Butylphthalide Inhibits TLR4/NF-κB Pathway by Upregulation of miR-21 to Have the Neuroprotective Effect

Stroke is a disease with the highest incidence rate and the highest mortality rate in the world. The study aims to verify the neuroprotective effect of Butylphthalide. The mice were divided into sham group, MCAO group, and MCAO + Butylphthalide-treated group. The mice in MCAO + Butylphthalide-treated group were administered with 70 mg/kg Butylphthalide injection intraperitoneally after cerebral ischemia-reperfusion. The normal saline with the same volume was administered intraperitoneally for the mice in the MCAO group and sham group. The levels of miR-21 in brain tissue and cells were detected by qPCR. The OGD/R injury model of Neuro2A cells was used to simulate the hypoxic-ischemic environment of neurons in vitro. The proliferation rate of Neuro2A cells was detected with CCK-8. The production of ROS was detected with DCFH-DA. Compared with the mice in MCAO group, a decrease ( P < 0.01 ) was observed in the functional neurologic impairment scoring, cerebral infarction volume, and brain loss volume in the mice treated with MCAO + Butylphthalide, but an increase ( P < 0.01 ) was observed in the level of miR-21, which was positively correlated with functional neurologic impairment scoring (r = −0.8933, P < 0.001 ). MTT assay showed that the cell viability of OGD/R + Butylphthalide group was significantly higher than that of other groups ( P < 0.001 ), and the activity of ROS was significantly decreased ( P < 0.001 ). The WB results showed that, compared with OGD/R + miR-NC and control groups, the ratio of Bcl-2/Bax in OGD/R + Butylphthalide group and OGD/R + miR-21 mimics group was significantly higher ( P < 0.05 ), while the ratio of caspase-3/GAPDH was significantly lower ( P < 0.05 ). In conclusion, Butylphthalide has neuroprotective effect on the mouse model of MCAO. It may upregulate the level of miR-21 to inhibit neuronal apoptosis and ROS production and improve the proliferation activity. The specific mechanism may lie in inhibiting TLR4/NF-κB pathway.

Transmembrane 29 (Tmem29), a Newly Identified Molecule Showed Downregulation in Hypoxic-Ischemic Brain Damage

Transmembrane 29 (Tmem29) gene with unknown function is a gene located on the X chromosome of the mouse genome. The gene showed differential expression in the Vannucci neonatal hypoxic-ischemic mouse brain model. We found the gene expresses with different molecular forms, including a group of long non-coding RNA forming a family of transcripts. It was predominantly expressed in the testes, brain, and kidney of mouse. In vitro identification and functional characterization were carried out in Neuro2a cells. Using fluorescence microscopy, Tmem29 protein was found to be constitutively expressed in mouse cell lines of different origins. Oxygen glucose deprivation (OGD) induced apoptotic cell death in Neuro2a cells and was confirmed by activations of caspase 3. Tmem29 protein was found to be associated with cell death especially at the time points of caspase 3 activations. A similar response was obtained in glucose deprivation (GD) cultures suggesting Tmem29 response to a common mechanism induced by OGD and GD. Downregulation of Tmem29 was induced by OGD and GD, further validating its response to hypoxia-ischemia (HI) insults. Our findings contributed to further understanding of molecular events after hypoxic-ischemic insults and opens new avenues for developing protective and therapeutic strategies for hypoxic-ischemic encephalopathy or even pathological programmed cell death.

1,800 MHz Radiofrequency Electromagnetic Irradiation Impairs Neurite Outgrowth With a Decrease in Rap1-GTP in Primary Mouse Hippocampal Neurons and Neuro2a Cells

Background: With the global popularity of communication devices such as mobile phones, there are increasing concerns regarding the effect of radiofrequency electromagnetic radiation (RF-EMR) on the brain, one of the most important organs sensitive to RF-EMR exposure at 1,800 MHz. However, the effects of RF-EMR exposure on neuronal cells are unclear. Neurite outgrowth plays a critical role in brain development, therefore, determining the effects of 1,800 MHz RF-EMR exposure on neurite outgrowth is important for exploring its effects on brain development.Objectives: We aimed to investigate the effects of 1,800 MHz RF-EMR exposure for 48 h on neurite outgrowth in neuronal cells and to explore the associated role of the Rap1 signaling pathway.Material and Methods: Primary hippocampal neurons from C57BL/6 mice and Neuro2a cells were exposed to 1,800 MHz RF-EMR at a specific absorption rate (SAR) value of 4 W/kg for 48 h. CCK-8 assays were used to determine the cell viability after 24, 48, and 72 h of irradiation. Neurite outgrowth of primary hippocampal neurons (DIV 2) and Neuro2a cells was observed with a 20 × optical microscope and recognized by ImageJ software. Rap1a and Rap1b gene expressions were detected by real-time quantitative PCR. Rap1, Rap1a, Rap1b, Rap1GAP, and p-MEK1/2 protein expressions were detected by western blot. Rap1-GTP expression was detected by immunoprecipitation. The role of Rap1-GTP was assessed by transfecting a constitutively active mutant plasmid (Rap1-Gly_Val-GFP) into Neuro2a cells.Results: Exposure to 1,800 MHz RF-EMR for 24, 48, and 72 h at 4 W/kg did not influence cell viability. The neurite length, primary and secondary neurite numbers, and branch points of primary mouse hippocampal neurons were significantly impaired by 48-h RF-EMR exposure. The neurite-bearing cell percentage and neurite length of Neuro2a cells were also inhibited by 48-h RF-EMR exposure. Rap1 activity was inhibited by 48-h RF-EMR with no detectable alteration in either gene or protein expression of Rap1. The protein expression of Rap1GAP increased after 48-h RF-EMR exposure, while the expression of p-MEK1/2 protein decreased. Overexpression of constitutively active Rap1 reversed the decrease in Rap1-GTP and the neurite outgrowth impairment in Neuro2a cells induced by 1,800 MHz RF-EMR exposure for 48 h.Conclusion: Rap1 activity and related signaling pathways are involved in the disturbance of neurite outgrowth induced by 48-h 1,800 MHz RF-EMR exposure. The effects of RF-EMR exposure on neuronal development in infants and children deserve greater focus.

Ethanolic Fruit Extract of Emblica officinalis Suppresses Neuroinflammation in Microglia and Promotes Neurite Outgrowth in Neuro2a Cells

Inhibiting neuroinflammation and modulating neurite outgrowth could be a promising strategy to prevent neurological disorders. Emblica officinalis (EO) may be a potent agent against them. Although EO extract reportedly has anti-inflammatory properties in macrophages, there is limited knowledge about its neuroprotective activity by suppressing microglia-mediated proinflammatory cytokine production and inducing neurite outgrowth. The present study aimed to elucidate the effect of EO fruit extract on the lipopolysaccharide- (LPS-) induced neuroinflammation using microglial (BV2) and neuroblastoma (Neuro2a) cells. The results demonstrated that, in LPS-treated BV2 cells, EO fruit extract reduced nitric oxide, interleukin-6, and tumor necrotic factor-α production. It also enhanced the neurite length of Neuro2a cells, which was linked to the upregulation of TuJ1 and MAP2 expressions. In conclusion, these findings indicate that the ethanolic extract of EO fruits has promising neuroprotective potential to exhibit antineuroinflammation activity and accelerative effect on neurite outgrowth in vitro. Therefore, EO fruit extract can be considered a novel herbal medicine candidate for managing neuroinflammatory diseases.

Lidocaine, an anesthetic drug, protects Neuro2A cells against cadmium toxicity

Purpose: To investigate the neuroprotective effect of lidocaine in Neuro2A cells Methods: Differentiated N2a cells were used in this study. Cell viability and neuroprotection were assessed using dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue assays, while Bax/Bcl-2 expression was assayed by western blotting. Mitochondrial membrane potential, reactive oxygen species and calcium levels were measured using flow cytometry. Results: Lidocaine protected differentiated N2a cells against cadmium-induced toxicity, and also attenuated cadmium toxicity-induced changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and calcium (Ca2+) levels. Furthermore, Bax/Bcl-2 ratio, which was disrupted by cadmium, and cadmium-induced apoptosis, were reversed by lidocaine. Conclusion: Lidocaine protects differentiated N2a cells against cadmium-induced toxicity by reversing apoptosis. Thus, lidocaine is a potential neuroprotective agent.

Decoding the Neuroprotective Potential of Methyl Gallate-Loaded Starch Nanoparticles against Beta Amyloid-Induced Oxidative Stress-Mediated Apoptosis: An In Vitro Study

Alzheimer’s disease (AD) is a multifaceted neuronal disorder and a challenge to medical practitioners, as the blood–brain barrier (BBB) acts as a major obstacle for drug delivery to the brain. Development of a nanomaterial-based drug delivery system (DDS) paved a way to penetrate the BBB. Starch, a ubiquitous natural biopolymer, has received much attention as a DDS due to its biocompatibility, biodegradability and eco-friendly nature. The present study focuses on encapsulating methyl gallate (MG) within starch nanoparticles (starch-encapsulated MG (SEMG)) and assesses its neuroprotective potential against β-amyloid (Aβ)-induced toxicity, the key factor for AD pathogenesis in Neuro2A cells. SEMG showed potent acetylcholinesterase inhibitory, antioxidant activity and anti-amyloidogenic activity by attenuating the fibrillation of Aβ and destabilizing the preformed mature fibrils. Furthermore, SEMG also attenuated the cytotoxic effect induced by Aβ in Neuro2A cells (50% inhibitory concentration 18.25 ± 0.025 μg/mL) by mitigating reactive oxygen species (ROS)-mediated macromolecular damage, restoring mitochondrial membrane potential and attenuating apoptosis. Characterization of SEMG revealed amorphous rock-shaped structure with average particle size of 264.6 nm, exhibiting 83% loading efficiency and sustained release of drug, with 73% release within 24 h at physiological pH. Overall, the outcome of the present study signifies starch as a promising nanocarrier for the delivery of drugs for the treatment of AD.

BTBD9 is a novel component of IGF signaling and regulates manganese-induced dopaminergic dysfunction

Restless legs syndrome (RLS) is a common neurological disorder associated with iron deficiency and dopaminergic (DAergic) neuronal dysfunction. BTBD9 is a genetic risk factor for RLS. However, its molecular function remains largely unknown. Here, we report the interaction between BTBD9, manganese (Mn) and insulin/insulin-like growth factor (IGF) signaling in Caenorhabditis elegans, mouse Neuro2a cells and humans. We found that elevated Mn downregulated BTBD9 mRNA levels; in turn, BTBD9 expression attenuated Mn-induced cellular stress and dopaminergic neurodegeneration. As Mn is a known co-factor for insulin receptor and IGF-1 receptor, which activates IGF signaling, we posited that BTBD9 negatively regulates IGF signaling. Our results showed that the protective effects of BTBD9 against Mn toxicity were dependent on the forkhead box O (FOXO) protein. Furthermore, BTBD9 overexpression significantly elevated FOXO level and decreased PKB level, while phosphoinositide-dependent kinase-1 (PDK1) level remained unchanged. We conclude that BTBD9 acts as a key component in the IGF signaling pathway. Meanwhile, the roles of Mn in DAergic neurotoxicity and regulating BTBD9 shed new light on the etiology of RLS.

Molecular Characterization of Mouse CREB3 Regulatory Factor in Neuro2a Cells

We performed expression and functional analysis of mouse CREB3 regulatory factor (CREBRF) in Neuro2a cells by constructing several expression vectors. Overexpressed full-length CREBRF protein was stabilized by MG132; however, the intrinsic CREBRF expression in Neuro2a cells was negligible under all conditions. On the other hand, N- or C-terminal deletion of CREBRF influenced its stability. Cotransfection of CREBRF together with GAL4-tagged full-length CREB3 increased luciferase reporter activity, and only the N-terminal region of CREBRF was sufficient to potentiate luciferase activity. Furthermore, this positive effect of CREBRF was also observed in cells expressing GAL4-tagged cleaved CREB3, although CREBRF hardly influenced the protein stability of NanoLuc-tagged cleaved CREB3 or intracellular localization of EGFP-tagged one. In conclusion, this study suggests that CREBRF, a quite unstable proteasome substrate, positively regulates the CREB3 pathway, which is distinct from the canonical ER stress pathway in Neuro2a cells.