Downregulation of miR-337-3p in hypoxia/reoxygenation neuroblastoma cells increases KCTD11 expression.

Neurodegeneration is linked to the progressive loss of neural function and is associated with several diseases. Hypoxia is a hallmark in many of these diseases, and several therapies have been developed to treat this disease, including gene expression therapies that should be tightly controlled to avoid side effects. Cells experiencing hypoxia undergo a series of physiological responses that are induced by the activation of various transcription factors. Modulation of microRNA (miRNA) expression to alter transcriptional regulation has been demonstrated to be beneficial in treating multiple diseases, and in this study, we therefore explored potential miRNA candidates that could influence hypoxia-induced nerve cell death. Our data suggest that in mouse neuroblasts Neuro-2a cells with hypoxia/reoxygenation (H/R), miR-337-3p is downregulated to increase the expression of Potassium channel tetramerization domain containing 11 (KCTD11) and subsequently promote apoptosis. Here, we demonstrate for the first time that KCTD11 plays a role in the cellular response to hypoxia, and we also provide a possible regulatory mechanism by identifying the axis of miR-337-3p/KCTD11 as a promising candidate modulator of nerve cell survival after H/R exposure.

Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro.

BACKGROUND: Rabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro. METHODS: The mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay. RESULTS: CDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence. CONCLUSION: CDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

GPR137 Inhibits Cell Proliferation and Promotes Neuronal Differentiation in the Neuro2a Cells.

The orphan receptor, G protein-coupled receptor 137 (GPR137), is an integral membrane protein involved in several types of cancer. GPR137 is expressed ubiquitously, including in the central nervous system (CNS). We established a GPR137 knockout (KO) neuro2A cell line to analyze GPR137 function in neuronal cells. KO cells were generated by genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and cultured as single cells by limited dilution. Rescue cells were then constructed to re-express GPR137 in GPR137 KO neuro2A cells using an expression vector with an EF1-alpha promoter. GPR137 KO cells increased cellular proliferation and decreased neurite outgrowth (i.e., a lower level of neuronal differentiation). Furthermore, GPR137 KO cells exhibited increased expression of a cell cycle regulator, cyclin D1, and decreased expression of a neuronal differentiation marker, NeuroD1. Additionally, GPR137 KO cells exhibited lower expression levels of the neurite outgrowth markers STAT3 and GAP43. These phenotypes were all abrogated in the rescue cells. In conclusion, GPR137 deletion increased cellular proliferation and decreased neuronal differentiation, suggesting that GPR137 promotes cell cycle exit and neuronal differentiation in neuro2A cells. Regulation of neuronal differentiation by GPR137 could be vital to constructing neuronal structure during brain development.

Inhibition of AGEs formation, antioxidative, and cytoprotective activity of Sumac (Rhus typhina L.) tannin under hyperglycemia: molecular and cellular study.

It is well known that accumulation of advanced glycation ends products (AGEs) lead to various diseases such as diabetes and diabetic complications. In this study we showed that hydrolysable tannin from Sumac (Rhus typhina L.)-3,6-bis-O-di-O-galloyl-1,2,4-tri-O-galloyl-ß-D-glucose (C55H40O34) inhibited generation of glycation markers in bovine serum albumin such as AGEs, dityrosine, N'-formylkynurenine and kynurenine under high glucose treatment. This effect was accompanied by stabilization of the protein structure, as was shown using ATR-FT-IR spectroscopy and fluorescence methods. C55H40O34 exhibited also a neuroprotective effect in high glucose-exposed Neuro2A cells suppressing ROS formation and expression of phospho NF-κß and iNOS. At the same time C55H40O34 increased expression of heme oxygenase-1 and NAD(P)H: quinone oxidoreductase and mitochondrial complex I and V activities. Results from this study demonstrates a potent antiglycation activity of C55H40O34 in vitro and indicates its possible therapeutic application in glycation related diseases.

Toxic dinoflagellate Karenia mikimotoi induces apoptosis in Neuro-2a cells through an oxidative stress-mediated mitochondrial pathway.

The dinoflagellate Karenia mikimotoi is a toxic bloom-forming species that threatens aquaculture and public health worldwide. Previous studies showed that K. mikimotoi induces neurotoxicity; however, the underlying mechanism is poorly understood. In this study, three neural cell lines were used to investigate the potential neurotoxicity of K. mikimotoi. The tested cells were exposed to a ruptured cell solution (RCS) of K. mikimotoi at different concentrations (0.5 × 105, 1.0 × 105, 2.0 × 105, 4.0 × 105, and 6 × 105 cells mL-1) for 24 h, and the RCS decreased cell viabilities and promoted Neuro-2a (N2A) cell apoptosis in a dose-dependent manner. The underlying mechanism was further investigated in N2A cells. At the biochemical level, the RCS stimulated reactive oxygen species (ROS) and malondialdehyde (MDA) formation, decreased SOD activity, and reduced mitochondrial membrane potential (MMP). At the gene level, the moderate RCS treatment (2.0 × 105 cells mL-1) upregulated antioxidant response genes (e.g., nrf-2, HO-1, NQO-1, and cat) to alleviate RCS-induced oxidative stress, while the high RCS treatment (4.0 × 105 cells mL-1) downregulated these genes, thereby aggravating oxidative stress. Meanwhile, apoptosis-related genes (e.g., p53, caspase 3, and bax2) were significantly upregulated and the anti-apoptotic gene bcl2 was suppressed after RCS treatment. Western blotting results for Caspase 3, Bax2 and Bcl2 were consistent with the mRNA trends. These results revealed that K. mikimotoi RCS can induce neural cell apoptosis via the oxidative stress-mediated mitochondrial pathway, providing novel insights into the neurotoxicity of K. mikimotoi.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E.

OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Co-delivery of paclitaxel and etoposide prodrug by human serum albumin and PLGA nanoparticles: synergistic cytotoxicity in brain tumour cells.

The aims of this study were to develop co-delivery systems of paclitaxel (PTX) and etoposide prodrug (4'-O-benzyloxycarbonyl-etoposide, ETP-cbz) based on non-cross-linked human serum albumin (HSA) and poly(lactide-co-glycolide) nanoparticles and to evaluate the synergistic potential of these drugs in vitro. The nanoformulations were prepared by the high-pressure homogenisation technique and characterised using DLS, TEM, SEM, AFM, HPLC, CZE, in-vitro release, and cytotoxicity in human and murine glioma cells. All nanoparticles had 90-150 nm in size and negative ζ-potentials. The Neuro2A cells were the most sensitive to both HSA- and PLGA-based co-delivery systems (IC50 0.024 µM and 0.053 µM, respectively). The drugs' synergistic effect (combination index < 0.9) was observed in the GL261 cells for both types of co-delivery formulations and in the Neuro2A cells for the HSA-based system. These nanodelivery systems may be useful to improve combination chemotherapy for brain tumour treatment. To our knowledge, this is the first report describing the non-cross-linked HSA-based co-delivery nanosuspension which was prepared using nab™ technology.

CDK5 inhibition improves glucose uptake in insulin-resistant neuronal cells via ERK1/2 pathway.

Role of CDK5 and its inhibition in various neuronal processes and functions are well established. However, role of CDK5 and its inhibition in neuronal insulin-signaling and-resistance is not yet explored. In the present study, we investigated the effect of CDK5 inhibition in neuronal insulin signaling, specifically insulin-stimulated glucose uptake. CDK5 expression in neuro-2a cells was increased under insulin-resistant state, developed by chronic treatment of insulin, confirming the crucial role of CDK5 in insulin resistance in neuronal cells. However, whether increased expression of CDK5 in hyperinsulinemia-mediated insulin-resistant conditions is a cause or a consequence, is still an unanswered question. We showed that CDK5 inhibition did not affect basal insulin signaling; however, insulin-stimulated glucose uptake enhanced in insulin-resistant cells. Moreover, CDK5 inhibition could improve glucose uptake, the ultimate outcome of insulin signaling, in insulin-resistant neuro-2a cells. We first time showed that CDK5 inhibition by roscovitine could ameliorate insulin resistance and increase glucose uptake in neuronal cells via ERK1/2 pathway. Our study provides intriguing insights about the effect of CDK5 inhibition on neuronal insulin resistance and opens up a new paradigm to develop new therapeutic strategies for neuronal insulin resistance and associated pathophysiological conditions.

Carvacrol abates NLRP3 inflammasome activation by augmenting Keap1/Nrf-2/p62 directed autophagy and mitochondrial quality control in neuropathic pain.

Objectives: We aimed to evaluate the effect of carvacrol (CRC), a phenolic monoterpene with high nutritional value on NLRP3 activation against chronic constriction injury (CCI) of sciatic nerve induced neuropathic pain (NP) in rats and in lipopolysacharide (LPS) induced neuroinflammation in neuro2a (N2A) cells. Methods: NP was induced in male SD rats by performing CCI and CRC (30 and 60 mg/kg, p.o) was administered for 14 days. Behavioural and functional parameters were evaluated using standard procedures. Various molecular experimentations were conducted to evaluate the efficacy of CRC against CCI induced neuropathy and in LPS (1 µg/ml) primed and ATP (5 µM) treated N2A cells.Results: CCI resulted in marked development of hyperalgesia and allodynia. Further, CCI rats, LPS and ATP treated N2A cells showed enhanced expression of NLRP3, ASC, Caspase-1 and IL-1ß. In addition, CCI rats exhibited diminished levels of Nrf-2 with an increase in Keap1 expression. Also, CCI animals manifested with compromised mitochondrial function along with decreased autophagy markers and enhanced p62 levels when compared to sham rats. However, CRC administration significantly ameliorated these changes suggesting NLRP3 inhibition by CRC may be attributed to activation of autophagy via Keap1/Nrf-2/p62 forward feedback loop and augmentation of mitochondrial quality control. Intriguingly, pretreatment of CRC (50 and 100 µM) to LPS and ATP treated N2A cells resulted in decreased colocalization of NLRP3 and ASC.Discussion: These findings revealed the neuroprotective potential of CRC against CCI induced NP and delineate the critical role of autophagy and mitochondrial quality control in NLRP3 regulation.

Evaluation of BPA effects on autophagy in Neuro-2a cells.

Bisphenol A (BPA), which is used for the industrial production of polycarbonate plastics and epoxy resins, is found in many commercially available products. Plasticizer BPA produces chemical substances worldwide, and knowledge of its effects on humans and animals is increasing. In the present work, the morphology of cells was observed by optical microscopy and phalloidin staining to evaluate the toxic effect of BPA on Neuro-2a cells. Autophagy has an important role in the regulation of cell metabolism. To study the effect of BPA on the autophagy in Neuro-2a cells, the expression distribution of LC3 was detected by immunofluorescence, and the expression levels of p62 and Beclin1 were determined using western blot and quantitative real-time PCR (qRT-PCR), respectively. Optical microscopy and phalloidin staining revealed that the cells became rounded and small and that the dendritic spine of the cells were reduced at high BPA doses. Immunofluorescence analysis demonstrated that the expression of LC3 fluorescence intensity was weak at increasing BPA concentrations. Western blot results showed that the relative expression of protein p62 increased significantly and that the relative expression levels of the Beclin1 and the LC3 proteins significantly decreased with increasing BPA concentration. qRT-PCR results showed that the relative expression level of autophagy-related p62 mRNA increased significantly and that the relative expression level of Beclin1 mRNA decreased significantly with increasing BPA concentration. The above results indicated that BPA treatment exerted dose-dependent toxic effects on Neuro-2a cells, and BPA inhibited the autophagy level of Neuro-2a cells, thereby providing a new perspective in studying the toxic effect of BPA on Neuro-2a cells.

Amylin Protein Expression in the Rat Brain and Neuro-2a Cells.

The localization and expression of amylin protein in the rodent brain and mouse neuroblastoma Neuro-2a (N2a) are less widely known. Thus, this study investigated the expression distribution of amylin in the rat brain and N2a treated with steroid hormones. Amylin protein was identified in the olfactory bulb, cerebral cortex, dentate gyrus, thalamus, hypothalamus, ventral tegmental area (VTA), cerebellum, and brain stem in the rat brain. Additionally, the amylin protein was localized with the mature neurons of the cerebral cortex and dopaminergic neurons of the VTA. Progesterone (P4) and dexamethasone (Dex) significantly decreased, and 17ß-estradiol (E2) increased the amylin protein level in the cerebral cortex. The P4 receptor antagonist RU486 significantly influenced the effects of P4 and Dex, and the E2 receptor antagonist ICI 182,780 slightly changed E2's effect. Amylin protein expression was significantly reduced in the VTA by P4 and Dex, and its expression was changed only following P4 plus RU486 treatment. It was confirmed for the first time that amylin protein is strongly expressed in the cytoplasm in N2a cells using immunofluorescent staining. P4 increased the levels of amylin, and RU486 treatment decreased them. Dex significantly increased the levels of amylin protein. RU486 treatment reversed the effects of Dex. Therefore, amylin protein is expressed in the cerebral cortex neurons and dopaminergic neurons of the VTA of the immature rat brain. P4 and Dex influence the expression of amylin protein in the rat brain and N2a cells.

Pax3 inhibits Neuro-2a cells proliferation and neurite outgrowth.

Pax3 and Pax7 are closely related transcription factors that are widely expressed in the developing nervous system and somites. During the normal development in the central nervous system (CNS), Pax3 and Pax7 are mainly expressed in the dorsal part of the neural tube. Further analysis revealed that Pax3 and Pax7 shared redundant functions in the spinal cord development. However, it is still unknown whether Pax3 and Pax7 play a role in neuronal differentiation. In this study, Pax3 and Pax7 genes were overexpressed in Neuro-2a, the mouse neuroblastoma cell line. CCK-8 and EdU assay results showed that overexpression of Pax3 inhibited cell viability and proliferation of Neuro-2a cells, whereas the overexpression of Pax7 had no significant difference on their cell viability and proliferation. Overexpression of Pax3 not only increased the percentage of cells in the S phase and G0/G1 phase, but also decreased that in the G2 phase. Moreover, the total neurite lengths of Neuro-2a cells were significantly shorter in Pax3 overexpressed group than those in negative control group and showed no significant difference between Pax7 overexpressed group and negative control group. These results suggested that Pax3 not only inhibited the cell viability and proliferation but also affected the cell cycle and the neurite outgrowth of Neuro-2a cells. RNA sequencing analysis showed up-regulated genes in Pax3 overexpressed group were involved in cell cycle machinery, which may reveal the potential mechanism of Neuro-2a cells proliferation.

The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory molecules.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a member of a family of lipid phosphatase related proteins, integral membrane proteins characterized by six transmembrane domains. This family of proteins is enriched in the brain and recent data indicate potential pleiotropic functions in several different contexts. An inherent ability of this family of proteins is to induce morphological changes, and we have previously reported that members of this family interact with each other and may function co-operatively. However, the function of PLPPR1 is not yet understood. Here we show that the expression of PLPPR1 reduces the inhibition of neurite outgrowth of cultured mouse hippocampal neurons by chondroitin sulfate proteoglycans and the retraction of neurites of Neuro-2a cells by lysophosphatidic acid (LPA). Further, we show that PLPPR1 reduces the activation of Ras homolog family member A (RhoA) by LPA in Neuro-2a cells, and that this is because of an association of PLPPR1with the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI1). These results establish a novel signaling pathway for the PLPPR1 protein.

AUF1, an mRNA decay factor, has a discordant role in Cpeb1 expression.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.

Phospholipid phosphatase related 1 (PLPPR1) increases cell adhesion through modulation of Rac1 activity.

Phospholipid Phosphatase-Related Protein Type 1 (PLPPR1) is a six-transmembrane protein that belongs to the family of plasticity-related gene proteins, which is a novel brain-specific subclass of the lipid phosphate phosphatase superfamily. PLPPR1-5 have prominent roles in synapse formation and axonal pathfinding. We found that PLPPR1 overexpression in the mouse neuroblastoma cell line (Neuro2a) results in increase in cell adhesion and reduced cell migration. During migration, these cells leave behind long fibrous looking extensions of the plasma membrane causing a peculiar phenotype. Cells expressing PLPPR1 showed decreased actin turnover and decreased disassembly of focal adhesions. PLPPR1 also reduced active Rac1, and expressing dominant negative Rac1 produced a similar phenotype to overexpression of PLPPR1. The PLPPR1-induced phenotype of long fibers was reversed by introducing constitutively active Rac1. In summary, we show that PLPPR1 decreases active Rac1 levels that leads to cascade of events which increases cell adhesion.

Sulfo-Gambierones, Two New Analogs of Gambierone Produced by Gambierdiscus excentricus.

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa. Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic Gambierdiscus species using Neuro2a cell-based assay identified G. excentricus as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated G. excentricus extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of G. excentricus due to their sensitive detection by mass spectrometry.

Discovery of a Novel Acetylcholinesterase Inhibitor by Fragment-Based Design and Virtual Screening.

Despite extensive and intensive research efforts in recent decades, there is still no effective treatment for neurodegenerative diseases. On this background, the use of drugs inhibiting the enzyme acetylcholinesterase (AChE) remains an eternal evergreen in the symptomatic treatment of mild to moderate cognitive impairments. Even more, the cholinergic hypothesis, somewhat forgotten in recent years due to the shift in focus on amyloid cascade, is back to life, and the search for new, more effective AChE inhibitors continues. We generated a fragment-based library containing aromatic moieties and linkers originating from a set of novel AChE inhibitors. We used this library to design 1220 galantamine (GAL) derivatives following the model GAL (binding core) - linker (L) - aromatic fragment (Ar). The newly designed compounds were screened virtually for blood-brain barrier (BBB) permeability and binding to AChE. Among the top 10 best-scored compounds, a representative lead molecule was selected and tested for anti-AChE activity and neurotoxicity. It was found that the selected compound was a powerful non-toxic AChE inhibitor, 68 times more active than GAL, and could serve as a lead molecule for further optimization and development.

Neurotoxicity of ß-HgS differs from environmental mercury pollutants (MeHgCl and HgCl2) in Neuro-2a cell.

ß-HgS, differing from environmental mercury pollutants (MeHgCl and HgCl2) in chemical form, is used as traditional medicine in Asian countries for thousands of years. In this study, Neuro-2a cells were exposed to ß-HgS, MeHgCl and HgCl2 (5 µM) for 6-24 h. The cell viability of ß-HgS was higher than MeHgCl with 25.9% and 72.4% in 12 h and 24 h respectively. As the incubation time increased, MeHgCl had obvious damage to cell morphology, decreased the ratio of Bcl-2 and Bak and increased the expressions of TNF-α, IL-6 and IL-1ß significantly. Furthermore, the expressions of IL-1ß and IL-6 in HgCl2 group were increased significantly in 6 h and 24 h. The apoptotic rates in MeHgCl and HgCl2 group were respectively higher than ß-HgS with 32.2% and 7.30% in 24 h. Our findings indicate that ß-HgS is much less neurotoxicity than MeHgCl and HgCl2 in Neuro-2a cells.

Synthesis, Cytotoxic Activity Evaluation and Quantitative Structure-Activity Analysis of Substituted 5,8-Dihydroxy-1,4-Naphthoquinones and their O- and S-Glycoside Derivatives Tested Against Neuro-2a Cancer Cells.

Based on 6,7-substituted 2,5,8-trihydroxy-1,4-naphtoquinones (1,4-NQs) derived from sea urchins, five new acetyl-O-glucosides of NQs were prepared. A new method of conjugation of per-O-acetylated 1-mercaptosaccharides with 2-hydroxy-1,4-NQs through a methylene spacer was developed. Methylation of 2-hydroxy group of quinone core of acetylthiomethylglycosides by diazomethane and deacetylation of sugar moiety led to 28 new thiomethylglycosidesof 2-hydroxy- and 2-methoxy-1,4-NQs. The cytotoxic activity of starting 1,4-NQs (13 compounds) and their O- and S-glycoside derivatives (37 compounds) was determined by the MTT method against Neuro-2a mouse neuroblastoma cells. Cytotoxic compounds with EC50 = 2.7-87.0 µM and nontoxic compounds with EC50 > 100 µM were found. Acetylated O- and S-glycosides 1,4-NQs were the most potent, with EC50 = 2.7-16.4 µM. Methylation of the 2-OH group innaphthoquinone core led to a sharp increase in the cytotoxic activity of acetylated thioglycosidesof NQs, which was partially retained for their deacetylated derivatives. Thiomethylglycosides of 2-hydroxy-1,4-NQs with OH and MeO groups in quinone core at positions 6 and 7, resprectively formed a nontoxic set of compounds with EC50 > 100 µM. A quantitative structure-activity relationship (QSAR) model of cytotoxic activity of 22 1,4-NQ derivatives was constructed and tested. Descriptors related to the cytotoxic activity of new 1,4-NQ derivatives were determined. The QSAR model is good at predicting the activity of 1,4-NQ derivatives which are unused for QSAR models and nontoxic derivatives.