Exosomes isolated from citrus lemon: a promising candidate for the treatment of Alzheimer's disease.

Aim: To investigate the efficacy of exosome-like nanovesicles from citrus lemon (EXO-CLs) in combating oxidative stress associated with Alzheimer's disease. Materials & methods: EXO-CLs were isolated through differential ultracentrifugation, characterized for particle size and evaluated for antioxidant activity. Results: EXO-CLs exhibited a mean size of 93.77 ± 12.31 nm, demonstrated permeability across the blood-brain barrier (BBB) and displayed antioxidant activity comparable to ascorbic acid. Additionally, they were found to be non-toxic, with over 80% cell viability observed in SH-SY5Y cells. Conclusion: The study proposes that EXO-CLs could serve as an effective treatment for neurodegenerative diseases. This suggests a promising approach for targeted interventions in brain-related disorders, owing to the antioxidant properties and BBB permeability exhibited by EXO-CLs.

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Anticancer Potential of Novel Cinnamoyl Derivatives against U87MG and SHSY-5Y Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is probably the most malignant and aggressive brain tumor belonging to the class of astrocytomas. The considerable aggressiveness and high malignancy of GBM make it a tumor that is difficult to treat. Here, we report the synthesis and biological evaluation of eighteen novel cinnamoyl derivatives (3a-i and 4a-i) to obtain more effective antitumor agents against GBM. METHODS: The chemical structures of novel cinnamoyl derivatives (3a-i and 4a-i) were confirmed by NMR and MS analyses. The physicochemical properties and evaluation of the ADME profile of 3a-i and 4a-i were performed by the preADMETlab2.0 web program. Cinnamoyl derivatives 3a-i and 4a-i were tested in vitro for their cytotoxicity against the human healthy fibroblast (HDFa) cells using an MTT cell viability assay. Derivatives with no toxicity on HDFa cells were tested both on human glioblastoma (U87MG) and neuroblastoma (SHSY- 5Y) cells, chosen as an experimental model of brain tumors. Cell death mechanisms were analyzed by performing flow cytometry analyses. RESULTS: Cinnamoyl derivatives 3a-i and 4a-i showed good physicochemical and ADME properties suggesting that these compounds could be developed as oral drugs endowed with a high capability to cross the blood-brain barrier. Compounds (E)-1-methoxy-4-(2-(phenylsulfonyl)vinyl)benzene (2c) and (E)-N-benzyl-N-(2- (cyclohexylamino)-2-oxoethyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (3e) did not show cytotoxicity on healthy human fibroblast cells up to 100 µg/mL. The most anticarcinogenic molecule, compound 3e, emerged as the most potent anticancer candidate in this study. Flow cytometry results showed that compound 3e (25 µg/mL) application resulted in nearly 86% and 84% cytotoxicity in the U87MG and the SHSY-5Y cell lines, respectively. Compound 2c (25 µg/mL) resulted in 81% and 82% cytotoxicity in the U87MG and the SHSY-5Y cell lines, respectively. CONCLUSION: Cinnamoyl derivative 3e inhibits the proliferation of cultured U87MG and SHSY-5Y cells by inducing apoptosis. Further detailed research will be conducted to confirm these data in in vivo experimental animal models.

Neuroprotective effect of chlorogenic acid on Parkinson's disease like symptoms through boosting the autophagy in zebrafish.

Parkinson's disease (PD) is characterized by both motor and non-motor symptoms, including hypokinesia, postural instability, dopaminergic (DA) neurons loss, and α-synuclein (α-syn) accumulation. A growing number of patients show negative responses towards the current therapies. Thus, preventative or disease-modifying treatment agents are worth to further research. In recent years, compounds extracted from natural sources become promising candidates to treat PD. Chlorogenic acid (CGA) is a phenolic compound appearing in coffee, honeysuckle, and eucommia that showed their potential as antioxidants and neuroprotectors. In this study, we investigated the anti-PD activity of CGA by testing its effect on 1-methyl-4-phenyl-1-1,2,3,6-tetrahydropyridine (MPTP) zebrafish model of PD. It was shown that CGA relieved MPTP-induced PD-like symptoms including DA neurons and blood vessel loss, locomotion reduction, and apoptosis events in brain. Moreover, CGA modulated the expression of PD- and autophagy-related genes (α-syn, lc3b, p62, atg5, atg7, and ulk1b), showing its ability to promote the autophagy which was interrupted in the PD pathology. The unblocked effect of CGA on autophagy was further verified in 6-hydroxydopamine (6-OHDA)-modeled SHSY5Y cells. Our findings indicated that CGA might relieve PD by boosting the autophagy in neuronal cells that makes CGA a potential candidate for anti-PD treatment.

Development and Evaluation of Solid Lipid Nanoparticles for the Clearance of Aß in Alzheimer's Disease.

Aggregation of Amyloid-ß (Aß) leads to the formation and deposition of neurofibrillary tangles and plaques which is the main pathological hallmark of Alzheimer's disease (AD). The bioavailability of the drugs and their capability to cross the BBB plays a crucial role in the therapeutics of AD. The present study evaluates the Memantine Hydrochloride (MeHCl) and Tramiprosate (TMPS) loaded solid lipid nanoparticles (SLNs) for the clearance of Aß on SHSY5Y cells in rat hippocampus. Molecular docking and in vitro Aß fibrillation were used to ensure the binding of drugs to Aß. The in vitro cell viability study showed that the M + T SLNs showed enhanced neuroprotection against SHSY5Y cells than the pure drugs (M + T PD) in presence of Aß (80.35µM ± 0.455 µM) at a 3:1 molar ratio. The Box-Behnken Design (BBD) was employed to optimize the SLNs and the optimized M + T SLNs were further characterized by %drug entrapment efficiency (99.24 ± 3.24 of MeHCl and 89.99 ± 0.95 of TMPS), particle size (159.9 ± 0.569 nm), PDI (0.149 ± 0.08), Zeta potential (-6.4 ± 0.948 mV), Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and in vitro drug release. The TEM & AFM analysis showed irregularly spherical morphology. In vitro release of SLNs was noted up to 48 h; whereas the pure drugs released completely within 3 hrs. M + T SLNs revealed an improved pharmacokinetic profile and a 4-fold increase in drug concentration in the brain when compared to the pure drug. Behavioral tests showed enhanced spatial memory and histological studies confirmed reduced Aß plaques in rat hippocampus. Furthermore, the levels of Aß decreased in AlCl3-induced AD. Thus, all these noted results established that the M + T SLNs provide enhanced neuroprotective effects when compared to pure and individual drugs and can be a promising therapeutic strategy for the management of AD.

Ameliorative Effects by Hexagonal Boron Nitride Nanoparticles against Beta Amyloid Induced Neurotoxicity.

Alzheimer's disease (AD) is considered as the most common neurodegenerative disease. Extracellular amyloid beta (Aß) deposition is a hallmark of AD. The options based on degradation and clearance of Aß are preferred as promising therapeutic strategies for AD. Interestingly, recent findings indicate that boron nanoparticles not only act as a carrier but also play key roles in mediating biological effects. In the present study, the aim was to investigate the effects of different concentrations (0−500 mg/L) of hexagonal boron nitride nanoparticles (hBN-NPs) against neurotoxicity by beta amyloid (Aß1-42) in differentiated human SH-SY5Y neuroblastoma cell cultures for the first time. The synthesized hBN-NPs were characterized by X-ray diffraction (XRD) measurements, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Aß1-42-induced neurotoxicity and therapeutic potential by hBN-NPs were assessed on differentiated SH-SY5Y cells using MTT and LDH release assays. Levels of total antioxidant capacity (TAC) and total oxidant status (TOS), expression levels of genes associated with AD and cellular morphologies were examined. The exposure to Aß1-42 significantly decreased the rates of viable cells which was accompanied by elevated TOS level. Aß1-42 induced both apoptotic and necrotic cell death. Aß exposure led to significant increases in expression levels of APOE, BACE 1, EGFR, NCTSN and TNF-α genes and significant decreases in expression levels of ADAM 10, APH1A, BDNF, PSEN1 and PSENEN genes (p < 0.05). All the Aß1-42-induced neurotoxic insults were inhibited by the applications with hBN-NPs. hBN-NPs also suppressed the remarkable elevation in the signal for Aß following exposure to Aß1-42 for 48 h. Our results indicated that hBN-NPs could significantly prevent the neurotoxic damages by Aß. Thus, hBN-NPs could be a novel and promising anti-AD agent for effective drug development, bio-nano imaging or drug delivery strategies.

LncRNA-MIAT regulates the growth of SHSY5Y cells by regulating the miR-34-5p-SYT1 axis and exerts a neuroprotective effect in a mouse model of Parkinson's disease.

To examine the neuroprotective roles of lncRNA-MIAT in Parkinson's disease (PD). RNA sequencing expression profiles were utilized to screen the dysregulated lncRNAs in patients with PD and to explore the underlying molecular mechanisms by which the lncRNAs regulate the pathogenesis of PD. 6-hydroxydopamine-induced SH-SY5Y cell lines and a PD mouse model were used to prove how the overexpressing or knocking-down of MIAT produce a marked effect in both in vitro and in vivo experiments. Subsequently, the subcellular localization of MIAT was detected via RNA fluorescence in situ hybridization (FISH) assays. Quantitative PCR, as well as western blotting, were used to determine the expression levels of the associated genes and proteins. We utilized Cell Counting Kit-8 (CCK8) assays to measure the viability of the cells, and the apoptotic rate was determined using Annexin V-FITC/PI double staining. The expressions of tyrosine hydroxylase (TH) and Parkin were quantified in the substantia nigra using immunohistochemical staining. Also, TUNEL staining was performed to visualize the apoptotic cells in the substantia nigra. Compared with the normal rats, the downregulation of MIAT was observed in the cortex, hippocampus, substantia nigra, and striatum of the PD rats. Overexpression of MIAT exhibited a neuroprotective effect on the SH-SY5Y cells. Through RNA-sequencing of the PD mice treated with an overexpression of MIAT and through a differentially expressed genes analysis, it was hypothesized that MIAT could upregulate the expression of synaptotagmin-1 (SYT1) through sponging of miR-34-5p. Interactions between MIAT, miR-34-5p, and SYT1 were confirmed using RIP and dual-luciferase reporter assays. At the same time, the MIAT overexpression group exhibited elevated Parkin and TH protein levels, increased cell viability but a decreased apoptosis rate of the SH-SY5Y cells in contrast with the negative control (NC) group. In vivo, compared with the NC group, the overexpression of MIAT resulted in an increase in the positive rates of Parkin and TH, and the apoptosis was decreased in the PD mice. The behavioral test results showed that the motor coordination and autonomous activity of the mice were enhanced in the MIAT overexpression group compared with the NC group. LncRNA-MIAT regulates the growth of SHSY5Y cells by sponging miR-34-5p which targets SYT1 and exerts a neuroprotective effect in a mouse model of PD.

Venlafaxine Inhibits the Apoptosis of SHSY-5Y Cells Through Active Wnt/ß-Catenin Signaling Pathway.

OBJECTIVE: This study aimed to explore the mechanism of venlafaxine in regulating the apoptosis of SHSY-5Y cells induced by hypoxia. METHODS: The CoCl2-induced neuronal hypoxia model was established based on SHSY-5Y cells. The morphology and related protein expression of SHSY-5Y cells were detected by qPCR, ELISA and Western blot. RESULTS: Under the condition of hypoxia-induced by CoCl2, the expression of HIF-1α in SHSY-5Y cells was up-regulated and the expression of ß-catenin was down-regulated. After adding siRNA targeting HIF-1 α to the culture cell system, down-regulation of ß -catenin expression in SHSY-5Y cells was restored. This confirmed the existence of the "hypoxia-HIF-1α-Wnt/ß-catenin-depression" axis. Further studies have shown that venlafaxine can alleviate neuronal apoptosis induced by hypoxia by upregulating the Wnt/ß-catenin signaling pathway. CONCLUSION: Venlafaxine regulates apoptosis induced by hypoxia through the Wnt/ß-catenin signaling pathway, which provides a new theoretical basis for the treatment of depression.

Larrea tridentata Extract Mitigates Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Creosote bush (Larrea tridentata; LT) leaves extracts were tested for their potential efficacy to mitigate cellular oxidative stress on human SH-SY5Y cells. Here, the differential nuclear staining assay, a bioimager system, and flow cytometric protocols, concurrently with several specific chemicals, were used to measure the percentage of cell viability and several facets implicated in the cytoprotective mechanism of LT extracts. Initially, three LT extracts, prepared with different solvents, ethanol, ethanol:water (e/w), and water, were tested for their capacity to rescue the viability of cells undergoing aggressive H2O2-induced oxidative stress. Results indicate that the LT extract prepared with a mixture of ethanol:water (LT-e/w; 60:40% v/v) displayed the most effective cytoprotection rescue activity. Interestingly, by investigating the LT-e/w mechanism of action, it was found that LT-e/w extract decreases the levels of H2O2-provoked reactive oxidative species (ROS) accumulation, mitochondrial depolarization, phosphatidylserine externalization, caspase-3/7 activation, and poly (ADP-ribose) polymerase (PARP) cleavage significantly, which are hallmarks of apoptosis. Thus, out of the three LT extracts tested, our findings highlight that the LT-e/w extract was the most effective protective reagent on SH-SY5Y cells undergoing oxidative stress in vitro, functioning as a natural anti-apoptotic extract. These findings warrant further LT-e/w extract examination in a holistic context.

The Anti-Tumor Agent Sodium Selenate Decreases Methylated PP2A, Increases GSK3ßY216 Phosphorylation, Including Tau Disease Epitopes and Reduces Neuronal Excitability in SHSY-5Y Neurons.

Selenium application as sodium selenate was repeatedly shown to have anti-carcinogenic properties by increasing levels of the serine/ threonine protein phosphatase 2A (PP2A) in cancer cells. PP2A has a prominent role in cell development, homeostasis, and in neurons regulates excitability. PP2A, GSK3ß and Tau reside together in a complex, which facilitates their interaction and (dys)-function as has been reported for several neurological disorders. In this study we recorded maximum increase in total PP2A at 3 µM sodium selenate in a neuron cell line. In conjunction with these data, whole-cell electrophysiological studies revealed that this concentration had maximum effect on membrane potentials, conductance and currents. Somewhat surprisingly, the catalytically active form, methylated PP2A (mePP2A) was significantly decreased. In close correlation to these data, the phosphorylation state of two substrate proteins, sensitive to PP2A activity, GSK3ß and Tau were found to be increased. In summary, our data reveal that sodium selenate enhances PP2A levels, but reduces catalytic activity of PP2A in a dose dependent manner, which fails to reduce Tau and GSK3ß phosphorylation under physiological conditions, indicating an alternative route in the rescue of cell pathology in neurological disorders.

Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome.

Activated nucleotide binding to the oligonucleotide receptor protein 3 (NLRP3) inflammasome is possibly involved in the pathogenesis of Alzheimer's disease through oxidative stress and neurogenic inflammation. Low expression of the signal transducer and activator of transcription 3 (STAT3) gene may promote the occurrence of neurodegenerative diseases to some extent. To clarify the roles of the NLRP3 inflammasome and STAT3 expression in oxidative stress, (1) SHSY5Y cells were incubated with 1 mM H2O2 to induce oxidative stress injury, and the expression of human-cell-specific signal transduction, STAT3-shRNA silencing signal transduction and STAT3 were detected. Cells were pretreated with Ca2+ chelator BAPATA-AM (0.1 mM) for 30 minutes as a control. (2) Western blot assay was used to analyze the expression of caspase-1, NLRP3, signal transduction and STAT3. Enzyme-linked immunosorbent assay was used to analyze interleukin-1ß levels. Flow cytometry was carried out to calculate the number of apoptotic cells. We found that H2O2 treatment activated NLRP3 inflammasomes and decreased phosphorylation of signal transduction and STAT3 serine 727. BAPTA-AM pretreatment abolished the H2O2-induced activation of NLRP3 inflammasomes, caspase-1 expression, interleukin-1ß expression and apoptosis in SHSY5Y cells, and had no effect in cells with downregulated STAT3 expression by RNAi. The findings suggest that downregulation of signal transduction and STAT3 expression may enhance the oxidative stress mediated by NLRP3, which may not depend on the Ca2+ signaling pathway.

Modulation of IGF1R Signaling Pathway by GIGYF1 in High Glucose-Induced SHSY-5Y Cells.

Grb10 (growth factor receptor-bound protein 10)-interacting GYF protein 1 (GIGYF1) can modulate insulin-like growth factor 1 receptor (IGF1R) signaling pathway, which plays an important role in regulating diabetes-associated cognitive impairment, by linking to Grb10 adapter. However, it remains unclear whether endogenous GIGYF1 expression is associated with the development of diabetes-related cognitive impairment. In this study, we measured the expression level of GIGYF1, Grb10, phosphorylated IGF1R/IGF1R, phosphorylated AKT serine/threonine protein kinase/protein kinase B (AKT)/AKT, and phosphorylated extracellular signal-regulated kinase (ERK)/ERK in human neuroblastoma SHSY-5Y cells. Meanwhile, we detected cell apoptosis, proliferation, and migration. Our results showed that the percentage of apoptotic cells increased along with the increasing concentrations of glucose (0-200 mM). The expression of GIGYF1 had a significant increase in the presence of 25 mM concentration of glucose in SHSY-5Y cells. In addition, high glucose augmented the expression of IGF1R and Grb10, but decreased the expression of p-IGF1R, p-AKT, and p-ERK. However, GIGYF1 knockdown reversed the decline in the expression of p-IGF1R, p-AKT, and p-ERK. In addition, knocking down GIGYF1 promoted the proliferation and migration of SHSY-5Y cells, but inhibited the apoptosis in SHSY-5Y cells. These results demonstrate that the expression of GIGYF1 can regulate IGF1R signaling pathway in high glucose-induced SHSY-5Y cells.

Emulsified isoflurane induces release of cytochrome C in human neuroblastoma SHSY-5Y cells via JNK (c-Jun N-terminal kinases) signaling pathway.

A large number of studies have demonstrated that inhalation anesthetic isoflurane induced neural cell death by apoptosis in various cell and animal models. Emulsified isoflurane (EIso) is a new type of intravenous preparation of isoflurane that attracts increasing research attention as a promising clinical agent due to its both advantages as an intravenous and inhalation anesthetics medication. However, its safety and underlying molecular mechanism of neurotoxicity largely remain unknown. Therefore, it is meaningful to investigate the safety of EIso and to further elucidate its mechanism of anesthetic neurotoxicity. Human neuroblastoma SHSY-5Y cells were cultured, followed by a random exposure to one of three doses of EIso (0.56mmol/l, 1.12mmol/l, and 2.24mmol/l) or the corresponding intralipid as vehicle (0.3956µl/ml, 0.7912µl/ml and 1.5824µl/ml) for 6h, 12h or 24h. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay and the morphological changes were determined by a light microscope. We detect JNK, p-JNK and cytochrome C (cyto C) protein levels by western blotting. SP600125, a specific inhibitor of JNK, was used to detect the role of JNK pathway in the neurotoxicity of EIso. Our study showed that EIso reduced the viability of SHSY-5Y cells in a dose- and time-dependent manner. 0.56mmol/l EIso has no significant effects on cell viability, while 1.12mmol/l of EIso with 24-h and 2.24mmol/l of EIso with over 12-h exposure notably reduced cell viability. EIso dramatically increased the levels of p-JNK and cyto C. The JNK pathway inhibitor SP600125 significantly increased the cell viability of SHSY-5Y cells induced by EIso. These findings suggest that EIso induces damage in human neuroblastoma SHSY-5Y cells by JNK signaling pathway activation and cyto C release. SP600125 protects neural cells against EIso-induced injury. Our findings provide a new insight in the exploration of potential novel therapeutic strategies for the treatment of EIso-induced neurotoxicity and other neurodegenerative diseases.

Zinc oxide nanoparticles induce toxic responses in human neuroblastoma SHSY5Y cells in a size-dependent manner.

Due to the widespread applications of zinc oxide nanoparticles (ZnO NPs), the potential exposure of workers, consumers, and scientists to these particles has increased. This potential for exposure has attracted extensive attention in the science community. Many studies have examined the toxicological profile of ZnO NPs in the immune system, digestive system, however, information regarding the toxicity of ZnO NPs in the nervous system is scarce. In this study, we detected the cytotoxicity of two types of ZnO NPs of various sizes - ZnOa NPs and ZnOb NPs - and we characterized the shedding ability of zinc ions within culture medium and the cytoplasm. We found that reactive oxygen species played a crucial role in ZnO NP-induced cytotoxicity, likely because zinc ions were leached from ZnO NPs. Apoptosis and cytoskeleton changes were also toxic responses induced by the ZnO NPs, and ZnOb NPs induced more significant toxic responses than ZnOa NPs in SHSY5Y cells. In conclusion, ZnO NPs induced toxic responses in SHSY5Y cells in a size-dependent manner, which can probably be attributed to their ion-shedding ability.

Neurorestorative effects of eugenol, a spice bioactive: Evidence in cell model and its efficacy as an intervention molecule to abrogate brain oxidative dysfunctions in the streptozotocin diabetic rat.

Eugenol (EU), an active principle of cloves, is also widely distributed in various other plants (eg. basil, cinnamon, etc). While its antioxidant and anti-inflammatory properties are well established, biochemical insights related to its neuromodulatory potential in diabetic conditions are not clear. In the present study, initially we investigated its potential to modulate specific biochemical responses in SHSY5Y cells under experimentally -induced hyperglycemic condition. Co-exposure of cells with EU (5-10 µM) not only enhanced the cell viability, but significantly offset glucose -associated oxidative stress (as evidenced by diminished levels of reactive oxygen species and hydroperoxides). Further EU enhanced the reduced glutathione (GSH) levels and also ameliorated the levels of 3 - nitrotyrosine and expression of HSP70. We subsequently examined its efficacy to attenuate biochemical aberrations in brain regions of a streptozotocin (STZ) diabetic rat employing an intervention approach. Brain regions of EU treated (10 mg/kg bw/d, post 6 weeks of STZ) diabetic rats showed diminished levels of oxidative markers and protein carbonyls in both cytosolic and mitochondrial fractions. EU treatment caused enhanced activities of enzymic antioxidants and diminished both GSH and total thiols. Further, activities of complex I - III, succinate dehydrogenase and citrate synthase in brain regions were also significantly restored. Interestingly, EU treatment differentially attenuated the elevated activity of acetylcholinesterase and levels of calcium in brain regions. Collectively, based on the data obtained in in vitro and in vivo models, we hypothesize that EU may be employed as an adjuvant therapeutic molecule to alleviate complications under diabetic conditions.

N-acetylcysteineamide protects against manganese-induced toxicity in SHSY5Y cell line.

Manganese (Mn) is an essential trace element required for normal cellular functioning. However, overexposure of Mn can be neurotoxic resulting in the development of manganism, a syndrome that resembles Parkinson׳s disease. Although the pathogenetic basis of this disorder is unclear, several studies indicate that it is mainly associated with oxidative stress and mitochondrial energy failure. Therefore, this study is focused on (1) investigating the oxidative effects of Mn on neuroblastoma cells (SHSY5Y) and (2) elucidating whether a novel thiol antioxidant, N-acetylcysteineamide (NACA), provides any protection against Mn-induced neurotoxicity. Reactive oxygen species (ROS) were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and activities of glutathione reductase (GR) and glutathione peroxidase (GPx) were altered in the Mn-treated groups. Loss of mitochondrial membrane potential, as assessed by flow cytometry and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. However, pretreatment with NACA protected against Mn-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and mitochondrial membrane potential. NACA can potentially be developed into a promising therapeutic option for Mn-induced neurotoxicity. This article is part of a Special Issue entitled SI: Metals in neurodegeneration.