNK/c-Jun activation induced by Cd, as celastrol prevented Cd from reducing PP5 expression, and over-expression of wild-type PP5 or dominant negative c-Jun strengthened celastrol's inhibition of Cd-induced phosphorylation of JNK and/or c-Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX2 with apocynin or silencing NOX2 by RNA interference enhanced the inhibitory effects of celastrol on Cd-induced inactivation of PP5, activation of JNK/c-Jun, ROS, and apoptosis in the cells. Furthermore, we noticed that expression of wild-type PP5 or dominant negative c-Jun, or pretreatment with JNK inhibitor SP600125 reinforced celastrol's suppression of Cd-induced NOX2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd-induced neuronal apoptosis via targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Pulsed microwave exposure real time and dynamically reduces reactive oxygen species in rat pheochromocytoma cells in vivo

ABSTRACTWith the widespread and ever‐increasing application of microwave technology in our daily lives, the potential adverse effects on biological systems from exposure to microwaves are extremely interesting based on thermal and nonthermal effects, especially nonthermal effects on the nervous system. To real time and dynamically elucidate the diversity of reactive oxygen species (ROS) in rat pheochromocytoma (PC12) cells using a real‐time‐pulsed microwave device combining laser confocal microscopy (LSCM). A LSCM system was designed, and its physical properties, including irradiation cavity simulation, field intensity distribution, and average specific absorption rate (SAR), were analyzed and a stable field intensity distribution as well as a precise average SAR were observed. Subsequently, the diversity of ROS in PC12 cells under pulsed microwave exposure was determined by LSCM online. Additionally, the total ROS in PC12 cells was determined by flow cytometry after pulsed microwave exposure. The ROS signal in single PC12 cell was rapidly quenched as the irradiation time was extended, whereas the total ROS showed no changes in flow cytometry after microwave exposure. This indicates that the change of intracellular ROS in PC12 cells by microwave exposure is a real‐time and dynamic process, and provides a significant reference for studying the biological effects of microwaves. © 2015 Wiley Periodicals, Inc. Microwave Opt Technol Lett 57:2395–2400, 2015

Effects of the combination of active component extracts from Astragalus membranaceus and Panax notoginseng on apoptosis, reactive oxygen species and mitochondrial membrane potential of PC12 cells with oxidative injury

To explore the effects and mechanisms of combining astragaloside IV (the effective component of Astragalus membranaceus) with notoginsenoside R1, ginsenoside Rb1 and ginsenoside Rg1 (the effective components of Panax notoginseng) against oxidative injury in PC12 cells induced by cobalt chloride (CoCl₂). CoCl₂ was used to stimulate PC12 cells to induce injury after transdifferentiation with nerve growth factor. Then the PC12 cells were divided into 10 groups and cultured with corresponding drugs. After culture, apoptotic cells were tested by using Hocchst 33258 fluorescent staining, the level of mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 fluorescent staining and the content of reactive oxygen species (ROS) in PC12 cell was measured by dichlorofluorescin diacetate fluorescent staining. CoCl₂ induced apoptosis along with the obvious decrease of MMP as well as overproduction of ROS in PC12 cells. Astragaloside IV, ginsenosides Rg1, ginsenosides Rb1 and notoginsenoside R1 had inhibition effects in different degree on PC12 cell apoptosis induced by CoCl₂, reduced the overproduction of ROS and the decrease of MMP. The effects of the combination were better than those of active component alone. Active components extracted from Astragalus and Panax notoginseng can inhibit PC12 cell apoptosis induced by oxidative injury, furthermore, the effects were enhanced by combination of these components, which may be associated with jointly antagonizing the generation of ROS and raising MMP.

Investigations of Oxidative Stress, Antioxidant Response, and Protein Binding in Chlorpyrifos Exposed Rat Neuronal PC12 Cells

ABSTRACTChlorpyrifos (CPF) is a widely used organophosphate insecticide. In addition to its known properties of cholinesterase inhibition, the production of reactive oxygen species (ROS) has been suggested as a possible toxic mechanism. To investigate CPF-generated ROS, rat neuronal PC12 cells were exposed to CPF concentrations of 0 to 5000 μg/mL in Krebs buffered media (KRH), KRH + 4% bovine serum albumin (BSA), and KRH + 25 μM of the antioxidant Trolox for 0 to 5 h. Paraquat served as a positive control for ROS. The fluorescent probe 2,7-dichlorodihydro-fluorescein and the MTS assay were used to measure ROS and cytotoxicity, respectively. Examinations into CPF-albumin binding were also conducted. CPF was not strongly cytotoxic to PC12 cells, causing only mild cytotoxicity at 5000 μg/ml. In KRH media, CPF-generated ROS was observed at 4 and 5 h at 500 and 1000 μg/mL, and at 1 to 5 h at 5000 μg/mL CPF. In KRH + 4% BSA, ROS was seen only at 5 h in 5000 μg/mL CPF. Trolox significantly reduced CPF- and paraquat-induced ROS. Calculated CPF-albumin binding at 1, 10, and 100 μg/mL CPF in 4% BSA was 96%, 75%, and 15%. These data show CPF at ≥500 μg/mL induced ROS in PC12 cells, but the addition of the antioxidant Trolox and 4% BSA dramatically reduced ROS levels.

Tea catechins protect against lead-induced ROS formation, mitochondrial dysfunction, and calcium dysregulation in PC12 cells.

Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species (ROS) and reducing the antioxidant defense system of cells, which suggests that antioxidants may play an important role in the treatment of lead poisoning. The present study was designed to elucidate whether tea catechins had any protective effects on altered oxidative stress parameter in PC12 cells exposed to lead. The experimental results showed that lead decreased PC12 cell viability and induced a rapid elevation of [Ca(2+)](i), which was followed by an accumulation of ROS and a decrease of mitochondrial membrane potential (MMP). Treatment by tea catechins significantly increased cell viability, decreased intracellular Ca(2+) levels and ROS formation, and improved MMP in PC12 cells exposed to lead. The galloylated catechins showed a greater effect on ROS formation and mitochondrial dysfunction than that of nongalloylated catechins, which was similar to the result of their scavenging ability on free radical. In view of the time course of ROS formation and mitochondrial dysfunction and their correlation, our results also suggested that the beneficial effects of tea catechins on MMP are related, at least in part, to its ability to scavenge ROS in PC12 cells exposed to 100 microM Pb(2+). The present results suggest that tea catechins supplementation may play a role for modulating oxidative stress in PC12 cells exposed to lead.

Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells

To investigate the involvement of reactive oxygen species (ROS) in neuronal apoptosis, we performed confocal and flow cytometric analysis with a ROS-specific fluorogen, 6-carboxy-2′,7′-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Serum deprivation significantly increased the level of ROS in PC12 cells and rat cortical neurons. N,N′ -diphenyl- p -phenylenediamine (DPPD), an antioxidant, reduced ROS production induced by serum deprivation and recovered cell survival. However, some survival factors such as nerve growth factor and Bcl-2, which prevented the apoptosis of PC12 cells, did not affect the up-regulation of ROS induced by serum deprivation. Epidermal growth factor which prevented the apoptosis of cortical neurons, did not affect the increase of ROS. These data suggest that survival factors rescue the serum deprivation-induced apoptosis independently of ROS production.

Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells

To investigate the involvement of reactive oxygen species (ROS) in neuronal apoptosis, we performed confocal and flow cytometric analysis with a ROS-specific fluorogen, 6-carboxy-2′,7′-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Serum deprivation significantly increased the level of ROS in PC12 cells and rat cortical neurons. N,N′-diphenyl- p-phenylenediamine (DPPD), an antioxidant, reduced ROS production induced by serum deprivation and recovered cell survival. However, some survival factors such as nerve growth factor and Bcl-2, which prevented the apoptosis of PC12 cells, did not affect the up-regulation of ROS induced by serum deprivation. Epidermal growth factor which prevented the apoptosis of cortical neurons, did not affect the increase of ROS. These data suggest that survival factors rescue the serum deprivation-induced apoptosis independently of ROS production.