1321N1 Cells Research Articles

Exploring P2X receptor activity: A journey from cellular impact to electrophysiological profiling

The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders.

JP4-039, a Mitochondria-Targeted Nitroxide, Mitigates the Effect of Apoptosis and Inflammatory Cell Migration in the Irradiated Mouse Retina.

We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.

Pharmacological differences between human and mouse P2X4 receptor explored using old and new tools

There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antagonists for P2X4, though agonist selectivity is less well studied. As there are some known pharmacological differences between P2X receptors from different species, it is important to understand these differences when designing a pharmacological strategy to probe P2X4 function in human tissue and mouse models. Here, we provide a systematic comparison of agonist and antagonist pharmacology in 1321N1 cells expressing either human or mouse P2X4 orthologues. We identify a rank order of agonist potency of ATP > 2-MeSATP > αβmeATP = BzATP > CTP = γ-[(propargyl)-imido]-ATP for human P2X4 and ATP > 2-MeSATP = CTP > ATPγS = γ-[(propargyl)-imido]-ATP = BzATP for mouse. Human P2X4 is not activated by ATPγS but can be activated by αβmeATP. We identify a rank order of antagonist potency of BAY-1797 = PSB-12062 = BX-430 > 5-BDBD > TNP-ATP = PPADS for human P2X4 and BAY-1797 > PSB-12062 = PPADS > TNP-ATP for mouse. Mouse P2X4 is not antagonised by 5-BDBD or BX-430. The study reveals key pharmacological differences between human and mouse P2X4, highlighting caution when selecting tools for comparative studies between human and mouse and ascribing cellular responses of some commonly used agonists to P2X4.

Protective roles of peroxiporins AQP0 and AQP11 in human astrocyte and neuronal cell lines in response to oxidative and inflammatory stressors.

In addition to aquaporin (AQP) classes AQP1, AQP4 and AQP9 known to be expressed in mammalian brain, our recent transcriptomic analyses identified AQP0 and AQP11 in human cortex and hippocampus at levels correlated with age and Alzheimer's disease (AD) status; however, protein localization remained unknown. Roles of AQP0 and AQP11 in transporting hydrogen peroxide (H2O2) in lens and kidney prompted our hypothesis that up-regulation in brain might similarly be protective. Established cell lines for astroglia (1321N1) and neurons (SHSY5Y, differentiated with retinoic acid) were used to monitor changes in transcript levels for human AQPs (AQP0 to AQP12) in response to inflammation (simulated with 10-100 ng/ml lipopolysaccharide [LPS], 24 h), and hypoxia (5 min N2, followed by 0 to 24 h normoxia). AQP transcripts up-regulated in both 1321N1 and SHSY5Y included AQP0, AQP1 and AQP11. Immunocytochemistry in 1321N1 cells confirmed protein expression for AQP0 and AQP11 in plasma membrane and endoplasmic reticulum; AQP11 increased 10-fold after LPS and AQP0 increased 0.3-fold. In SHSY5Y cells, AQP0 expression increased 0.2-fold after 24 h LPS; AQP11 showed no appreciable change. Proposed peroxiporin roles were tested using melondialdehyde (MDA) assays to quantify lipid peroxidation levels after brief H2O2. Boosting peroxiporin expression by LPS pretreatment lowered subsequent H2O2-induced MDA responses (∼50%) compared with controls; conversely small interfering RNA knockdown of AQP0 in 1321N1 increased lipid peroxidation (∼17%) after H2O2, with a similar trend for AQP11 siRNA. Interventions that increase native brain peroxiporin activity are promising as new approaches to mitigate damage caused by aging and neurodegeneration.

Pioneering first-in-class FAAH-HDAC inhibitors as potential multitarget neuroprotective agents.

Aiming to simultaneously modulate the endocannabinoid system (ECS) functions and the epigenetic machinery, we selected the fatty acid amide hydrolase (FAAH) and histone deacetylase (HDAC) enzymes as desired targets to develop potential neuroprotective multitarget-directed ligands (MTDLs), expecting to achieve an additive or synergistic therapeutic effect in oxidative stress-related conditions. We herein report the design, synthesis, and biological evaluation of the first-in-class FAAH-HDAC multitarget inhibitors. A pharmacophore merging strategy was applied, yielding 1-phenylpyrrole-based compounds 4a-j. The best-performing compounds (4c, 4f, and 4h) were tested for their neuroprotective properties in oxidative stress models, employing 1321N1 human astrocytoma cells and SHSY5 human neuronal cells. In our preliminary studies, compound 4h stood out, showing a balanced nanomolar inhibitory activity against the selected targets and outperforming the standard antioxidant N-acetylcysteine in vitro. Together with 4f, 4h was also able to protect 1321N1 cells from tert-butyl hydroperoxide or glutamate insult. Our study may provide the basis for the development of novel MTDLs targeting the ECS and epigenetic enzymes.

Helicobacter pylori infection contributes to the expression of Alzheimer's disease-associated risk factors and neuroinflammation

Over time, mounting evidence has demonstrated extra-gastric manifestations of Helicobacter pylori infection. As such, a number of studies demonstrated the potential contribution of H. pylori infection to the incidence and progression of Alzheimer's disease (AD). Considering unanswered questions regarding the effect of H. pylori infection on brain activity, we sought to investigate the impact of H. pylori infection on the expression of AD-associated risk factors. We used two H. pylori clinical strains obtained from two patients with peptic ulcer and evaluated their influence on the expression level of AD-associated genes (APP, ApoE2, ApoE4, ABCA7, BIN1, Clu, CD33) and genes for inflammatory markers (TLR-4, IL-8, TNF-α) by RT-qPCR in human glioblastoma (U87MG) and astrocyte (1321N1) cell lines. The expression of inflammatory cytokines was further assessed by ELISA assay. The exposure of U97MG and 1321N1 cells to H. pylori strains resulted in a significant enhancement in the expression level of the risk allele ApoE4, while reducing the expression of the protective allele ApoE2. H. pylori infection remarkably increased the expression level of main AD-associated risk genes, and also pro-inflammatory cytokines. Furthermore, we noticed a substantial elevation in the mRNA expression level of transmembrane receptor TLR-4 following H. pylori infection. Our findings presented the potential for H. pylori to stimulate the expression of AD-associated risk genes and trigger neuroinflammation in the brain tissue. This, in principle, leads to the recommendation that AD patients should perhaps test for H. pylori infection and receive treatments upon positive detection.

Suppressive Effect of Chemically Induced Hypoxia on Glioblastoma Cell Proliferation.

Glioblastoma is a tumor characterized by pronounced hypoxia. Hypoxia produces diverse effects on tumor cells, and the results of experimental studies available so far are contradictory. In vitro hypoxia can be modeled in two ways: by reducing the level of atmospheric oxygen (physically induced hypoxia) or by using hypoxia-inducing chemicals such as cobalt chloride (II) (CoCl2) (chemically induced hypoxia). In the present work, we analyzed the effect of CoCl2 on the viability, proliferation, and apoptosis of cells of three glioblastoma cell lines: 1321N1, T98g, and U373 MG. It was shown that CoCl2 induced a dose-dependent decrease in cell viability and proliferation, and at high concentrations (200 and 400 μM) stimulated cell death. CoCl2 had no effect on the cytotoxic activity of doxorubicin in two cell lines T98g and U373 MG, and enhanced the effect of the chemotherapeutic agent on the 1321N1 cell line, though no synergistic cytotoxic effect of the two agents was observed.

Antidepressant effects of p-coumaric acid isolated from Vaccinium bracteatum leaves extract on chronic restraint stress mouse model and antagonism of serotonin 6 receptor in vitro

BackgroundVaccinium bracteatum Thunb. leaves (VBL) are used in traditional herbal medicines to treat various biological diseases. p-coumaric acid (CA), the main active component of VBL, has neuroprotective effects against corticosterone-induced damage in vitro. However, the effects of CA on immobility induced by chronic restraint stress (CRS) in a mouse model and 5-HT receptor activity have not been investigated. Hypothesis/PurposeWe investigated the antagonistic effects of VBL, NET-D1602, and the three components of Gαs protein-coupled 5-HT receptors. Additionally, we identified the effects and mechanism of action of CA, the active component of NET-D1602, in the CRS-exposed model. MethodsFor in vitro analyses, we used 1321N1 cells stably expressing human 5-HT6 receptors and CHO-K1 expressing human 5-HT4 or 5-HT7 receptors cell lines to study the mechanism of action. For in vivo analyses, CRS-exposed mice were orally administered CA (10, 50, or 100 mg/kg) daily for 21 consecutive days. The effects of CA were analyzed by assessing behavioral changes using a forced swim test (FST), measuring levels of hypothalamic-pituitary-adrenal (HPA) axis-related hormones in ntial therapeutic effects as 5-HT6 receptor antagonists for neurodegenerative diseases and depressioserum, and acetylcholinesterase (AChE), monoamines, including 5-HT, dopamine, and norepinephrine, using enzyme-linked immunosorbent assay kits. The underlying molecular mechanisms of the serotonin transporter (SERT), monoamine oxidase A (MAO-A), and extracellular signal-regulated kinase (ERK)/protein kinase B (Akt)/mTORC1 signaling were detected using western blotting. ResultsCA was confirmed to be an active component in the antagonistic effects of NET-D1602 on 5-HT6 receptor activity through decreases in cAMP and ERK1/2 phosphorylation. Moreover, CRS-exposed mice treated with CA showed a significantly reduced immobility time in the FST. CA also significantly decreased corticosterone, corticotropin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) levels. CA enhanced 5-HT, dopamine, and norepinephrine levels in the hippocampus (HC) and prefrontal cortex (PFC) but decreased MAO-A and SERT protein levels. Similarly, CA significantly upregulated the ERK, Ca2+/calmodulin-dependent protein kinase II (CaMKII), Akt/mTOR/p70S6K/S6 signaling pathways in both HC and the PFC. ConclusionCA contained in NET-D1602 may play the antidepressant effects against CRS-induced depression-like mechanism and the selective antagonist effect of 5-HT6 receptor.

Quercetin and Mercury In Vitro Anti-Proliferative Effect in Human Astrocytoma Cells

Mercury (Hg) is a toxic heavy metal to which we are exposed in everyday life. Exposure to environmental Hg may lead to toxicity in the human body associated with major health issues. Quercetin (QE) on the other hand, is a natural flavonoid widely distributed in higher plants and is part of the human diet. Several studies demonstrated the therapeutic and protective effects of QE against multiple diseases and health problems. The aim of this study is to investigate the effect of QE and Hg on the proliferation of human astrocytoma 1321N1 cell line. This study is a continuation of our previous work in which we investigated cadmium (Cd) instead of Hg. The 1321N1 cells were either treated with Hg alone, or pre- or co-treated with QE. Cell viabilities were determined by MTT assay. Results indicated that simultaneous treatment of the cells with 200 µM and 16 µM Hg for 48 hrs significantly reduced cell viability to 11.7 ± 3.1 % compared to the DMSO vehicle-treated cells. Other experiments of QE pre-treatment followed by exposure to Hg alone or with QE indicated a significant ability to reduce proliferation compared to treatment with Hg alone. In conclusion, our study suggested a synergistic anti-proliferative interaction of Hg and QE in malignantly transformed cells. However, this effect is higher when combining Cd and QE as indicated in our previous work. These data may be beneficial in exploiting the biological effect of QE for treating the malignantly transformed cells.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

A human-derived neurovascular unit in vitro model to study the effects of cellular cross-talk and soluble factors on barrier integrity.

The blood-brain barrier (BBB) restricts paracellular and transcellular diffusion of compounds and is part of a dynamic multicellular structure known as the "neurovascular unit" (NVU), which strictly regulates the brain homeostasis and microenvironment. Several neuropathological conditions (e.g., Parkinson's disease and Alzheimer's disease), are associated with BBB impairment yet the exact underlying pathophysiological mechanisms remain unclear. In total, 90% of drugs that pass animal testing fail human clinical trials, in part due to inter-species discrepancies. Thus, in vitro human-based models of the NVU are essential to better understand BBB mechanisms; connecting its dysfunction to neuropathological conditions for more effective and improved therapeutic treatments. Herein, we developed a biomimetic tri-culture NVU in vitro model consisting of 3 human-derived cell lines: human cerebral micro-vascular endothelial cells (hCMEC/D3), human 1321N1 (astrocyte) cells, and human SH-SY5Y neuroblastoma cells. The cells were grown in Transwell hanging inserts in a variety of configurations and the optimal setup was found to be the comprehensive tri-culture model, where endothelial cells express typical markers of the BBB and contribute to enhancing neural cell viability and neurite outgrowth. The tri-culture configuration was found to exhibit the highest transendothelial electrical resistance (TEER), suggesting that the cross-talk between astrocytes and neurons provides an important contribution to barrier integrity. Lastly, the model was validated upon exposure to several soluble factors [e.g., Lipopolysaccharides (LPS), sodium butyrate (NaB), and retinoic acid (RA)] known to affect BBB permeability and integrity. This in vitro biological model can be considered as a highly biomimetic recapitulation of the human NVU aiming to unravel brain pathophysiology mechanisms as well as improve testing and delivery of therapeutics.

ADAMTS9-AS1 Long Non‑coding RNA Sponges miR‑128 and miR-150 to Regulate Ras/MAPK Signaling Pathway in Glioma.

Glioma is a malignancy of the central nervous system with a poor prognosis. Therefore, the elaboration of its molecular features creates therapeutic opportunities. Looking for the regulatory non-coding RNAs (lncRNAs and miRNAs) that are involved in glioma incidence/progression, RNA-seq analysis introduced upregulated ADAMTS9-AS1 as a bona fide candidate that sponges miR-128 and miR-150 and shows the negative correlation of expression with them. Then, RT-qPCR verified the upregulation of ADAMTS9-AS1 in glioma tissues and cell lines. Furthermore, dual-luciferase assay supported that cytoplasmic ADAMTS9-AS1 is capable of sponging miR-128 and miR-150, which are known as regulators of Ras/MAPK, PI3K, and Wnt pathways. Following the overexpression of ADAMTS9-AS1 in 1321N1 and U87 glioma cells, tyrosine kinase receptors (IGF1R and TrkC), as well as Wnt receptors (Lrp6 and Fzd) were upregulated, detected by RT-qPCR. Furthermore, downstream genes of both Ras/MAPK and Wnt pathways were upregulated. Finally following the ADAMTS9-AS1 overexpression, upregulation of Ras/MAPK and Wnt signaling pathways was verified through western blotting and Top/Fop flash assay, respectively. At the cellular level, ADAMTS9-AS1 overexpression brought about reduced sub-G1 cell population, increased proliferation rate, reduced apoptosis level, increased migration rate, shortened Bax/Bcl2 ratio, induced EMT, and stemness characteristics of transfected cells, detected by flow cytometry, MTT assay, scratch test, and RT-qPCR. Overall, these results introduced ADAMTS9-AS1 as an oncogene that upregulates Ras/MAPK and Wnt pathways through sponging of the miR-128 and miR-150 in glioma cells. The outcome of ADAMTS9-AS1 expression is more aggression of the glioma cells through increased EMT and stemness characteristics. These features candidate ADAMTS9-AS1 locus for glioma therapy. As a result, we discovered the oncogenic properties of ADAMTS9-AS1 in glioma cancer. It sponges miR-128 and miR-150 and subsequently overstimulates RAS/MAPK and Wnt signaling pathways, particularly at the receptors level. Thus, ADAMTS9-AS1 increases proliferation, migration, and stemness in glioma cell lines. A schematic representation showing the functional effect of ADAMTS9-AS1.

Neurotrophic and Immunomodulatory Lanostane Triterpenoids from Wood-Inhabiting Basidiomycota.

Neurotrophins such as nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) play important roles in the central nervous system. They are potential therapeutic drugs for the treatment of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In this study, we investigated the neurotrophic properties of triterpenes isolated from fruiting bodies of Laetiporus sulphureus and a mycelial culture of Antrodia sp. MUCL 56049. The structures of the isolated compounds were elucidated based on nuclear magnetic resonance (NMR) spectroscopy in combination with high-resolution electrospray mass spectrometry (HR-ESIMS). The secondary metabolites were tested for neurotrophin (ngf and bdnf) expression levels on human astrocytoma 1321N1 cells. Neurite outgrowth activity using rat pheochromocytoma (PC-12) cells was also determined. Twelve triterpenoids were isolated, of which several potently stimulated the expression of neurotrophic factors, namely, ngf (sulphurenic acid, 15α-dehydroxytrametenolic acid, fomefficinic acid D, and 16α-hydroxyeburicoic acid) and bdnf (sulphurenic acid and 15α-dehydroxytrametenolic acid), respectively. The triterpenes also potentiated ngf-induced neurite outgrowth in PC-12 cells. This is, to the best of our knowledge, the first report on the compound class of lanostanes in direct relation to bdnf and ngf enhancement. These compounds are widespread in medicinal mushrooms; hence, they appear promising as a starting point for the development of drugs and mycopharmaceuticals to combat neurodegenerative diseases. Interestingly, they do not show any pronounced cytotoxicity and may, therefore, be better suited for therapy than many other neurotrophic compounds that were previously reported.

The 'Danse Macabre'-Neutrophils the Interactive Partner Affecting Oral Cancer Outcomes.

Over the past few decades, tremendous advances in the prevention, diagnosis, and treatment of cancer have taken place. However for head and neck cancers, including oral cancer, the overall survival rate is below 50% and they remain the seventh most common malignancy worldwide. These cancers are, commonly, aggressive, genetically complex, and difficult to treat and the delay, which often occurs between early recognition of symptoms and diagnosis, and the start of treatment of these cancers, is associated with poor prognosis. Cancer development and progression occurs in concert with alterations in the surrounding stroma, with the immune system being an essential element in this process. Despite neutrophils having major roles in the pathology of many diseases, they were thought to have little impact on cancer development and progression. Recent studies are now challenging this notion and placing neutrophils as central interactive players with other immune and tumor cells in affecting cancer pathology. This review focuses on how neutrophils and their sub-phenotypes, N1, N2, and myeloid-derived suppressor cells, both directly and indirectly affect the anti-tumor and pro-tumor immune responses. Emphasis is placed on what is currently known about the interaction of neutrophils with myeloid innate immune cells (such as dendritic cells and macrophages), innate lymphoid cells, natural killer cells, and fibroblasts to affect the tumor microenvironment and progression of oral cancer. A better understanding of this dialog will allow for improved therapeutics that concurrently target several components of the tumor microenvironment, increasing the possibility of constructive and positive outcomes for oral cancer patients. For this review, PubMed, Web of Science, and Google Scholar were searched for manuscripts using keywords and combinations thereof of “oral cancer, OSCC, neutrophils, TANs, MDSC, immune cells, head and neck cancer, and tumor microenvironment” with a focus on publications from 2018 to 2021.

LINC02381-ceRNA exerts its oncogenic effect through regulation of IGF1R signaling pathway in glioma

LncRNAs play essential roles in the cellular and molecular biology of glioma. Some LncRNAs exert their role through sponging miRNAs and regulating multiple signaling pathways. LINC02381 is involved in several cancer types as either oncogene or tumor suppressor. Here, we intended to find the molecular mechanisms of the LINC02381 effect during the glioma progression in related cell lines. RNA-seq data analysis indicated the oncogenic characteristics of LINC02381, and RT-qPCR results confirmed its upregulation compared to normaltissues. Besides its expression was relatively stronger in invasive glioma cell lines. Furthermore, in silico analysis revealed LINC02381 is concentrated in the cytoplasm and predicted its sponging effect against miR-128 and miR-150, which was verified through dual luciferase assay. When LINC02381 was overexpressed in 1321N1, U87, and A172 cell lines, IGF1R and TrkC receptors as well as their downstream pathways (PI3K and RAS/MAPK), were upregulated, detected by RT-qPCR, and verified by western analysis. Consistently, LINC02381 overexpression was followed by an increased proliferation rate of transfected glioma cell lines, detected by flow cytometry and MTT assay, and RT-qPCR. It also resulted in elevated EMT and stemness markers expression level, increased migration rate, and reduced apoptosis rate, detected by RT-qPCR, western analysis, scratch test, and Annexin/PI flow cytometry analysis, respectively. The overall results indicated that LINC02381 exerts its oncogenic effect in glioma cells through sponging miR-128 and miR-150 to upregulate the IGF1R signaling pathway. Our results introduce LINC02381 and miR-128, and miR-150 as potential prognosis and therapy targets for the treatment of glioma.

Inhibitory Mechanism of Combined Hydroxychavicol With Epigallocatechin-3-Gallate Against Glioma Cancer Cell Lines: A Transcriptomic Analysis.

Emerging reports have shown therapeutic potential of hydroxychavicol (HC) and epigallocatechin-3-gallate (EGCG) against cancer cells, however high concentrations are required to achieve the anticancer activity. We reported the synergy of low combination doses of EGCG+HC in glioma cell lines 1321N1, SW1783, and LN18 by assessing the effects of EGCG+HC through functional assays. Using high throughput RNA sequencing, the molecular mechanisms of EGCG+HC against glioma cell lines were revealed. EGCG/HC alone inhibited the proliferation of glioma cell lines, with IC50 values ranging from 82 to 302 µg/ml and 75 to 119 µg/ml, respectively. Sub-effective concentrations of combined EGCG+HC enhanced the suppression of glioma cell growth, with SW1783 showing strong synergism with a combination index (CI) of 0.55 and LN18 showing a CI of 0.51. A moderate synergistic interaction of EGCG+HC was detected in 1321N1 cells, with a CI value of 0.88. Exposure of 1321N1, SW1783, and LN18 cells to EGCG+HC for 24 h induces cell death, with caspase-3 activation rates of 52%, 57%, and 9.4%, respectively. However, the dose for SW1783 is cytotoxic to normal cells, thus this dose was excluded from other tests. EGCG+HC induced cell cycle arrest at S phase and reduced 1321N1 and LN18 cell migration and invasion. Combined EGCG+HC amplified its anticancer effect by downregulating the axon guidance process and metabolic pathways, while simultaneously interfering with endoplasmic reticulum unfolded protein response pathway. Furthermore, EGCG+HC exerted its apoptotic effect through the alteration of mitochondrial genes such as MT-CO3 and MT-RNR2 in 1321N1 and LN18 cells respectively. EGCG+HC dynamically altered DYNLL1 alternative splicing expression in 1321N1 and DLD splicing expression in LN18 cell lines. Our work indicated the pleiotropic effects of EGCG+HC treatment, as well as particular target genes that might be investigated for future glioma cancer therapeutic development.

Dihydropyridines Potentiate ATP-Induced Currents Mediated by the Full-Length Human P2X5 Receptor.

The P2X5 receptor, an ATP-gated cation channel, is believed to be involved in tumor development, inflammatory bone loss and inflammasome activation after bacterial infection. Therefore, it is a worthwhile pharmacological target to treat the corresponding diseases, especially in minority populations that have a gene variant coding for functional homotrimeric P2X5 channels. Here, we investigated the effects of dihydropyridines on the human full-length P2X5 receptor (hP2X5FL) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage clamp method. Agonist dependency, kinetics and permeation behavior, including Cl− permeability, were similar to hP2X5FL expressed in HEK293 or 1321N1 cells. Additionally, 1,4-dihydropyridines have been shown to interact with various other purinergic receptors, and we have examined them as potential hP2X5 modulators. Of seven commercially available and four newly synthesized dihydropyridines tested at hP2X5FL, only amlodipine exerted an inhibitory effect, but only at a high concentration of 300 µM. Isradipine and—even more—nimodipine stimulated ATP-induced currents in the low micromolar range. We conclude that common dihydropyridines or four new derivatives of amlodipine are not suitable as hP2X5 antagonists, but amlodipine might serve as a lead for future synthesis to increase its affinity. Furthermore, a side effect of nimodipine therapy could be a stimulatory effect on inflammatory processes.

Emodin relieves the inflammation and pyroptosis of lipopolysaccharide-treated 1321N1 cells by regulating methyltransferase-like 3 -mediated NLR family pyrin domain containing 3 expression

ABSTRACT Sepsis brain injury (SBI) is a major cause of death in critically ill patients. The present study aimed to investigate the role of emodin in SBI development. Human astrocyte 1321N1 cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) to establish an SBI model in vitro. Flow cytometry was performed to measure the cell pyroptosis. The protein expression levels of syndecan-1 (SDC-1), NLR family pyrin domain containing 3 (NLRP3), Caspase-1, and the N-terminal fragment of gasdermin D (GSDMD-N) were measured using Western blotting. Interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α levels in cells were measured using enzyme-linked immunosorbent assay kits. The N6-methyladenosine (m6A) modification was analyzed using the methylated RNA immunoprecipitation assay. NLRP3 activator, nigericin, was used to overexpress NLRP3. LPS treatment significantly enhanced the pyroptosis in 1321N1 cells, increased the levels of TNF-α, IL-1β, and IL-6, and decreased the levels of IL-10. The protein expression levels of NLRP3, SDC-1, GSDMD-N, and Caspase-1 were also increased. Emodin treatment decreased the levels of TNF-α, IL-1β, IL-6, NLRP3, SDC-1, GSDMD-N, and Caspase-1, while increasing the levels of IL-10 in LPS-treated 1321N1 cells. Nigericin reversed the effects of emodin. Furthermore, emodin upregulated m6A levels in NLRP3 by increasing the expression of methyltransferase-like 3 (METTL3). Meanwhile, knockdown of METTL3 reversed the effects of emodin on the mRNA expression and stability of NLRP3. Therefore, emodin inhibits the inflammation and pyroptosis of LPS-treated 1321N1 cells by inactivating METTL3-mediated NLRP3 expression.

Anti-obesity effects of Ishophloroglucin A from the brown seaweed Ishige okamurae (Yendo) via regulation of leptin signal in ob/ob mice

Brown seaweeds, which are macroalgae, are common foods and traditional medicines for Asians. Ishige okamurae (Yendo), found in Korea's shallow coastal areas, is one of the edible and medicinal brown seaweeds. Recent studies verified the pharmacological activities of the valuable properties from brown seaweeds such as ishophloroglucin A, a phlorotannin from I. okamurae. Recently, many studies have presented the anti-obesity effects of brown seaweeds and their secondary metabolites, such as phlorotannins. However, studies that target the appetite hormone leptin are few. In this study, we explored the natural anti-obesity agents from brown seaweeds targeting leptin. Also, we investigated the anti-obesity effects of ishophloroglucin A, using in vivo and in vitro experiments. The prospective anti-obesity agents were explored in silico among diverse phlorotannins from brown seaweeds. The anti-obesity effects of ishophloroglucin A (2.5 mg/kg, oral administration for seven weeks) were evaluated in ob/ob mice, serving as a leptin-deficiency model. To verify the leptin signal regulation efficacy of ishophloroglucin A, the key molecules of leptin signal were measured in the leptin receptor-silenced N1 hypothalamic neuron cell line using immunocytochemistry. According to the in silico simulation, ishophloroglucin A favorably docks with the leptin receptor. In in vivo results, ishophloroglucin A treatment reduced body weight, fat mass, and food intake. The leptin signaling in the hypothalamus and the insulin signaling in peripheral organs were stimulated by ishophloroglucin A treatment. Moreover, in leptin receptor-silenced N1 cells, ishophloroglucin A treatment failed to induce the leptin signals, unlike in general N1 cells. These scientific results present that ishophloroglucin A from I. okamurae possesses the anti-obesity effect through the leptin signaling pathway and suggest that ishophloroglucin A can be used as sustainable and natural therapeutic ingredients to reduce obesity in leptin-deficient individuals.

Ca2+/Calcineurin‐mediated release of exosomal miR‐23a regulates astrocyte‐neuron cross‐talk and protects against neuroinflammation and neurodegeneration

AbstractBackgroundAstrocyte dysfunction and associated neuroinflammation is well implicated in various neurodegenerative disorders like Alzheimer’s (AD) and Parkinson’s diseases (PD). Importantly, Ca2+/Calcineurin signaling is reported to induce astrocyte dysfunction in AD (1) but very little is known regarding PD. So, our objective was to study the role of Ca2+/Calcineurin signaling in astrocyte‐neuron cross‐talk during PD‐related neuroinflammation which is unreported till date.Method1.Mammalian cell culture 2.Immunoblotting. 3.qRT‐PCR. 4.Exosome isolation. 5.Intracellular Ca2+ measurement. 6.Transfection (miRNA mimics). 7. 3’UTR cloning. 8.Luciferase assay.ResultWe treated human astrocytic 1321N1 cells with Rotenone, a neuro‐toxin known to induce PD‐associated neuroinflammation (2). At the dose of Rotenone which caused about 40% death in 24 hrs, simultaneous activation of the Ca2+‐dependent protein phosphatase Calcineurin and intracellular surge in the Ca2+ levels were also observed. Now, miR‐23a is an anti‐apoptotic miRNA and Calcineurin/NFAT‐mediated upregulation of miR‐23a has been reported in cardiac hypertrophy (3). Surprisingly, in our study, Rotenone caused a Calcineurin‐mediated decrease of intracellular miR‐23a levels in astrocytes. Now this decrease, we found, was due to a concomitant release of miR‐23a via exosomes. Can this anti‐apoptotic miR‐23a released by astrocytes protect the neurons? To answer this, we treated neuronal SH‐SY5Y cells with Rotenone and found that the pro‐apoptotic protein PUMA was upregulated although the miR‐23a levels remained unchanged. Interestingly, over‐expression of miR‐23a caused attenuation of the Rotenone‐mediated death in neurons. PUMA being a predicted target of miR‐23a, we cloned the 3’UTR of PUMA and subsequent luciferase assay revealed that indeed PUMA is a direct target of miR‐23a under our experimental conditions.ConclusionOur results suggest a novel mechanism by which Ca2+/Calcineurin‐mediated release of miR‐23a through exosomes regulates astrocyte‐neuron cross‐talk and plays a probable role in protection against neuroinflammation and neurodegeneration.