Rat Adrenal Pheochromocytoma Cell Line Research Articles

TDP-43 is a potential marker of dopaminergic neuronal damage caused by atrazine exposure

Atrazine (ATR) is one of the herbicides widely used worldwide. Meanwhile, it is an environmental endocrine disruptor that can cross the blood-brain barrier and cause damage to the endocrine-nervous system, especially by affecting the normal secretion of dopamine (DA). Regrettably, effector markers and cascade response mechanisms in damaged dopaminergic neurons induced by ATR exposure remain elusive. In this paper, we focus on investigating aggregation and position change of transactive response DNA-binding protein-43 (TDP-43) after ATR exposure, and illustrating whether TDP-43 can serve as a potential marker of mitochondrial dysfunction which causes damage to dopaminergic neurons. In our study, we used rat adrenal pheochromocytoma cell line 12 (PC12) to establish an in vitro model of dopaminergic neurons. After PC12 was intervened by ATR, we found reduced DA cycling and DA levels, and that TDP-43 aggregated continuously in the cytoplasm and then translocated to mitochondria. Furthermore, the studies we have performed showed that the translocation can cause mitochondrial dysfunction through activating the unfolded mitochondrial protein response (UPRmt), ultimately causing damage to dopaminergic neuron. The research we have done suggests that TDP-43 can serve as a potential effector marker of dopaminergic neuron damaged caused by ATR exposure.

Transcriptome Analysis of PC12 Cells Reveals That <i>trans</i>-Banglene Upregulates <i>RT1-CE1</i> and Downregulates <i>abca1</i> in the Neurotrophic Pathway

trans-Banglene and cis(c)-banglene possess neurotrophin-like activity in rat neurons. However, the molecular mechanisms underlying t-banglene-induced neurotrophic activity in rat and human neurons remain unclear. Here, we performed transcriptome analysis in PC12 cells, a rat adrenal gland pheochromocytoma cell line treated with t-banglene, using comprehensive RNA sequencing. The differentially expressed gene analysis of the sequencing data revealed that the expression of RT1 class I, locus CE1 (RT1-CE1) was upregulated, and that of ATP binding cassette subfamily A member 1 (abca1), myosin light chain 6, and hippocampus abundant transcript 1 was downregulated in t-banglene-treated PC12 cells, with statistically significant differences. We also confirmed the RT1-CE1 upregulation and abca1 downregulation in t-banglene-treated PC12 cells by real-time reverse transcription quantitative PCR. RT1-CEl is a major histocompatibility complex class I (MHCI) protein. ABCAl is a major cholesterol transporter that regulates efflux of intracellular cholesterol and phospholipids. Thus, our results suggest an exciting link between MHCI, cholesterol regulation, and neural development.

Chemoenzymatic Total Synthesis of the Neuritogenic Echinoderm Ganglioside LLG‐5 and Related Analogues

AbstractLLG‐5 1 is a Neu5Gc8Me‐containing echinoderm ganglioside (EG) isolated from starfish Linckia laevigata. It showed neuritogenic activity toward the rat adrenal pheochromocytoma cell line PC‐12. The first chemoenzymatic total synthesis of the trisialoganglioside LLG‐5 1 has been accomplished by utilizing a convergent [2+3] coupling between a Neu5Gc8Meα(2→5)Neu5Gc‐linked disialoside and a C5‐amino GM3 ceramide, Neu5NH2(2→3)Galβ(1→4)Glcβ‐Cer. A photoactivatable lactosyl phytosphingosine bearing a sulphonate moiety was developed to permit aqueous buffer solubility of the hydrophobic glycolipid acceptor for enzymatic α(2,3) sialylation. In addition, the synthetic route is flexible, allowing non‐natural LLG‐5 analogues in native lengths with or without modification at C8 of the terminal Neu5Gc and/or N‐acyl component, further expanding the diversity of LLG‐5 structures that could be achieved. The developed synthetic strategy may enable access to other EGs.magnified image

Notoginsenoside R1 alleviates spinal cord injury through the miR-301a/KLF7 axis to activate Wnt/β-catenin pathway.

Spinal cord injury (SCI) is a devastating incident that induces neuronal loss and dysfunction. Notoginsenoside R1 (NGR1) has been reported to exhibit a neuroprotective role after SCI. In this study, the effect and molecular mechanisms of NGR1 in models of SCI were further investigated. Rat adrenal pheochromocytoma cell line (PC-12) were stimulated with lipopolysaccharide (LPS) to establish a cell model of SCI-like condition. The changes of proinflammatory cytokines and associated proteins were analyzed using enzyme linked immunosorbent assay (ELISA) and western blotting. A rat model of SCI was established. Nissl staining were used to observe the morphological characteristics of spinal cord tissues. reverse transcription-quantitative PCR (RT-qPCR) was used to measure the expression of miR-301a andKrüppel-like factor 7 (KLF7). Our results showed that NGR1 alleviated LPS-triggered apoptosis and inflammation in PC-12 cells. MiR-301a was upregulated in LPS-stimulated PC-12 cells and was downregulated by NGR1 treatment. MiR-301a overexpression reversed the effect of NGR1 in LPS-treated PC-12 cells. KLF7 was verified to be targeted by miR-301a. NGR1 activated Wnt/β-catenin signaling in LPS-treated PC-12 cells by inhibiting miR-301a and upregulating KLF7. Moreover, blocking wingless/integrated (Wnt)/β-catenin signaling eliminated the protective effect of NGR1 against SCI in vitro and in vivo. Overall, NGR1 could reduce inflammation and apoptosis and promote functional recovery of SCI rats by activating Wnt/β-catenin pathway.

MiRNA-99a alleviates inflammation and oxidative stress in lipopolysaccharide-stimulated PC-12 cells and rats post spinal cord injury

ABSTRACT Spinal cord injury (SCI) is caused by spinal fracture after the displacement of the spine or broken bone fragments protruding into the spinal canal, resulting in different degrees of injury to the spinal cord or spinal nerves. Expression levels of miR-99a and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in cerebrospinal fluid of SCI patients were analyzed. Rat adrenal gland pheochromocytoma cell line PC-12 were stimulated with lipopolysaccharide (LPS) to mimic the in vitro environment of SCI. A rat mode of SCI was established by laminectomy. Reactive oxygen species (ROS) levels were measured by 2’,7’-Dichlorodihydrofluorescein diacetate staining assay. Western blot was conducted to evaluate the expression levels of apoptotic indexes and proinflammatory cytokines. The interaction between miR-99a and NOX4 was verified by dual-luciferase reporter assay. The expression level of miR-99a was reduced while NOX4 expression was upregulated in cerebrospinal fluid of SCI patients and LPS-treated PC-12 cells. LPS impeded cell viability and promoted inflammation, apoptosis and ROS levels of PC-12 cells. Overexpression of miR-99a significantly promoted cell viability and reduced inflammation, apoptosis and oxidative stress in LPS-stimulated PC-12 cells. Dual-luciferase reporter assays verified that NOX4 was a target of miR-99a. Moreover, the expression of NOX4 was reduced in PC-12 cells after transfection with miR-99a mimic. Overexpression of NOX4 partly abolished the protective effect of miR-99a in LPS-treated PC-12 cells. To sum up, miR-99a suppresses NOX4 expression to relieve the LPS-induced inflammation, apoptosis and the progression of oxidative stress in SCI.

Adipose Mesenchymal Stem Cell-Derived Exosomes Enhance PC12 Cell Function through the Activation of the PI3K/AKT Pathway.

Transplantation of mesenchymal stem cells has been considered as an auspicious treatment for repairing nerve injuries. The rat adrenal pheochromocytoma cell line (PC12) is one of the traditional models for the study of neuronal differentiation and neuroregeneration in vitro. However, the effects of adipose mesenchymal stem cell-derived exosomes (ADSC-exo) on PC12 cells remain unclear and to be elucidated. In our study, the effects of ADSC-exo on PC12 cells were investigated. ADSC-exo were isolated by ultracentrifugation and characterized by transmission electron microscopy, flow nanoanalysis, and western blot. The effects of ADSC-exo on PC12 cell proliferation, migration, apoptosis, and the protein levels were analyzed using CCK-8 assay and EdU incorporation assay, transwell migration assay and scratch wound assay, flow cytometry, and western blot, respectively. We successfully isolated and purified exosomes from ADSC supernatant and found that ADSC-exo treatment significantly promoted PC12 cell proliferation and migration, inhibited their apoptosis, and activated the PI3K/AKT pathway, while PI3K/AKT signaling repression using LY294002 exhibited the opposite effects. The results showed that ADSC-exo promoted proliferation and migration and inhibited apoptosis of PC12 through the activation of the PI3K/AKT pathway. Thus, the effect of ADSC-exo on PC12 cells may suggest ADSC-exo may be a promising therapeutic for nerve damage.

(Keynote) Electrochemical Interface Using a New Double Mediator System for Intracellular NADH Sensing and Acute Cytotoxicity Test

Introduction Nicotinamide Adenine Dinucleotide (reduced form), NADH is a very important redox compound that is essential for metabolic reactions and ATP production in all living cells. Intracellular NADH metabolism is also important to consider the cancer treatment. Intracellular NADH has been conventionally measured by WST assay using a cell membrane-permeable redox mediator, 1-methoxy PMS (1-Methoxy-5-methylphenazinium methylsulfate) and a water soluble tetrazolium to form formazan. This colorimetric method is very easy and useful for cell counting and cytotoxicity test. However, WST assay is time-consuming and then it is not able to apply for evaluation of acute cytotoxicity by chemical compounds including drugs and pollutants. On the other hand, some research groups have already shown that electrochemical method with double mediator system is useful to monitor rapidly intracellular NADH level corresponding to the cell viability [1-5].In this paper, we would like to report the usefulness of the electrochemical interface using a small screen printed carbon electrode (SPCE) and a new double mediator system combining 1-methoxy PMS and ferricyanide. It was demonstrated that our electrochemical interface was available for faster and wide range of mammalian cell counting as compared with WST assay. Furthermore our method could be applied to acute cytotoxicity test on mammalian cells. The optimization of measurement condition is mentioned in the presentation and the advantages of our new double mediator system against other double mediator system are also discussed in the presentation. Experimental method We used PC12 cell (Rat adrenal pheochromocytoma cell line) as a mammalian cell sample. For the cell counting, the medium was exchanged to HBSS before experiments. After measurement of cell density of the original cell suspension with a hemocytometer, cell suspension was exactly diluted to prepare various density of cell suspension. Each diluted cell suspension was taken in a well of 96 well plate and final concentration of 500 μM potassium ferricyanide and 10 μM 1-metoxy PMS solution were added in a drop wise to the cell suspension. After incubation for 10 minutes, a SPCE (working electrode area is 1 mm2) was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V vs. Ag/AgCl reference electrode on the SPCE.We further tried to test the acute cytotoxicity of oxamic acid that is a famous inhibitor of anaerobic glycolysis. Each concentration of oxamic acid/HBSS was prepared and added into PC12 cell/HBSS suspension (6 x 105 cells /well) in a well (final concentration of oxamic acid: 0 to 20 mM) and the cell suspension was incubated for 1hour. And then potassium ferricyanide (final 500 mM) and 1-metoxy PMS (final 10 μM) solutions were added into the cell suspension. After incubation for more 10 minutes, a SPCE was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V. Results and c onclusions In the first stage it was demonstrated that our electrochemical interface with a new combination of double mediator system was very useful for very wide range of cell counting. The amperometric oxidation current showed very good linearity vs. the number of viable cells in the range from 7500 to 96400 cells/well (Data is not shown). The result strongly supported the combination of 1-methoxy PMS and ferricyanide is effective to monitor intracellular NADH level and to speculate the cell viability.Therefore, in the second stage, we applied our electrochemical interface to test the acute toxicity by oxamic acid. Figure 1 shows the decrease of oxidation current of the double mediator system after 1 hour oxamic acid treatment. The decrease of oxidation current was clearly dependent on the concentration of oxamic acid as shown in Figure 2. It indicated that intracellular NADH level corresponding to the cell viability was rapidly decreased by oxamic acid treatment. This result suggested that our electrochemical interface with a new double mediator system might be useful to evaluate acute cytotoxicity of drugs.

Electrochemical Interface Using a New Double Mediator System for Intracellular NADH Sensing and Acute Cytotoxicity Test

Introduction Nicotinamide Adenine Dinucleotide (reduced form), NADH is a very important redox compound that is essential for metabolic reactions and ATP production in all living cells. Intracellular NADH metabolism is also important to consider the cancer treatment. Intracellular NADH has been conventionally measured by WST assay using a cell membrane-permeable redox mediator, 1-methoxy PMS (1-Methoxy-5-methylphenazinium methylsulfate) and a water soluble tetrazolium to form formazan. This colorimetric method is very easy and useful for cell counting and cytotoxicity test. However, WST assay is time-consuming and then it is not able to apply for evaluation of acute cytotoxicity by chemical compounds including drugs and pollutants. On the other hand, some research groups have already shown that electrochemical method with double mediator system is useful to monitor rapidly intracellular NADH level corresponding to the cell viability [1-5].In this paper, we would like to report the usefulness of the electrochemical interface using a small screen printed carbon electrode (SPCE) and a new double mediator system combining 1-methoxy PMS and ferricyanide. It was demonstrated that our electrochemical interface was available for faster and wide range of mammalian cell counting as compared with WST assay. Furthermore our method could be applied to acute cytotoxicity test on mammalian cells. The optimization of measurement condition is mentioned in the presentation and the advantages of our new double mediator system against other double mediator system are also discussed in the presentation. Method We used PC12 cell (Rat adrenal pheochromocytoma cell line) as a mammalian cell sample. The cells were grown in culture flasks containing DMEM with 5% FBS and 10%HS at 37℃ with 5% CO2 atmosphere. For the cell counting experiment, the medium was exchanged to HBSS before experiments. Cells were removed from the bottom of culture flask by trypsin treatment and suspended in HBSS. After measurement of cell density of the original cell suspension with a hemocytometer, cell suspension was exactly diluted to prepare various density of cell suspension. Each diluted cell suspension was taken in a well of 96 well plate and final concentration of 500 μM potassium ferricyanide and 10 μM 1-metoxy PMS solution were added in a drop wise to the cell suspension. After incubation for 10 minutes, a SPCE (working electrode area is 1 mm2) was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V vs. Ag/AgCl reference electrode on the SPCE. The electrochemical measurement was carried out using a potentiostat (Multi-Autolab Cabinet, Metrohm Autolab).We further tried to test the acute cytotoxicity of oxamic acid that is a famous inhibitor of anaerobic glycolysis. Acute cytotoxicity test was carried out with the same electrochemical instrument by following procedure. Each concentration of oxamic acid/HBSS was prepared and added into PC12 cell/HBSS suspension (5.5 x 105 cells /well) in a well (final concentration of oxamic acid: 0 to 20 mM) and the cell suspension was incubated for 1hour. And then potassium ferricyanide (final 500 mM) and 1-metoxy PMS (final 10 μM) solutions were added into the cell suspension. After incubation for more 10 minutes, a SPCE was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V. Results and Conclusions In the first place, it was demonstrated that our electrochemical interface with a new combination of double mediator system was very useful for very wide range of cell counting. The amperometric oxidation current showed very good linearity vs. the number of viable cells in the range from 7500 to 964000 cells/well (Data is not shown). The result strongly supported the combination of 1-methoxy PMS and ferricyanide is effective to monitor intracellular NADH level and to speculate the cell viability.Therefore, in the second place, we applied our electrochemical interface to test the acute toxicity by oxamic acid. The inset of Figure 1 shows the decrease of oxidation current of the double mediator system after 1 hour oxamic acid treatment. The decrease of oxidation current was clearly dependent on the concentration of oxamic acid. It indicated that intracellular NADH level corresponding to the cell viability was rapidly decreased by oxamic acid treatment. This result suggested that our electrochemical interface with a new double mediator system might be useful to evaluate acute cytotoxicity of drugs.

Tormentic acid confers protection against oxidative stress injury in rats with Parkinson's disease by targeting the Wnt/β-catenin signaling pathway

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). Studies have shown that oxidative stress (OS) may contribute to the cascade of reactions leading to the degeneration of dopaminergic neurons in the brain. The present study investigated the protective effect of tormentic acid (TMA) on OS-induced injury in rat model of PD, and the underlying mechanism. Evaluation of learning and memorizing ability was done using Morris water maze (MWM) test. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx), and level of malondialdehyde (MDA) in substantia nigra were determined using enzyme-linked immunosorbent assay (ELISA). The protein and mRNA expressions of β-catenin, GSK-3β, and GSK-3β-Ser9 were determined using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting. The effect of TA on cell viability and proliferation was determined in vitro on rat adrenal pheochromocytoma (PC12) cell line using MTT assay. The results showed that the escape latency of rats in negative control group was significantly higher than that in normal control group (p < 0.05). However, treatment with TMA significantly and time-dependently reduced the escape latency of the rats (p < 0.05). The extent of apoptosis was significantly reduced after treatment with TMA (p < 0.05). Besides, treatment with TMA significantly increased the viability of brain cells (p < 0.05). The activities of SOD and GPx were significantly lower in negative control group than in normal control group, but were significantly increased after treatment with TMA (p < 0.05). The level of MDA was significantly higher in negative control group than in normal control group, but was significantly reduced after treatment with TMA (p < 0.05). The results of qRT-PCR and Western blotting showed that treatment with TMA significantly activated Wnt/β-catenin signaling pathway (p < 0.05). TMA treatment significantly reversed the effect of 6-hydroxydopamine on the expression levels of these proteins and their mRNAs (p < 0.05). These results suggest that TMA confers protection against OS-induced injury in rats with PD by targeting the Wnt/β-catenin signaling pathway.

LncRNA ANRIL protects against oxygen and glucose deprivation (OGD)-induced injury in PC-12 cells: potential role in ischaemic stroke

lncRNA ANRIL was reported to be closely related to ischaemic stroke (IS). In this study, we used oxygen-glucose deprivation (OGD) to stimulate rat adrenal medulla-derived pheochromocytoma cell line PC-12 to construct an in vitro IS cell model and investigated the role of ANRIL and the underlying mechanism. PC-12 cells were stimulated by OGD and/or transfected with pc-ANRIL, si-ANRIL, miR-127 mimic, miR-127 inhibitor, pEX-Mcl-1, sh-Mcl-1 and their negative controls. Cell viability, apoptosis, mRNA and protein expression was detected using CCK-8 assay, flow cytometry assay, qRT-PCR and western blot, respectively. Results showed that OGD-induced PC-12 cell injury and decreased ANRIL expression. ANRIL overexpression significantly reduced OGD-induced PC-12 cell injury evidenced by increasing cell viability and decreasing apoptosis, while ANRIL silence led to the opposite results. Meanwhile, dysregulation of ANRIL altered the expression of apoptotic proteins. Furthermore, ANRIL negatively regulated miR-127 expression. miR-127 overexpression significantly enhanced OGD-induced PC-12 cell injury. In addition, Mcl-1 expression was negatively regulated by miR-127, besides ANRIL up-regulated Mcl-1 expression by down-regulation of miR-127. Mcl-1 overexpression alleviated cell injury and miR-127 silence up-regulated Mcl-1 expression. In conclusion, lncRNA ANRIL alleviated OGD-induced PC-12 cell injury as evidenced. PI3K/AKT pathway might be involved in this regulating progression.

Da-Bu-Yin-Wan Improves the Ameliorative Effect of DJ-1 on Mitochondrial Dysfunction Through Augmenting the Akt Phosphorylation in a Cellular Model of Parkinson's Disease.

Da-Bu-Yin-Wan (DBYW) is recorded originally in China over six centuries ago, and it is used to treat Parkinson’s disease (PD) clinically in recent decades. DJ-1 is a homodimeric protein linked to early-onset PD, and found in the mitochondria. In addition, DJ-1 could protect the cells by regulating gene transcription and modulating the Akt signal pathways. Therefore, in this research, we aimed to investigate the ameliorative effect of DBYW on mitochondria in the view of the DJ-1 and Akt signaling. Rat adrenal pheochromocytoma cell line PC-12 was transfected with the plasmid pcDNA3-Flag-DJ-1 (pDJ-1). Subsequently, PC-12 cells were exposed to the PD-related mitochondrial toxin (1-methyl-4-phenylpyridinium) without/with the DBYW. After transfected with the plasmid pDJ-1, the 1-methyl-4-phenylpyridinium-induced toxicity was decreased, and the DJ-1 expression in protein level was increased. DJ-1 overexpression not only increased the mitochondrial mass, but also improved the total ATP content. Moreover, Akt phosphorylation was augmented by DJ-1 overexpression. Additionally, DBYW enhanced the above effects. Conclusively, these findings indicate that DBYW promotes the ameliorative effects of DJ-1 on mitochondrial dysfunction at least through augmenting the Akt phosphorylation in 1-methyl-4-phenylpyridinium-treated PC-12 cells.

Genesis of Neuroprotective Peptoid from Aβ30–34 Inhibits Aβ Aggregation and AChE Activity

Aβ peptide and hyper-phosphorylated microtubule associated protein (Tau) aggregation causes severe damage to both the neuron membrane and key signal processing microfilament (microtubule) in Alzheimer's disease (AD) brains. To date, the key challenge is to develop nontoxic, proteolytically stable amyloid inhibitors, which can simultaneously target multiple pathways involved in AD. Various attempts have been made in this direction; however, clinical outcomes of those attempts have been reported to be poor. Thus, we choose development of peptoid (N-substituted glycine oligomers)-based leads as potential AD therapeutics, which are easy to synthesize, found to be proteolytically stable, and exhibit excellent bioavailability. In this paper, we have designed and synthesized a new short peptoid for amyloid inhibition from 30-34 hydrophobic pocket of amyloid beta (Aβ) peptide. The peptoid selectively binds with 17-21 hydrophobic region of Aβ and inhibits Aβ fibril formation. Various in vitro assays suggested that our AI peptoid binds with tubulin/microtubule and promotes its polymerization and stability. This peptoid also inhibits AChE-induced Aβ fibril formation and provides significant neuroprotection against toxicity generated by nerve growth factor (NGF) deprived neurons derived from rat adrenal pheochromocytoma (PC12) cell line. Moreover, this peptoid shows serum stability and is noncytotoxic to primary rat cortical neurons.

Punicalagin reduces H2O2-induced cytotoxicity and apoptosis in PC12 cells by modulating the levels of reactive oxygen species

Background: Oxidative stress has long been linked to neuronal cell death in many neurodegenerative diseases. Antioxidant conventional supplements are poorly effective in preventing neuronal damage caused by oxidative stress due to their inability to cross the blood brain barrier. Hence the use of molecules extracted from plants and fruits such as phenolics, flavonoids, and terpenoids compounds constitute a new wave of antioxidant therapies to defend against free radicals.Objective: In this study we examined the effects of punicalagin, a ellagitannin isolated from the pomegranate juice, on a rat adrenal pheochromocytoma cell line, treated with hydrogen peroxide, evaluating the viability, oxidation potential, mitochondrial function, and eventual apoptosis.Methods: This study was performed on PC12 cells pretreated with punicalagin (0.5, 1, 5, 10 e 20 µM) 24 hours before of the damage by hydrogen peroxide (H2O2). H2O2 concentration (300 µM) used in our study was determined by preliminary experiments of time course. The cell viability and ROS production were evaluated by MTS assay and cytofluorometry assays, respectively. Subsequently, the number of apoptotic-positive cells and mitochondrial transmembrane potential, were measured by flow cytometry, in the same experimental paradigm. Finally, the expression of Bax and enzymatic activity of Caspase 3, some of the principle actors of programmed cell death, were investigated by semiquantitative PCR and utilizing a colorimetric assay kit, respectively.Results: We found that pretreatment with punicalagin protected the cells from H2O2-induced damage. In particular, the protective effect seemed to be correlated with a control both in radical oxygen species production and in mitochondrial functions. In fact the cells treated with H2O2 showed an altered mitochondrial membrane integrity while the pretreatment with punicalagin retained both the cellular viability and the mitochondrial membrane potential similar to the control. Furthermore, the punicalagin, modulated the apoptotic cascade triggered reducing Bax gene expression and Caspase 3 activity.Discussion: Results of the present study demonstrated a neuroprotective effect of punicalagin on H2O2-induced PC12 cell death, including mitochondria damage and expression of apoptotic gene Bax; therefore we hypothesize a possible prevent role for this molecule in neurodegenerative diseases related to oxidative stress.

Biatractylolide Modulates PI3K-Akt-GSK3β-Dependent Pathways to Protect against Glutamate-Induced Cell Damage in PC12 and SH-SY5Y Cells.

Biatractylolide, isolated from the ethyl acetate extract of Atractylodes macrocephala, has shown various pharmacological activities such as antitumor and antioxidant activities. In this work, we aim to study the protective effect of biatractylolide on glutamate-induced rat adrenal pheochromocytoma cell (PC12) and human bone marrow neuroblastoma cell line (SH-SY5Y) injury and preliminarily explore its mechanism. The results showed that glutamate was cytotoxic with an inhibitory concentration 50% (IC50) of 8.5 mM in PC12 and 10 mM in SH-SY5Y cells. In this work, the preincubation with biatractylolide (10, 15, and 20 μM) observably improved cell viability, inhibited the apoptosis of cells induced by glutamate, and reduced the activity of LDH. AO staining revealed that apoptosis of cells was decreased. Additionally, the results of western blotting manifested that pretreatment with biatractylolide could downregulate GSK3β protein expression and upregulate p-Akt protein expression, thereby protecting PC12 and SH-SY5Y cells from injury. All these findings indicate that biatractylolide has a neuroprotective effect on glutamate-induced injury in PC12 and SH-SY5Y cells through a mechanism of the PI3K-Akt-GSK3β-dependent pathways.

Bryonolic Acid, a Triterpenoid, Protect Against N-methyl-d-Aspartate-Induced Neurotoxicity in PC12 Cells.

Calcium overload is considered to be one of the mechanisms of cerebral ischemia. Ca2+ influx and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cAMP response element-binding protein (CREB) phosphorylation are considered to be involved in N-Methyl-d-aspartate (NMDA)-induced apoptosis process. This study investigated the neuroprotective effects of bryonolic acid (BA) in an NMDA-induced rat adrenal pheochromocytoma cell line (PC12) cells and the potential mechanism. PC12 was treated by NMDA to establish an excitotoxicity model. BA (110,100 and 1000 μM final concentration) was added to the medium 24 h prior to the addition of NMDA. Subsequently, a methyl thiazolyl tetrazolium (MTT) assay and a lactate dehydrogenase (LDH) release were performed. Ca2+ concentration was demonstrated using a scanning-dual wavelength fluorimetric method. In addition, protein and mRNA levels were determined via Western blot and real-time PCR. In the presence of BA, MTT assay and LDH assay showed that more cells were viable in comparison with the NMDA group. Moreover, the concentration of Ca2+ decreased with the addition of BA in culture. Furthermore, BA could upregulate protein expressions of Bcl-2, p-CREB, and p-CaMKII and downregulate protein expression of Bax. The mRNA results showed that the pattern of mRNA expression were similar to their respective protein levels. All these results indicate that BA protected PC12 cells against NMDA-induced apoptosis by inhibiting Ca2+ influx and regulating gene expression in the Ca2+-CaMKII-CREB signal pathway. Therefore, the present study supports the notion that BA may be a promising neuroprotective agent for the treatment of cerebral ischemia disease.

Chemical constituents from the roots and stems of Litsea cubeba

A new monoterpene and a new lignan, named litsecols A and B (1 and 2), respectively, together with nine known compounds (3–11), were isolated in a continuous investigation on the roots and stems of Litsea cubeba. Their structures were elucidated on the basis of extensive spectroscopic data analysis, and the absolute configuration of 1 was resolved by X-ray diffraction analysis. Compounds 2–5 and 7–9 showed significant inhibitory activity against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine microglial (Bv-2) cell line. Compounds 10 and 11 exhibited significant neuroprotective effect against hydrogen peroxide-induced oxidative damage in rat adrenal pheochromocytoma (PC12) cell line.

Neuroprotective Effects of Etidronate and 2,3,3-Trisphosphonate Against Glutamate-Induced Toxicity in PC12 Cells.

Etidronate is one of the best known bisphosphonates (BP) derivatives. It is often used as a reference drug in research related to hypercalcaemia and other common bone diseases. 2,3,3-trisphosphonate (TrisPP) is brand new analogue of BP, that also contains a 'germinal bisphosphonate' unit with an additional phosphoryl group attached in proximity to the BP unit. It is known that BPs bind to calcium by chemisorptions to form Ca-BP complexes through (O)P-C-P(O) moiety and hydrogen coordinations, and so they suppress calcium flow by interfering with Ca(2+) channel operations. The mechanistic actions of BP, involving interactions and regulations of Ca(2+), are somewhat similar to the pathogenesis of well-known neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and Huntington's disease. To investigate if neuroprotective effects are exhibited by the compounds of interests, we used a rat adrenal pheochromocytoma cell line (PC12) as our in vitro model to observe any occurrence of neuron inter-reflection. We pre-treated these PC12 cells with etidronate and TrisPP before challenging the cells with a high concentration of the neurotoxin, glutamate. Our data showed that pre-treatment with 100 μM etidronate partially ameliorated the glutamate-induced decrease in cell viability (47 %), whereas pre-treating cells with 10-100 μM TrisPP showed remarkable cell protection (78-86 %). Moreover, pre-treatments of the cells with etidronate or TrisPP attenuated cell apoptosis, reactive oxygen species generation, Ca(2+) overloading and caspase-3 protein expression, which were associated with a remarkable increase in superoxide dismutase activity in our glutamate-injured PC12 cells. Therefore, this study supports the notion that etidronate and TrisPP may be promising neuroprotective agents.

Total Synthesis and Neuritogenic Activity Evaluation of Ganglioside PNG‐2A from the Starfish Protoreaster nodosus

AbstractThe total synthesis of ganglioside PNG‐2A from the starfish Protoreaster nodosus and its neuritogenic activity toward neuron‐like cells is described. The unique galactose‐enriched glycan moiety, in which an 8‐O‐methyl sialic acid residue is sandwiched between galactose residues, was constructed through an efficient 8‐O‐methylation and subsequent di‐tert‐butylsilylene‐assisted α‐glycosylation at the 4‐OH of the sialic acid residue with a Galfβ(1,3)Gal donor. In vitro biological evaluation in a rat adrenal pheochromocytoma (PC12) cell line revealed that the synthesized PNG‐2A potentiated the neurite outgrowth of PC12 cells in the presence of nerve growth factor (NGF).

CysLT2 receptor mediates lipopolysaccharide-induced microglial inflammation and consequent neurotoxicity in vitro

Neuroinflammation induced by microglial activation plays a critical role in many neurodegenerative diseases, including Parkinson׳s disease (PD). Recent studies have indicated that cysteinyl leukotriene receptor 2 (CysLT2R) is involved in inflammation and brain injury after cerebral ischemia. However, the role of CysLT2R in microglial responses associated with PD remains unclear. In the present study, we determined the regulatory roles of CysLT2R in microglial inflammation and subsequent neurotoxicity in an in vitro brain inflammation model induced by the microglial activator lipopolysaccharide (LPS). We found that LPS induced phagocytosis of a murine microglial cell line (BV-2 cells) and increased production of the proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β). The expression of CysLT2R protein was up-regulated and the nuclear translocation of CysLT2R was induced in LPS-activated BV-2 cells. CysLT2R selective antagonist HAMI 3379 significantly inhibited LPS-induced phagocytosis and overproduction of the cytokines in BV-2 cells. Similarly, the CysLT2R silencing by specific short hairpin RNA (shRNA) had the same effects as those of HAMI 3379, suggesting that the effect might be CysLT2R-dependent. Furthermore, the conditioned medium (CM) derived from LPS-treated BV-2 cells induced the cell death of a rat adrenal pheochromocytoma cell line (PC12). HAMI 3379 and CysLT2R shRNA attenuated neuronal death by suppressing the production of neurotoxic cytokines released from LPS-activated microglia. Collectively, these results suggest that CysLT2R mediates LPS-induced microglial inflammation and consequent neurotoxicity. CysLT2R may be a promising molecular target that modulates microglia-related neuroinflammation in neurodegenerative disorders, such as PD.

Neuritogenic Activity of Tetradecyl 2,3-Dihydroxybenzoate Is Mediated through the Insulin-Like Growth Factor 1 Receptor/Phosphatidylinositol 3 Kinase/Mitogen-Activated Protein Kinase Signaling Pathway

Tetradecyl 2,3-dihydroxybenzoate (ABG-001) is a lead compound derived from neuritogenic gentisides. In the present study, we investigated the mechanism by which ABG-001 induces neurite outgrowth in a rat adrenal pheochromocytoma cell line (PC12). Inhibitors of insulin-like growth factor 1 (IGF-1) receptor, phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated kinase (ERK) 1/2 significantly decreased ABG-001-induced neurite outgrowth. Western blot analysis revealed that ABG-001 significantly induced phosphorylation of IGF-1 receptor, protein kinase B (Akt), ERK, and cAMP responsive element-binding protein (CREB). These effects were markedly reduced by addition of the corresponding inhibitors. We also found that ABG-001-induced neurite outgrowth was reduced by protein kinase C inhibitor as well as small-interfering RNA against the IGF-1 receptor. Furthermore, like ABG-001, IGF-1 also induced neurite outgrowth of PC12 cells, and low-dose nerve growth factor augmented the observed effects of ABG-001 on neurite outgrowth. These results suggest that ABG-001 targets the IGF-1 receptor and activates PI3K, mitogen-activated protein kinase, and their downstream signaling cascades to induce neurite outgrowth.