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Background: Alzheimer's disease (AD) develops as a result of oxidative damage to neurons and chronic inflammation of microglia. These processes can be influenced by the use of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs). The CM contains a wide range of factors that have neurotrophic, antioxidant, and anti-inflammatory effects. In addition, the therapeutic potential of the CM can be further enhanced by pretreating the MSCs to increase their paracrine activity. The current study aimed to investigate the neuroprotective effects of CM derived from MSCs, which were either activated by a TLR3 ligand or exposed to CoCl2, a hypoxia mimetic (pCM or hCM, respectively), in an in vitro model of AD. Methods: We have developed a novel in vitro model of AD that allows us to investigate the neuroprotective and anti-inflammatory effects of MSCs on induced neurodegeneration in the PC12 cell line and the activation of microglia using THP-1 cells. Results: This study demonstrates for the first time that pCM and hCM exhibit more pronounced immunosuppressive effects on proinflammatory M1 macrophages compared to CM derived from untreated MSCs (cCM). This may help prevent the development of neuroinflammation by balancing the M1 and M2 microglial phenotypes via the decreased secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased secretion of IL-4, as well as the expression of IL-10 and TGF-β by macrophages. Moreover, a previously unknown increase in the neurotrophic properties of hCM was discovered, which led to an increase in the viability of neuron-like PC12 cells under H2O2-induced oxidative-stress conditions. These results are likely associated with an increase in the production of growth factors, including vascular endothelial growth factor (VEGF). In addition, the neuroprotective effects of CM from preconditioned MSCs are also mediated by the activation of the Nrf2/ARE pathway in PC12 cells. Conclusions: TLR3 activation in MSCs leads to more potent immunosuppressive effects of the CM against pro-inflammatory M1 macrophages, while the use of hCM led to increased neurotrophic effects after H2O2-induced damage to neuronal cells. These results are of interest for the potential treatment of AD with CM from preactivated MSCs. [ABSTRACT FROM AUTHOR]

Elemental analysis at the single-cell level is an emerging technique in the field of inductively coupled plasma mass spectrometry (ICP-MS). In comparison to the analysis of cell suspensions by fast time-resolved analysis, single-cell sampling by laser ablation (LA) allows the discriminatory analysis of single cells according to their size and morphology. In this study, we evaluated the changes in elemental contents in a single cell through differentiation of rat adrenal pheo-chromo-cytoma into neuron-like cells by LA-ICP-MS. The contents of seven essential minerals were increased about 2–3 times after the differentiation. In addition, we evaluated the degree of differentiation at the single-cell level by means of imaging cytometry after immuno-fluorescence staining of microtubule-associated protein 2 (Map2), a neuron-specific protein. The fluorescence intensities of Alexa Fluor 488-conjugated secondary antibody against the anti-Map2 primary antibody showed large variations among the cells after the onset of differentiation. Although the average fluorescence intensity was increased through the differentiation, there were still less-matured neuron-like cells that exhibited a lower fluorescence intensity after 5 days of differentiation. Since a positive correlation between the fluorescence intensity and the cell size in area was found, we separately measured the elemental contents in the less-matured smaller cells and well-matured larger cells by LA-ICP-MS. The larger cells had higher elemental contents than the smaller cells, indicating that the essential minerals are highly required at a later stage of differentiation. [ABSTRACT FROM AUTHOR]

Objectives: Plant polysaccharides have attracted increasing attention due to their high efficiency and low toxicity. Codonopsis pilosula polysaccharide (CPP) is an essential substance extracted from Codonopsis pilosula, known for its excellent antioxidant and neuroprotective effects. However, it is still unclear how CPP improves nerve protection and what its underlying molecular mechanisms are. This study aimed to investigate the neuroprotective effect of CPP on Aβ25-35-induced damage in PC12 cells and its underlying molecular mechanisms. Methods: The neuroprotective effect of CPP was evaluated using Aβ25-35-induced damage in pheochFfromocytoma (PC12) cells as an in vitro cell model. The cells were treated with CPP alone or in combination with SB203580 (an inhibitor of p38MAPK) in Aβ25-35 culture. The cell viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,diphenyltetrazolium (MTT) assay. Furthermore, reactive oxygen species (ROS) were detected using flow cytometry. The production levels of intracellular superoxide dismutase (SOD), dismutase (SOD), glutathione (GSH), catalase (CAT), and malondialdehyFde (MDA) were determined using the colorimetric method. Annexin V-FITC and propidium iodide (PI) staining, as well as 33258 were performed using fluorescence microscopy. Moreover, the effect of adding SB203580 was studied to determine the changes in cell apoptosis induced by CPP treatment and Aβ25-35 induction. Results: The CPP markedly inhibited Aβ25-35-induced reduction in the viability and apoptosis of PC12 cells. CPP also reduced the Aβ25-35-induced increase in the expression of the apoptosis factors and the levels of free radicals (ROS and MDA) and reversed the Aβ25-35-induced suppression of antioxidant activity. Additionally, inhibition of p38MAPK via the addition of their antagonists reversed the observed anti-apoptosis effects of CPP. Conclusions: CPP can efficiently provide neuroprotection against Aβ25-35-induced damage in PC12 cells brought about via oxidation and apoptosis reactions, and the underlying mechanisms involve the p38MAPK pathways. Therefore, CPP could potentially be useful as a neuroprotective agent in natural medicine, pharmacy, and the food industry. [ABSTRACT FROM AUTHOR]

Injuries to the nervous system continue to be a problem on a global scale due to the limited capacity of the nervous system to self-repair. Today, electrospun nanofibrous membranes (NFMs) are widely used in nerve tissue engineering due to their advanced properties such as low-cost, being uncomplicated, the potential to give direction to neurite outgrowth, and their highly manageable properties. Recently, the fabrication of functionalized NFMs has been proposed as a viable strategy to help restore the function of the nervous system. This would be accomplished by creating the ideal microenvironment that could mimic the features of the extracellular matrix of neural cells such as conductivity. The main objective of this project was to construct a biocompatible and electro-conductive NFM with the potential to promote proliferation and induce differentiation into neuron-like cells in PC12 cells. Basic PCL and gelatin based scaffolds seem to lack the highly desired properties of cellular implants for neural tissue engineering such as high biocompatibility, tailored biodegradability, high antibacterial, and ROS scavenging properties. For this purpose, Poly(ε-caprolactone)/gelatin (PG) electrospun nanofibrous scaffolds were coated with GO and GQD through Mussel-inspired polydopamine (DOPA) (PG-DOPA-GO and PG-DOPA-GQD). There is a dearth of research on the application of GQD in neural tissue engineering, and there is no comparative assessment of GO and GQD's effectiveness when coated through DOPA on the surface of PG NFM, in PC12 differentiation. For the first time, the outcomes of these NFMs, as neural tissue engineering scaffolds are assessed and contrasted from the standpoints of surface, structure, mechanical, and biological aspects. Apart from that, as far as we know, this is the first work using graphene-based nanomaterials via polydopamine mediated coatings in PG NFMs for nerve tissue engineering. The NFMs structural analysis through SEM, FTIR, and EDAX determined that the nanofibrous membranes are porous and truly coated by DOPA, GO, and GQD. It was also demonstrated that PG-DOPA-GO and PG-DOPA-GQD NFMs are highly conductive, hemo-compatible, antibacterial, possessing good hydrophilicity. At the same time, they are displayed to be biodegradable with adjustable structural integrity and stiffness. The NFMs potential to induce the expression of neuron-like differentiation factors in the PC12 cells cultured on the scaffold was determined by introducing neurofilament-200 (NF200) and Nestin antibodies after 7 days' cell ceding. Simultaneously, qRT-PCR analysis confirmed that the NF-200 and Nestin genes, both among the important genes regulating neural differentiation, are highly expressed when the conductivity and biocompatibility of the scaffold are increased through GO coating. Overall, the PG-DOPA-GO NFM was determined to outperform the other scaffolds regarding increased proliferation, viability, and neuron-like differentiation-related factors in PC12 cells, both assessed by ICC and qRT-PCR, in vitro. [ABSTRACT FROM AUTHOR]

Objective: To explore the neuroprotective effects of the Shaoyao Gancao decoction (SGD) against excitatory damage in PC12 cells and the role of the Src-NR2-nNOS pathway mediation by SGD in regulating γ-aminobutyric acid (GABA)-glutamate (Glu) homeostasis. Methods: N-Methyl-d-aspartic acid (NMDA) was used to establish a PC12 cell excitability injury model. To investigate the neuroprotective effect of SGD, a cell counting kit-8 (CCK-8) assay was used to determine PC12 cell viability, Annexin V/Propidium Iodide (Annexin V/PI) double staining was used to determine PC12 cell apoptosis, and Ca2+ concentration was observed using laser confocal microscopy. GABA receptor agonists and antagonists were used to analyze the neuroprotective interactions between γ-aminobutyric acid (GABA) and NMDA receptors. Additionally, molecular biology techniques were used to determine mRNA and protein expression in the Src-NR2-nNOS pathway. We analyzed the correlations between the regulatory sites of GABA and NMDA interactions, excitatory neurotoxicity, and brain damage at the molecular level. Results: NMDA excitotoxic injury manifested as a significant decrease in cell activity, increased apoptosis and caspase-3 protein expression, and a significant increase in intracellular Ca2+ concentration. Administration of SGD, a GABAA receptor agonist (muscimol), or a GABAB receptor agonist (baclofen) decreased intracellular Ca2+ concentrations, attenuated apoptosis, and reversed NMDA-induced upregulation of caspase-3, Src, NMDAR2A, NMDAR2B, and nNOS. Unexpectedly, a GABAA receptor antagonist (bicuculline) and a GABAB receptor antagonist (saclofen) failed to significantly increase excitatory neurotoxicity. Conclusions: Taken together, these results not only provide an experimental basis for SGD administration in the clinical treatment of central nervous system injury diseases, but also suggest that the Src-NR2A-nNOS pathway may be a valuable target in excitotoxicity treatment.

The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment. NEW & NOTEWORTHY: This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation. [ABSTRACT FROM AUTHOR]

Nymphaea candida Presl (NC), traditionally used in medicine for heat syndrome-related ailments, possesses antioxidative, anti-inflammatory, hepatoprotective, and neuroprotective properties. This research investigates the antidepressant and neuroprotective effects and mechanisms of Nymphaea candida Presl ethyl acetate (NCEA). Primary components of NCEA were identified as phenolic acids and flavonoids through UPLC-MS/MS analysis. The depression mouse model was induced via intracerebroventricular injection of Lipopolysaccharide (LPS), followed by oral administration of fluoxetine and NCEA for one week. Biochemical assays and HE staining confirmed NCEA's non-toxicity and protective effects on the liver and lungs. NCEA administration mitigated LPS-induced depressive behaviors, decreased IL-1β, TNF-α levels in the hippocampus, suppressed microglial activation, reduced Iba-1 expression, and increased NA, brain-derived neurotrophic factor (BDNF), and dendritic spine density in the hippocampus. Furthermore, NCEA enhanced cell viability in a CORT-induced PC12 cell model, decreased lactate dehydrogenase (LDH) release rate, total superoxide dismutase (SOD) inhibition rate, intracellular nitric oxide (NO) release, and reduced reactive oxygen species (ROS) production. Our research findings suggest that NCEA exhibits significant antidepressant effects, which may be attributed to its reduction of neuroinflammation, improvement in neurotransmitter levels, neuronal protection, and antioxidative stress properties.

Background/purpose: Peripheral neural regeneration is an interesting and challenging field. The aim of this study was to investigate the interactions of neural-like PC12 cells and Poly-D-Lysine (PDL)-coated 3D-printed polycaprolactone (PCL) scaffolds with different inner diameters of half tubular array (HTA) (0, 200, 300, and 400 μm), respectively. Materials and methods: This study used the fused deposition modeling (FDM) technique with 3D-printing to fabricate the thermoplastic polymer. Scaffold properties were measured by mechanical testing, and coating quality was observed under a scanning electron microscope (SEM). PC12 cell biocompatibility was examined by an MTT assay. Cell differentiation was evaluated by immunofluorescence staining. Results: The cell viability of PC12 cells on PDL-coated PCL scaffolds with a 200-μm inner diameter of HTA was shown with significant differences (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001) than other PCL groups at all experimental dates. The SEM observation showed that PDL-coated PCL scaffolds with 200-μm inner diameters of HTA promoted cell adhesion. An immunofluorescence staining of PC12 cells on the PDL-coated PCL scaffold with a 200-μm inner diameter of the HTA group showed that it stimulated PC12 cells for neurite formation much better than the other groups.A PDL-coated PCL scaffold with a 200-μm inner diameter of HTA can promote the growth and differentiation of PC12 cells better than other groups. It indicated that PDL-coated PCL scaffolds with a 200-μm inner diameter HTA can be used for further neural regeneration application.

Rotenone is a pesticide used in research for its ability to induce changes similar, in vivo and in vitro, to those observed in Parkinson's disease (PD). This includes a selective death of dopaminergic neurons in the substantia nigra. Nonetheless, the precise mechanism through which rotenone modifies structure and function of neurons remains unclear. The PC12 cells closely resemble dopamine terminal neurons. This makes it a preferred model for studying the morphology of central dopamine neurons and predicting neurotoxicity. In this paper, we investigated the effects of 0.5 µM rotenone for 24–48 h on PC12 cell viability and ultrastructure (TEM), trying to identify primary and more evident alterations that can be related to neuronal damages similar to that seen in animal PD models. Cell viability decreased after 24 h rotenone treatment, with a further decrease after 48 h. Ultrastructural changes included vacuolar degeneration, mitochondrial mild swelling, decrease in the number of neuropeptide granules, and the loss of cell-to-cell adhesion. These findings are in agreement with previous research suggesting that rotenone, by inhibiting energy production and increasing ROS generation, is responsible for significant alterations of the ultrastructure and cell death of PC12 cells. Our data confirm the link between rotenone exposure, neuronal damage, and changes in dopamine metabolism, suggesting its role in the pathogenesis of PD. [ABSTRACT FROM AUTHOR]

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)‐dependent neuronal cell survival and differentiation. The small GTPase ADP‐ribosylation factor 6 (Arf6) is implicated in NGF‐dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6‐specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF‐dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N‐terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF‐dependent TrkA trafficking compared with wild‐type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF‐dependent TrkA trafficking and signaling. [ABSTRACT FROM AUTHOR]

Study Design Animal and cellular models of spinal cord injury (SCI) were used to explore the role of miR-335 in regulating cell viability and apoptosis. Objective. To investigate the role and the target of miR-335 in SCI. Summary of Background. Based on analysis of the GSE19890 data set, miR-335 was identified as a downregulated microRNA (miRNAs) following SCI. Thus, this study investigated whether downregulation of miR-335 is important in the pathological process of SCI. Materials and Methods. The GSE19890 data set investigating the expression profiles of miRNAs after SCI was downloaded from the GEO database. GSE45006 and GSE4550 data sets were used to identify differentially expressed genes between normal samples and SCI samples. The targets of rno-miR-335 were predicted using the TargetScan database. An experimental model of SCI was established, and agomir-miR-335 was intrathecally injected into rats with SCI. Functional recovery was evaluated by assessment of Basso-Beattie-Bresnahan scores and spinal cord water content and performing hematoxylin-eosin staining. Apoptosis was estimated by TUNEL staining. The H2O2-treated PC12 cells were used as in vitro models of SCI. Cell viability and apoptosis were examined by cell counting kit-8 and flow cytometry. Caspase-3 expression was evaluated by immunofluorescence staining. Levels of miRNAs and mRNAs were measured by reverse transcriptase quantitative polymerase chain reaction. Western blotting was performed to measure Bcl-2, Bax, cleaved caspase-3, and specificity protein 1 (SP1) protein levels. Results. For in vivo studies, miR-335 was downregulated following SCI, and agomir-miR-335 delivery improved functional recovery through suppressing neuronal apoptosis by inactivating the SP1-Bax/Bcl-2/caspase-3 signaling. During in vitro analysis, miR-335 protected PC12 cells against H2O2-induced damage by negatively regulating the SP1-Bax/Bcl-2/caspase-3 signaling axis. Moreover, upregulation of SP1 abolished the apoptosis suppressive effects of miR-335 upregulation. Conclusion. MiR-335 ameliorates locomotor impairment in rats with SCI through the suppression of neuronal apoptosis by inactivating SP1-Bax/Bcl-2/caspase-3 signaling. [ABSTRACT FROM AUTHOR]

Peripheral neural regeneration is an interesting and challenging field. The aim of this study was to investigate the interactions of neural-like PC12 cells and Poly-D-Lysine (PDL)-coated 3D-printed polycaprolactone (PCL) scaffolds with different inner diameters of half tubular array (HTA) (0, 200, 300, and 400 μm), respectively. This study used the fused deposition modeling (FDM) technique with 3D-printing to fabricate the thermoplastic polymer. Scaffold properties were measured by mechanical testing, and coating quality was observed under a scanning electron microscope (SEM). PC12 cell biocompatibility was examined by an MTT assay. Cell differentiation was evaluated by immunofluorescence staining. The cell viability of PC12 cells on PDL-coated PCL scaffolds with a 200-μm inner diameter of HTA was shown with significant differences (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001) than other PCL groups at all experimental dates. The SEM observation showed that PDL-coated PCL scaffolds with 200-μm inner diameters of HTA promoted cell adhesion. An immunofluorescence staining of PC12 cells on the PDL-coated PCL scaffold with a 200-μm inner diameter of the HTA group showed that it stimulated PC12 cells for neurite formation much better than the other groups. A PDL-coated PCL scaffold with a 200-μm inner diameter of HTA can promote the growth and differentiation of PC12 cells better than other groups. It indicated that PDL-coated PCL scaffolds with a 200-μm inner diameter HTA can be used for further neural regeneration application. [ABSTRACT FROM AUTHOR]

Background: Alzheimer’s disease (AD) develops as a result of oxidative damage to neurons and chronic inflammation of microglia. These processes can be influenced by the use of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs). The CM contains a wide range of factors that have neurotrophic, antioxidant, and anti-inflammatory effects. In addition, the therapeutic potential of the CM can be further enhanced by pretreating the MSCs to increase their paracrine activity. The current study aimed to investigate the neuroprotective effects of CM derived from MSCs, which were either activated by a TLR3 ligand or exposed to CoCl2, a hypoxia mimetic (pCM or hCM, respectively), in an in vitro model of AD. Methods: We have developed a novel in vitro model of AD that allows us to investigate the neuroprotective and anti-inflammatory effects of MSCs on induced neurodegeneration in the PC12 cell line and the activation of microglia using THP-1 cells. Results: This study demonstrates for the first time that pCM and hCM exhibit more pronounced immunosuppressive effects on proinflammatory M1 macrophages compared to CM derived from untreated MSCs (cCM). This may help prevent the development of neuroinflammation by balancing the M1 and M2 microglial phenotypes via the decreased secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased secretion of IL-4, as well as the expression of IL-10 and TGF-β by macrophages. Moreover, a previously unknown increase in the neurotrophic properties of hCM was discovered, which led to an increase in the viability of neuron-like PC12 cells under H2O2-induced oxidative-stress conditions. These results are likely associated with an increase in the production of growth factors, including vascular endothelial growth factor (VEGF). In addition, the neuroprotective effects of CM from preconditioned MSCs are also mediated by the activation of the Nrf2/ARE pathway in PC12 cells. Conclusions: TLR3 activation in MSCs leads to more potent immunosuppressive effects of the CM against pro-inflammatory M1 macrophages, while the use of hCM led to increased neurotrophic effects after H2O2-induced damage to neuronal cells. These results are of interest for the potential treatment of AD with CM from preactivated MSCs.

Objectives: Plant polysaccharides have attracted increasing attention due to their high efficiency and low toxicity. Codonopsis pilosula polysaccharide (CPP) is an essential substance extracted from Codonopsis pilosula, known for its excellent antioxidant and neuroprotective effects. However, it is still unclear how CPP improves nerve protection and what its underlying molecular mechanisms are. This study aimed to investigate the neuroprotective effect of CPP on Aβ25-35-induced damage in PC12 cells and its underlying molecular mechanisms. Methods: The neuroprotective effect of CPP was evaluated using Aβ25-35-induced damage in pheochFfromocytoma (PC12) cells as an in vitro cell model. The cells were treated with CPP alone or in combination with SB203580 (an inhibitor of p38MAPK) in Aβ25-35 culture. The cell viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,diphenyltetrazolium (MTT) assay. Furthermore, reactive oxygen species (ROS) were detected using flow cytometry. The production levels of intracellular superoxide dismutase (SOD), dismutase (SOD), glutathione (GSH), catalase (CAT), and malondialdehyFde (MDA) were determined using the colorimetric method. Annexin V-FITC and propidium iodide (PI) staining, as well as 33258 were performed using fluorescence microscopy. Moreover, the effect of adding SB203580 was studied to determine the changes in cell apoptosis induced by CPP treatment and Aβ25-35 induction. Results: The CPP markedly inhibited Aβ25-35-induced reduction in the viability and apoptosis of PC12 cells. CPP also reduced the Aβ25-35-induced increase in the expression of the apoptosis factors and the levels of free radicals (ROS and MDA) and reversed the Aβ25-35-induced suppression of antioxidant activity. Additionally, inhibition of p38MAPK via the addition of their antagonists reversed the observed anti-apoptosis effects of CPP. Conclusions: CPP can efficiently provide neuroprotection against Aβ25-35-induced damage in PC12 cells brought about via oxidation and apoptosis reactions, and the underlying mechanisms involve the p38MAPK pathways. Therefore, CPP could potentially be useful as a neuroprotective agent in natural medicine, pharmacy, and the food industry.

PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-β cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells. [ABSTRACT FROM AUTHOR]

Background: The goal of tissue engineering is to create biological solutions that restore, maintain, and improve the function of damaged tissue. Scaffolds are structures based on extracellular matrix materials that have undergone various treatments. Objectives: This study aimed to prepare polycaprolactone/silymarin and polycaprolactane/tragacanth scaffolds and compare the morphology and viability of PC12 cell lines. Methods: A 7% polycaprolactane solution (dissolved in acetic acid) was mixed with a 0.9% silymarin solution to prepare the polycaprolactane scaffold and silymarin loading, and a 7% polycaprolactane solution was mixed to prepare the polycaprolactane and tragacanth scaffold. Tragacanth solution was mixed at a concentration of 0.7% by weight, and then two scaffolds were prepared by electrospinning. Themorphology of the scaffoldswasstudied by scanning electron microscopy (SEM), and the chemical structure of the scaffolds was studied by ATR-FTIR spectroscopy. The biocompatibility of the scaffolds and cell survival of PC12 cells was investigated by MTT assay. Results: The morphology of the scaffolds and their chemical structure showed good porosity and successful loading of silymarin onto PCL and a suitable combination of tragacanth with PCL. The biocompatibility of the scaffolds was evaluated at 24, 48, and 72 hours after PC12 cell culture. Cell survival was found to increase on polycaprolactane/silymarin scaffolds compared to polycaprolactane/tragacanth. Conclusions: From the results of this study, loading polycaprolactane scaffold with silymarin increases the proliferation and survivability of PC12 cells compared to polycaprolactane/supporting scaffold, which may be a good candidate for neural tissue engineering. [ABSTRACT FROM AUTHOR]

Background and purpose: It is well established that valproic acid (VPA) is teratogenic associated with oxidative stress in humans and in all animal species tested. In this study, considering the chemical composition of catechins, we investigated its protective effect on the survival of PC12 nerve cells exposed to valproic acid. Materials and methods: The protective effects of epicatechin (EC) at different concentrations (10, 50, 100, 500, 1000 μg/ml) on survival and viability of PC12 neuronal cells exposed to valproic acid were measured by MTT assay and oxidative stress tests (GPx, SOD, ROS, and lipid peroxidation). All data were analyzed in GraphPad Prism 8.0 using one-way ANOVA and Tukey post-test. Results: According to findings, EC reduced the cytotoxic effects of valproic acid at 500 and 1000 µg/ml (P

Accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs) is indispensable for efficient cholinergic transmission. Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs. However, the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear. Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5 (SNX5) as its novel interacting protein. SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells. Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5. Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway. More importantly, we found that, in PC12 cells, the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles (SVLVs) localization of VAChT. Therefore, the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.

Rotenone is a pesticide used in research for its ability to induce changes similar, in vivo and in vitro, to those observed in Parkinson’s disease (PD). This includes a selective death of dopaminergic neurons in the substantia nigra. Nonetheless, the precise mechanism through which rotenone modifies structure and function of neurons remains unclear. The PC12 cells closely resemble dopamine terminal neurons. This makes it a preferred model for studying the morphology of central dopamine neurons and predicting neurotoxicity. In this paper, we investigated the effects of 0.5 µM rotenone for 24–48 h on PC12 cell viability and ultrastructure (TEM), trying to identify primary and more evident alterations that can be related to neuronal damages similar to that seen in animal PD models. Cell viability decreased after 24 h rotenone treatment, with a further decrease after 48 h. Ultrastructural changes included vacuolar degeneration, mitochondrial mild swelling, decrease in the number of neuropeptide granules, and the loss of cell-to-cell adhesion. These findings are in agreement with previous research suggesting that rotenone, by inhibiting energy production and increasing ROS generation, is responsible for significant alterations of the ultrastructure and cell death of PC12 cells. Our data confirm the link between rotenone exposure, neuronal damage, and changes in dopamine metabolism, suggesting its role in the pathogenesis of PD.

A variety of different signals induce specific responses through a common, extracellular-signal regulated kinase (ERK)-dependent cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubtion of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we have expanded the toolbox of Förster Resonance Energy Transfer (FRET)-based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytoplasm and nucleus. Furthermore, EGF-induced plasma membrane ERK activity involves Rap1, a noncanonical activator, and controls cell morphology and EGF-induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.

Nerve growth factor (NGF) plays a key role in controlling growth, maintenance and survival of neurons as well as nociceptor development. Many of its critical actions are believed to be mediated by activation of corresponding transcription factors. Here I attempt to reveal signaling cascades that relay NGF stimulation to NFAT, a family of Ca2+/calcineurin-dependent transcription factors that are implicated in playing a pivotal role in neuronal development and survival, and pain sensation. With a luciferase gene reporter assay, it was confirmed that NGF can trigger NFAT-dependent gene expression in PC12 cells. Subsequent research with pharmacological tools uncovers that this effect was mediated by the TrkA-MEK1/2 pathway. Furthermore, it was determined that NFATc3 is the predominant isoform by calculating an absolute copy number of each NFAT isoform with quantitative PCR. Taken together, it is proposed that elucidating cellular connections linking NGF and NFAT may help reveal novel therapeutic targets, which can be exploited to devise innovative remedies for the treatment of NGF and NFAT-associated clinical disorders such as anomalous neuronal development and hyperalgesia.

Brassica rapa L. (BR), a traditional biennial herb belonging to the Brassica species of Brassicaceae, has been widely used for functions of anti-inflammatory, antitumor, antioxidation, antiaging, and regulation of immunity. In this study, antioxidant activity and protective effects on H2O2-induced oxidative damage in PC12 cells of the active fractions of BR were investigated in vitro. Among all active fractions, the ethyl acetate fraction of ethanol extract from BR (BREE-Ea) showed the strongest antioxidant activity. Additionally, it was noted that BREE-Ea and n-butyl alcohol fraction of ethanol extract from BR (BREE-Ba) both have protective effects in oxidatively damaged PC12 cells, while BREE-Ea displayed the best protective effect in all determined experimental doses. Furthermore, flow cytometry (DCFH-DA staining) analysis indicated that BREE-Ea could reduce the H2O2-induced apoptosis in PC12 cells by reducing the production of intracellular reactive oxygen species (ROS) and increasing enzymatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Moreover, BREE-Ea could decrease the malondialdehyde (MDA) content and reduce the release of extracellular lactic dehydrogenase (LDH) from H2O2-induced PC12 cells. All these results demonstrate that BREE-Ea has a good antioxidant capacity and protective effect on PC12 cells against apoptosis induced by H2O2 and that it can be used as a good edible antioxidant to improve the body's endogenous antioxidant defense. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study is to establish a prognostic risk model based on ferroptosis to prognosticate the severity of Alzheimer's disease (AD) through gene expression changes. Methods: The GSE138260 dataset was initially downloaded from the Gene expression Omnibus database. The ssGSEA algorithm was used to evaluate the immune infiltration of 28 kinds of immune cells in 36 samples. The up-regulated immune cells were divided into Cluster 1 group and Cluster 2 group, and the differences were analyzed. The LASSO regression analysis was used to establish the optimal scoring model. Cell Counting Kit-8 and Real Time Quantitative PCR were used to verify the effect of different concentrations of Aβ1-42 on the expression profile of representative genes in vitro. Results: Based on the differential expression analysis, there were 14 up-regulated genes and 18 down-regulated genes between the control group and Cluster 1 group. Cluster 1 and Cluster 2 groups were differentially analyzed, and 50 upregulated genes and 101 down-regulated genes were obtained. Finally, nine common differential genes were selected to establish the optimal scoring model. In vitro, CCK-8 experiments showed that the survival rate of cells decreased significantly with the increase of Aβ1-42 concentration compared with the control group. Moreover, RT-qPCR showed that with the increase of Aβ1-42 concentration, the expression of POR decreased first and then increased; RUFY3 was firstly increased and then decreased. Discussion: The establishment of this research model can help clinicians make decisions on the severity of AD, thus providing better guidance for the clinical treatment of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Background and purpose: It is well established that valproic acid (VPA) is teratogenic associated with oxidative stress in humans and in all animal species tested. In this study, considering the chemical composition of catechins, we investigated its protective effect on the survival of PC12 nerve cells exposed to valproic acid. Materials and methods: The protective effects of epicatechin (EC) at different concentrations (10, 50, 100, 500, 1000 μg/ml) on survival and viability of PC12 neuronal cells exposed to valproic acid were measured by MTT assay and oxidative stress tests (GPx, SOD, ROS, and lipid peroxidation). All data were analyzed in GraphPad Prism 8.0 using one-way ANOVA and Tukey post-test. Results: According to findings, EC reduced the cytotoxic effects of valproic acid at 500 and 1000 µg/ml (P<0.0001). EC significantly reduced the oxidative stress induced by valproic acid via decreasing reactive oxygen species (ROS) and malondialdehyde (MDA) production (P<0.001 and P<0.0001, respectively). Conclusion: The present study showed that epicatechin could increase antioxidant potential and exhibits significant cytoprotective effect against valproic acid toxicity in PC12 cells. [ABSTRACT FROM AUTHOR]

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3β, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5–120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992. [ABSTRACT FROM AUTHOR]

Since tectorigenin has been reported to possess anti-inflammation, redox balance restoration, and antiapoptosis properties, we determine to unravel whether tectorigenin has potential in alleviating spinal cord injury (SCI). Herein, PC12 cells were induced by lipopolysaccharide (LPS) to establish in vitro SCI models. The cell viability and apoptosis were detected through cell counting kit-8 and flow cytometry assays. The caspase-3/8/9 content was measured by colorimetric method. Western blot was conducted to quantify the expressions of cleaved caspse3/8/9, IGFBP6, TLR4, IκBα, p-IκBα, RELA proto-oncogene, p65, and p-p65. Enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction were carried out to quantitate expressions of IGFBP6, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). SwissTargetPrediction and GSE21497 database were utilized to predict the potential therapeutic targets of tectorigenin. Comparison of IGFBP6 expression in SCI tissues and normal tissues was analyzed by GEO2R. Our study found that LPS induced the declined cell viability, elevated cell apoptosis, upregulation of caspase-3/8/9, cleaved caspase-3/8/9, IL-1β, IL-6, TNF-α, IGFBP6, and TLR4, and the activation of IκBα and p65 in PC12 cells. Tectorigenin reversed the above effects of LPS. IGFBP6 was predicted to be the potential therapeutic target of tectorigenin and was overexpressed in SCI tissues. Notably, IGFBP6 overexpression offset the effects of tectorigenin on PC12 cells. In conclusion, tectorigenin could alleviate the LPS-induced apoptosis, inflammation, and activation of NF-κB signaling in SCI cell models via inhibiting IGFBP6. [ABSTRACT FROM AUTHOR]

Background: Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4) suppresses hypoxia/re-oxygenation-induced injury in hepatocytes, and protects against hepatic and myocardial ischemia/reperfusion (I/R)-induced injury. The role of LGR4 on cerebral I/R injury was investigated in this study. Objective: Providing potential therapeutic target of cerebral I/R injury. Results: Expression of LGR4 was reduced in OGD/R-induced PC12. Over-expression of LGR4 attenuated OGD/R-induced decrease of cell viability and increase of apoptosis. Increased levels of LDH, ROS, MDA, and MPO, as well as decreased SOD, in OGD/R-induced PC12, were restored by LGR4 over-expression. Over-expression of LGR4 counteracted with the suppressive effects of OGD/R on protein expression of AKT and GSK3β phosphorylation in PC12. Conclusion: The protective effect of LGR4 against OGD/R-induced oxidative stress and apoptosis in PC12 was mediated by activation of AKT/GSK3β pathway. [ABSTRACT FROM AUTHOR]

Nymphaea candida Presl (NC), traditionally used in medicine for heat syndrome-related ailments, possesses antioxidative, anti-inflammatory, hepatoprotective, and neuroprotective properties. This research investigates the antidepressant and neuroprotective effects and mechanisms of Nymphaea candida Presl ethyl acetate (NCEA). Primary components of NCEA were identified as phenolic acids and flavonoids through UPLC-MS/MS analysis. The depression mouse model was induced via intracerebroventricular injection of Lipopolysaccharide (LPS), followed by oral administration of fluoxetine and NCEA for one week. Biochemical assays and HE staining confirmed NCEA's non-toxicity and protective effects on the liver and lungs. NCEA administration mitigated LPS-induced depressive behaviors, decreased IL-1β, TNF-α levels in the hippocampus, suppressed microglial activation, reduced Iba-1 expression, and increased NA, brain-derived neurotrophic factor (BDNF), and dendritic spine density in the hippocampus. Furthermore, NCEA enhanced cell viability in a CORT-induced PC12 cell model, decreased lactate dehydrogenase (LDH) release rate, total superoxide dismutase (SOD) inhibition rate, intracellular nitric oxide (NO) release, and reduced reactive oxygen species (ROS) production. Our research findings suggest that NCEA exhibits significant antidepressant effects, which may be attributed to its reduction of neuroinflammation, improvement in neurotransmitter levels, neuronal protection, and antioxidative stress properties. [Display omitted] • For the first time, UPLC-MS/MS was used to qualitatively analyze NCEA. • Selecting multiple intraventricular injections of LPS to induce depression model. • NCEA has low toxicity. • NCEA improves LPS-induced depressive behavior in mice. • Inhibited CORT-induced oxidative stress in PC12 cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Different bioactive effects of Thymus vulgaris and Allium cepa was investigated. • Thymus vulgaris and Allium cepa had protective, anti-apoptotic, and antioxidant effects. • Thymus vulgaris had high effects on the expression of genes involved in Alzheimer's. • Food additive with the studied plants proposed for protection against Alzheimer's. Thymus vulgaris and Allium cepa are plants with great medicinal importance. Thymol monoterpene and quercetin, which are present in these plants, have anti-Alzheimer's and antioxidant effects. The objectives of this research were investigating the effects of these compounds on the pathogenesis and progress of Alzheimer's disease in cells modeled by formaldehyde. MTT, flow cytometry, and RT-PCR were used to investigate the toxicity, survival rate and apoptosis of the cells, and the expression level of PP2A , GSK3 , NMDAR , BACE1 , and APP genes, respectively. Also, the total antioxidant capacity of the modeled cells was measured. The results showed that the two compounds as well as the plants extract and essential oil were able to increase the percentage of cell survival; among them, Thymus vulgaris essential oil had the greatest effect (93.55316 % in 48 h exposure). In addition, quercetin was able to reduce the rate of apoptosis in Alzheimer's cells (4.73 %) which was greater than the effects of other compounds. In general, the essential oil of Thymus vulgaris compared to thymol; and quercetin compared to Allium cepa extract showed more improving effects on the expression of genes involved in the disease. All four compounds increased the antioxidant capacity of the modeled cells compared to the control group, and these effects were almost equal between the compounds. According to the obtained results, both plants, especially Thymus vulgaris can be proposed as candidates to be included in the diet of Alzheimer's patients. In addition, polyphenols thymol and quercetin as derivates from the studied plants can be used in new drugs development for Alzheimer's disease, with greater safety than currently used drugs. These results are significant because most of the drug for Alzheimer's treatments such as cholinesterases (e.g. rivastigmine and donepezil) and memantine are chemically based and have many side effects. [ABSTRACT FROM AUTHOR]

Abstract Background Parkinson’s disease (PD) is a multifactorial movement disorder with the progressive degeneration of the nigrostriatal system that impairs patients’ movement ability. Oxidative stress has been found to affect the etiology and pathogenesis of PD. Thymol, a monoterpenic phenol, is one of the most important dietary constituents in thyme species. It has been used in traditional medicine and possesses some properties including antioxidant, free radical scavenging, anti-inflammatory. In this study, in vitro and in vivo experiments were performed with the thymol in order to investigate its potential neuroprotective effects in models of PD. Methods The present study aimed to evaluate the therapeutic potential of thymol in 6-hydroxydopamine (6-OHDA)-induced cellular and animal models of PD. Results Post-treatment with thymol in vitro was found to protect PC12 cells from toxicity induced by 6-OHDA administration in a dose-dependent manner by (1) increasing cell viability and (2) reduction in intracellular reactive oxygen species, intracellular lipid peroxidation, and annexin-positive cells. In vivo, post-treatment with thymol was protective against neurodegenerative phenotypes associated with systemic administration of 6-OHDA. Results indicated that thymol improved the locomotor activity, catalepsy, akinesia, bradykinesia, and motor coordination and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Increased level of reduced glutathione content and a decreased level of MDA (malondialdehyde) in striatum were observed in the 6-OHDA rats post-treated with thymol. Conclusions Collectively, our findings suggest that thymol exerts protective effects, possibly related to an anti-oxidation mechanism, in these in vitro and in vivo models of Parkinson’s disease.

Pheochromocytomas and paragangliomas (PPGLs) are classified into 3 major categories with distinct driver genes: pseudohypoxia, kinase signaling, and Wnt-altered subtypes. PPGLs in the Wnt-altered subtype are sporadic and tend to be aggressive with metastasis, where somatic gene fusions affecting mastermind-like 3 (MAML3) and somatic mutations in cold shock domain containing E1 (CSDE1) cause overactivation of Wnt-β-catenin signaling. However, the relation between Wnt-β-catenin signaling and the biological behavior of PPGLs remains unexplored. In rat pheochromocytoma PC12 cells, Wnt3a treatment enhanced cell proliferation and suppressed mRNA expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, and dopamine secretion. We identified the expression of sclerostin in PC12 cells, which is known as an osteocyte-derived negative regulator for Wnt signaling-driven bone formation. Inhibition of endogenous Wnt pathway by XAV939 or sclerostin resulted in attenuated cell proliferation and increased TH expression. Furthermore, Wnt3a pretreatment suppressed bone morphogenetic protein (BMP)-induced Smad1/5/9 phosphorylation whereas BMPs enhanced sclerostin expression in PC12 cells. In the Wnt-altered subtype, the increased Wnt-β-catenin pathway may contribute the aggressive clinical behavior with reduced catecholamine production. Furthermore, upregulated expression of sclerostin by BMPs may explain the osteolytic metastatic lesions observed in metastatic PPGLs. [ABSTRACT FROM AUTHOR]

Many patients suffer from peripheral nerve injury, which can impair their quality of life. Restoring nerve tissue is difficult due to the low ability of nerves to regenerate. Nerve conduits are designed to help peripheral nerve regeneration by providing a scaffold that can match the tissue characteristics, facilitate cellular activities, and be easily implanted. In order to provide a nerve conduit having scaffolding properties, conductance cytocompatibility, we have investigated the potential of channeled structures made of poly (glycerol-sebacate) (PGS) elastomer containing carbon nanofibers (CNFs) in the regeneration of nerve tissue. The first step was to synthesize PGS elastomer and tune its properties to match the nerve tissue. Then, a carbon dioxide laser was used to create micro channels on the elastomer surface for guiding nerve cells. The PGS elastomer was blended with carbon nanofiber (CNF), which was functionalized to bond with the elastomer, to form a conductive structure. The constructs were investigated in terms of cell behavior using PC12 and S42 cell lines. A statistically significant increase in cell proliferation was observed in both cell lines. It was found that the cells began to grow along the canal in places. In terms of elasticity, conductance and cell response, these constructs may be a potential candidate for nerve tissue engineering., (© 2024 Wiley Periodicals LLC.)

TrkB and TrkC are quite common neurotrophin receptors found on the same cells in CNS. In the C-terminal tail, TrkB and TrkC differ only in two amino acid residues at positions immediately preceding the tyrosine residue, which, upon phosphorylation, becomes the docking site for phospholipase Cγ1 (PLCγ1). The question arose whether such a difference near the PLCγ1 docking site might contribute to differential response to neurotrophin. PC12 clones with the following receptors were obtained: wild-type TrkC, TrkC-Y820F with a defective PLCγ1 binding site, TrkC-T817S-I819V with two amino acid residues replaced with those in the TrkB tail. The outgrowth of neurite-like processes from TrkC-Y820F-containing cells appeared to be impaired, while the TrkC-T817S-I819V variant appeared more effective than wild-type TrkC in promoting the outgrowth of neurite-like processes after neurotrophin stimulation, at least in the compared PC12 cell clones. Taken together, both the tyrosine residue at the PLCγ1 docking site and the amino acid residues immediately preceding it appear important for TrkC-supported outgrowth of neurite-like processes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Since tectorigenin has been reported to possess anti-inflammation, redox balance restoration, and anti-apoptosis properties, we determine to unravel whether tectorigenin has potential in alleviating spinal cord injury (SCI). Herein, PC12 cells were induced by lipopolysaccharide (LPS) to establish in vitro SCI models. The cell viability and apoptosis were detected through cell counting kit-8 and flow cytometry assays. The caspase-3/8/9 content was measured by colorimetric method. Western blot was conducted to quantify the expressions of cleaved caspse-3/8/9, IGFBP6, TLR4, IκBα, p-IκBα, RELA proto-oncogene, p65, and p-p65. Enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction were carried out to quantitate expressions of IGFBP6, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). SwissTargetPrediction and GSE21497 database were utilized to predict the potential therapeutic targets of tectorigenin. Comparison of IGFBP6 expression in SCI tissues and normal tissues was analyzed by GEO2R. Our study found that LPS induced the declined cell viability, elevated cell apoptosis, upregulation of caspase-3/8/9, cleaved caspase-3/8/9, IL-1β, IL-6, TNF-α, IGFBP6, and TLR4, and the activation of IκBα and p65 in PC12 cells. Tectorigenin reversed the above effects of LPS. IGFBP6 was predicted to be the potential therapeutic target of tectorigenin and was overexpressed in SCI tissues. Notably, IGFBP6 overexpression offset the effects of tectorigenin on PC12 cells. In conclusion, tectorigenin could alleviate the LPS-induced apoptosis, inflammation, and activation of NF-κB signaling in SCI cell models via inhibiting IGFBP6.

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Brassica rapa L. (BR), a traditional biennial herb belonging to the Brassica species of Brassicaceae, has been widely used for functions of anti-inflammatory, antitumor, antioxidation, antiaging, and regulation of immunity. In this study, antioxidant activity and protective effects on H2O2-induced oxidative damage in PC12 cells of the active fractions of BR were investigated in vitro. Among all active fractions, the ethyl acetate fraction of ethanol extract from BR (BREE-Ea) showed the strongest antioxidant activity. Additionally, it was noted that BREE-Ea and n-butyl alcohol fraction of ethanol extract from BR (BREE-Ba) both have protective effects in oxidatively damaged PC12 cells, while BREE-Ea displayed the best protective effect in all determined experimental doses. Furthermore, flow cytometry (DCFH-DA staining) analysis indicated that BREE-Ea could reduce the H2O2-induced apoptosis in PC12 cells by reducing the production of intracellular reactive oxygen species (ROS) and increasing enzymatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Moreover, BREE-Ea could decrease the malondialdehyde (MDA) content and reduce the release of extracellular lactic dehydrogenase (LDH) from H2O2-induced PC12 cells. All these results demonstrate that BREE-Ea has a good antioxidant capacity and protective effect on PC12 cells against apoptosis induced by H2O2 and that it can be used as a good edible antioxidant to improve the body’s endogenous antioxidant defense.

[Display omitted] • Natural products were extracted and isolated from Odontonema strictum. • They showed cell proliferation- and neurite outgrowth-promoting effects. • The results indicate they are useful in preventing neurodegenerative diseases. The leaves of Odontonema strictum , a tropical plant used for its antihypertensive properties, are rich in nutrients and biologically active phytochemicals, such as β-sitosterol, stigmasterol, umuravumbolide, deacetylumuravumbolide, dideacetylboronolide, deacetylboronolide, verbascoside, and isoverbascoside. In addition, its roots are rich in β-sitosterol, stigmasterol, and the iridoid glycoside β- O -methyl-unedoside. Ingestion of the roots was reported to have a sedative effect in a dog was previously reported on a dog eating the roots of this plant. In the present study, we report for the first time the cell proliferation- and neurite outgrowth-promoting effects in PC12 neuronal cells of the isolated organic compounds and crude extracts from O. strictum. Pituitary adenylate cyclase-activating peptide (PACAP) and quercetin were used as positive controls. At the concentration of 0.2 μg/mL, β-sitosterol was more potent than quercetin and displayed the same activity (>45 μm/cell) as PACAP (100 nM). At a low concentration (0.04 μg/mL), verbascoside and isoverbascoside showed the strongest neurite outgrowth-promoting effect (neurite length of 30 to 35 μm/cell). Our results indicate that phytomedicines made from O. strictum may be useful in preventing neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Oxidative stress has been proven to play a critical role in the pathogenesis of neuronal injury. As a novel adipocytokine, omentin is produced by visceral adipose with insulin sensitizing effects and has been revealed to possess anti-inflammatory effects. However, the possible effect of omentin on oxidative stress remains unknown. The present study aimed to detect the potential protective effect of omentin against hydrogen peroxide (H2O2)-induced cytotoxicity of PC12 cells. The results showed that no cytotoxic effect was shown in PC12 cells co-cultured with omentin alone at a concentration of 50–1000 ng/mL. The CCK8 and TUNEL assays suggested that omentin could remarkably attenuate apoptosis induced by 100 μM H2O2. The PCR and western blotting showed that the expression levels of Bax was significantly inhibited by omentin via the upregulation of miR-128-3p at its 3′-UTR. Taken together, these results indicated that omentin protects PC12 cells against H2O2-induced apoptosis, and further studies need to be conducted before utilization in the clinic for the treatment of neurodegenerative diseases.

Background: Parkinson's disease (PD) is a multifactorial movement disorder with the progressive degeneration of the nigrostriatal system that impairs patients' movement ability. Oxidative stress has been found to affect the etiology and pathogenesis of PD. Thymol, a monoterpenic phenol, is one of the most important dietary constituents in thyme species. It has been used in traditional medicine and possesses some properties including antioxidant, free radical scavenging, anti-inflammatory. In this study, in vitro and in vivo experiments were performed with the thymol in order to investigate its potential neuroprotective effects in models of PD. Methods: The present study aimed to evaluate the therapeutic potential of thymol in 6-hydroxydopamine (6-OHDA)-induced cellular and animal models of PD. Results: Post-treatment with thymol in vitro was found to protect PC12 cells from toxicity induced by 6-OHDA administration in a dose-dependent manner by (1) increasing cell viability and (2) reduction in intracellular reactive oxygen species, intracellular lipid peroxidation, and annexin-positive cells. In vivo, post-treatment with thymol was protective against neurodegenerative phenotypes associated with systemic administration of 6-OHDA. Results indicated that thymol improved the locomotor activity, catalepsy, akinesia, bradykinesia, and motor coordination and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Increased level of reduced glutathione content and a decreased level of MDA (malondialdehyde) in striatum were observed in the 6-OHDA rats post-treated with thymol. Conclusions: Collectively, our findings suggest that thymol exerts protective effects, possibly related to an anti-oxidation mechanism, in these in vitro and in vivo models of Parkinson's disease. [ABSTRACT FROM AUTHOR]

Objective: To explore the mechanism of dexmedetomidine (DEX)-mediated miR-134 inhibition in hypoxia-induced damage in PC12 cells. Methods: Hydrogen peroxide (H2O2)-stimulated PC12 cells were divided into control, H2O2, DEX + H2O2, miR-NC/inhibitor + H2O2, and miR-NC/ mimic + DEX + H2O2 groups. Cell viability and apoptosis were assessed by the 3-(4,5-dimethylthiazol(-2-y1)-2,5-diphenytetrazolium bromide (MTT) assay and Annexin V-FITC/PI staining, while gene and protein expression levels were detected by qRT-PCR and western blotting. Reactive oxygen species (ROS) levels were tested by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and malondialdehyde (MDA) content was determined with a detection kit. Results: DEX treatment decreased H2O2-elevated miR-134 expression. H2O2-induced PC12 cell damage was improved by DEX and miR-134 inhibitor; additionally, cell viability was increased, while cell apoptosis was reduced. In addition, both DEX and miR-134 inhibitor reduced the upregulated expression of cleaved caspase-3 and increased the downregulated expression of Bcl-2 in H2O2-induced PC12 cells. However, compared to that in the DEX + H2O2 group, cell viability in the mimic + DEX + H2O2 group was decreased, and the apoptotic rate was elevated with increased cleaved caspase-3 and decreased Bcl-2 expression. Inflammation and oxidative stress were increased in H2O2-induced PC12 cells but improved with DEX or miR-134 inhibitor treatment. However, this improvement of H2O2-induced inflammation and oxidative stress induced by DEX in PC12 cells could be reversed by the miR-134 mimic. Conclusion: DEX exerts protective effects to promote viability and reduce cell apoptosis, inflammation, and oxidative stress in H2O2-induced PC12 cells by inhibiting the expression of miR-134. [ABSTRACT FROM AUTHOR]

Objective(s): Rutin is a flavonoid with potent antioxidant property, which exhibited cytoprotective effects in several models of neuronal injury. This work aimed to examine whether rutin can protect neurons against oxidative DNA damage caused by serum/glucose deprivation (SGD) as an in vitro model of neurodegeneration and ischemia. Materials and Methods: The PC12 cells were cultured for 2 hr in normal culture medium containing different concentrations of rutin or α-tocopherol (positive control) and then further incubated for 12 hr in SGD condition. Then, cell viability, DNA fragmentation, lipid peroxidation, generation of reactive oxygen species (ROS), and the expression of proteins involved in apoptosis were determined. Results: The SGD condition significantly decreased viability of the cells, which was accompanied by a significant rise in the generation of ROS and lipid peroxidation. Rutin enhanced the viability of PC12 cells in SGD condition and reduced the production of ROS and lipid peroxidation. In addition, rutin decreased DNA damage and inhibited apoptotic cell death by decreasing the levels of proapoptotic proteins (Bax, caspase-3, caspase-9) and increasing the level of anti-apoptotic protein Bcl-2.Conclusion: This study demonstrated that rutin inhibits oxidative DNA damage and neuronal death induced by nutrients deprivation condition. Further studies may warrant the use of rutin as an appropriate neuroprotective agent for ischemic attacks and other neurodegenerative disorders.

Abstract Background The embryos of Nelumbo nucifera Gaertn seeds, lianzixin, are used in China as food and traditional herbal medicine. Principal therapeutic indications are insomnia, anxiety and pyrexia. Caffeine is a psychostimulant and excessive use predisposes to cell damage and neurotoxicity. We aimed to investigate the potential protect effect of Neferine and lianzixin extracts on undifferentiated caffeine-damaged phaeochromocytoma cells (PC12 cells). Methods A cell damage model based on undifferentiated PC12 was established with caffeine. Effect of Lianzixin extracts (total alkaloids, alcohol extract and water extract) and neferine on caffeine-damaged PC12 cells was evaluated. Cell viability was assessed using the methyl thiazolyl tetrazolium (MTT) assay, cellular morphology by inverted microscope, the nucleus by Hoechst 33342 staining and cleaved poly ADP-ribose polymerase (PARP) expression by western blot analysis. Results Lianzixin extracts (total alkaloids, alcohol extract and water extract) and neferine improved the viability of PC12 cells damaged by caffeine. The morphology of PC12 cells pretreated with neferine, or alcohol or water extract of lianzixin aggregated and attached better than caffeine-damaged cells, but cells pretreated with total alkaloids of lianzixin showed abnormal morphology. Compared with caffeine-damaged cells, cells pretreated with neferine, or alcohol or water extract of lianzixin showed a notable increase in nucleus staining and an obvious decrease in cleaved PARP expression. Conclusions Lianzixin extracts and neferine have protective effects against caffeine-induced damage in PC12 cells, which laid a foundation for finding a new medicine value of Lianzixin.

Recently, scientists have used the metallic nanoparticles especially copper nanoparticles for formulating many new neuroprotective supplements in the field of neurology. Also, we know the role of Nigella sativa L. in increasing the physiological activities of the central nervous system in traditional medicine. In the present study, we decided to prepare and formulate a new neuroprotective supplement (copper nanoparticles in aqueous medium using N. sativa seed aqueous extract) in the in vitro condition. The organometallic chemistry tests such as Fourier Transformed Infrared Spectroscopy (FT‐IR), UV–Visible Spectroscopy (UV-Vis), Field Emission Scanning Electron Microscopy (FE‐SEM), and Transmission Electron Microscopy (TEM) were used for characterizing of copper nanoparticles. In the FT-IR test, the presence of many antioxidant compounds with related bonds caused excellent condition for reducing copper in the copper nanoparticles. In UV-Vis, the clear peak in the wavelength of 569 nm showed the copper nanoparticles formation. Also, in the TEM and FE-SEM images, the copper nanoparticles had the size of 19.5 nm. In the biological part of the current study, methadone significantly (p ≤ 0.01) decreased cell viability, mitochondrial membrane potential, and increased inflammatory cytokines concentrations, caspase-3 activity, and DNA fragmentation. CuNPs-treated cell cutlers significantly (p ≤ 0.01) increased cell viability and mitochondrial membrane potential, and decreased inflammatory cytokines concentrations, caspase-3 activity, and DNA fragmentation in the high concentration of methadone-treated adrenal phaeochromocytoma (PC12) cells. For investigating the antioxidant properties of copper nanoparticles, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used in the presence of butylated hydroxytoluene as the positive control. The copper nanoparticles inhibited half of the DPPH molecules in the concentration of 171 µg/mL. In this study, we concluded that copper nanoparticles biosynthesized by N. sativa L. seed suppressed methadone-induced cell death in PC12 cells.

Transient receptor potential melastatin 2 (TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mechanism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma (PC12) cells injured by oxygen-glucose deprivation (OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reoxygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2 (CXCL2), NACHT, LRR, and PYD domain–containing protein 3 (NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.

Objective: Sleep disorders are among the most common psychiatric and medical conditions. In the present study, the hypnotic effect of Tanacetum parthenium was studied in mice. Material and Methods: The hydro-alcoholic extract (HAE) of T. parthenium and three fractions of it, namely water fraction (WF), ethyl acetate fraction (EAF), and n-hexane fraction (NHF), were intraperitoneally (ip) administrated to mice 30 min before injection of sodium pentobarbital (30 mg/kg, ip). Then, 30 min after administration of HAE, motor coordination (rota-rod test) was evaluated. Besides, LD50 of HAE was determined and the cytotoxicity of HAE was evaluated in PC12 cells using the MTT assay. Results: HAE 50-200 mg/kg increased the sleeping time. EAF was the only fraction which could prolong the sleep duration and decrease sleep latency. The LD50 value was 4.8 g/kg. The extract induced no cytotoxic effects in PC12 cell line. Conclusion: The results suggested that T. parthenium potentiates pentobarbital hypnosis without causing toxic effects. Probably, its effects are mediated by the components present in EAF of this plant.

(1) Background: Nanotechnology is an emerging field that can be applied in the biomedical area. In this study, Eudragit nanocapsules (NCs) containing nicotine were produced. Nicotine is the main alkaloid found in tobacco and has anti-inflammatory properties. NCs containing nicotine may be used as an adjuvant therapy in the treatment of inflammation in the central nervous system. (2) Methods: Nanocapsules were prepared by the interfacial deposition of the pre-formed polymer method and characterized in terms of zeta potential, diameter, polydispersity index, pH, encapsulation efficiency (EE), stability and sustained release profile. In vitro tests with the PC12 cell line were performed, such as MTT, LIVE/DEAD and ELISA assays, to verify their cytotoxic and anti-inflammatory effects. (3) Results: The nanocapsules presented satisfactory values of the characterization parameters; however, poor encapsulation was obtained for nicotine (8.17% ± 0.47). The in vitro tests showed that the treatment with nanocapsules reduced cell viability, which suggests that the Eudragit or the amount of polymer on top of the cells may be detrimental to them, as the cells were able to survive when treated with bulk nicotine. ELISA showed an increment in the expression of IL-6 and IL-β, corroborating the hypothesis that NCs were toxic to the cells because of the increase in the levels of these pro-inflammatory cytokines. (4) Conclusions: This study demonstrates that NCs of Eudragit present toxicity. It is therefore necessary to improve NC formulation to obtain better values for the encapsulation efficiency and reduce toxicity of these nanodevices. [ABSTRACT FROM AUTHOR]

Accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs) is indispensable for efficient cholinergic transmission. Previous studies have suggested that the dileucine motif within the Cterminus of the transporter is sufficient for its targeting to SVs. However, the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear. Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5 (SNX5) as its novel interacting protein. SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells. Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5. Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway. More importantly, we found that, in PC12 cells, the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles (SVLVs) localization of VAChT. Therefore, the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) stimulates numerous cellular physiological processes, including growth, differentiation, and survival, and maintains the phenotype of several neuronal types. Most of these NGF-induced processes require adaptation of the secretory pathway since they involve extensive remodeling of membranes and protein redistribution along newly formed neuritic processes. CREB3 transcription factors have emerged as signaling hubs for the regulation of numerous genes involved in the secretory pathway and Golgi homeostasis, integrating stimuli from multiple sources to control secretion, posttranslational modifications and trafficking of proteins. Although recent studies have focused on their role in the central nervous system, little is known about their participation in cell differentiation. Therefore, we aimed to analyze the expression and signaling mechanism of CREB3 transcription factor family members, using the NGF-induced PC12 cell differentiation model. Results show that NGF treatment causes Golgi enlargement and a parallel increased expression of proteins and mRNAs encoding for proteins required for membrane transport (transport factors). Additionally, a significant increase in CREB3L2 protein and mRNA levels is detected in response to NGF. Both MAPK and cAMP signaling pathways are required for this response. Interestingly, CREB3L2 overexpression hampers the NGF-induced neurite outgrowth while its inhibition enhances the morphological changes driven by NGF. In agreement, CREB3L2 overexpressing cells display higher immunofluorescence intensity of Rab5 GTPase (a negative regulator of PC12 differentiation) than control cells. Also, Rab5 immunofluorescence levels decrease in CREB3L2-depleted cells. Taken together, our findings imply that CREB3L2 is an important downstream effector of NGF-activated pathways, leading to neuronal differentiation.