Rat Pheochromocytoma Cell Line Research Articles

Gentiana capitata Buch.-Ham. ex D.Don Cell Suspension Culture as a New Source of Isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside and Their Effect on PC-12 Cell Viability.

Some species of the Gentianaceae family are a valuable source of secondary metabolites. However, the phytochemical knowledge of some of these species remains insufficient. Therefore, this work focused on the isolation of the two main secondary metabolites in the methanolic extract from a Gentiana capitata cell suspension using preparative HPLC and the determination of their structure using UHPLC-DAD-IT-MS/MS and NMR methods. Their content in the methanolic extract was quantified using a previously validated HPLC method. The toxicity of the extract and two isolated compounds was also tested on the PC-12 cell line. The structures of the main secondary metabolites were identified as isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside by comparing the UHPLC-DAD-IT-MS/MS and NMR results with the literature data. The content of isosaponarin was determined to be 0.76 ± 0.04%, and the content of 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside was found to be 0.31 ± 0.02% in the dry extract. Additionally, a two-fold increase in the viability of the PC-12 cell line was observed compared to the control after treatment with the methanolic extract at a concentration of 500 µg/mL. These results suggest the potential use of G. capitata cell suspension methanolic extract as a new source of isosaponarin and 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-β-D-ribopyranosyl-β-D-allopyranoside, highlighting their lack of toxicity to the PC-12 (rat pheochromocytoma) cell line.

Synthesis of Novel Plant-Derived Encapsulated Radiolabeled Compounds for the Diagnosis of Parkinson's Disease and the Evaluation of Biological Effects with In Vitro/In Vivo Methods.

Parkinson's disease (PD) is a neurodegenerative disorder that affects millions of individuals globally. It is characterized by the loss of dopaminergic neurons in Substantia Nigra pars compacta (SNc) and striatum. Neuroimaging techniques such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) help diagnosing PD. In this study, the focus was on developing technetium-99m ([99mTc]Tc) radiolabeled drug delivery systems using plant-derived compounds for the diagnosis of PD. Madecassoside (MA), a plant-derived compound, was conjugated with Levodopa (L-DOPA) to form MA-L-DOPA, which was then encapsulated using Poly Lactic-co-Glycolic Acid (PLGA) to create MA-PLGA and MA-L-DOPA-PLGA nanocapsules. Extensive structural analysis was performed using various methods such as Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), dynamic light scattering (DLS), and scanning electron microscopy (SEM) to characterize the synthesized products. Radiochemical yields of radiolabeled compounds were determined using thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC) methods. In vitro cell culture studies were conducted on human neuroblastoma (SH-SY5Y) and rat pheochromocytoma (PC-12) cell lines to assess the incorporation of [99mTc]Tc radiolabeled compounds ([99mTc]Tc-MA, [99mTc]Tc-MA-L-DOPA, [99mTc]Tc-MA-PLGA and [99mTc]Tc-MA-L-DOPA-PLGA) and the cytotoxicity of inactive compounds (MA and MA-L-DOPA compounds and encapsulated compounds (MA-PLGA and MA-L-DOPA-PLGA). Additionally, the biodistribution studies were carried out on healthy male Sprague-Dawley rats and a Parkinson's disease experimental model to evaluate the compounds' bioactivity using the radiolabeled compounds. The radiochemical yields of all radiolabeled compounds except [99mTc]Tc-L-DOPA-PLGA were above 95% and had stability over 6h. The cytotoxic effects of all substances on SH-SY5Y and PC-12 cells increase with increasing concentration values. The uptake values of PLGA-encapsulated compounds are statistically significant in SH-SY5Y and PC-12 cells. The biodistribution studies showed that [99mTc]Tc-MA is predominantly retained in specific organs and brain regions, with notable uptake in the prostate, muscle, and midbrain. PLGA-encapsulation led to higher uptake in certain organs, suggesting its biodegradable nature may enhance tissue retention, and surface modifications might further optimize brain penetration. Overall, the results indicate that radiolabeled plant-derived encapsulated drug delivery systems with [99mTc]Tc hold potential as diagnostic agents for PD symptoms. This study contributes to the advancement of drug delivery agents in the field of brain research.

A Neurofilament-L reporter cell line for the quantification of early neuronal differentiation: A Bioassay for neurotrophic activities

BackgroundNeurotrophic activity constitutes a crucial factor in the recovery from neurological injuries and is impaired in neurodegenerative disorders. Preclinical studies of neurotrophic factors to improve outcome of neurodegenerative diseases have yielded promising results. However, due to the complexity of these therapies, the clinical translation of this approach was so far not successful and more feasible treatments with neurotrophic activity may be promising alternatives. Therefore, highly sensitive and robust assays for compound screening are required. New methodNerve growth factor is known to induce Neurofilament-L (NF-L) expression in a rat pheochromocytoma cell line (PC12 cells) during early neuronal differentiation. We generated and characterized an enhanced green fluorescent protein (EGFP)-NF-L reporter PC12 cell line for the development of a cell-based assay (designated Neurofilament-L Bioassay) that allows straightforward quantification of early neuronal differentiation based on NF-L expression. ResultsUsing Cerebrolysin® as a role model for a pharmacological compound that stimulates neurotrophic activity in the central nervous system, the Neurofilament-L Bioassay was proved to be a robust, specific, and reproducible method. Comparison with existing method(s)It was already shown that NF-L expression correlates with neurite outgrowth in PC12 cells. Currently, quantification of neurite outgrowth is the most commonly used method to evaluate neuronal differentiation in PC12 cells, an approach that is time-consuming and of high variability. ConclusionsThis work describes the development of an EGFP-NF-L reporter PC12 cell-based assay as a robust and reproducible tool for “high throughput” compound screening for neurotrophic activity.

SİNİR BÜYÜME FAKTÖRÜ İLE FARKLILAŞTIRILMIŞ PC12 HÜCRELERİNİN MORFOMETRİK VE FLORESANS ANALİZİ

Objective
 PC12 is a rat pheochromocytoma cell line. These
 cells characteristically undergo differentiation when
 cultured with nerve growth factor (NGF). Depending
 on the dose of NGF, the length of neurite extensions
 changes. Thanks to this differentiation property,
 the cells are used in neuroscience and in modeling
 pathophysiological diseases such as Alzheimer's,
 Parkinson's, and Amyotrophic Lateral Sclerosis.
 However, literature studies showing the effect of NGF
 on neurite extensions formed in PC12 cells are very
 limited. This study aimed to investigate the effect of
 NGF on neurite extensions and cell viability depending
 on dose and incubation time.
 Materials and Methods
 In this study, PC12 cells were incubated with 50 ng/ml
 and 100 ng/ml NGF for 3, 6 and 7 days. The lengths
 of neurite outgrowths and dead cell ratios were
 calculated in incubated cells.
 Results
 The results showed that the length of neurite
 extensions and dead cell ratio increased depending
 on NGF doses and incubation time. When NGF
 incubation times were compared, no difference was
 found between 50 ng/ml NGF 6 days and 100 ng/ml
 NGF 3 days groups.
 Conclusion
 When the dead cell ratios and sizes of neurite
 extensions in the experimental groups are evaluated,
 it is thought that 100 ng/ml NGF and 3 days incubation
 time parameters are ideal for PC12 cell differentiation.

Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Neuroprotective Actions of Different Exogenous Nucleotides in H2O2-Induced Cell Death in PC-12 Cells.

Exogenous nucleotides (NTs) are considered conditionally essential nutrients, and the brain cannot synthesize NTs de novo. Therefore, the external supplementation of exogenous NTs is of great significance for maintaining normal neuronal metabolism and function under certain conditions, such as brain aging. This study, therefore, sets out to assess the neuroprotective effect of four kinds of single exogenous NTs and a mixture of the NTs, and to elucidate the potential mechanism. A rat pheochromocytoma cell line PC-12 was treated with different concentrations of exogenous NTs after 4 h of exposure to 200 µM H2O2. We found that the exogenous NTs exerted significant neuroprotection through decreasing neuron apoptosis and DNA damage, ameliorating inflammation and mitochondrial dysfunction, promoting cell viability, and augmenting antioxidant activity, and that they tended to up-regulate the NAD+/SIRTI/PGC-1α pathway involved in mitochondrial biogenesis. Among the different NTs, the neuroprotective effect of AMP seemed to be more prominent, followed by the NT mixture, NMN, and CMP. AMP also exhibited the strongest antioxidant activity in H2O2-treated PC-12 cells. UMP was excellent at inhibiting neuronal inflammation and improving mitochondrial function, while GMP offered major advantages in stabilizing mitochondrial membrane potential. The mixture of NTs had a slightly better performance than NMN, especially in up-modulating the NAD+/SIRTI/PGC-1α pathway, which regulates mitochondrial biogenesis. These results suggest that antioxidant activity, anti-inflammatory activity, and protection of mitochondrial function are possible mechanisms of the neuroprotective actions of exogenous NTs, and that the optimization of the mixture ratio and the concentration of NTs may achieve a better outcome.

Anti-inflammatory activity of novel natural plant extracts composition—LevidorTM

Abstract Introduction Due to the severe side effects of non-steroidal anti-inflammatory drugs, there is an urgent need to find new natural or synthetic chemicals that could act as selective inhibitors of cyclooxygenase-2 (COX-2). The most valuable source of new bioactive compounds and potential drugs are plants. The present study aimed at the investigation of LevidorTM, a patented composition based on oil from the seeds of Nigella sativa and extract from the root of Angelica archangelica, regarding its potential anti-inflammatory effects on the molecular level. Materials and Methods Studies were carried out with the use of murine macrophages (RAW 264.7), human monocytes (U937), and rat pheochromocytoma (PC-12) cell lines. The cytotoxicity of the LevidorTM extract was evaluated by MTT assay. The concentration of pro-inflammatory cytokines (IL-6, TNF-α) was evaluated by ELISA. Moreover, the analysis of COX-1 and COX-2 mRNA expression levels by RT-PCR, as well as protein level by Western blot and immunofluorescent staining, were assessed. Results The LevidorTM extract did not impact cell growth and showed anti-inflammatory effect in the present study. It was able to decrease the level of IL-6, TNF-α and suppress transcription of COX-2, whereas it had no effect on COX-1. Conclusions Obtained results showed that LevidorTM exhibits strong anti-inflammatory properties and may provide a significant alternative to traditionally used medicines in acute pain and chronic inflammation.

Neuritogenic steroid glycosides from crown-of-thorns starfish: Possible involvement of p38 mitogen-activated protein kinase and attenuation of cognitive impairment in senescence-accelerated mice (SAMP8) by peripheral administration

Novel steroid glycosides, acanthasterosides A1, B1, and B3, have been isolated from the crown-of-thorns starfish Acanthaster planci. Acanthasterosides B1 and B3 having two separated xyloses induced neurite outgrowth as like as nerve growth factor (NGF) in the rat pheochromocytoma cell line PC12, whereas acanthasteroside A1, having one xylose, did not induce neurite outgrowth. The acanthasteroside B3 induced neuritogenesis via the significant activation of p38 mitogen-activated protein kinase after the activation of the small G-protein Cdc42 rather than via Ras–MEK–ERK pathway that is predominantly activated by NGF. Following subcutaneous administration, acanthasteroside B3 attenuated cognitive impairment of senescence-accelerated mice (SAMP8) in two different cognitive tests. Liquid chromatography–mass spectrometry-assisted quantitative analysis demonstrated that acanthasteroside B3 could be transported into the brain via the circulatory system in mice. Thus, acanthasteroside B3 (and possibly B1) are a novel class of potential drug candidates for neurodegenerative diseases.

The Relationship between Procyanidin Structure and Their Protective Effect in a Parkinson's Disease Model.

This study evaluated the effect of grape seed-derived monomer, dimeric, and trimeric procyanidins on rat pheochromocytoma cell line (PC12) cells and in a zebrafish Parkinson’s disease (PD) model. PC12 cells were cultured with grape seed-derived procyanidins or deprenyl for 24 h and then exposed to 1.5 mm 1-methyl-4-phenylpyridinium (MPP+) for 24 h. Zebrafish larvae (AB strain) 3 days post-fertilization were incubated with deprenyl or grape seed-derived procyanidins in 400 µM 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 4 days. The results showed that the procyanidin dimers procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), procyanidin B2-3-O-gallate (B2-G), and the procyanidin trimer procyanidin C1 (C1) had a protective effect on PC12 cells, decreasing the damaged dopaminergic neurons and motor impairment in zebrafish. In PC12 cells and the zebrafish PD model, procyanidin (B1, B2, B3, B4, B1-G, B2-G, C1) treatment decreased the content of reactive oxygen species (ROS) and malondialdehyde (MDA), increased the activity of antioxidant enzymes glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and upregulated the expression of nuclear factor-erythroid 2-related factor (Nrf2), NAD(P)H: quinone oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1). These results suggest that in PC12 cells and the zebrafish PD model, the neuroprotective effects of the procyanidins were positively correlated with their degree of polymerization.

Organic cation transporter 3 mediates the non-norepinephrine transporter driven uptake of meta-[211At]astato-benzylguanidine

IntroductionMeta-[211At]astato-benzylguanidine ([211At]MABG) accumulates in pheochromocytoma via norepinephrine transporter (NET) and leads to a strong antitumor effect, but it also distributed in normal tissues non-specifically. Meta-[131I]iodo-benzylguanidine ([131I]MIBG), an iodine-labeled analog of [211At]MABG, is known to be transported by not only NET but also organic cation transporter (OCT). The involvement of OCT in [211At]MABG uptake is still largely unknown. We investigated the involvement of OCT in the non-NET-driven uptake of [211At]MABG both in vitro and in vivo. Methods[123I]MIBG and [211At]MABG uptake was investigated in PC-12 (rat pheochromocytoma cell line), NIH/3T3 (mouse fibroblasts cell line), ACHN (human renal cancer cell line), and BxPC-3 (human pancreatic cancer cell line). Herein, we used desipramine and dl-norepinephrine to inhibit NET, and we used steroids (hydrocortisone and prednisolone) to inhibit OCT3. The [211At]MABG uptake in OCT3-knockdown cells established with OCT3-selective siRNA was also investigated. We investigated the biodistribution of [211At]MABG in PC-12 tumor-bearing mice after a preloading of phosphate-buffered saline (PBS) or hydrocortisone solution. ResultsThe uptake of both [123I]MIBG and [211At]MABG was significantly inhibited by desipramine in PC-12 cells but not the other cell lines. The expression of OCT3 was relatively higher than those of the other OCT subtypes in ACHN and BxPC-3 cells. The expression of OCTs was not observed in NIH/3T3 cells. The uptake of both [123I]MIBG and [211At]MABG in ACHN and BxPC-3 cells was significantly inhibited by the steroid treatments. The [211At]MABG uptake was also reduced in OCT3-knockdown cells (p < 0.001). The radioactivity of [211At]MABG was significantly reduced in normal tissues by the preloading of hydrocortisone. In contrast, there was an increasing trend of [211At]MABG uptake in the PC-12 tumors. The tumor-to-normal tissue ratio was significantly increased by the preloading of hydrocortisone compared to that of PBS. ConclusionOur results suggest that OCT3 is involved in non-NET-driven [211At]MABG uptake. The preloading of hydrocortisone selectively reduced [211At]MABG accumulation in normal organs in vivo. OCT3 inhibition may therefore be beneficial for a reduction of the radiation risk in healthy organs in the treatment of malignant pheochromocytomas.

SIRT1 exerts anti-hypertensive effect via FOXO1 activation in the rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM) is a pivotal region in the central regulation of blood pressure (BP). It has been documented that silent information regulator 2 homolog 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent multifunctional transcription regulatory factor, has many cardiovascular protective effects. However, the role and significance of SIRT1 in the central regulation of cardiovascular activity, especially in RVLM, remains unknown. Therefore, the aim of this study was to explore the role and underlying mechanism of SIRT1 in the central regulation of cardiovascular activity in hypertension. Spontaneously hypertensive rats (SHRs) were given resveratrol (RSV) via intracerebroventricular (ICV) infusion or injected with SIRT1-overexpressing lentiviral vectors into the RVLM. In vitro experiments, angiotensin II (Ang II)-induced rat pheochromocytoma cell line (PC12 cells) were transfected with forkhead box protein O1 (FOXO1) small interfering RNA (siRNA) before treatment with RSV. Our results showed that SIRT1 activation with RSV or overexpression in the RVLM significantly decreased BP and sympathetic outflow of SHRs. Furthermore, SIRT1 overexpression in the RVLM significantly decreased reactive oxygen species (ROS) production and facilitated the forkhead box protein O1 (FOXO1) activation, accompanied by upregulation of the ROS-detoxifying enzyme superoxide dismutases 1 (SOD1) in the RVLM of SHRs. In PC12 cells, it was found that Ang II could induce oxidative stress and downregulate the SIRT1-FOXO1-SOD1 signaling pathway, which indicated that the suppressed expression of SIRT1 in the RVLM of SHRs might relate to the elevated central Ang II level. Furthermore, the enhanced oxidative stress and decreased SIRT1-FOXO1-SOD1 axis induced by Ang II were restored by treatment with RSV. However, these favorable effects mediated by SIRT1 activation were blocked by FOXO1 knockdown. Based on these findings, we concluded that SIRT1 activation or overexpression in the RVLM exerts anti-hypertensive effect through reducing oxidative stress via SIRT1-FOXO1-SOD1 signaling pathway, which providing a new target for the prevention and intervention of hypertension.

Hispolon alleviates oxidative damage by stimulating the Nrf2 signaling pathway in PC12 cells

Natural products derived from the daily diet are garnering increasing attention for neurodegenerative disease (ND) treatment. Hispolon (His), a small molecule from Phellinus linteus, has been reported to have various pharmacological activities. Here, we evaluated its protective effect on a neuron-like rat pheochromocytoma cell line (PC12). Results showed that His could restore cell death induced by oxidative damage. Nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) plays a significant role in maintaining cellular redox homeostasis. After treatment with His, some Nrf2-governed antioxidant genes were upregulated in a dose-dependent manner. However, the protective effect of His on PC12 cells was easily terminated by Nrf2 knockdown, demonstrating that Nrf2 is a critical component in this cytoprotective process. Taken together, our study showed that His was not only an effective activator of Nrf2 but also a promising candidate for ND treatment.

Relationship between Neuroprotective Effects and Structure of Procyanidins.

This study evaluated the relationship between the neuroprotective effects of procyanidins and their structural characteristics. In vitro, a rat pheochromocytoma cell line (PC12) was exposed to the grape seed-derived procyanidin monomers: catechin (C), epicatechin (EC), and epicatechin gallate (ECG); the procyanidin dimers: procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), and procyanidin B2-3-O-gallate (B2-G); and the procyanidin trimers: procyanidin C1 (C1) and N-acetyl-l-cysteine (NAC) for 24 h. Cells were then incubated with 200 μM H2O2 for 24 h. In vivo, zebrafish larvae (AB strain) 3 days post-fertilization were incubated with NAC or procyanidins (C, EC, ECG, B1, B2, B3, B4, B1-G, B2-G, C1) in 300 µM H2O2 for 4 days. Different grape seed procyanidins increased the survival of PC12 cells challenged with H2O2, improved the movement behavior disorder of zebrafish caused by H2O2, inhibited the increase of ROS and MDA and the decrease of GSH-Px, CAT, and SOD activities, and up-regulated the Nrf2/ARE pathway. The neuroprotective effects of the procyanidin trimer C1 treatment group were greater than the other treatment groups. These results suggest that the neuroprotective effect of procyanidins is positively correlated with their degree of polymerization.

Tankyrase Regulates Neurite Outgrowth through Poly(ADP-ribosyl)ation-Dependent Activation of β-Catenin Signaling.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase (TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin’s ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.

Emodin ameliorates antioxidant capacity and exerts neuroprotective effect via PKM2-mediated Nrf2 transactivation

Pyruvate kinase M2 (PKM2) is overexpressed in neuronal cells. However, there are few studies on the involvement of PKM2 modulators in neurodegenerative diseases. Emodin, a dominating anthraquinone derivative extracting from the rhizome of rhubarb, has received expanding consideration due to its pharmacological properties. Our data reveal that emodin could resist hydrogen peroxide- or 6-hydroxydopamine-mediated mitochondrial fission and apoptosis in PC12 cells (a neuron-like rat pheochromocytoma cell line). Notably, emodin at nontoxic concentrations significantly inhibits PKM2 activity and promotes dissociation of tetrameric PKM2 into dimers in cells. The PKM2 dimerization enhances the interaction of PKM2 and NFE2-related factor 2 (Nrf2), which further triggers the activation of the Nrf2/ARE pathway to upregulate a panel of cytoprotective genes. Modulating the PKM2/Nrf2/ARE axis by emodin unveils a novel mechanism for understanding the pharmacological functions of emodin. Our findings indicate that emodin is a potential candidate for the treatment of oxidative stress-related neurodegenerative disorders.

Assessing physicochemical, mechanical, and in vitro biological properties of polycaprolactone/poly(glycerol sebacate)/hydroxyapatite composite scaffold for nerve tissue engineering

The ability to mimic the extracellular matrix (ECM) by electrospun fibers has opened new practical ways in nerve tissue engineering. In the current research, polycaprolactone (PCL)/poly(glycerol sebacate) (PGS) scaffold containing 5, 10, and 15 wt% hydroxyapatite (HAp) particles were electrospinned. The morphology, surface contact angle, mechanical properties, in vitro degradation behavior, and water uptake of the PCL/PGS/HAp fibers were studied. The viability and adhesion of the rat pheochromocytoma cell line (PC12) on each scaffold were investigated using MTT assay and scanning electron microscope (SEM), respectively. SEM images revealed that the fibers were uniform and aligned in general, while by increasing the HAp particles loading from 0 to 15%, the fiber diameter decreased from 831 to 382 nm. According to transmission electron microscopy (TEM) images, needle-like HAp particles were distributed along the fibers. Fourier-transform infrared spectroscopy (FTIR) and X-ray analyses confirmed the chemical interactions between PCL, PGS, and HAp. Young's modulus of all fibers was about 0.16–0.3 MPa, which is practically suitable for nerve tissue engineering. HAp particles displayed a positive effect on PC12 viability and adhesion. To conclude, the results suggested that electrospun PCL/PGS/HAp fibers can be a promising biodegradable scaffold for serving in nerve tissue engineering.

Evaluation of PC12 Cells' Proliferation, Adhesion and Migration with the Use of an Extracellular Matrix (CorMatrix) for Application in Neural Tissue Engineering.

The use of extracellular matrix (ECM) biomaterials for soft tissue repair has proved extremely successful in animal models and in some clinical settings. The aim of the study was to investigate the effect of the commercially obtained CorMatrix bioscaffold on the viability, proliferation and migration of rat pheochromocytoma cell line PC12. PC12 cells were plated directly onto a CorMatrix flake or the well surface of a 12-well plate and cultured in RPMI-1640 medium and a medium supplemented with the nerve growth factor (NGF). The surface of the culture plates was modified with collagen type I (Col I). The number of PC12 cells was counted at four time points and then analysed for apoptosis using a staining kit containing annexin V conjugate with fluorescein and propidium iodide (PI). The effect of CorMatrix bioscaffold on the proliferation and migration of PC12 cells was tested by staining the cells with Hoechst 33258 solution for analysis using fluorescence microscopy. The research showed that the percentage of apoptotic and necrotic cells was low (less than 7%). CorMatrix stimulates the proliferation and possibly migration of PC12 cells that populate all levels of the three-dimensional architecture of the biomaterial. Further research on the mechanical and biochemical capabilities of CorMatrix offers prospects for the use of this material in neuro-regenerative applications.

Osteocalcin promotes proliferation, differentiation, and survival of PC12 cells

Involvement of the bone matrix protein osteocalcin (OC) in the development of learning and memory, and the prevention of anxiety-like behaviors in mice. However, the direct effects of OC on neurons are still unknown comparing to the mechanism how OC affects systemic energy expenditure and glucose homeostasis. In this study, we investigated the effect of OC on proliferation, differentiation, and survival of neurons using the rat pheochromocytoma cell line PC12.RT-PCR analysis for OC receptor candidates revealed that Gpr158, but not Gprc6a, mRNA was expressed in PC12 cells. The growth of PC12 cells cultured in the presence of 5–50 ng/mL of either uncarboxylated (GluOC) or carboxylated (GlaOC) OC was increased compared to cells cultured in the absence of OC. In addition, NGF-induced neurite outgrowth was enhanced by OC, and H2O2-induced cell death was suppressed by pretreatment with OC. All of these results were observed for both GluOC and GlaOC at comparable levels, suggesting that OC may directly affect cell proliferation, differentiation, and survival by binding to its candidate receptor, GPR158.

Neuroprotective roles of HAX-1 in ischemic neuronal injury

Hematopoietic cell-specific protein 1 associated protein X-1 (HAX-1) is a novel mitochondrial protein that regulates oxidative stress-induced apoptosis. However, the roles of HAX-1 in ischemic neuronal injury have not been thoroughly elucidated. In this study, the expression and roles of HAX-1 after ischemic stress were investigated using in vivo and in vitro models. The effect of oxidative stress on the regulation of HAX-1 was examined using knockout mice lacking nicotinamide-adenine dinucleotide phosphate oxidase 2 (NOX2), which is a major source of reactive oxygen species (ROS) after cerebral ischemia. Male C57BL/6 J mice were subjected to transient forebrain ischemia induced by 22-min occlusion of the bilateral common carotid arteries, and striatum samples were analyzed. For in vitro ischemic experiments, oxygen and glucose deprivation (OGD) in a rat pheochromocytoma cell line was utilized. Western blotting and immunofluorescence analysis revealed HAX-1 expression in neuronal mitochondria, which was significantly decreased after ischemia in vivo and in vitro. In NOX2 knockout mice, ischemia-induced decrease in HAX-1 expression and ischemic neuronal injury was significantly alleviated compared to those in wild-type mice. Inhibition of HAX-1 using small interfering RNA significantly increased injury in cultured cells after OGD. These findings suggest that HAX-1 has a neuroprotective effect against ischemic neuronal injury, and downregulation of HAX-1 by NOX2-produced ROS induces apoptosis after cerebral ischemia.

Synthesis and anticancer activity of chalcone–quinoxalin conjugates

Two series of quinoxaline–chalcone conjugates have been prepared by aldolic condensation and aromatic nucleophilic substitution reaction, and their anticancer activity against three cancer cell lines including benign prostatic hyperplasia epithelial cell (BPH-1), neuron-like rat pheochromocytoma cell line (PC12) and human breast cancer cell line (MCF-7) were evaluated in vitro. All of the synthesized compounds exhibited moderate to good activity against the cancer cell lines selected. Particularly, Compound A5 showed the excellent potent activity against BPH-1 and MCF-7 with IC50 values of 10.4 and 9.1 μM, respectively, which is similar to doxorubicin (14.1 and 9.2 μM, respectively). As well as compound B6 exhibited most excellent activity toward PC12 with IC50 values of 16.4 μM. Compound A10 exhibited 55.4, 36.8 and 54.5 folds higher selectivity for BPH-1, PC12 and MCF-7 cells than for HEK-293 cell, respectively. In addition, theoretical biological activities of compounds A5 and A10 were evaluated by SwissADME.