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Neurodegenerative diseases (NDs) are characterized by the progressive loss of neurons. As to developing effective therapeutic interventions, it is crucial to understand the underlying mechanisms of NDs. Cellular models have become invaluable tools for studying the complex pathogenesis of NDs, offering insights into disease mechanisms, determining potential therapeutic targets, and aiding in drug discovery. This review provides a comprehensive overview of various cellular models used in ND research, focusing on Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Cell lines, such as SH-SY5Y and PC12 cells, have emerged as valuable tools due to their ease of use, reproducibility, and scalability. Additionally, co-culture models, involving the growth of distinct cell types like neurons and astrocytes together, are highlighted for simulating brain interactions and microenvironment. While cell lines cannot fully replicate the complexity of the human brain, they provide a scalable method for examining important aspects of neurodegenerative diseases. Advancements in cell line technologies, including the incorporation of patient-specific genetic variants and improved co-culture models, hold promise for enhancing our understanding and expediting the development of effective treatments. Integrating multiple cellular models and advanced technologies offers the potential for significant progress in unraveling the intricacies of these debilitating diseases and improving patient outcomes. [ABSTRACT FROM AUTHOR]

Latroeggtoxin-VI (LETX-VI) is a proteinaceous toxin found in the eggs of spider Latrodectus tredecimguttatus and was previously shown to promote the synthesis and release of dopamine that has been shown to be an endogenous tumor modulator. Here, the anticancer activity of LETX-VI was investigated from both direct and indirect aspects. LETX-VI was shown to have no obvious direct effects on the tested cancer cell lines including B16-F10, Hela, MDA-MB-231, SMMC7721, A549, U251 and U87-MG cells, but has indirect inhibitory effects on the glioma cells U87-MG and U251 by stimulating the synthesis and release of dopamine and cancer-inhibitory proteins from PC12 cells. Culture of U251 and U87-MG cells with the PC12 cell conditional medium (CM) prepared after LETX-VI treatment remarkably inhibited the proliferation and migration ability of the glioma cells. Dopamine quantitative determination and exogenous dopamine addition experiment demonstrated that the inhibitory effect of the CM on U87-MG and U251 cells was at least partially mediated by the LETX-VI-induced increase in dopamine of the CM. Besides, proteomics analysis of the exosomes secreted from PC12 cells into the CM after LETX-VI treatment suggested that some cancer-inhibitory proteins in the exosomes, particularly the proteins encoded by ROCK2, PIK3R1 and TGFβ1 genes, respectively, might synergize with dopamine to mediate the inhibitory effect of CM on glioma cells. All the observations not only increased our understanding of the biological properties and application prospects of LETX-VI, but also provided new ideas for the prevention and treatment of brain gliomas via the endogenous pathways. [ABSTRACT FROM AUTHOR]

Latroeggtoxin‐VI (LETX‐VI), a peptide toxin discovered from the eggs of spider Latrodectus tredecimguttatus, was previously shown to promote the synthesis and release of dopamine in rat pheochromocytoma (PC12) cells, showing potential applications in the neurobiology and medicine. To further understand the structure and properties of LETX‐VI, the key residues were identified and their roles in the structure, function, and stability of LETX‐VI were analyzed in the present work. Based on the protein molecular docking, our previous work, and the relevant literature, the potential key residues of LETX‐VI were selected and identified by alanine‐scanning mutagenesis. The wild‐type LETX‐VI and its 13 mutants, including a double mutant, were prepared by gene cloning and heterologous expression in Escherichia coli, followed by activity, structure, and stability determination. The results demonstrated that the activity of the mutants K25A, R35A, K40A/R41A, and L45A, particularly R35A, to promote dopamine release from PC12 cells was significantly decreased compared with that of the wild‐type LETX‐VI, indicating that these mutated residues are the key residues. Circular dichroism (CD) analysis showed that the secondary structure of these mutants was not obviously different from that of wild‐type LETX‐VI, suggesting that mutation‐caused decrease in the activity of LETX‐VI is due to the changes in the binding site on the molecule surface, rather than the abnormal alternation of the molecular conformation of LETX‐VI. Acetonitrile (ACN) did not obviously influence the activity of LETX‐VI; however, 0.1% trifluoroacetic acid (TFA) treatment for 2 h significantly reduced its activity. Treatment with weakly acidic and basic buffers (pH ≥ 6.6) for 12 h was favorable for LETX‐VI and R35A to exert their activity. Higher temperatures (>37°C) decreased the activity of both wild‐type LETX‐VI and R35A. In conclusion, K25, R35, K40, R41, and L45 particularly R35 are the important functional site residues; during experiments, care should be taken to avoid the adverse influence of strong acid and high temperature on LETX‐VI. These observations have enhanced our understanding of the structure and properties of LETX‐VI and provided references for the subsequent modification of structure and function of LETX‐VI. [ABSTRACT FROM AUTHOR]

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BACKGROUND: Mesenchymal stem cell-derived exosomes may play a crucial role in tissue damage repair, and miRNA is an important component of exosomes for therapeutic effects. Among them, miR-29b-3p has the effect of reducing cell apoptosis, promoting axonal regeneration, and angiogenesis. OBJECTIVE: To study the protective effect of adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p on a neural cell injury model simulated by H2O2-treated PC12 cells, and explore the relevant mechanisms. METHODS: (1) First, the collagenase digestion method was used to extract rat adipose-derived mesenchymal stem cells. Adipose-derived mesenchymal stem cells were transfected with miR-29b-3p mimics and inhibitors. Exosomes were extracted from the culture supernatant by ultracentrifugation and identified so as to construct adipose-derived mesenchymal stem cell-derived exosomes with high expression and knockdown miR-29b-3p. (2) By constructing a neural cell injury model simulated by PC12 cells treated with H2O2, the relevant mechanisms of the protective effect of adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p on the simulated neuronal cell injury model were studied. RESULTS AND CONCLUSION: (1) Adipose-derived mesenchymal stem cell-derived exosome had a typical cup-shaped shape and a diameter distribution in the range of 50-140 nm, expressed membrane proteins Alix, CD63, and TSG101, which were specific markers on the surface of exosomes, and could be successfully ingested by PC12 cells. (2) Adipose-derived mesenchymal stem cell-derived exosome pretreatment could reduce cell apoptosis induced by H2O2 treatment in PC12 cells, and this protective effect was enhanced with the increase of miR-29b-3p expression in the exosomes and weakened with the decrease of miR-29b-3p expression in the exosomes. The mechanism of its effect was related to adipose-derived mesenchymal stem cell-derived exosome via miR-29b-3p promoting the expression of anti-apoptotic protein Bcl-2 and inhibiting the expression of apoptotic protein Bax. [ABSTRACT FROM AUTHOR]

Naringin exists in a wide range of Chinese herbal medicine and has proven to possess several pharmacological properties. In this study, PC12, HepG2 cells, and female Drosophila melanogaster were used to investigate the antioxidative and anti-aging effects of naringin and explore the underlying mechanisms. The results showed that naringin inhibited H2O2-induced decline in cell viability and decreased the content of reactive oxygen species in cells. Meanwhile, naringin prolonged the lifespan of f lies, enhanced the abilities of climbing and the resistance to stress, improved the activities of antioxidant enzymes, and decreased malondialdehyde content. Naringin also improved intestinal barrier dysfunction and reduced abnormal proliferation of intestinal stem cells. Moreover, naringin down-regulated the mRNA expressions of inr, chico, pi3k, and akt-1, and up-regulated the mRNA expressions of dilp2, dilp3, dilp5, and foxo, thereby activating autophagy-related genes and increasing the number of lysosomes. Furthermore, the mutant stocks assays and computer molecular simulation results further indicated that naringin delayed aging by inhibiting the insulin signaling (IIS) pathway and activating the autophagy pathway, which was consistent with the result of network pharmacological predictions.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A novel double network (DN) hydrogel nanocomposite with good compressive strength has been prepared by using Carboxymethyl Cellulose-g-Acrylic acid-Iron Oxides Nanoparticles (CMC-g-AA-IONs) as the primary network and polyacrylamide as the secondary network. The structure of the synthesized DN hydrogel nanocomposite was investigated by Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), and mechanical strength measurement. The synthesized DN hydrogel showed high mechanical strength (σ of 0.238 MPa), despite the water content of 90%. Its biocompatibility effect on sensitive nerve cells was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) test. The results showed that the DN hydrogel nanocomposite has no cytotoxicity on these cells and did not cause any disturbance or inhibit their growth and proliferation. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The aggregation of tau hyperphosphorylation (p-tau) into neurofibrillary tangles (NFT) is a hallmark in the histopathology of Alzheimer's disease (AD). Our previous experiments found that β-asarone could prevent injury of PC12 cells induced by A 1–42, but could it fight cell damage of p-tau induced by okadaic acid (OA) is poorly understood. Objectives: The emphasis of this study lies in β-asarone's therapeutical effect on p-tau inhibition stimulated by OA. Materials and Methods: 175 nmol OA was used to establish AD cells. Cell viability rate and cell toxicity were evaluated by the CCK-8 kit and LDH kit, respectively. The p-tau, Aβ42, β-secretase, and protein phosphatase 2A (PP2A) were examined by ELISA. Proteins closely related to the pathogenesis of AD are involved p-tau, Beclin-1, p-Akt, and p-mTOR were analyzed by western-blotting and immunofluorescence detection. Results: The results revealed that β-asarone enhanced cell viability induced by OA in a dose-dependent manner. Moreover, compared to the OA model, p-tau, Aβ42, β-secretase, and Beclin-1 were reduced, while PP2A, p-Akt, and p-mTOR increased after treatment with β-asarone. Conclusion: All data suggested that β-asarone decreased p-tau, Aβ42, and β-secretase levels, and activated PP2A levels by inhibiting Beclin-1-dependent autophagy in OA model cells, involving Akt/mTOR/Beclin-1 pathway. [ABSTRACT FROM AUTHOR]

Studies on ischemia/reperfusion (I/R) injury suggest that exogenous neural stem cells (NSCs) are ideal candidates for stem cell therapy reperfusion injury. However, NSCs are difficult to obtain owing to ethical limitations. In addition, the survival, differentiation, and proliferation rates of transplanted exogenous NSCs are low, which limit their clinical application. Our previous study showed that neuregulin1β (NRG1β) alleviated cerebral I/R injury in rats. In this study, we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1β on PC12 cells injured by oxygen-glucose deprivation/reoxygenation (OGD/R). Our results found that 5 and 10 nM NRG1β promoted the generation and proliferation of NSCs. Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1β improved the level of reactive oxygen species, malondialdehyde, glutathione, superoxide dismutase, nicotinamide adenine dinucleotide phosphate, and nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial damage in injured PC12 cells; these indexes are related to ferroptosis. Research has reported that p53 and solute carrier family 7 member 11 (SLC7A11) play vital roles in ferroptosis caused by cerebral I/R injury. Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4 (GPX4) was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells, but this change was alleviated after co-culturing NSCs with damaged PC12 cells. These findings suggest that NSCs pretreated with NRG1β exhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.

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.

Objective To study the effect of miR-499a-5p on injury induced by chemical hypoxia of pheochromocytoma cell(PC12). Methods PC12 cells were divided into control group, hypoxia group (treated with CoCl2 for 24 h), hypoxia+miR-NC or miR-mimic groups. Cell viability was detected by MTT assay; the expression of miR-499a-5p was detected by RT-qPCR; lactate dehydrogenase (LDH) activity was detected by kit; apoptosis was detected by flow cytometry; the expression of phosphatase and tensin homolog (PTEN) protein was detected by Western blot. The luciferase reporter gene was used to detect whether Pten was a direct target of miR-499a-5p. Results Compared with control group, the cell viability and miR-499a-5p expression decreased (P＜0.01 or P＜0.001), LDH activity and apoptosis increased (P＜0.01 or P＜0.001) in hypoxia group. Over-expression of miR-499a-5p enhanced survival rate and inhibited apoptosis of PC12 cells under hypoxia (P＜0.05 or P＜0.01). Pten was a direct target of miR-499a-5p. Conclusions miR-499a-5p alleviates hypoxic injury of PC12 cells through targeting at Pten.

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Apelin/APJ exhibits functions in regulating cell proliferation, apoptosis, inhibiting inflammatory responses and vascular remodeling. Therefore, it is speculated that Apelin has similar functions in spinal cord injury. OBJECTIVE: To evaluate the effects of Apelin in rats with spinal cord injury. METHODS: A rat model of spinal cord transection was used in an in vivo experiment. Forty-five female Sprague-Dawley rats were randomly divided into sham group, spinal cord injury group and Apelin-13 group. Rats in the latter two groups were submitted to make animal models of spinal cord transection. Rats were given the intraperitoneal injection of 0.2 mg/kg Apelin-13 in the Apelin group or the same amount of normal saline in the other two groups for 14 consecutive days. The motor function of rat’s hind limbs was assessed by Basso, Beattie, Bresnahan locomotor rating scale at the 1st, 3rd, 7th, and 14th days after modeling. The spinal cord tissues were collected at the 14th day after spinal cord injury and used for immunohistochemical, immunofluorescence and RT-PCR analyses. In in vitro experiments, H2O2 was used to induce PC12 cell injury followed by treatment with different concentrations (1, 2, 4 μmol/L) of Apelin-13. We then used cell counting kit-8 to detect the effect of Apelin-13 on the viability of injured PC12 cells, thereby exploring the neuroprotective effect of Apelin. RESULTS AND CONCLUSION: (1) After spinal cord injury, the hindlimb function of the rats was completely lost and motor recovery was observed 3 days after injury. However, there was no significant difference in the spinal cord injury group at different time points. Apelin promoted motor function recovery post spinal cord injury. (2) Spinal cord injury disrupted the local continuity of the spinal cord, and Apelin restored the injured morphology induced by spinal cord injury. (3) There were only a few cells positive for ionized calcium binding adapter protein 1 in the sham group. Spinal cord injury led to a sharp increase in the number of local positive cells and astrocytes. Apelin could alleviate the local activation of astrocytes and microglia (P < 0.05). (4) Severe demyelination occurred after spinal cord injury and the positive area of Luxol Fast Blue staining was significantly reduced. Apelin could attenuate demyelination caused by spinal cord injury. (5) The expression of growth-related protein 43 was significantly increased after spinal cord injury (P < 0.001). Apelin could further promote the expression of growth-related protein 43 (P < 0.05). (6) Apelin increased the cell viability of H2O2-stimulated PC12 cells in vitro (P < 0.05). To conclude, Apelin promotes locomotor recovery and spinal cord morphology repair in rats after spinal cord injury. [ABSTRACT FROM AUTHOR]

Reperfusion is the fundamental treatment for ischaemic stroke; however, many ischaemic stroke patients cannot undergo reperfusion treatment. Furthermore, reperfusion can cause ischaemic reperfusion injuries. This study aimed to determine the effects of reperfusion in an in vitro ischaemic stroke model—oxygen and glucose deprivation (OGD) (0.3% O2)—with rat pheochromocytoma (PC12) cells and cortical neurons. In PC12 cells, OGD resulted in a time-dependent increase in cytotoxicity and apoptosis, and reduction in MTT activity from 2 h onwards. Reperfusion following shorter periods (4 and 6 h) of OGD recovered apoptotic PC12 cells, whereas after 12 h, OGD increased LDH release. In primary neurons, 6 h OGD led to significant increase in cytotoxicity, reduction in MTT activity and dendritic MAP2 staining. Reperfusion following 6 h OGD increased the cytotoxicity. HIF-1a was stabilised by 4 and 6 h OGD in PC12 cells and 2 h OGD onwards in primary neurons. A panel of hypoxic genes were upregulated by the OGD treatments depending on the duration. In conclusion, the duration of OGD determines the mitochondrial activity, cell viability, HIF-1a stabilization, and hypoxic gene expression in both cell types. Reperfusion following OGD of short duration is neuroprotective, whereas OGD of long duration is cytotoxic. [ABSTRACT FROM AUTHOR]

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)‐1β, 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. [ABSTRACT FROM AUTHOR]

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.

Background: PC12 pheochromocytoma tumor cell lines are widely used to decipher the intracellular signaling mechanisms mediating the effects of some growth factors. Nevertheless, the disparity in appearance of some PC12 cell lines used in the different publications questions our ability to compare the results obtained by the numerous laboratories which use them. This led us to analyze the phenotypic aspect and transcriptomic expression of 5 PC12 cell lines from different origins under control conditions and after treatment with nerve growth factor (NGF) or pituitary adenylate cyclase-activating polypeptide (PACAP). Methods: Characterization of the 5 PC12 cell lines was conducted using imaging techniques and high-throughput real-time PCR combined with bioinformatics analysis. Results: The results show that the 5 cell lines are very variable in terms of shape, proliferation rate, motility, adhesion to the substrate, and gene expression. This high heterogeneity of the cell lines is also found when looking at their response to NGF or PACAP on gene expression or differentiation, with even in some cases opposite effects, as, for example, on cell proliferation. Actually, only 2 of the cell lines tested exhibited some phenotypic similarities with each other, even though the transcriptomic analyses show that they are far from identical. Discussion/Conclusion: As this issue of cell heterogenicity is not restricted to PC12 cells, the present results highlight the need to facilitate the supply of cell lines at low cost, the necessity to standardize practices regarding the use of cell lines, and the requirement to define precise markers of established cell lines which should be monitored in every publication. Regarding this latter point, the present data show that transcriptomic analysis by real-time PCR using a panel of genes of interest is easy to implement and provides a reliable method to control the possible drift of the cells over time in culture. Transcriptomic phenotyping combined with bioinformatics analysis can also be a useful approach to predict the response of the cells to treatments in terms of cell signaling activation, which can help to choose among several cell lines the most appropriate one for the investigation of a particular mechanism. Taken together, the results from this study highlight the need to use well-characterized cell lines with standardized protocols to generate reproducible results from 1 laboratory to the other. [ABSTRACT FROM AUTHOR]

Parkinson's disease is the second most common neurodegenerative disease after Alzheimer's disease. This study was aimed at investigating the neuroprotective mechanisms of the ethyl acetate fraction of Terminalia chebula ethanol-soluble extract using PC12 cells treated with paraquat as a model system. The ethyl acetate fraction of T. chebula ethanol-soluble extract was prepared using 95% ethanol and ethyl acetate as described in our previous study. The neuroprotective potential of the ethyl acetate fraction of T. chebula ethanol-soluble extract was analyzed by evaluating the intracellular reactive oxygen species and calcium levels, apoptosis, caspase-3 and α-synuclein activities, as well as Bcl-2 and Bax expressions. The ethyl acetate fraction of T. chebula ethanol-soluble extract exhibited neuroprotective effects against paraquat-induced apoptosis in PC12 cells at concentrations ranging from 0.4 μg/mL to 0.6 μg/mL. The various neuroprotective mechanisms identified for the extract included reduction in reactive oxygen species levels; inhibition of increased calcium ion concentration; increased Bcl-2 expression; reduced Bax, caspase-3 and α-synuclein expression; and reduced nuclear shrinkage. This study elucidates the various mechanisms underlying the neuroprotective effects of the ethyl acetate fraction of T. chebula ethanol-soluble extract that may further aid the Parkinson's disease pharmacological application of T. chebula. [ABSTRACT FROM AUTHOR]

BACKGROUND: Ginsenoside Rg3 can protect the brain from ischemia injury. However, it is unclear whether ginsenoside Rg3 can protect PC12 cell against oxygen-glucose deprivation/reoxygenation (OGD/R) injury. OBJECTIVE: To investigate the protective role and underlying mechanism of ginsenoside Rg3 in OGD/R-injured PC12 cells. METHODS: A model of OGD/R-injured PC12 cells was constructed. Protective effects of ginsenoside Rg3 at different concentrations on OGD/R-injured PC12 cells were detected using MTT, lactic dehydrogenase, Calcein-AM/PI cell apoptosis double staining assay, western blot assay, and flow cytometry. PI3K/AKT pathway inhibitor LY294002 and AKT activator SC79 were involved to reveal the potential mechanisms of ginsenoside Rg3. RESULTS AND CONCLUSION: (1) In this study, OGD/R exposure successfully induced cell injury, which reduced cell survival and promoted cell apoptosis in a time-dependent manner, up-regulated p-AKT/AKT and p-PI3K/PI3K ratio, increased the levels of NLRP3 inflammasomes, tumor necrosis factor-α, interleukin1β and BAX, and promoted the release of lactic dehydrogenase. (2) Ginsenoside Rg3 at a certain range enhanced the viability and reduced apoptosis and lactic dehydrogenase release of OGD/R-injured PC12 cells. Ginsenoside Rg3 down-regulated the ratio of p-AKT/AKT and p-PI3K/PI3K, reduced NLRP3, interleukin-1β, tumor necrosis factor-α and BAX levels. Ginsenoside Rg3 also suppressed the expression of activated caspase-9 and up-regulated the expression of activated caspase-3. (3) LY294002 shared the same protective effect as ginsenoside Rg3 whereas SC79 reversed this outcome. Therefore, ginsenoside Rg3 protects PC12 cells from OGD/R injury mainly through inhibiting the PI3K/AKT pathway and phosphorylation of PI3K and AKT, which may exert an anti-inflammatory and antiapoptotic effect. [ABSTRACT FROM AUTHOR]

The development of amyotrophic lateral sclerosis (ALS) may be related to the abnormal alterations of multiple proteins. Our previous study revealed that the expression of phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4) was decreased in ALS. However, the role of PIK3R4 in ALS pathogenesis remains unknown. This study was the first to find that transfection of PC12 cells with small interfering RNA against the PIK3R4 gene significantly decreased the expression levels of PIK3R4 and the autophagy-related proteins p62 and LC3. Additionally, in vivo experiments revealed that the PIK3R4 protein was extensively expressed in the anterior horn, posterior horn, central canal, and areas surrounding the central canal in cervical, thoracic, and lumbar segments of the spinal cord in adult mice. PIK3R4 protein was mainly expressed in the neurons within the spinal lumbar segments. PIK3R4 and p62 expression levels were significantly decreased at both the pre-onset and onset stages of ALS disease in Tg(SOD1*G93A)1Gur mice compared with control mice, but these proteins were markedly increased at the progression stage. LC3 protein expression did not change during progression of ALS. These findings suggest that PIK3R4 likely participates in the prevention of ALS progression. This study was approved by the Ethics Committee for Animal Care and Use of Jiangxi Provincial People’s Hospital, Affiliated People’s Hospital of Nanchang University (approval No. 2020025) on March 26, 2020.

Honokiol, the main bioactive extract of Magnolia officinalis, exhibits extensive therapeutic actions. Its treatment for advanced non-small cell lung cancer is undergoing clinical trials in China. However, the published safety evaluation studies have focused on extract mixtures of Magnolia officinalis in which the honokiol content was well below the reported clinical dose of the honokiol monomer. Therefore, safety assessment of the honokiol monomer is urgently needed. Our previous studies have already demonstrated that a high dose of the honokiol microemulsion (0.6 μg/mL) induces developmental toxicity in rats and zebrafish by inducing oxidative stress. By exploring the relationship between time and toxicity, we found that developmental toxic responses were stage-dependent. They mainly occurred within the first 24 h post fertilization (hpf) especially the first 12 hpf. In zebrafish, low doses of honokiol microemulsion (0.15, 0.21 μg/mL) significantly decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increased the mRNA expression of bcl-2. In contrast, high dose (0.6 μg/mL) increased the levels of ROS and MDA, decreased activities and mRNA expression of superoxide dismutase (SOD) and catalase (CAT), and increased mRNA expression of bax, c-jnk, p53 and bim. By acridine orange staining, we found that a high dose of honokiol microemulsion induced apoptosis mainly in zebrafish brain. In rat pheochromocytoma cells (PC12 cells), low doses of the honokiol microemulsion (1, 5, 10 µM) exerted a protective effect against H2O2-induced oxidative damage while high doses (≥20 µM) induced oxidative stress, which further confirms the dual effects of honokiol microemulsion on nerve cells. These dual roles of the honokiol microemulsion in oxidation–reduction reactions and apoptosis may be regulated by the forkhead box class O (FoxO) signaling pathway. Due to the potential of developmental toxicity, we recommend that the administration of high dose honokiol microemulsion in pregnant women should be considered with caution. [ABSTRACT FROM AUTHOR]

Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin mined from the egg transcriptome of spider L. tredecimguttatus , was previously found to promote the release of dopamine from PC12 cells. However, the relevant molecular mechanism has not been fully clear. Here LETX-VI was demonstrated to rapidly penetrate the plasma membrane of PC12 cells via the vesicle exocytosis/endocytosis cycle, during which vesicular transmembrane protein synaptotagmin 1 (Syt1) functions as a receptor, with its vesicle luminal domain interacting with the C-terminal region of LETX-VI. The C-terminal sequence of LETX-VI is the functional region for both entering cells and promoting dopamine release. After gaining entry into the PC12 cells, LETX-VI down-regulated the phosphorylation levels of Syt1 at T201 and T195, thereby facilitating vesicle fusion with plasma membrane and thus promoting dopamine release. The relevant mechanism analysis indicated that LETX-VI has a protein phosphatase 2A (PP2A) activator activity. The present work has not only probed into the Syt1-mediated action mechanism of LETX-VI, but also revealed the structure-function relationship of the toxin, thus suggesting its potential applications in the drug transmembrane delivery and treatment of the diseases related to dopamine release and PP2A activity deficiency. [ABSTRACT FROM AUTHOR]

The root of Cynanchum auriculatum Royle ex Wight is a traditional Chinese medicine, which is rich in C21 steroidal glycosides by phytochemistry research. In this study, the antioxidant effect of 27 C21 steroidal glycosides isolated from the root of C. auriculatum by our group was evaluated using the H2O2-treated PC12 cells. As the result, all tested compounds altered the activities of lactate dehydrogenase, superoxide dismutase, catalase and glutathione peroxidase at concentrations as low as 1 μM in H2O2-treated PC12 cells. They also decreased the levels of intracellular reactive oxygen species and Ca2+. Further, the correlation between their structural features described by molecular descriptors and the indicators of bioactivity was analyzed by partial least squares analysis, displaying those six bioindicators were positive correlated with 13 molecular descriptors and providing some guidance for further study of relationships between steroid structure and antioxidant activity. [ABSTRACT FROM AUTHOR]

It has been suggested that oxidative stress (OS) has a role in the development of aging and neurodegenerative disorders. Biological molecules are easily damaged by reactive oxygen species, which can ultimately result in necrotic or apoptotic cell death. Foods containing phytochemicals, such as phenolic compounds, may have potential preventive effects against several diseases, including alzheimer’s disease (AD), according to epidemiological and in vitro research. Gastrodia elata is a well-known homology of medicine and food plant that has been used for centuries in China and other East Asian countries to treat central nervous system disorders. In this study, we focused on the potential of the extract, Gastrodia elata polyphenols (GPP), for the prevention and treatment of AD. H2O2 induced PC12 cell damage was used to simulate the oxidative stress of AD. The effects of GPP on the injury model were evaluated by cell survival rate, lactate dehydrogenase (LDH), lipid peroxidation (MDA), production of intracellular antioxidant enzymes, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cellular inflammation level and apoptosis level. The results showed that GPP pretreatment had a protective effect by increasing cell viability, reducing lactate dehydrogenase infiltration, decreasing MDA and increasing intracellular antioxidant enzymes, diminishing reactive oxygen species production and decreasing mitochondrial membrane potential, reducing cell inflammation and decreasing apoptosis. Accordingly, it is suggested that GPP possessed promising neuroprotective benefits which enabled the prevention or therapeutic implementation of AD along with serving as a reference towards the exploitation of functional foods or drugs derived from Gastrodia elata.

Background & objectives: Research on intelligent nanomaterials that accelerate the process of nerve regeneration and treatment by different methods such as antioxidant effects, stimulation of nerve cell proliferation, modulation of the immune system and inflammatory factors is great importance. The aim of this study was to prepare cinnamon-coated cerium oxide nanoparticles and evaluate its antioxidant and cytotoxicity effects on PC12 cell line. Methods: Cinnamon-coated cerium oxide nanoparticles were prepared and their structural properties were evaluated by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). To evaluate the antioxidant properties of the compounds, free radical trapping methods 2 and 2 diphenyl-1-picryl hydrazyl were used. Cell viability in the presence of compounds was measured by a toxicity test (MTT) [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Data were analyzed by one-way analysis of variance and Tukey's post hoc test. Results: FTIR spectra and TEM images showed the processing of nanoparticles with an average size of less than 100 nm with cinnamon coating on their surface. Also, the antioxidant capacity of cinnamon-coated cerium oxide nanoparticles was significantly higher (*p

Dopamine is an important neurotransmitter in the body and closely related to many neurodegenerative diseases. Therefore, the detection of dopamine is of great significance for the diagnosis and treatment of diseases, screening of drugs and unraveling of relevant pathogenic mechanisms. However, the low concentration of dopamine in the body and the complexity of the matrix make the accurate detection of dopamine challenging. Herein, an electrochemical sensor is constructed based on ternary nanocomposites consisting of one-dimensional Pt nanowires, two-dimensional MXene nanosheets, and three-dimensional porous carbon. The Pt nanowires exhibit excellent catalytic activity due to the abundant grain boundaries and highly undercoordinated atoms; MXene nanosheets not only facilitate the growth of Pt nanowires, but also enhance the electrical conductivity and hydrophilicity; and the porous carbon helps induce significant adsorption of dopamine on the electrode surface. In electrochemical tests, the ternary nanocomposite-based sensor achieves an ultra-sensitive detection of dopamine (S/N = 3) with a low limit of detection (LOD) of 28 nM, satisfactory selectivity and excellent stability. Furthermore, the sensor can be used for the detection of dopamine in serum and in situ monitoring of dopamine release from PC12 cells. Such a highly sensitive nanocomposite sensor can be exploited for in situ monitoring of important neurotransmitters at the cellular level, which is of great significance for related drug screening and mechanistic studies. [Display omitted] • Pt/MX binary composites by solvothermal synthesis. • Pt/MXPC ternary nanocomposites by mixing Pt/MX binary composites and PC. • Pt/MX/PC-modified SPCEs for electrochemical detection of dopamine. • In situ monitoring of dopamine released from PC12 cells by K+ stimulation. [ABSTRACT FROM AUTHOR]

Background: Oxidative stress is associated with the pathogenesis of ischemic stroke (±)-5-bromo-2-(5-fluoro-1-hydroxyamyl) benzoate (BFB) is a novel compound modified by dl-3-n-butylphthalide (NBP). Here, we hypothesized that BFB may protect the PC12 cells against H2O2-induced oxidative stress injury through activation of the Nrf2 pathway. Methods: We measured the cell viability and levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) to determine the construction of the H2O2-induced models of oxidative stress in PC12 cells. Additionally, apoptotic cell death, mitochondrial membrane potential, and cellular morphology were examined to determine the effect of BFB on oxidative stress injury in H2O2-treated PC12 cells. The expression levels of Nrf2-related and autophagy-related genes and proteins were detected using real time quantative PCR (RT-qPCR), Western Blot, and immunofluorescence analyses. Results: Our study showed that BFB treatment reduced the elevated levels of MDA, LDH, and ROS, and decreased cell viability and GSH in H2O2-treated PC12 cells. We also observed the elevated expression of Nrf2 pathway-related factors and intranuclear transitions and found that Nrf2 inhibitors (ML385) could block the protective effect of BFB. The inhibitory effect of BFB on oxidative stress may be partially regulated by Nrf2 activation, and the initiation and induction of autophagy. Conclusion: BFB inhibited H2O2-induced oxidative stress injury in PC12 cells by activating the Nrf2 pathway, initiating and inducing autophagy, suggesting that BFB may be a promising therapeutic agent in treating neurological disorders like cerebral ischemia. [ABSTRACT FROM AUTHOR]

Background: Oxidative stress is associated with the pathogenesis of ischemic stroke (±)-5-bromo-2-(5-fluoro-1-hydroxyamyl) benzoate (BFB) is a novel compound modified by dl-3-n-butylphthalide (NBP). Here, we hypothesized that BFB may protect the PC12 cells against H2O2-induced oxidative stress injury through activation of the Nrf2 pathway.Methods: We measured the cell viability and levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) to determine the construction of the H2O2-induced models of oxidative stress in PC12 cells. Additionally, apoptotic cell death, mitochondrial membrane potential, and cellular morphology were examined to determine the effect of BFB on oxidative stress injury in H2O2-treated PC12 cells. The expression levels of Nrf2-related and autophagy-related genes and proteins were detected using real time quantative PCR (RT-qPCR), Western Blot, and immunofluorescence analyses.Results: Our study showed that BFB treatment reduced the elevated levels of MDA, LDH, and ROS, and decreased cell viability and GSH in H2O2-treated PC12 cells. We also observed the elevated expression of Nrf2 pathway-related factors and intranuclear transitions and found that Nrf2 inhibitors (ML385) could block the protective effect of BFB. The inhibitory effect of BFB on oxidative stress may be partially regulated by Nrf2 activation, and the initiation and induction of autophagy.Conclusion: BFB inhibited H2O2-induced oxidative stress injury in PC12 cells by activating the Nrf2 pathway, initiating and inducing autophagy, suggesting that BFB may be a promising therapeutic agent in treating neurological disorders like cerebral ischemia.

Most degenerative diseases are caused by free radicals. Antioxidin-RL peptide is a free radical scavenger found in the skin of plateau frog Odorrana livida, which is more stable than vitamin C as it resists light-induced degradation. However, whether and how antioxidin-RL protects cells from oxidative stress was not clear. Here we addressed this issue, and in addition, we designed a series of antioxidin cognates by adding tyrosine residues to enhance free radical-binding capability. We performed free radical-clearing assays in solution to screen the mutants, and found a mutant antioxidin-2 that was as stable as antioxidin-RL and cleared free radical faster. By using PC-12 cells as a model, we demonstrated that both antioxidin-2 and antioxidin-RL inhibited the accumulation of intracellular free radicals triggered by H2O2, reduced mitochondria membrane potential dissipation, maintained mitochondrial morphology, and decreased the expression of dynamin-related protein-1 in mitochondria, with antioxidin-RL more effective. Antioxidin-RL also attenuated the changes in SOD1 and GPx1 expression induced by H2O2. These findings provide insight into the anti-oxidative mechanisms of antioxidin-RL and its derivatives, which will provide rational basis for the development of more effective antioxidants to cure diseases.

Background Lidocaine was an anesthetic commonly used for analgesia, but the neurotoxicity could not be ignored. However, benzodiazepines could alleviate the toxicity when combined with other drugs. Purpose To explore the molecular mechanism of benzodiazepines in protecting nerve cells after the induction of lidocaine. Methods PC12 cells were induced by lidocaine (0 mM, 0.1 mM, 0.5 mM and 1 mM) first and then treated by benzodiazepines (0 μM–200 μM). RT-qPCR assays measured RNA expressions of epidermal growth factor receptor (EGFR) and microRNA-133a-3p (miR-133a-3p) in PC12 cell line, respectively. Western blot was for protein detections of EGFR and caspase-3. Flow cytometry assay assessed apoptosis and cellular viability was validated via Cell Counting Kit-8 (CCK-8) test. Bioinformatics analysis predicted the potential link between miR-133a-3p and EGFR and the binding was verified using the Dual luciferase reporter experiment. Results Benzodiazepines increased cellular viability of PC12 cells up to 100 μM while suppressed viability between 100 and 200 μM. Benzodiazepines (0 μM, 10 μM, 50 μM and 100 μM) did not regulate PC12 cell viability but promoted the viability of lidocaine-treated PC12 cells. Lidocaine downregulated miR-133a-3p RNA expression but facilitated EGFR mRNA expression, which was reversed after treated by benzodiazepines. MiR-133a-3p targeted and negatively regulated EGFR expressions in mRNA and protein levels. Furthermore, miR-133a-3p inhibitor and overexpressed EGFR transfection both restrained the decreased PC12 cell viability and prompted cell apoptosis caused by benzodiazepines. Conclusion Benzodiazepines restrained lidocaine-induced toxicity in PC12 cells which secured viability and reduced apoptosis via miR-133a-3p/EGFR pathway. [ABSTRACT FROM AUTHOR]

Genipin has attracted much attention for its hepatoprotective, anti-inflammatory, and neuroprotection activities. However, poor water solubility and active chemical properties limit its application in food and pharmaceutical industries. This article aimed to develop a lipid-based microemulsion delivery system to improve the stability and bioavailability of genipin. The excipients for a genipin microemulsion (GME) preparation were screened and a pseudo-ternary phase diagram was established. The droplet size (DS), zeta potential (ZP), polydispersity index (PDI), physical and simulated gastrointestinal digestion stability, and in vitro drug release properties were characterized. Finally, the effect of the microemulsion on its cellular uptake by Caco-2 cells and the protective effect on PC12 cells were investigated. The prepared GME had a transparent appearance with a DS of 16.17 ± 0.27 nm, ZP of −8.11 ± 0.77 mV, and PDI of 0.183 ± 0.013. It exhibited good temperature, pH, ionic strength, and simulated gastrointestinal digestion stability. The in vitro release and cellular uptake data showed that the GME had a lower release rate and better bioavailability compared with that of free genipin. Interestingly, the GME showed a significantly better protective effect against amyloid-β (Aβ1-42)-induced PC12 cell cytotoxicity than that of the unencapsulated genipin. These findings suggest that the lipid-based microemulsion delivery system could serve as a promising approach to improve the application of genipin. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that microRNAs play a pivotal role in neural remodeling after spinal cord injury (SCI). This study aimed to investigate the mechanisms of miR-135a-5p in regulating the functional recovery of SCI by impacting its target genes and downstream signaling. The gene transfection assay and luciferase reporter assay confirmed the target relationship between miR-135a-5p and its target genes (specificity protein 1 [SP1] and Rho-associated kinase [ROCK]1/2). By establishing the H2O2-induced injury model, miR-135a-5p transfection was found to inhibit the apoptosis of PC12 cells by downregulating the SP1 gene, which subsequently induced downregulation of pro-apoptotic proteins (Bax, cleaved caspase-3) and upregulation of anti-apoptotic protein Bcl-2. By measuring the neurite lengths of PC12 cells, miR-135a-5p transfection was found to promote axon outgrowth by downregulating the ROCK1/2 gene, which subsequently caused upregulation of phosphate protein kinase B (AKT) and phosphate glycogen synthase kinase 3β (GSK3β). Use of the rat SCI models showed that miR-135a-5p could increase the Basso, Beattie, and Bresnahan (BBB) scores, indicating neurological function recovery. In conclusion, the miR-135a-5p-SP1-Bax/Bcl-2/caspase-3 and miR-135a-5p-ROCK-AKT/GSK3β axes are involved in functional recovery of SCI by regulating neural apoptosis and axon regeneration, respectively, and thus can be promising effective therapeutic strategies in SCI.

Introduction: Ropivacaine has been regularly used because of its good anesthetic and analgesic effects, but it may exert neurotoxic effects on neurocyte. Dexmedetomidine has presented special advantages in the fields of neuroprotection, and it also could improve peripheral nerve block combining with ropivacaine. However, if dexmedetomidine could repair neurocyte injury induced by ropivacaine, and the specific mechanism remain unclear. Methods: Western blotting and qRT-PCR were applied for measuring expression of protein and mRNA, respectively. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8) and colony formation assays. Transwell assay was applied to measure the migration and invasion of cells. Dual luciferase reporter assay was applied for confirming the binding site between microRNA-381 (miR-381) and Leucine-rich repeat C4 protein (LRRC4). Results: The viability of PC12 cells increased with raising the concentration of dexmedetomidine (0 μM, 10 μM, 50 μM, 100 μM). Dexmedetomidine reversed role of ropivacaine (0 mM, 0.1 mM, 0.5 mM, 1 mM) by upragulating the expression of miR-381 and suppressing the expression of LRRC4 in PC12 cells. miR-381 can directly interact with target gene LRRC4 and negatively regulate its expression. Dexmedetomidine promoted the proliferation, migration, and invasion and inhibited apoptosis of PC12 cells by suppressing LRRC4 via up-regulating the expressions of miR-381 and further activated SDF-1/CXCR4 signaling pathway. Conclusions: Dexmedetomidine could protect PC12 cells from ropivacaine injury through miR-381/LRRC4/SDF-1/CXCR4 signaling pathway. This study may provide new therapeutic strategy targeting miR-381/LRRC4/SDF-1/CXCR4 signaling pathway about the prevention of ropivacaine induced neurocyte injury.

The present study preliminarily evaluated neurotoxicity injuries induced by chlorpyrifos in PC12 ​cell, which were used as a model for nervous cell system. In cultured PC12 ​cell, application of soy isoflavones (genistein, daidzein, monomer and mixture) significantly reduced chlorpyrifos induced toxicity, a widely used pesticide, and resulted in a better cell survival rate. Treatments with isoflavones reduced malondialdehyde content, reactive oxygengeneration and acetylcholine level in medium, and maintained mitochondrial membrane potential integrity. Daidzein enhanced endogenous antioxidant system in PC12 ​cell with an increasing in superoxide dismutase per-unit activity. Genistein reduced acetylcholine content in the medium. Daidzein and genistein showed different effects, and their combined effect were greater than individual. In conclusion, soy isoflavones as an antioxidant and neuroprotectant, enhanced choline metabolism, which effectively mitigated disadvantageous influence of PC12 ​cell caused by chlorpyrifos.

One main fraction of Selenium-enriched Pleurotus ostreatus (P. ostreatus) polysaccharide (Se-POP-1) was extracted and purified by DEAE-52 and sephadex G-100. Se-POP-1 was an approximate homogenous polysaccharide with an average molecular weight of 1.62 × 104 Da, and mainly composed of glucose, mannose and galactose, with molar ratio of 5.30:1.55:2.14. The absorption peaks at 941 cm−1 and 1048 cm−1 in FT-IR analysis were ascribed as C-O-Se and Se=O bonds. Pr-treatment of Se-POP-1 (400 μg/mL) increased the cell survival of H2O2-stimulated PC12 cells and inhibited intrinsic apoptosis and oxidative stress in H2O2-stimulated PC12 cells via limiting DNA degradation and decreasing the reactive oxygen species (ROS) generation. In addition, up-regulation of anti-apoptotic protein Bcl-2, down- regulation of pro-apoptotic protein Bax, cleaved caspase 3, and cytochrome c were also observed. Se-POP-1 presented an obvious effect to alleviate oxidative damage and apoptosis in PC12 cells induced by H2O2. Therefore, Se-POP-1 possessed potent antioxidant and biological activities with the ability to prevent oxidation via scavenging ROS and free radicals in cells. It could be developed as organic selenium dietary supplement and functional food.

The gene product of ocular albinism 1 (OA1)/G-protein-coupled receptor (GPR)143 is a receptor for L-3,4-dihydroxyphenylanine (l-DOPA), the most effective agent for Parkinson's disease. When overexpressed, human wild-type GPR143, but not its mutants, inhibits neurite outgrowth in PC12 cells. We investigated the downstream signaling pathway for GPR143-induced inhibition of neurite outgrowth. Nifedipine restored GPR143-induced neurite outgrowth inhibition to the level of control transfectant but did not affect outgrowth in GPR143-knockdown cells. Cilnidipine and flunarizine also suppressed the GPR143-induced inhibition, but their effects at higher concentrations still occurred even in GPR143-knockdown cells. These results suggest that GPR143 regulates neurite outgrowth via L-type calcium channel(s)., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

The neuroprotective mechanisms of Crataegus pinnatifida extracts and crataegolic acid were studied using paraquat induced cytotoxicity in PC12 cells. C. pinnatifida extracts were prepared using hexane, ethyl acetate, and 95% ethanol. Additionally, crataegolic acid (also known as maslinic acid) was found in C. pinnatifida extracts. Assessment methods included the examinations of cytotoxicity, intracellular reactive oxygen species and calcium changes, activity of caspase-3 and a-synuclein, apoptotic cell death, and the expression levels of the B-cell lymphoma 2 (Bcl-2) and BCL2-associated X (Bax) proteins to investigate the neuroprotective mechanisms of C. pinnatifida extracts and its active component, crataegolic acid. The three extracts and crataegolic acid exhibited potent neuroprotective actions against paraquat induced PC12 cell apoptosis at 5--20 μg/mL and 80--100 μM concentrations, respectively. The key protective mechanisms included decreasing cell apoptosis, upregulating Bcl-2 protein levels, and downregulating Bax protein levels. The 95% ethanol extract also decreased paraquat induced reactive oxygen species production, calcium overloading, and caspase-3 and a-synuclein activities. The beneficial effects of these extracts could be explained by the active component, crataegolic acid that also inhibited paraquat-induced apoptosis through the suppression of reactive oxygen species generation and the caspase-3 signaling pathway. [ABSTRACT FROM AUTHOR]

Fipronil and its metabolites (fipronil sulfone, fipronil sulfide and fipronil desulfinyl) adversely affect the environment and human health. Targeted metabolomics and lipidomics based on ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to analyse the alterations of glycerophospholipids and amino acids after exposure to fipronil and its metabolites at dosages of 0.5, 12.5 and 50 μM for 72 h and to evaluate their different toxic effects. Results showed that fipronil sulfone and fipronil desulfinyl are more toxic than their parent compound, with fipronil desulfinyl as the most toxic and fipronil sulfide as the least toxic. Fipronil and its metabolites affected the metabolism of PC18:1/16:0, PI18:0/20:4, arginine, leucine and tyrosine and the “phenylalanine, tyrosine and tryptophan biosynthesis” pathway, indicating their possible inducing role in cellular macromolecule damage, nerve signal transmission disturbance and energy metabolism disruption caused by oxidative stress. Importantly, fipronil sulfone and fipronil desulfinyl more strongly influenced lipid and amino acid metabolism, mainly reflected in the number of changed glycerophospholipids and differential metabolites associated with oxidative stress, including PS18:0/20:4, glutamate, phenylalanine and histidine for fipronil sulfone and PS18:0/20:4, glutamate, phenylalanine, serine and aspartic acid for fipronil desulfinyl. Therefore, the higher toxicity of fipronil desulfinyl and fipronil sulfone may be also related to oxidative stress. This study provides implications for risk assessment and toxic mechanism research on fipronil and its metabolites.

Objective To explore the role and molecular mechanism of Neferine (Nef) on PC12 cells injury induced by Aβ1-42. Methods PC12 cells were divided into control group, model group(4 μg/mL Aβ1-42 cultured for 24 h) and low, medium and high-dose neferine intervention group. The PC12 cell viability was determined by MTT method and cell apoptosis was detected by flow cytometry. Protein expression of aquaporin4 (AQP4), Bcl-2 and Bax was analyzed by Western Blot, and miR-29a-3p, AQP4 mRNA were detected by qPCR. Biological information prediction and dual luciferase gene report analyzed the targeting relationship between miR-29a-3p and AQP4. miR-29a-3p, si-AQP4 were transfected into PC12 cells induced by Aβ1-42, or anti-miR-29a-3p was transfected into cells and performed high-dose Nef intervention to investigate the effects on cell survival and apoptosisin PC12 cells injury induced by Aβ1-42. Results Compared with the model group, the cell viability rate, Bcl-2 and miR-29a-3p expression of neferine group were significantly increased, and the cell apoptosis rate, Bax, AQP4 mRNA and protein of neferine group expression were significantly decreased (P＜0.05). AQP4 was the target gene of miR-29a-3p. Both over-expression of miR-29a-3p and inhibition of AQP4 expression significantly increased the viability rate of PC12 cells injury induced by Aβ1-42 and the expression level of Bcl-2, and decreased the apoptosis rate and Bax protein level. Inhibitiion of expression of miR-29a-3p reversed the promotion effect of Neferine on the survival of PC12 cell injury induced by Aβ1-42 and the level of Bcl-2 protein, as well as the inhibition effect of Neferine on the apoptosis rate and Bax protein expression. Conclusions Neferine inhibits PC12 cell injury induced by Aβ1-42, promotes cell survival and inhibits apoptosis by miR-29a-3p/AQP4.