Pheochromocytoma 12 Research Articles

Bioinformatic Identification of Signaling Pathways and Hub Genes in Vascular Dementia.

Vascular dementia (VaD) is a prevalent neurodegenerative disease characterized by cognitive impairment due to cerebrovascular factors, affecting a significant portion of the aging population and highlighting the critical need to understand specific targets and mechanisms for effective prevention and treatment strategies. We aimed to identify pathways and crucial genes involved in the progression of VaD through bioinformatics analysis and subsequently validate these findings. We conducted differential expression analysis, Weighted Gene Co-expression Network Analysis (WGCNA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) analysis. We utilized pheochromocytoma 12 (PC12) cells to create an in vitro oxygen-glucose deprivation (OGD) model. We investigated the impact of overexpression and interference of adrenoceptor alpha 1D (ADRA1D) on OGD PC12 cells using TdT-mediated dUTP nick-end labeling (TUNEL), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and Fluo-3-pentaacetoxymethyl ester (Fluo-3 AM) analysis. We found 187 differentially expressed genes (DEGs) in the red module that were strongly associated with VaD and were primarily enriched in vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and cell adhesion. Among these pathways, we identified ADRA1D as a gene shared by vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction. The TUNEL assay revealed a significant decrease in PC12 cell apoptosis with ADRA1D overexpression (p < 0.01) and a significant increase in apoptosis upon silencing ADRA1D (p < 0.01). RT-qPCR and WB analysis revealed elevated ADRA1D expression (p < 0.001) and decreased phospholipase C beta (PLCβ) and inositol 1,4,5-trisphosphate receptor (IP3R) expression (p < 0.05) with ADRA1D overexpression. Moreover, the Fluo-3 AM assessment indicated significantly lower intracellular Ca2+ levels with ADRA1D overexpression (p < 0.001). Conversely, interference with ADRA1D yielded opposite results. Our study provides a new perspective on the pathogenic mechanisms of VaD and potential avenues for therapeutic intervention. The results highlight the role of ADRA1D in modulating cellular responses to OGD and VaD, suggesting its potential as a target for VaD treatment.

Effects of cinnamon oil and its main constituents, cinnamic acid and cinnamaldehyde, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurodegeneration in PC-12 cells

Cinnamon (Cinnamomum verumJ.Presl) is a well-known medicinal plant considered as an effective treatment for neurological disorders based on Persian medicine. The aim of the present study was assessing the effect of cinnamon oil, cinnamic acid, and cinnamaldehyde, on the in vitromodel of Parkinson's disease (PD). Cinnamon oil, prepared in sesame oil, was phytochemically analyzed using high performance liquid chromatography (HPLC). Pheochromocytoma-12 (PC-12) cells were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as an in vitromodel of neurodegeneration in PD. Cell viability, activity of caspase enzymes, and formation of reactive oxygen species (ROS) were evaluated. MPTP significantly decreased cell viability and increased Casp activity, as well asROS formation. Cinnamon oil and cinnamic acid at 200 μg/mLcould significantly reverse MPTP-induced abnormalities in PC-12 cells including Casp activity and ROS formation. Our study supports the beneficial effect of cinnamon oil in neurodegeneration. Further investigations are needed to clarify the mechanisms and main active components.

Hierarchically patterned polyurethane microgrooves featuring nanopillars or nanoholes for neurite elongation and alignment

Surface micro- and nanostructures profoundly affect the functional performance of nerve regeneration implants by modulating neurite responses. However, few studies have investigated the impact of discrete nanostructures, such as nanopillars and nanoholes, and their combination with microgrooves on neurite outgrowth and alignment. Furthermore, numerous techniques have been developed for surface micro-/nanopatterning, but simple and low-cost approaches are quite limited. In this work, we show that nanopillars and nanoholes, and their combination with microgrooves, can be patterned on polyurethane (PU) films using a low-cost, reusable photoresist master mold prepared via nanosphere lens lithography and UV-LED photolithography, with specific “reinforcement” methods for overcoming the inherent drawbacks of using photoresist masters. We show that the PU nanopillars and nanoholes increase the neurite length of pheochromocytoma 12 (PC12) cells through unique growth cone interactions. Moreover, we demonstrate, for the first time, that hierarchically patterned nano-/microstructured PU films enhance both PC12 neurite elongation and alignment, showing the potential use of our proposed method for the micro-/nanopatterning of polymers for nerve tissue engineering.

Fucoidan and microtopography on polyvinyl alcohol hydrogels guided axons and enhanced neuritogenesis of pheochromocytoma 12 (PC12) cells.

Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. Polyvinyl alcohol (PVA) nerve conduits have been clinically approved, but the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neuritogenesis and axon guidance. PVA was conjugated with extracellular matrix proteins and fucoidan, a bioactive sulfated polysaccharide, to improve cell adhesion. Micro-sized topographies, including 1.8 μm convex lenses, 2 μm gratings, and 10 μm gratings were successfully fabricated on PVA by nanofabrication, and the synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0% to 2.8% and further increased to 5% by presenting laminin on the surface. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. The incorporation of 2 μm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration.

Piceatannol Protects PC-12 Cells against Oxidative Damage and Mitochondrial Dysfunction by Inhibiting Autophagy via SIRT3 Pathway.

Oxidative stress has been identified as a major cause of cellular injury in a variety of neurodegenerative disorders. This study aimed to investigate the cytoprotective effects of piceatannol on hydrogen peroxide (H2O2)-induced pheochromocytoma-12 (PC-12) cell damage and explore the underlying mechanisms. Our findings indicated that piceatannol pre-treatment significantly attenuated H2O2-induced PC-12 cell death. Furthermore, piceatannol effectively improved mitochondrial content and mitochondrial function, including enhancing mitochondrial reactive oxygen species (ROS) elimination capacity and increasing mitochondrial transcription factor (TFAM), peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α) and mitochondria Complex IV expression. Meanwhile, piceatannol treatment inhibited mitochondria-mediated autophagy as demonstrated by restoring mitochondrial membrane potential, reducing autophagosome formation and light chain 3B II/I (LC3B II/I) and autophagy-related protein 5 (ATG5) expression level. The protein expression level of SIRT3 was significantly increased by piceatannol in a concentration-dependent manner. However, the cytoprotective effect of piceatannol was dramatically abolished by sirtuin 3 (SIRT3) inhibitor, 3-(1H-1,2,3-Triazol-4-yl) pyridine (3-TYP), which led to an exacerbated mitochondrial dysfunction and autophagy in PC-12 cells under oxidative stress. In addition, the autophagy activator (rapamycin) abrogated the protective effects of piceatannol on PC-12 cell death. These findings demonstrated that piceatannol could alleviate PC-12 cell oxidative damage and mitochondrial dysfunction by inhibiting autophagy via the SIRT3 pathway.

USP22 knockdown protects against cerebral ischemia/reperfusion injury via destabilizing PTEN protein and activating the mTOR/TFEB pathway.

Ubiquitin-specific protease 22 (USP22) expression was reported to be increased in response to ischemic brain damage, but the biological role and underlying mechanism remain little understood. USP22 shRNA was intravenously injected into the mouse brain, and then a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was constructed, and the infarct volume, neurobehavioral deficit score, cell apoptosis, oxidative stress, and autophagy in vivo were evaluated. Oxygen-glucose deprivation/reperfusion (OGD/R) treated pheochromocytoma-12 (PC12) cells were used as an in vitro model of ischemia/reperfusion. The effects of USP22 on proliferation, apoptosis, oxidative stress, and autophagy were explored by CCK-8, flow cytometry, ELISA, and Western blot assays. The relationship between USP22 and the phosphatase and tensin homolog (PTEN) was measured by Co-IP and Western blot assays. Both USP22 and PTEN were highly expressed in MCAO/R mouse brain tissues and OGD/R-induced PC12 cells. In vitro, USP22 knockdown strongly improved OGD/R-mediated changes in cell viability, apoptosis, oxidative stress, and lactate dehydrogenase (LDH) production in PC12 cells. USP22 bound to PTEN and stabilized PTEN expression by decreasing its ubiquitination. PTEN overexpression reversed the promoting effect of USP22 knockdown on cell viability and the inhibitory effects of USP22 knockdown on apoptosis, oxidative stress, and LDH release rate in PC12 cells subjected to OGD/R. PTEN silencing elevated the protein levels of p62, p-mTOR, TFEB, and LAMP1 and reduced the protein levels of LC3-II/LC3-I. USP22 expression levels were negatively correlated with mTOR expression levels, and USP22-shRNA-mediated expression of p62, p-mTOR, TFEB, and LAMP1 was reversed by rapamycin, an inhibitor of mTOR. In vivo, USP22 silencing significantly alleviated infarct volume, neurobehavioral impairments, cell apoptosis, oxidative stress, and autophagy in MCAO/R mice. USP22 knockdown exerts neuroprotective effects in cerebral ischemia/reperfusion injury by downregulating PTEN and activating the mTOR/TFEB pathway.

Heat Treatment Enhances the Neuroprotective Effects of Crude Ginseng Saponin by Increasing Minor Ginsenosides.

Ginsenoside is the primary active substance of ginseng and has many pharmacological effects, such as anti-cancer, immune, regulating sugar and lipid metabolism, and antioxidant effects. It also protects the nervous and cardiovascular systems. This study analyzes the effects of thermal processing on the bioactivities of crude ginseng saponin. Heat treatment increased the contents of minor ginsenosides in crude saponins, such as Rg3, and heat-treated crude ginseng saponin (HGS) had better neuroprotective effects than non-treated crude saponin (NGS). HGS reduced glutamate-induced apoptosis and reactive oxygen species generation in pheochromocytoma 12 (PC12) cells, significantly more than NGS. HGS protected PC12 cells against glutamate-induced oxidative stress by upregulating Nrf2-mediated antioxidant signaling and downregulating MAPK-mediated apoptotic signaling. HGS has the potential for the prevention and treatment of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease.

Stem cells alleviate OGD/R mediated stress response in PC12 cells following a co-culture: modulation of the apoptotic cascade through BDNF-TrkB signaling.

Apoptosis mediated by endoplasmic reticulum (ER) stress plays a crucial role in several neurovascular disorders, including ischemia/reperfusion injury (I/R injury). Previous in vitro and in vivo studies have suggested that following I/R injury, ER stress is vital for mediating CCAT-enhancer-binding protein homologous protein (CHOP) and caspase-12-dependent apoptosis. However, its modulation in the presence of stem cells and the underlying mechanism of cytoprotection remains elusive. In vivo studies from our lab have reported that post-stroke endovascular administration of stem cells renders neuroprotection and regulates apoptosis mediated by ER stress. In the current study, a more robust in vitro validation has been undertaken to decipher the mechanism of stem cell-mediated cytoprotection. Results from our study have shown that oxygen-glucose deprivation/reoxygenation (OGD/R) potentiated ER stress and apoptosis in the pheochromocytoma 12 (PC12) cell line as evident by the increase of protein kinase R (PKR)-like ER kinase (p-PERK), p-Eukaryotic initiation factor 2α subunit (EIF2α), activation transcription factor 4 (ATF4), CHOP, and caspase 12 expressions. Following the co-culture of PC12 cells with MSCs, ER stress was significantly reduced, possibly via modulating the brain-derived neurotrophic factor (BDNF) signaling. Furthermore, inhibition of BDNF by inhibitor K252a abolished the protective effects of BDNF secreted by MSCs following OGD/R. Our study suggests that inhibition of ER stress-associated apoptotic pathway with MSCs co-culture following OGD/R may help to alleviate cellular injury and further substantiate the use of stem cells as a therapeutic modality toward neuroprotection following hypoxic injury or stroke in clinical settings.

Analyzing Angiotensin II Receptor Type 1 Clustering in PC12 Cells in Response to Hypoxia Using Direct Stochastic Optical Reconstruction Microscopy (dSTORM).

Angiotensin II (Ang II) is a hormone that plays a major role in maintaining homeostasis. The Ang II receptor type 1 (AT1R) is expressed in acute O2 sensitive cells, including carotid body (CB) type I cells and pheochromocytoma 12 (PC12) cells, and Ang II increases cell activity. While a functional role for Ang II and AT1Rs in increasing the activity of O2 sensitive cells has been established, the nanoscale distribution of AT1Rs has not. Furthermore, it is not known how exposure to hypoxia may alter the single-molecule arrangement and clustering of AT1Rs. In this study, the AT1R nanoscale distribution under control normoxic conditions in PC12 cells was determined using direct stochastic optical reconstruction microscopy (dSTORM). AT1Rs were arranged in distinct clusters with measurable parameters. Across the entire cell surface there averaged approximately 3 AT1R clusters/μm2 of cell membrane. Cluster area varied in size ranging from 1.1×10-4 to 3.9×10-2μm2. Twenty-four hours of exposure to hypoxia (1% O2) altered clustering of AT1Rs, with notable increases in the maximum cluster area, suggestive of an increase in supercluster formation. These observations could aid in understanding mechanisms underlying augmented Ang II sensitivity in O2 sensitive cells in response to sustained hypoxia.

Downregulation of ELAVL1 attenuates ferroptosis-induced neuronal impairment in rats with cerebral ischemia/reperfusion via reducing DNMT3B-dependent PINK1 methylation.

Ferroptosis is a non-apoptotic form of programmed cell death and has been found in ischemic stroke. Increasing evidence revealed that ELAVL1 is associated with ferroptosis, but it remains largely unclear whether ELAVL1 is involved in ischemic stroke. Here, we aimed to investigate the biological role and mechanism of ELAVL1 in cerebral ischemia/reperfusion (I/R) injury. ELAVL1 shRNA were intravenously injected into rat brain, and then ischemic/reperfusion (I/R) model was constructed in rats to detect infarct volume, neurobehavioral deficit, and several ferroptosis factors (GSH, GPX4, SLC7A11, MDA, ROS, iron ion) in vivo. Oxygen-glucose deprivation/reperfusion (OGD/R) treated pheochromocytoma-12 (PC12) cells were used as in vitro models of I/R. RIP, biotin pull-down and ChIP assays was used to explore the relationship among ELAVL1, DNMT3B, and PINK1. ELAVL1 was highly expressed in rat brain tissue after I/R injury. Compared with those in the I/R group, the injection of RSL3 (30mg/kg) or ferrostatin-1 (10mg/kg) aggravated or alleviated infarct volume, neurobehavioral impairments, and increased or decreased ferroptosis factor levels, respectively. ELAVL1 silencing ameliorated brain damage in I/R-treated rats by inhibiting ferroptosis. Moreover, ELAVL1 silencing observably facilitated cell viability, GSH content, GPX4 and SLC7A11 expression, and reduced iron ion concentration, ROS and MDA levels in OGD/R-treated PC12 cells. ELAVL1 bound with DNMT3B mRNA 3'UTR and promoted DNMT3B expression. ELAVL1 inhibited PINK1 expression through stabilizing DNMT3B mRNA and blocking DNMT3B-mediated DNA methylation of PINK1 promoter. PINK1 knockdown reversed the effects of ELAVL1 inhibition on cell viability, GSH, GPX4, SLC7A11, iron ion concentration, ROS and MDA levels in OGD/R-treated PC12 cells. ELAVL1 plays a critical role in protecting against ferroptosis-induced cerebral I/R and subsequent brain damage via DNMT3B/PINK1 axis, thus providing a new potential target for ischemic stroke treatment.

Piezoelectric conduit combined with multi-channel conductive scaffold for peripheral nerve regeneration

Due to the complex structural hierarchy and regenerative microenvironment of peripheral nerves, developing a nerve guidance conduit (NGC) that can replace autografts for repairing long-distance nerve defects remains a challenge. To overcome this limitation, a multi-channel NGC with dual electrical activity of piezoelectricity and conductivity was constructed to enhance peripheral nerve regeneration. Silk fibroin (SF) was adopted to prepare a multi-channel cryogel scaffold by gradient freezing method, and then poly(3,4-ethylenedioxythiophene) (PEDOT) in situ polymerization based on the porous structure of the cryogel was carried out to achieve good conductivity. Meanwhile, a polyvinylidene fluoride (PVDF)/poly (l-lactic acid-co-caprolactone) (PLCL) film with spontaneous electrical properties and high mechanical strength was constructed and wrapped in the outer layer of SF/PEDOT cryogel to construct an NGC with spontaneous electrical stimulation properties and surgical suture resistance, providing a biomimetic electrophysiological microenvironment for peripheral nerve regeneration. In vitro cell experiments indicated that the multi-channel electroactive NGC not only promoted the proliferation, maturation and myelination of Schwann cells (SCs), but also effectively promoted neuronal differentiation and axonal regeneration of Pheochromocytoma 12 (PC12) cells. Furthermore, a rat sciatic nerve defect model was constructed to evaluate the effect of multi-channel electroactive NGC on peripheral nerve repair and the results showed that electroactive NGC could significantly promote peripheral nerve regeneration and functional recovery after 12 weeks, achieving the repair effect of autografts through the positive synergistic effect of piezoelectric and conductive properties of NGC. These results demonstrated the great potential of multi-channel electroactive NGCs in repairing peripheral nerve injury (PNI).

Sasa borealis Ethanol Extract Protects PC12 Neuronal Cells against Oxidative Stress

The overproduction of reactive oxygen species (ROS) can cause oxidative stress to biomolecules such as nucleic acids, proteins, and lipids, leading to neurodegenerative disorders. Sasa borealis (SB) has antioxidant, anti-inflammatory, antidiabetic, and anti-obesity effects. We evaluated the neuroprotective activity of SB on hydrogen peroxide (H2O2)-induced oxidative stress. We investigated the antioxidant and neuroprotective effects of SB water extract (SBW) and SB ethanol extract (SBE) by measuring the radical scavenging activities and intracellular ROS production. SBE, which had a high level of isoorientin, had higher antioxidative activities than SBW in 2,2′-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS+) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. It also reduced ROS generation in pheochromocytoma 12 (PC12) cells more significantly than SBW. It increased the translation of heme oxygenase-1 (HO-1), superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase (GPx) with a corresponding increase in the translation of NF-E2-related factor-2 (Nrf-2). In conclusion, SBE with high levels of phenolic compounds such as isoorientin shows promise for preventing neurodegenerative diseases.

Protein phosphatase 1 regulates reactive oxygen species-dependent degradation of histone deacetylase 5 by intermittent hypoxia.

We recently reported pheochromocytoma 12 (PC12) cells and rats subjected to intermittent hypoxia (IH), a hallmark manifestation of obstructive sleep apnea (OSA), exhibit reduced histone deacetylase activity and HDAC5 protein. Our study further suggested that posttranslational modifications rather than transcriptional mechanism(s) mediate IH-induced HDAC5 degradation. These observations prompted our current study to investigate the mechanism(s) underlying HDAC5 degradation by IH in PC12 cell cultures. IH-induced HDAC5 degradation was blocked by an antioxidant, and reactive oxygen species (ROS) mimetics decreased HDAC5 protein, suggesting that ROS mediates HDAC5 degradation by IH. NADPH oxidases (NOX) 2 and 4 were identified as sources of ROS that mediate the effects of IH. HDAC5 degradation during IH was associated with dephosphorylation of HDAC5 at serine259, and this response was blocked by a NOX inhibitor, suggesting that ROS-dependent dephosphorylation mediates HDAC5 degradation. IH-induced dephosphorylation of HDCA5 was inhibited by calyculin A, an inhibitor of protein phosphatase (PP)-1 and -2, or by the overexpression of nuclear inhibitor of PP1 (NIPP1). HDAC5 dephosphorylation by IH lead to augmented hypoxia-inducible factor (HIF)-1α protein and an increase in its transcriptional activity. These data suggest that PP1-dependent dephosphorylation of S259 destabilizes HDAC5 protein in response to IH, resulting in HIF-1α stabilization and transcriptional activity. Our findings highlight hither to unexplored role of protein phosphatases, especially PP1 in regulating HDAC5 protein, which is an upstream activator of HIF-1 signaling by IH.

A Living 3D In Vitro Neuronal Network Cultured inside Hollow Electrospun Microfibers

Micro/nanofiber-based scaffolds are widely used as a physical graft for axon growth in vitro for nerve tissue engineering. In this study, the fabrication of hollow coaxial microfibers with a 3D structure for directional neuronal cell culture is reported. The coaxial microfibers with poly-ε-caprolactone (PCL) sheath and polyvinyl alcohol (PVA) mixed with PC 12 cells as core are prepared using a coaxial double cylinder based on the liquid–liquid coflowing electrospinning method. By optimizing the electrospinning condi-tions, uniform microfibers with an average diameter of 54.6 µm are obtained using a PCL concentration of 20% and a tip-to-collector distance of 6 cm with a voltage of 6 kV. After selectively dissolving the PVA core, pheochro-mocytoma 12 (PC12) cells are successfully cultured inside these fibers. It is experimentally confirmed that such 3D hollow fibers can support and guide PC12 cells to grow, proliferate, and differentiate inside the hollow fibers and form 3D connected neuronal networks with axons extended along the hollow fibers. This implies that complex nerve connection could be constructed using the low cost and powerful liquid–liquid coflowing electrospinning method, which open the door to culture well-controlled nerve connections as in vitro models for neural tissue regeneration, brain disease models, and so on.

MiR-10b-3p alleviates cerebral ischemia/reperfusion injury by targeting Krüppel-like factor 5 (KLF5).

Although miR-10b-3p has been identified to be involved in cerebral ischemia injury, its impact and specific mechanism in cerebral ischemia injury remain unclear. The effects of Mir-10b-3p were investigated by establishing rat and cell models of ischemia/reperfusion (I/R) injury. Oxygen-glucose deprivation/reperfusion (OGD/R) was performed on pheochromocytoma-12 (PC12) cells. MiR-10b-3p expression levels in brain tissues and PC12 cells were detected by qRT-PCR. The impacts of miR-10b-3p on neurological deficits, infarct volume, inflammatory factor expression, in vivo brain water content, cell viability, and cell apoptosis were assessed. The relationship between miR-10b-3p and KLF5 was determined by TargetScan and luciferase reporter assay. The rescue experiments were performed to confirm the role of this axis in cerebral ischemia injury. Mir-10b-3p levels in rat brain tissue and PC12 cells were significantly decreased after I/R injury. MiR-10b-3p overexpression obviously reduced neurological deficits, infarct volume, brain water content, inflammatory factors expression, and neuronal apoptosis in the brain of ischemia-stroked rats. Meanwhile, miR-10b-3p upregulation also inhibited cell viability and apoptosis of OGD/R-induced PC12 cells. Besides, KLF5 was identified as a target of miR-10b-3p, and rescue experiments revealed that KLF5 was involved in the regulation of miR-10b-3p in ischemic injury. Our results demonstrated that miR-10b-3p had the neuroprotective effects against ischemia injury by targeting KLF5 and provided a potential underlying target for ischemic stroke treatment.

Grape seed proanthocyanidins protect PC12 cells from hydrogen peroxide-induced damage via the PI3K/AKT signaling pathway

Grape seed proanthocyanidins (GSP) are natural flavonoids with strong antioxidant and anti-apoptotic effects. Oxidative stress and neuronal apoptosis are major contributors to spinal cord injury (SCI). In this study, we assessed the potential protective effects of GSP on hydrogen peroxide (H2O2)-damaged pheochromocytoma-12 (PC12) cells in an in vitro model of SCI as well as the putative mechanism of action. We established a model using PC12 cells with oxidative damage induced by H2O2. Cells were treated with various concentrations of GSP (control group, 200 μmol/L H2O2 group, 5 μM GSP + H2O2 group, 10 μM GSP + H2O2 group, and 25 μM GSP + H2O2 group). The CCK-8 assay was used to determine cell activity. Dichloro-dihydro-fluorescein diacetate was used to detect intracellular reactive oxygen species (ROS), and flow cytometry was used to determine apoptosis rate. Western blot analysis was used to detect the expression of caspase-3, Bax, Bcl-2, and PI3K/AKT proteins. The results showed that GSP reduced H2O2-induced intracellular ROS and inhibited apoptosis. Furthermore, GSP inhibited the expression of caspase-3 and Bax, while promoting the expression of Bcl-2. In addition, GSP promoted the phosphorylation of PI3K and AKT. Moreover, a PI3K inhibitor (LY294002) weakened the protective effects of GSP on H2O2-induced PC12 cells. In conclusion, GSP pretreatment can protect PC12 cells from oxidative damage induced by H2O2 via the PI3K/AKT signaling pathway.

MicroRNA-184 enhances the sensitivity of pheochromocytoma-12 cells to doxorubicin by targeting ADAM22

Pheochromocytoma (PCC) is a catecholamine-producing and neuroendocrine tumor with the 5-year overall survival of advanced stage PCC lower than 40%. Increasing evidence has shown that aberrant expression of microRNAs (miRNAs) plays important roles in the development and chemotherapy resistance of cancers including PCC. The tumor-suppressive function of miR-184 has been identified in several types of cancers. The aim of this study is to explore the function and the underlying mechanism of miR-184 in the chemo-resistance of PCC. miR-184 expression was significantly lower in doxorubicin (Dox)-resistant pheochromocytoma-12 (PC-12) cells and PCC patients. Consistently, in vitro analysis showed that overexpression of miR-184 obviously improved the sensitivity of PC-12/Dox cells, while knockdown of miR-184 sensitised PC-12/Dox cells to chemotherapeutics. To further understand the possible functional mechanism of miR184 in the chemo-resistance of PCC, the targets of miR-184 were predicted. The results of miRDB database suggested A disintegrin and metalloproteinase 22 (ADAM22) carrying the potential complementary binding sites of miR-184 within its 3′-untranslated region (UTR). Further experiments confirmed that miR-184 bound the 3′-UTR of ADAM22 mRNA and down-regulated the expression of ADAM22 in PC-12/Dox cells. Moreover, ADAM22 was overexpressed in Dox-resistant PC-12 cells and PCC patients. Additionally, overexpression of ADAM22 attenuated miR-184-mediated chemo-sensitivity of PC-12/Dox cells. miR-184 played a role in the chemo-sensitivity of PC-12/Dox cells at least partially via negatively regulating ADAM22. These results suggested miR-184 as a possible novel target to attenuate the chemo-resistance of PCC.

Rational design of a highly porous electronic scaffold with concurrent enhancement in cell behaviors and differentiation under electrical stimulation.

Conductive polymers (CPs) have received increasing attention as promising materials for studying electrophysiological signals in cell and tissue engineering. The combination of CPs with electrical stimulation (ES) could possibly enhance neurogenesis, osteogenesis, and myogenesis. To date, research has been prioritized on capitalizing CPs as two-dimensional (2D) structures for guiding the differentiation. In contrast, relatively little is conducted on the implementation of 3D conductive scaffolds. In this research, we report the synergic assembly of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) and multi-walled carbon nanotubes (MWCNTs) as a biocompatible, electrically conductive, mechanically robust and structurally porous 3D scaffold. To showcase the bioelectronic utilization, a proof-of-concept demonstration of electrically stimulated cell culture under ES is conducted. The ES effects coupled with the 3D scaffold are promising on pheochromocytoma 12 (PC12), a neuronal cell line, and the ES effect on osteogenesis of human adipose-derived stem cells (hASC) was further studied. PC12 cultured on this PEDOT:PSS/MWCNT 3D scaffolds was induced to differentiate toward a more mature neuronal phenotype with the ES treatment. Furthermore, hASC osteogenesis could be highly promoted in this conductive scaffold with ES. Calcium deposition concentration and osteo-differentiated gene markers were significantly higher with ES. The facile assembly of 3D conductive scaffolds sheds light on both platforms for investigating the 3D microenvironment for electrophysiological simulation of cells and tissues under the ES treatment of in vivo tissue engineering.

Oxidative stress and mitochondrial membrane potential are involved in the cytotoxicity of perfluorododecanoic acid to neurons.

Perfluorododecanoic acid (PFDoA), used in numerous commercial products, was recently demonstrated to accumulate in the brain more easily than other perfluorinated compounds and to cause cognitive deficits. In this study, pheochromocytoma 12 (PC12) cells were exposed to doses of PFDoA to explore the cytotoxicity of this compound to neurons. The results showed that treatment with PFDoA decreased PC12 cell viability dose-dependently. Treatment with 50 and 100 µM PFDoA significantly increased reactive oxygen species (p < 0.01) and malondialdehyde (p < 0.01) and decreased total antioxidant capacity (p < 0.05 and p < 0.01, respectively) in PC12 cells. The administration of 50 and 100 µM PFDoA led to a loss of mitochondrial membrane potential (MMP) (p < 0.05 and p < 0.01, respectively) in PC12 cells. The activity of caspase 3 was obviously increased (p < 0.05) in 100 µM PFDoA-treated PC12 cells. In general, the results demonstrated that PFDoA exposure could result in the disruption of MMP, which may contribute to the increase of oxidative stress and activation of the apoptotic signaling cascade in PC12 cells.

MiR-211-5p alleviates focal cerebral ischemia-reperfusion injury in rats by down-regulating the expression of COX2

The present study was to investigate the role of microRNA (miR)-211-5p on cerebral ischemia–reperfusion injury (CIRI) and clarify its underlying mechanisms. Middle cerebral artery occlusion/reperfusion (MCAO/R) was operated on male Sprague Dawley (SD) rats, oxygen-glucose deprivation/reperfusion (OGD/R) was conducted on pheochromocytoma-12 (PC12) cells. Here, we found that miR-211-5p and Cyclooxygenase (COX2) expressions were altered in the plasma, cortex and hippocampus of MCAO/R-treated rats, as well as in the OGD/R-treaded PC12 cells. In vivo, overexpression of miR-211-5p resulted in decrease of infarct volumes, neurological deficit scores and histopathological damage. In vitro, miR-211-5p overexpression significantly decreased cell apoptosis and Lactate dehydrogenase (LDH) release rate, increased cell viability. Furthermore, our data showed that miR-211-5p overexpression markedly reduced the expressions of COX2 mRNA and protein, and the contents of Prostaglandin D2 (PGD2), PGE2, tumor necrosis factor-α (TNF-α) and Interleukin-1β (IL-1β). In addition, inhibition of COX2 significantly rescued the effects of miR-211-5p inhibitor. At last, dual luciferase experimental data showed that miR-211-5p regulated the mRNA stability of COX2 by directly binding to the 3′-untranslated region (3′-UTR) of COX2. In conclusion, our data suggested the neuroprotective effects of miR-211-5p on CIRI by targeting COX2.