Melatonin suppresses TLR4-mediated RSV infection in the central nervous cells by inhibiting NLRP3 inflammasome formation and autophagy.

Respiratory syncytial virus (RSV) infects neuronal cells in the central nervous system (CNS), resulting in neurological symptoms. In the present study, we intended to explore the mechanism of RSV infection-induced neuroinflammatory injury from the perspective of the immune response and sought to identify effective protective measures against the injury. The findings showed that toll-like receptor 4 (TLR4) was activated after RSV infection in human neuronal SY5Y cells. Furthermore, TLR4 activation induced autophagy and apoptosis in neuronal cells, promoted the formation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, and increased the secretion of downstream inflammatory cytokines such as interleukin-1ß (IL-1ß), interleukin-18 (IL-18) and tumour necrosis factor-α (TNF-α). Interestingly, blockade of TLR4 or treatment with exogenous melatonin significantly suppressed TLR4 activation as well as TLR4-mediated apoptosis, autophagy and immune responses. Therefore, we infer that melatonin may act on the TLR4 to ameliorate RSV-induced neuronal injury, which provides a new therapeutic target for RSV infection.

Mechanism by which HDAC3 regulates manganese induced H3K27ac in SH-SY5Y cells and intervention by curcumin.

Long-term excessive exposure to manganese can impair neuronal function in the brain, but the underlying pathological mechanism remains unclear. Oxidative stress plays a central role in manganese-induced neurotoxicity. Numerous studies have established a strong link between abnormal histone acetylation levels and the onset of various diseases. Histone deacetylase inhibitors and activators, such as TSA and ITSA-1, are often used to investigate the intricate mechanisms of histone acetylation in disease. In addition, recent experiments have provided substantial evidence demonstrating that curcumin (Cur) can act as an epigenetic regulator. Given these findings, this study aims to investigate the mechanisms underlying oxidative damage in SH-SY5Y cells exposed to MnCl2·4H2O, with a particular focus on histone acetylation, and to assess the potential therapeutic efficacy of Cur. In this study, SH-SY5Y cells were exposed to manganese for 24 h, were treated with TSA or ITSA-1, and were treated with or without Cur. The results suggested that manganese exposure, which leads to increased expression of HDAC3, induced H3K27 hypoacetylation, inhibited the transcription of antioxidant genes, decreased antioxidant enzyme activities, and induced oxidative damage in cells. Pretreatment with an HDAC3 inhibitor (TSA) increased the acetylation of H3K27 and the transcription of antioxidant genes and thus slowed manganese exposure-induced cellular oxidative damage. In contrast, an HDAC3 activator (ITSA-1) partially increased manganese-induced cellular oxidative damage, while Cur prevented manganese-induced oxidative damage. In summary, these findings suggest that inhibiting H3K27ac is a possible mechanism for ameliorating manganese-induced damage to dopaminergic neurons and that Cur exerts a certain protective effect against manganese-induced damage to dopaminergic neurons.

A rare olive compound oleacein functions as a TrkB agonist and mitigates neuroinflammation both in vitro and in vivo.

BACKGROUND: Neuroinflammation is widely acknowledged as a characteristic feature of almost all neurological disorders and specifically in depression- and anxiety-like disorders. In recent years, there has been significant attention on natural compounds with potent anti-inflammatory effects due to their potential in mitigating neuroinflammation and neuroplasticity. METHODS: In the present study, we aimed to evaluate the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. Our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response using human neuroblastoma cells (SH-SY5Y cells) and an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation. RESULTS: In SH-SY5Y cells, OC exhibited a significant dose-dependent increase in BDNF expression. This enhancement was absent when cells were co-treated with inhibitors of BDNF's receptor TrkB, as well as downstream molecules PI3K and MEK. Whole-transcriptomics analysis revealed that OC upregulated cell cycle-related genes under normal conditions, while downregulating inflammation-associated genes in LPS-induced conditions. Furthermore, surface plasmon resonance (SPR) assays demonstrated that OC exhibited a stronger and more stable binding affinity to TrkB compared to the positive control, 7,8-dihydroxyflavone. Importantly, bioluminescence imaging revealed that a single oral dose of OC significantly increased BDNF expression in the brains of Bdnf-IRES-AkaLuc mice. Furthermore, oral administration of OC at a dosage of 10 mg/kg body weight for 10 days significantly reduced immobility time in the tail suspension test compared to the LPS-treated group. RT-qPCR analysis revealed that OC significantly decreased the expression of pro-inflammatory cytokines Tnfα, Il6, and Il1ß, while simultaneously enhancing Bdnf expression, as well as both pro and mature BDNF protein levels in mice hippocampus. These changes were comparable to those induced by the positive control antidepressant drug fluoxetine. Additionally, microarray analysis of mouse brains confirmed that OC could counteract LPS-induced inflammatory biological events. CONCLUSION: Altogether, our study represents the first report on the potential antineuroinflammatory and antidepressant properties of OC via modulation of BDNF/TrkB neurotrophic activity. This finding underscores the potential of OC as a natural therapeutic agent for depression- and anxiety-related disorders.

Baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating ATP-sensitive potassium channels.

Dopaminergic neurons in the substantia nigra (SN) expressing SUR1/Kir6.2 type ATP-sensitive potassium channels (K-ATP) are more vulnerable to rotenone or metabolic stress, which may be an important reason for the selective degeneration of neurons in Parkinson's disease (PD). Baicalein has shown neuroprotective effects in PD animal models. In this study, we investigated the effect of baicalein on K-ATP channels and the underlying mechanisms in rotenone-induced apoptosis of SH-SY5Y cells. K-ATP currents were recorded from SH-SY5Y cells using whole-cell voltage-clamp recording. Drugs dissolved in the external solution at the final concentration were directly pipetted onto the cells. We showed that rotenone and baicalein opened K-ATP channels and increased the current amplitudes with EC50 values of 0.438 µM and 6.159 µM, respectively. K-ATP channel blockers glibenclamide (50 µM) or 5-hydroxydecanoate (5-HD, 250 µM) attenuated the protective effects of baicalein in reducing reactive oxygen species (ROS) content and increasing mitochondrial membrane potential and ATP levels in rotenone-injured SH-SY5Y cells, suggesting that baicalein protected against the apoptosis of SH-SY5Y cells by regulating the effect of rotenone on opening K-ATP channels. Administration of baicalein (150, 300 mg·kg-1·d-1, i.g.) significantly inhibited rotenone-induced overexpression of SUR1 in SN and striatum of rats. We conducted surface plasmon resonance assay and molecular docking, and found that baicalein had a higher affinity with SUR1 protein (KD = 10.39 µM) than glibenclamide (KD = 24.32 µM), thus reducing the sensitivity of K-ATP channels to rotenone. Knockdown of SUR1 subunit reduced rotenone-induced apoptosis and damage of SH-SY5Y cells, confirming that SUR1 was an important target for slowing dopaminergic neuronal degeneration in PD. Taken together, we demonstrate for the first time that baicalein attenuates rotenone-induced SH-SY5Y cell apoptosis through binding to SUR1 and activating K-ATP channels.

Natural pyrethrins induce cytotoxicity in SH-SY5Y cells and neurotoxicity in zebrafish embryos (Danio rerio).

Natural pyrethrins are widely used in agriculture because of their good insecticidal activity. Meanwhile, natural pyrethrins play an important role in the safety evaluation of pyrethroids as precursors for structural development of pyrethroid insecticides. However, there are fewer studies evaluating the neurological safety of natural pyrethrins on non-target organisms. In this study, we used SH-SY5Y cells and zebrafish embryos to explore the neurotoxicity of natural pyrethrins. Natural pyrethrins were able to induce SH-SY5Y cells damage, as evidenced by decreased viability, cycle block, apoptosis and DNA damage. The apoptotic pathway may be related to the involvement of mitochondria and the results showed that natural pyrethrins induced a rise in Capase-3 viability, Ca2+ overload, a decrease in adenosine triphosphate (ATP) and a collapse of mitochondrial membrane potential in SH-SY5Y cells. Natural pyrethrins may mediate DNA damage in SH-SY5Y cells through oxidative stress. The results showed that natural pyrethrins induced an increase in reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and catalase (CAT) activity, and induced a decrease in glutathione peroxidase (GPx) activity in SH-SY5Y cells. In vivo, natural pyrethrins induced developmental malformations in zebrafish embryos, which were mainly characterized by pericardial edema and yolk sac edema. Meanwhile, the results showed that natural pyrethrins induced damage to the Huc-GFP axis and disturbed lipid metabolism in the head of zebrafish embryos. Further results showed elevated ROS levels and apoptosis in the head of zebrafish embryos, which corroborated with the results of the cell model. Finally, the results of mRNA expression assay of neurodevelopment-related genes indicated that natural pyrethrins exposure interfered with their expression and led to neurodevelopmental damage in zebrafish embryos. Our study may raise concerns about the neurological safety of natural pyrethrins on non-target organisms.

In vitro exposure to PM2.5 of olfactory Ensheathing cells and SH-SY5Y cells and possible association with neurodegenerative processes.

PM2.5 exposure represents a risk factor for the public health. PM2.5 is able to cross the blood-alveolar and blood-brain barriers and reach the brain through three routes: nasal olfactory pathway, nose-brain pathway, blood-brain barrier pathway. We evaluated the effect of PM2.5 to induce cytotoxicity and reduced viability on in vitro cultures of OECs (Olfactory Ensheathing Cells) and SH-SY5Y cells. PM2.5 samples were collected in the metropolitan area of Catania, and the gravimetric determination of PM2.5, characterization of 10 trace elements and 16 polycyclic aromatic hydrocarbons (PAHs) were carried out for each sample. PM2.5 extracts were exposed to cultures of OECs and SH-SY5Y cells for 24-48-72 h, and the cell viability assay (MTT) was evaluated. Assessment of mitochondrial and cytoskeleton damage, and the assessment of apoptotic process were performed in the samples that showed lower cell viability. We have found an annual average value of PM2.5 = 16.9 µg/m3 and a maximum value of PM2.5 = 27.6 µg/m3 during the winter season. PM2.5 samples collected during the winter season also showed higher concentrations of PAHs and trace elements. The MTT assay showed a reduction in cell viability for both OECs (44%, 62%, 64%) and SH-SY5Y cells (16%, 17%, 28%) after 24-48-72 h of PM2.5 exposure. Furthermore, samples with lower cell viability showed a decrease in mitochondrial membrane potential, increased cytotoxicity, and also impaired cellular integrity and induction of the apoptotic process after increased expression of vimentin and caspase-3 activity, respectively. These events are involved in neurodegenerative processes and could be triggered not only by the concentration and time of exposure to PM2.5, but also by the presence of trace elements and PAHs on the PM2.5 substrate. The identification of more sensitive cell lines could be the key to understanding how exposure to PM2.5 can contribute to the onset of neurodegenerative processes.

Regulating effect of miR-132-3p on the changes of MAPK pathway in rat brains and SH-SY5Y cells exposed to excessive fluoride by targeting expression of MAPK1.

BACKGROUND: Although the changes of mitogen-activated protein kinase (MAPK) pathway in the central nervous system (CNS) induced by excessive fluoride has been confirmed by our previous findings, the underlying mechanism(s) of the action remains unclear. Here, we investigate the possibility that microRNAs (miRNAs) are involved in the aspect. METHODS: As a model of chronic fluorosis, SD rats received different concentrations of fluoride in their drinking water for 3 or 6 months and SH-SY5Y cells were exposed to fluoride. Literature reviews and bioinformatics analyses were used to predict and real-time PCR to measure the expression of 12 miRNAs; an algorithm-based approach was applied to identify multiply potential target-genes and pathways; the dual-luciferase reporter system to detect the association of miR-132-3p with MAPK1; and fluorescence in situ hybridization to detect miR-132-3p localization. The miR-132-3p inhibitor or mimics or MAPK1 silencing RNA were transfected into cultured cells. Expression of protein components of the MAPK pathway was assessed by immunofluorescence or Western blotting. RESULTS: In the rat hippocampus exposed with high fluoride, ten miRNAs were down-regulated and two up-regulated. Among these, miR-132-3p expression was down-regulated to the greatest extent and MAPK1 level (selected from the 220 genes predicted) was corelated with the alteration of miR-132-3p. Furthermore, miR-132-3p level was declined, whereas the protein levels MAPK pathway components were increased in the rat brains and SH-SY5Y cells exposed to high fluoride. MiR-132-3p up-regulated MAPK1 by binding directly to its 3'-untranslated region. Obviously, miR-132-3p mimics or MAPK1 silencing RNA attenuated the elevated expressions of the proteins components of the MAPK pathway induced by fluorosis in SH-SY5Y cells, whereas an inhibitor of miR-132-3p just played the opposite effect. CONCLUSION: MiR-132-3p appears to modulate the changes of MAPK signaling pathway in the CNS associated with chronic fluorosis.

Exploring the Regulation of Cytochrome P450 in SH-SY5Y Cells: Implications for the Onset of Neurodegenerative Diseases.

Human individual differences in brain cytochrome P450 (CYP) metabolism, including induction, inhibition, and genetic variation, may influence brain sensitivity to neurotoxins and thus participate in the onset of neurodegenerative diseases. The aim of this study was to explore the modulation of CYPs in neuronal cells. The experimental approach was focused on differentiating human neuroblastoma SH-SY5Y cells into a phenotype resembling mature dopamine neurons and investigating the effects of specific CYP isoform induction. The results demonstrated that the differentiation protocols using retinoic acid followed by phorbol esters or brain-derived neurotrophic factor successfully generated SH-SY5Y cells with morphological neuronal characteristics and increased neuronal markers (NeuN, synaptophysin, ß-tubulin III, and MAO-B). qRT-PCR and Western blot analysis showed that expression of the CYP 1A1, 3A4, 2D6, and 2E1 isoforms was detectable in undifferentiated cells, with subsequent increases in CYP 2E1, 2D6, and 1A1 following differentiation. Further increases in the 1A1, 2D6, and 2E1 isoforms following ß-naphthoflavone treatment and 1A1 and 2D6 isoforms following ethanol treatment were evident. These results demonstrate that CYP isoforms can be modulated in SH-SY5Y cells and suggest their potential as an experimental model to investigate the role of CYPs in neuronal processes involved in the development of neurodegenerative diseases.

Neuroprotection of Truncated Peptide IIAVE from Isochrysis zhanjiangensis: Quantum Chemical, Molecular Docking, and Bioactivity Studies.

Parkinson's disease (PD) is a progressive neurodegenerative disorder of the elderly for which there is no cure or disease-modifying therapy. Mitochondrial dysfunction and oxidative stress play a central role in dopaminergic neurodegeneration in PD. Therefore, antioxidants are considered a promising neuroprotective approach. In in vivo activity studies, 6-OHDA-induced oxidative stress in SH-SY5Y cells was established as a model of PD for cellular experiments. IIAVE (Ile-Ile-Ala-Val-Glu) was derived from Isochrysis zhanjiangensis octapeptide (IIAVEAGC), which has a small molecular weight. The structure and antioxidant activity of IIAVE were tested in a previous study and proved to have good antioxidant potential. In this study, the chemical properties of IIAVE were calculated using quantum chemical methods, including frontier molecular orbital (FMO), molecular electrostatic potential (MEP), natural population analysis (NPA), and global reactivity properties. The interaction of IIAVE with Bcl-2 and DJ-1 was investigated using the molecular docking method. The results showed that IIAVE promoted the activation of the Keap1/Nrf2 pathway and up-regulated the expression of the superoxide dismutase 1 (SOD-1) protein by inhibiting the level of reactive oxygen species (ROS) in cells. In addition, IIAVE inhibits ROS production and prevents 6-OHDA-induced oxidative damage by restoring mitochondrial membrane potential. Furthermore, IIAVE inhibited cell apoptosis by increasing the Bcl-2/Bax ratio and inhibiting the activation of Caspase-9 and Caspase-3. Thus, IIAVE may become a potential drug for the treatment and prevention of PD.

Different Directions of Effects of Polyclonal IgG Antibodies from Patients with Schizophrenia and Healthy Individuals on Cell Death In Vitro: A Pilot Study.

Numerous studies indicate the involvemen of oxidative stress in the pathogenesis of schizophrenia. It has been shown that the serum pool of antibodies in patients with schizophrenia contains catalytically active antibodies (abzymes) that have a wide range of activities, including redox properties. In the present work, the effects of IgGs-having oxidoreductase activities-isolated from the serum of patients with schizophrenia and healthy individuals were studied in vitro. The IgGs were purified by affinity chromatography followed by an SDS-PAGE analysis of homogeneity in a 4-18% gradient gel. The catalase and superoxide dismutase (SOD) activities of the IgGs were measured spectrophotometrically using a kinetic module. Human neuroblastoma SH-SY5Y cells were cultured with IgG at a final concentration of 0.2 mg/mL for 24 h. In a parallel experiment, tert-butyl hydroperoxide was used as an oxidative stressor. The number of dead cells after incubation was determined with fluorescent dyes, propidium iodide and Hoechst, by high-throughput screening on the CellInsight CX7 platform. A cytotoxic effect of the IgG from the schizophrenia patients on SH-SY5Y cells was detected after 24 h incubation. A correlation was found between the SOD activity of the IgGs and IgG-induced cell death. Under the induced oxidative stress, the cytotoxic effect of the IgG from the patients with schizophrenia on the SH-SY5Y cell line was five times stronger. Meanwhile, the IgG from the healthy individuals exerted a cytoprotective effect on the cultured cells, accompanied by high catalase activity. Thus, the observed influence on cell viability depends on the catalytic properties of the abzymes.

Diglycolic acid inhibits succinate dehydrogenase activity, depletes mitochondrial membrane potential, and induces inflammation in an SH-SY5Y neuroblastoma model of neurotoxicity in vitro.

Diethylene glycol is a toxic industrial solvent resulting in a well-defined toxidrome. Diglycolic acid (DGA) has been identified as the metabolite responsible for the nephrotoxicity and hepatotoxicity. These studies assess the mechanism of DGA-induced neurotoxicity, specifically addressing the known ability of DGA to chelate calcium (Ca2+) in solution and inhibit mitochondrial complex II. SH-SY5Y cells were seeded into 96-well plates to assess intracellular Ca2+ chelation, complex II activity, mitochondrial membrane potential (ΔΨm), ATP production, and release of inflammatory cytokines TNF-α and IL-1ß with 2-, 4-, 6-, 24-, and 48-h DGA exposure. Peak Ca2+ chelation occurred at 4 h in cells treated with 6.25-50 mM DGA; however, effects were transient. Complex II activity was significantly decreased at all DGA concentrations tested, with 12.5 mM DGA causing 80% inhibition and 25 and 50 mM DGA causing 97 and 100% inhibition, respectively. Subsequently, 12.5-50 mM DGA concentrations significantly decreased ΔΨm at all time points. 50 mM DGA significantly increased release of TNF-α and IL-1ß after 24 and 48 h with significantly decreased ATP production observed at the same time points and concentration. These studies demonstrate that the DGA-induced mechanism of SH-SY5Y cell death involves complex II inhibition leading to mitochondrial depolarization, and subsequent ATP depletion with accompanying inflammatory cytokine release. These results indicate a direct mechanism of DGA-induced neurotoxicity in vitro, similarly observed in other DEG-affected target organs.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease.

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aß1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aß1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aß1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

Development of a novel microfluidic perfusion 3D cell culture system for improved neuronal cell differentiation.

Three-dimensional (3D) cell cultures have recently gained popularity in the biomedical sciences because of their similarity to the in vivo environment. SH-SY5Y cells, which are neuronal cells and are commonly used to investigate neurodegenerative diseases, have particularly been reported to be differentiated as neuron-like cells expressing neuron-specific markers of mature neurons in static 3D culture environments when compared to static 2D environments, and those in perfusion environments have not yet been investigated. Microfluidic technology has provided perfusion environment which has more similarity to in vivo through mimicking vascular transportation of nutrients, but air bubbles entering into microchannels drastically increase instability of the flow. Furthermore, static incubation commonly used is incompatible with perfusion setup due to its air conditions, which is a critical huddle to the biologists. In the present study, we developed a novel microfluidic perfusion 3D cell culture system that overcomes the disturbance from air bubbles and intuitionally sets the incubation with the perfusion 3D culture. The system is capable of generating concentration gradients between 5 and 95% and air bubble traps were included to increase stability during incubation by collecting air bubbles. To evaluate the perfusion 3D culture, SH-SY5Y differentiation was examined in static 2D, static 3D, and perfusion 3D cultures. Our system supported significantly increased clustering of SH-SY5Y compared to static 2D and 3D methods, as well as increasing neurite growth rate. This novel system therefore supports differentiation of SH-SY5Y and can be used to more accurately model the in vivo environment during cell culture experiments.

Salsolinol Protects SH-SY5Y Cells Against MPP+ Damage and Increases Enteric S100-Immunoreactivity in Wistar Rats.

A dopamine derivative, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, known as salsolinol (SAL), has increasingly gained attention since its first detection in the urine of Parkinson's disease patients treated with levodopa, and has been proposed as a possible neurotoxic contributor to the disease. Yet, so far, the neurobiological role of SAL remains unclear. Thus, the main aims of our study were to compare the neurotoxic potential of SAL with MPP+ (1-methyl-4-phenylpyridinium ion) in vitro, and to examine intestinal and metabolic alterations following intraperitoneal SAL administration in vivo. In vitro, SH-SY5Y neuroblastoma cell line was monitored following MPP+ and SAL treatment. In vivo, Wistar rats were subjected to SAL administration by either osmotic intraperitoneal mini-pumps or a single intraperitoneal injection, and after two weeks, biochemical and morphological parameters were assessed. SH-SY5Y cells treated with MPP+ (1000 µM) and SAL (50 µM) showed increase in cell viability and fluorescence intensity in comparison with the cells treated with MPP+ alone. In vivo, we predominantly observed decreased collagen content in the submucosal layer, decreased neuronal density with comparable ganglionic area in the jejunal myenteric plexus, and increased glial S100 expression in both enteric plexuses, yet with no obvious signs of inflammation. Besides, glucose and triglycerides levels were lower after single SAL-treatment (200 mg/kg), and low- to high-density lipoprotein (LDL/HDL) ratio and aspartate to alanine aminotransferases (AST/ALT) ratio levels were higher after continuous SAL-treatment (200 mg/kg in total over 2 weeks). Low doses of SAL were non-toxic and exhibited pronounced neuroprotective properties against MPP+ in SH-SY5Y cell line, which supports the use of SAL as a reference compound for in vitro studies. In vivo results give insight into our understanding of gastrointestinal remodeling following intraperitoneal SAL administration, and might represent morphological correlates of a microglial-related enteric neurodegeneration and dopaminergic dysregulation.

Ursolic acid enhances autophagic clearance and ameliorates motor and non-motor symptoms in Parkinson's disease mice model.

Protein aggregation and the abnormal accumulation of aggregates are considered as common mechanisms of neurodegeneration such as Parkinson's disease (PD). Ursolic acid (UA), a natural pentacyclic triterpenoid compound, has shown a protective activity in several experimental models of brain dysfunction through inhibiting oxidative stress and inflammatory responses and suppressing apoptotic signaling in the brain. In this study, we investigated whether UA promoted autophagic clearance of protein aggregates and attenuated the pathology and characteristic symptoms in PD mouse model. Mice were injected with rotenone (1 mg · kg-1 · d-1, i.p.) five times per week for 1 or 2 weeks. We showed that rotenone injection induced significant motor deficit and prodromal non-motor symptoms accompanied by a significant dopaminergic neuronal loss and the deposition of aggregated proteins such as p62 and ubiquitin in the substantia nigra and striatum. Co-injection of UA (10 mg · kg-1 · d-1, i.p.) ameliorated all the rotenone-induced pathological alterations. In differentiated human neuroblastoma SH-SY5Y cells, two-step treatment with a proteasome inhibitor MG132 (0.25, 2.5 µM) induced marked accumulation of ubiquitin and p62 with clear and larger aggresome formation, while UA (5 µM) significantly attenuated the MG132-induced protein accumulation. Furthermore, we demonstrated that UA (5 µM) significantly increased autophagic clearance by promoting autophagic flux in primary neuronal cells and SH-SY5Y cells; UA affected autophagy regulation by increasing the phosphorylation of JNK, which triggered the dissociation of Bcl-2 from Beclin 1. These results suggest that UA could be a promising therapeutic candidate for reducing PD progression from the prodromal stage by regulating abnormal protein accumulation in the brain.

Neuroprotective effect of hyperoside in MPP+/MPTP -induced dopaminergic neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the pathological loss of nigrostriatal dopaminergic neurons, which causes an insufficient release of dopamine (DA) and then induces motor and nonmotor symptoms. Hyperoside (HYP) is a lignan component with anti-inflammatory, antioxidant, and neuroprotective effects. In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) were used to induce dopaminergic neurodegeneration. The results showed that HYP (100 µg/mL) reduced MPTP-mediated cytotoxicity of SH-SY5Y cells in vitro, and HYP [25 mg/(kg d)] alleviated MPTP-induced motor symptoms in vivo. HYP treatment reduced the contents of nitric oxide (NO), H2O2, and malondialdehyde (MDA), as well as the mitochondrial damage of dopaminergic neurons, both in vitro and in vivo. Meanwhile, HYP treatment elevated the levels of neurotrophic factors such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and recombinant cerebral dopamine neurotrophic factor in vivo, but not in vitro. Finally, Akt signaling was activated after the administration of HYP in MPP+/MPTP-induced dopaminergic neurodegeneration. However, the blockage of the Akt pathway with Akt inhibitor did not abolish the neuroprotective effect of HYP on DA neurons. These results showed that HYP protected the dopaminergic neurons from the MPP+- and MPTP-induced injuries, which did not rely on the Akt pathway.

PM2.5 induces autophagy-dependent ferroptosis by endoplasmic reticulum stress in SH-SY5Y cells.

Fine particulate matter (PM2.5 ) has been a global environmental problem threatening public health in recent years. PM2.5 exposure was associated with an increased risk of neurodegenerative diseases related to neuronal apoptosis. Ferroptosis is a nonapoptotic form of programmed the cell death, characterized by excess iron-dependent lipid peroxidation products. Whether PM2.5 could induce ferroptosis in cells and thus be involved in its neurotoxicity is unknown. In this study, we found that PM2.5 induced endoplasmic reticulum stress, apoptosis, autophagy, and ferroptosis in neuroblastoma human neuroblastoma cells (SH-SY5Y). Interestingly, ferroptosis was the predominant form of mortality in the presence of high doses of PM2.5 exposure. In addition, the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) inhibited PM2.5 -induced cellular autophagy, apoptosis, and ferroptosis. Autophagy inhibitors chloroquine (CQ) alleviated PM2.5 -induced ferroptosis but did not reverse apoptosis. We also found that inhibition of both endoplasmic reticulum stress and autophagy reversed the PM2.5 -induced increase in the expression level of cytophagy nuclear receptor coactivator 4 (NCOA4). Our results suggested that PM2.5 -induced ferroptosis in SH-SY5Y cells was autophagy-dependent ferroptosis due to endoplasmic reticulum stress, which might be associated with the elevation of iron content caused by NCOA4-mediated ferritin autophagy.

Organic extracts in PM2.5 are the major triggers to induce ferroptosis in SH-SY5Y cells.

As a major air pollutant, PM2.5 can induce apoptosis of nerve cells, causing impairment of the learning and memory capabilities of humans and animals. Ferroptosis is a newly discovered way of programmed cell death. It is unclear whether the neurotoxicity induced by PM2.5 is related to the ferroptosis of nerve cells. In this study, we observed the changes in ferroptosis hallmarks of SH-SY5Y cells after exposure to various doses (40, 80, and 160 µg/mL PM2.5) for 24 h, exposure to 40 µg/mL PM2.5 for various times (24, 48, and 72 h), as well as exposure to various components (Po, organic extracts; Pw, water-soluble extracts; Pc, carbon core component). The results showed that PM2.5 reduced the cell viability, the content of GSH, and the activity of GSH-PX and SOD in SH-SY5Y cells with exposure dose and duration increasing. On the other hand, PM2.5 increased the content of iron, MDA, and the level of lipid ROS in SH-SY5Y cells with exposure dose and duration increasing. Additionally, PM2.5 reduced the expression levels of HO-1, NRF2, SLC7A11, and GPX4. The ferroptosis inhibitors Fer-1 and DFO significantly increase the cells viabilities and significantly reversed the changes of other above ferroptosis hallmarks. We also observed the different effects on ferroptosis hallmarks in the SH-SY5Y cells exposed to PM2.5 (160 µg/mL) and its various components (organic extracts, water-soluble extracts, and carbon core) for 24 h. We found that only the organic extracts shared similar results with PM2.5 (160 µg/mL). This study demonstrated that PM2.5 induced ferroptosis of SH-SY5Y cells, and organic extracts might be the primary component that caused ferroptosis.

Mechanisms underlying citrinin-induced toxicity via oxidative stress and apoptosis-mediated by mitochondrial-dependent pathway in SH-SY5Y cells.

Citrinin (CIT) is a mycotoxin produced as a secondary product by the genera Aspergillus, Penicillium, Monascus, and other strains. CIT has the potential for contaminating animal feed and human food such as maize, wheat, rye, barley, oats, rice, cheese, and sake. Although CIT is primarily known as a nephrotoxic mycotoxin, it also affects other organs, including the liver and bone marrow, and its mechanisms of toxicity have not been clearly elucidated. There is a further lack of studies investigating the potential for CIT-induced neurotoxicity and its mechanisms. In the current study, SH-SY5Y human neuroblastoma cell line was treated with CIT for 24 h to evaluate various toxicological endpoints, such as reactive oxygen species (ROS) production and apoptosis induction. Results indicate that CIT has an IC50 value of 250.90 µM and cell proliferation decreased significantly at 50 and 100 µM CIT concentrations. These same concentrations also caused elevated ROS production (≥34.76%), apoptosis (≥9.43-fold) and calcium ion mobilization (≥36.52%) in the cells. Results show a significant decrease in the mitochondrial membrane potential (≥86.8%). We also found that CIT significantly upregulated the expression of some genes related to oxidative stress and apoptosis, while downregulating others. These results suggest that apoptosis and oxidative stress may be involved in the mechanisms underlying CIT-induced neurotoxicity.

Assessment of Global DNA Methylation in SH-SY5Y Cells Exposed to the Neonicotinoid Insecticides Imidacloprid and Thiamethoxam.

Neonicotinoid insecticides, known for their selectivity and low mammalian toxicity, have been widely used in recent years as alternatives to organophosphate insecticides. Although neonicotinoids are generally considered to be safe, data show that they can cause harmful effects on human and environmental health. Due to the lack of information on their mechanism of toxicity, the effects of imidacloprid and thiamethoxam on DNA methylation as the most used marker for epigenetic effects were investigated in human neuroblastoma (SH-SY5Y) cells. The cells were exposed to imidacloprid and thiamethoxam in concentrations of 100, 200, and 500 µM for 24 hours, then global DNA methylation and expression of genes involved in global DNA methylation (DNMT1, DNMT3a and DNMT3b) were investigated. Global DNA methylation significantly increased after imidacloprid exposure at 100 µM, and thiamethoxam exposures at 200 µM and 500 µM (>1.5-fold). Imidacloprid significantly decreased the expression of DNMT1 and DNMT3a, whereas thiamethoxam did not cause any significant changes in the expression of DNMT genes. Our findings suggested that alteration in global DNA methylation may be involved in the toxic mechanisms of imidacloprid and thiametoxam.