Study on the Neuroprotective Effects of Eight Iridoid Components Using Cell Metabolomics.

Iridoid components have been reported to have significant neuroprotective effects. However, it is not yet clear whether the efficacy and mechanisms of iridoid components with similar structures are also similar. This study aimed to compare the neuroprotective effects and mechanisms of eight iridoid components (catalpol (CAT), genipin (GE), geniposide (GEN), geniposidic acid (GPA), aucubin (AU), ajugol (AJU), rehmannioside C (RC), and rehmannioside D (RD)) based on corticosterone (CORT)-induced injury in PC12 cells. PC12 cells were randomly divided into a normal control group (NC), model group (M), positive drug group (FLX), and eight iridoid administration groups. Firstly, PC12 cells were induced with CORT to simulate neuronal injury. Then, the MTT method and flow cytometry were applied to evaluate the protective effects of eight iridoid components on PC12 cell damage. Thirdly, a cell metabolomics study based on ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was performed to explore changes in relevant biomarkers and metabolic pathways following the intervention of administration. The MTT assay and flow cytometry analysis showed that the eight iridoid components can improve cell viability, inhibit cell apoptosis, reduce intracellular ROS levels, and elevate MMP levels. In the PCA score plots, the sample points of the treatment groups showed a trend towards approaching the NC group. Among them, AU, AJU, and RC had a weaker effect. There were 38 metabolites (19 metabolites each in positive and negative ion modes, respectively) identified as potential biomarkers during the experiment, among which 23 metabolites were common biomarkers of the eight iridoid groups. Pathway enrichment analysis revealed that the eight iridoid components regulated the metabolism mainly in relation to D-glutamine and D-glutamate metabolism, arginine biosynthesis, the TCA cycle, purine metabolism, and glutathione metabolism. In conclusion, the eight iridoid components could reverse an imbalanced metabolic state by regulating amino acid neurotransmitters, interfering with amino acid metabolism and energy metabolism, and harmonizing the level of oxidized substances to exhibit neuroprotective effects.

Design, Synthesis, and Biological Evaluation of Novel Cinnamide Derivatives as Neuroprotective Agents for the Treatment of Cerebral Ischemia.

BACKGROUND: Ischemic stroke, the most common type of cerebrovascular accident, is a major cause of severe disability among adults worldwide. Although there has been progress in interventions for ischemic stroke in the past decades, there is no effective treatment to prevent brain damage in acute ischemic stroke. Therefore, it is urgent to develop novel neuroprotective agents with a wide therapeutic time window to provide a better prognosis for ischemic stroke patients. OBJECTIVE: The current study aimed to synthesize novel derivatives with substituent cinnamide scaffolds, evaluate biological activity, and obtain neuroprotective agents. METHODS: The target compounds were synthesized using classical methods of medicinal chemistry. The neuroprotective effects in vitro against Glu-induced neurotoxicity injury were evaluated in PC12 cells by MTT assay. The cell apoptosis was analyzed by flow cytometer. The proteins were detected by western blotting. The neuroprotective activities in vivo were determined in two in vivo models of global and focal cerebral ischemia. RESULTS: Among the title compounds, 9t, 9u, 9y, and 9z exhibited good neuroprotection in vivo and in vitro, which were selected and further studied to determine their mechanism of action. 9t, 9u, 9y and 9z protected PC12 cells against glutamate-induced apoptosis in a dose-dependent manner via caspase-3 pathway. Moreover, the four compounds significantly reduced brain infarct area and exhibited excellent neuroprotective activities in the in vivo MCAO model. CONCLUSION: Compounds 9t, 9u, 9y, and 9z, as potent neuroprotective agents with anti- neurotoxicity activity in vitro and anticerebral infarction efficacy in vivo, might serve as a useful molecular tool for further physiology and pathophysiology function studies, leading to potential clinical therapeutic agents for ischemic injury.

In vitro drug screening models derived from different PC12 cell lines for exploring Parkinson's disease based on electrochemical signals of catecholamine neurotransmitters.

Gold nanostructures and a Nafion modified screen-printed carbon electrode (Nafion/AuNS/SPCE) were developed to assess the cell viability of Parkinson's disease (PD) cell models. The electrochemical measurement of cell viability was reflected by catecholamine neurotransmitter (represented by dopamine) secretion capacity, followed by a traditional tetrazolium-based colorimetric assay for confirmation. Due to the  capacity to synthesize, store, and release catecholamines as well as their unlimited homogeneous proliferation, and ease of manipulation, pheochromocytoma (PC12) cells were used for PD cell modeling. Commercial low-differentiated and highly-differentiated PC12 cells, and home-made nerve growth factor (NGF) induced low-differentiated PC12 cells (NGF-differentiated PC12 cells) were included in the modeling. This approach achieved sensitive and rapid determination of cellular modeling and intervention states. Notably, among the three cell lines, NGF-differentiated PC12 cells displayed the enhanced neurotransmitter secretion level accompanied with attenuated growth rate, incremental dendrites in number and length that were highly resemble with neurons. Therefore, it was selected as the PD-tailorable modeling cell line. In short, the electrochemical sensor can be used to sensitively determine the biological function of neuron-like PC12 cells with negligible destruction and to explore the protective and regenerative impact of various substances on nerve cell model.

Effects of latroeggtoxin-VI on dopamine and α-synuclein in PC12 cells and the implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by death of dopaminergic neurons leading to dopamine deficiency, excessive α-synuclein facilitating Lewy body formation, etc. Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin discovered from the eggs of spider L. tredecimguttatus, was previously found to promote the synthesis and release of PC12 cells, showing a great potential as a drug candidate for PD. However, the relevant mechanisms have not been understood completely. The present study explored the mechanism underlying the effects of LETX-VI on dopamine and α-synuclein of PC12 cells and the implications for PD. RESULTS: After PC12 cells were treated with LETX-VI, the level of dopamine was significantly increased in a dose-dependent way within a certain range of concentrations. Further mechanism analysis showed that LETX-VI upregulated the expression of tyrosine hydroxylase (TH) and L-dopa decarboxylase to enhance the biosynthesis of dopamine, and downregulated that of monoamine oxidase B to reduce the degradation of dopamine. At the same time, LETX-VI promoted the transport and release of dopamine through modulating the abundance and/or posttranslational modification of vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT). While the level of dopamine was increased by LETX-VI treatment, α-synuclein content was reduced by the spider toxin. α-Synuclein overexpression significantly decreased the dopamine level and LETX-VI efficiently alleviated the inhibitory action of excessive α-synuclein on dopamine. In the MPTP-induced mouse model of PD, application of LETX-VI ameliorated parkinsonian behaviors of the mice, and reduced the magnitude of MPTP-induced α-synuclein upregulation and TH downregulation. In addition, LETX-VI displayed neuroprotective effects by inhibiting MPTP-induced decrease in the numbers of TH-positive and Nissl-stained neurons in mouse brain tissues. CONCLUSIONS: All the results demonstrate that LETX-VI promotes the synthesis and release of dopamine in PC12 cells via multiple mechanisms including preventing abnormal α-synuclein accumulation, showing implications in the prevention and treatment of PD.

Extraction, rapid preparation and neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir.

Searching for new anti-ischemic stroke (anti-IS) drugs has always been a hot topic in the pharmaceutical industry. Natural products are an important source of discovering anti-IS drugs. The aim of the present study is to extract, rapidly prepare and explore the neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir., which is a kind of Tibetan medicine with a clear anti-IS effect. The results showed that 95% ethanol was the optimal extraction solvent. A three-step rapid preparation method for texasin was successfully established, with a purity of 99.2%. Texasin at the concentration of 25-100 µM had no effect on the viability of normal cultured PC12 cells; 12.5 and 25 µM texasin could enhance the viability of PC12 cells damaged by oxygen and glucose deprivation/reoxygenation (OGD/R), and their effects are comparable to the positive drug edaravone at the concentration of 50 µM. Compared with the normal group, the expression of Bcl-2 protein in OGD/R-injured PC12 cells was downregulated (p < 0.01), and that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins were upregulated (p < 0.01, p < 0.001). Compared with the OGD/R group, 25 µM texasin could upregulate the expression of Bcl-2 protein (p < 0.01), and downregulate that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins (p < 0.01, p < 0.001). The 7-OH and 1-O of texasin formed H-bonds with residues Cys891 of the hinge ß-strand of PERK, which is crucial for kinase inhibitors. The above results suggest that the method established in the present study achieved rapid preparation of high-purity texasin. Texasin might inhibit neuronal apoptosis via the regulation of endoplasmic reticulum stress PERK/eIF2α/ATF4/CHOP signalling pathway to exert a protective effect on OGD/R-injured PC12 cells. Aiding by molecular docking, texasin was assumed to be a potential PERK inhibitor.

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

An ultra-sensitive platinized nanocavity electrode for analysis of cytosolic catecholamines in one living cell.

The catecholamines, mainly dopamine (DA), are present in the cellular cytosol with low abundance, while, play key roles in various neurodegenerative disorders. Here, platinized nanocavity carbon electrodes are employed to analyze cytosolic catecholamines in a single living PC12 cell, which is not easily quantified using the classic electrodes. The confined structure and excellent conductivity in the platinized nanocavity accelerate the electron transfer of the DA, resulting in a low detection limit down to 50 nM. The sensitivity of DA detection is improved to be 10.73 pA mM-1 nm-1 in the response range of 50 nM-100 µM, which guarantees quantitative analysis of cytosolic catecholamines with low abundance. Eventually, the platinized nanocavity electrode is employed to detect cytosolic catecholamines in a single PC12 cell without an obvious interruption of cellular catecholamine level. The cytosolic catecholamines in a single PC12 cell is measured in situ to be 0.1 µM, which is achieved for the first time at the single cell level using the electrochemical method. The results demonstrate that the nanocavity electrode with a high sensitivity could offer a promising means to dynamically track catecholamines in a single cell.

Effects of tricholoma matsutake polysaccharides on 1-methy-4-pehnyl-pyridine ion-induced PC12 cell damage / 解剖学报

Obｊective To investigate the protective mechanism of tricholoma matsutake polysaccharides(TMP) against 1-methy-4-pehnyl-pyridine ion (MPP

Enhancing the Study of Quantal Exocytotic Events: Combining Diamond Multi-Electrode Arrays with Amperometric PEak Analysis (APE) an Automated Analysis Code.

MicroGraphited-Diamond-Multi Electrode Arrays (µG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As µG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (µG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.

Ultrasonic neural regulation over two-dimensional graphene analog biomaterials: Enhanced PC12 cell differentiation under diverse ultrasond excitation.

Two-dimensional (2D) biomaterials, with unique planar topology and quantum effect, have been widely recognized as a versatile nanoplatform for bioimaging, drug delivery and tissue engineering. However, during the complex application of nerve repair, in which inflammatory microenvironment control is imperative, the gentle manipulation and trigger of 2D biomaterials with inclusion and diversity is still challenging. Herein, inspired by the emerging clinical progress of ultrasound neuromodulation, we systematically studied ultrasound-excited 2D graphene analogues (graphene, graphene oxide, reduced graphene oxide (rGO) and carbon nitride) to explore their feasibility, accessibility, and adjustability for ultrasound-induced nerve repair in vitro. Quantitative observation of cell differentiation morphology demonstrates that PC12 cells added with rGO show the best compatibility and differentiation performance under the general ultrasound mode (0.5 w/cm2, 2 min/day) compared with graphene, graphene oxide and carbon nitride. Furthermore, the general condition can be improved by using a higher intensity of 0.7 w/cm2, but it cannot go up further. Later, ultrasonic frequency and duty cycle conditions were investigated to demonstrate the unique and remarkable inclusion and diversity of ultrasound over conventional electrical and surgical means. The pulse waveform with power of 1 MHz and duty cycle of 50 % may be even better, while the 3 MHz and 100 % duty cycle may not work. Overall, various graphene analog materials can be regarded as biosafe and accessible in both fundamental research and clinical ultrasound therapy, even for radiologists without material backgrounds. The enormous potential of diverse and personalized 2D biomaterials-based therapies can be expected to provide a new mode of ultrasound neuromodulation.

Lactic Acid Bacteria-Derived Exopolysaccharides Mitigate the Oxidative Response via the NRF2-KEAP1 Pathway in PC12 Cells.

Parabiotics, including L-EPSs, have been administered to patients with neurodegenerative disorders. However, the antioxidant properties of L-EPSs against H2O2-induced oxidative stress in PC12 cells have not been studied. Herein, we aimed to investigate the antioxidant properties of the L-EPSs, their plausible targets, and their mechanism of action. We first determined the amount of L-EPSs in Lactobacillus delbrueckii ssp. bulgaricus B3 and Lactiplantibacillus plantarum GD2 using spectrophotometry. Afterwards, we studied their effects on TDH, TOS/TAS, antioxidant enzyme activities, and intracellular ROS level. Finally, we used qRT-PCR and ELISA to determine the effects of L-EPSs on the NRF2-KEAP1 pathway. According to our results, the L-EPS groups exhibited significantly higher total thiol activity, native thiol activity, disulfide activity, TAS levels, antioxidant enzyme levels, and gene expression levels (GCLC, HO-1, NRF2, and NQO1) than did the H2O2 group. Additionally, the L-EPS groups caused significant reductions in TOS levels and KEAP1 gene expression levels compared with those in the H2O2 group. Our results indicate that H2O2-induced oxidative stress was modified by L-EPSs. Thus, we revealed that L-EPSs, which regulate H2O2-induced oxidative stress, could have applications in the field of neurochemistry.

Analysis of differentially expressed genes discovers Latroeggtoxin VI-induced changes and SYNJ1 as a main target in PC12 cells.

BACKGROUND: Previous preliminary work found that Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin from the eggs of spider Latrodectus tredecimguttatus, could promote the synthesis and release of dopamine in PC12 cells. However, the underlying mechanisms have not been fully clear. Here, the effects of LETX-VI on the gene expression profile and dopamine in PC12 cells were analyzed with the differential transcriptome-based strategies. RESULTS: After treatment of PC12 cells with LETX-VI for 24 h, a total of 356 differentially expressed transcripts were identified. Of them 165 were up-regulated and 191 down-regulated. Relevant GO analysis indicated that LETX-VI modulated the expression of certain genes and thereby affected multiple biological processes in PC12 cells, including protein metabolism, nucleic acid metabolism, substance transport, signaling, neurotransmitter metabolism and release. When western blot analysis was employed to confirm the abundance levels of synaptojanin 1 and synuclein alpha interacting protein, the representatives of highly up- and down-regulated transcript-encoded proteins that are closely related with dopamine respectively, it was found that the level of synaptojanin 1 in the PC12 cells treated with LETX-VI was increased, whereas that of synuclein alpha interacting protein was not obviously altered, suggesting that synaptojanin 1 may be much more involved in the effects of LETX-VI on dopamine. After synaptojanin 1 level was knocked down using siRNA, the levels of both total and released dopamine were significantly decreased, indicating that synaptojanin 1 is a protein positively modulating the synthesis and secretion of dopamine. When the PC12 cells with knocked down synaptojanin 1 were treated by LETX-VI, the adverse effects of synaptojanin 1 knockdown on dopamine were attenuated, confirming that LETX-VI promotes the synthesis and secretion of dopamine at least partially by enhancing the expression of the gene SYNJ1 encoding synaptojanin 1. CONCLUSIONS: This work demonstrates that LETX-VI exerts multiple regulatory effects on the cellular processes in PC12 cells by altering the gene expression profile. LETX-VI modulates the expression of the genes closely related to the synthesis, transport and release of neurotransmitters especially dopamine in PC12 cells, with the gene SYNJ1 encoding synaptojanin 1 as a main target.

Insights into the mediation of Ca2+ signaling in the promoting effects of LETX-VI on the synthesis and release of dopamine.

Latroeggtoxin-VI (LETX-VI) is an active protein and was previously demonstrated to have effects on the synthesis and release of dopamine. Hererin, the involvement of Ca2+ signaling in the effects of LETX-VI on dopamine was systematically investigated, using PC12 cells as a neuron model. LETX-VI was shown to promote dopamine release from PC12 cells both in the presence and absence of extracellular Ca2+; however the presence of extracellular Ca2+ was favorable for enhancing the promoting effects of LETX-VI on dopamine, because LETX-VI facilitated the influx of extracellular Ca2+ through the L-type calcium channels in plasma membrane (PM) to increase cytosolic Ca2+ concentration. LETX-VI was able to penetrate the PM of PC12 cells to act on the Ca2+ channel proteins IP3Rs and RyRs in the endoplasm reticulum (ER) membrane, opening the Ca2+ channels and promoting the release of ER Ca2+ to elevate cytosolic Ca2+ level. With the help of intracellular Ca2+ chelator BAPTA, the elevated cytosolic Ca2+ level was proven to play crucial role for the enhanced promoting effects of LETX-VI on dopamine. Taken together, LETX-VI is able to open the Ca2+ channels in both PM and ER membrane simultaneously to facilitate extracellular Ca2+ influx and ER Ca2+ release, and thus increases the cytosolic Ca2+ concentration to enhance the promoting effects on the synthesis and release of dopamine.

The Role of NMDA Receptor Partial Antagonist, Carbamathione, as a Therapeutic Agent for Transient Global Ischemia.

Carbamathione (Carb), an NMDA glutamate receptor partial antagonist, has potent neuroprotective functions against hypoxia- or ischemia-induced neuronal injury in cell- or animal-based stroke models. We used PC-12 cell cultures as a cell-based model and bilateral carotid artery occlusion (BCAO) for stroke. Whole-cell patch clamp recording in the mouse retinal ganglion cells was performed. Key proteins involved in apoptosis, endoplasmic reticulum (ER) stress, and heat shock proteins were analyzed using immunoblotting. Carb is effective in protecting PC12 cells against glutamate- or hypoxia-induced cell injury. Electrophysiological results show that Carb attenuates NMDA-mediated glutamate currents in the retinal ganglion cells, which results in activation of the AKT signaling pathway and increased expression of pro-cell survival biomarkers, e.g., Hsp 27, P-AKT, and Bcl2 and decreased expression of pro-cell death markers, e.g., Beclin 1, Bax, and Cleaved caspase 3, and ER stress markers, e.g., CHOP, IRE1, XBP1, ATF 4, and eIF2α. Using the BCAO animal stroke model, we found that Carb reduced the brain infarct volume and decreased levels of ER stress markers, GRP 78, CHOP, and at the behavioral level, e.g., a decrease in asymmetric turns and an increase in locomotor activity. These findings for Carb provide promising and rational strategies for stroke therapy.

Design, synthesis and evaluation of novel 1-phenyl-pyrrolo[1,2-b]isoquinolin-3-one derivatives as antagonists for the glycine binding site of the NMDA receptor.

A new series of 1-phenyl-pyrrolo[1,2-b]isoquinolin-3-one derivatives were designed, synthesized and demonstrated to act as antagonists for the glycine binding site of the NMDA receptor. These new derivatives protected PC12 cells against NMDA-induced injury and cell apoptosis in vitro, among which compound 13b exhibited excellent cytoneuroprotective potency and shown a dose-dependent prevention. The increased intracellular Ca2+ influx caused by NMDA in PC12 cells was reversed when pretreated with compound 13b. Furthermore, the interaction between compound 13b and the glycine binding site of the NMDA receptor was validated via MST assay. It was observed that the stereochemistry of compound 13b did not influence the binding affinity, which was consistent with the neuroprotective result. Molecular docking study confirmed the observed activity of compound 13b by virtue of their Pi-stacking, cation-Pi, H-bonding and Pi-electron interactions with the key amino acids in the glycine binding pocket. These results confirm the potential of 1-phenyl-pyrrolo[1,2-b]isoquinolin-3-one derivatives as neuroprotective agents targeting the glycine binding site of the NMDA receptor.

Rapamycin reverses ferroptosis by increasing autophagy in MPTP/MPP+-induced models of Parkinson's disease.

Parkinson's disease is a neurodegenerative disorder, and ferroptosis plays a significant role in the pathological mechanism underlying Parkinson's disease. Rapamycin, an autophagy inducer, has been shown to have neuroprotective effects in Parkinson's disease. However, the link between rapamycin and ferroptosis in Parkinson's disease is not entirely clear. In this study, rapamycin was administered to a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mouse model and a 1-methyl-4-phenylpyridinium-induced Parkinson's disease PC12 cell model. The results showed that rapamycin improved the behavioral symptoms of Parkinson's disease model mice, reduced the loss of dopamine neurons in the substantia nigra pars compacta, and reduced the expression of ferroptosis-related indicators (glutathione peroxidase 4, recombinant solute carrier family 7, member 11, glutathione, malondialdehyde, and reactive oxygen species). In the Parkinson's disease cell model, rapamycin improved cell viability and reduced ferroptosis. The neuroprotective effect of rapamycin was attenuated by a ferroptosis inducer (methyl (1S,3R)-2-(2-chloroacetyl)-1-(4-methoxycarbonylphenyl)-1,3,4,9-tetrahyyridoindole-3-carboxylate) and an autophagy inhibitor (3-methyladenine). Inhibiting ferroptosis by activating autophagy may be an important mechanism by which rapamycin exerts its neuroprotective effects. Therefore, the regulation of ferroptosis and autophagy may provide a therapeutic target for drug treatments in Parkinson's disease.

Dextran-coated iron oxide nanoparticles in combination with ginger extract without NGF promote neurite outgrowth and PC12 cell branching.

Neurogenesis is decreased in the absence of nerve growth factor (NGF). It would be beneficial to discover substances that stimulate neurogenesis without NGF, given the high molecular weight and brief half-life of NGF. This work aims to assess the neurogenesis of ginger extract (GE) combined with superparamagnetic iron oxide nanoparticles (SPIONs) without NGF. Based on our research, GE and SPIONs start neurogenesis before NGF. In comparison to the control group, GE and SPIONs dramatically reduced the length and quantity of neurites, according to statistical analysis. Our findings also indicated that SPIONs and ginger extract together had an additive impact on one another. The total number significantly increased with the addition of GE and nanoparticles. In comparison to NGF, the mixture of GE and nanoparticles significantly enhanced the total number of cells with neurites (by about 1.2-fold), the number of branching points (by about 1.8-fold), and the length of neurites. The difference between ginger extract and nanoparticles with NGF was significant (about 3.5-fold), particularly in the case of cells with one neurite. The results of this study point to the possibility of treating neurodegenerative disorders via the combination of GE and SPIONs without NGF.

Latroeggtoxin-VI protects nerve cells and prevents depression by inhibiting NF-κB signaling pathway activation and excessive inflammation.

Depression has a high incidence and seriously endangers human health. Accumulated evidence indicates that targeting neuroinflammation is a potential avenue for neuroprotection and thus depression prevention. Herein, the effects of latroeggtoxin-VI (LETX-VI), a bioactive protein from the eggs of spider Latrodectus tredecimguttatus, on lipopolysaccharide (LPS)-induced inflammation and depression were systematically investigated using RAW264.7 macrophages and depression mouse model. Pretreatment with LETX-VI suppressed LPS-evoked NF-κB signaling pathway activation, inhibited LPS-induced over-production of NO, iNOS, IL-6 and TNF-α; at the same time LETX-VI mitigated the inhibitory effect of LPS on the expression of anti-inflammatory factors such as Arg-1, thereby suppressing oxidative stress and excessive inflammation. Culture of PC12 cells with the conditioned medium of RAW264.7 cells pretreated with LETX-VI demonstrated the neuroprotective effect of LETX-VI due to its anti-inflammation effect. In the LPS-induced depression mouse model, pretreatment with LETX-VI improved the LPS-induced depression-like behaviors, inhibited the activation of microglia and astrocytes, prevented the down-regulation of Nurr1 expression and alleviated the LPS-caused adverse changes in the brain tissues. Taken together, these in vitro and in vivo findings provide powerful insights into the anti-inflammation-based neuroprotective and antidepressant mechanisms of LETX-VI, which is helpful to deeply reveal the biological effects and potential applications of LETX-VI.

Honeycomb-like nickel oxide-reduced graphene oxide based sensor for the electrochemical tracking of norepinephrine in neuronal cells.

Highly sensitive and specific detection and monitoring of trace norepinephrine (NE) in biological fluids and neuronal cell lines is essential for the investigation of pathogenesis of certain neurological diseases. Herein, we constructed a novel electrochemical sensor for real-time monitoring of NE released by PC12 cells based on glassy carbon electrode (GCE) modified with honeycomb-like nickel oxide (NiO)-reduced graphene oxide (RGO) nanocomposite. The synthesized NiO, RGO and the NiO-RGO nanocomposite were characterized using X-ray diffraction spectrogram (XRD), Raman spectroscopy and scanning electron microscopy (SEM). The porous three-dimensional honeycomb-like structure of NiO and high charge transfer kinetics of RGO endowed the nanocomposite with excellent electrocatalytic activity, large surface area and good conductivity. The developed sensor exhibited superior sensitivity and specificity towards NE in a wide linear range from 20 nM to 14 µM and 14 µM-80 µM, with a low detection limit of 5 nM. The performances of the sensor in terms of excellent biocompatibility and high sensitivity allow it to be successfully employed in the tracking of NE release from PC12 cells under the stimulation of K+, providing an effective strategy for the real-time monitoring of cellular NE.

CircSmox knockdown alleviates PC12 cell apoptosis and inflammation in spinal cord injury by miR-340-5p/Smurf1 axis.

BACKGROUND: Spinal cord injury (SCI) is a traumatic central nervous system disorder that leads to irreversible neurological dysfunction. Emerging evidence has shown that differentially expressed circular RNAs (circRNAs) after SCI is closely associated with the pathophysiological process. Herein, the potential function of circRNA spermine oxidase (circSmox) in functional recovery after SCI was investigated. METHODS: Differentiated PC12 cells stimulated with lipopolysaccharide (LPS) were employed as an in vitro model for neurotoxicity research. Levels of genes and proteins were detected by quantitative real-time PCR and Western blot analysis. Cell viability and apoptosis were determined by CCK-8 assay and flow cytometry. Western blot analysis was used to detect the protein level of apoptosis-related markers. The levels of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α. Dual-luciferase reporter, RIP, and pull-down assays were used to confirm the target relationship between miR-340-5p and circSmox or Smurf1 (SMAD Specific E3 Ubiquitin Protein Ligase 1). RESULTS: LPS elevated the levels of circSmox and Smurf1, but decreased the levels of miR-340-5p in PC12 cells in a dose-dependent manner. Functionally, circSmox silencing alleviated LPS-induced apoptosis and inflammation in PC12 cells in vitro. Mechanistically, circSmox directly sponged miR-340-5p, which targeted Smurf1. Rescue experiments showed that miR-340-5p inhibition attenuated the neuroprotective effect of circSmox siRNA in PC12 cells. Moreover, miR-340-5p suppressed LPS-triggered neurotoxicity in PC12 cells, which was reversed by Smurf1 overexpression. CONCLUSION: CircSmox enhances LPS-induced apoptosis and inflammation via miR-340-5p/Smurf1 axis, providing an exciting view of the potential involvement of circSmox in SCI pathogenesis.