An Amphibian Host Defense Peptide Is Virucidal for Human H1 Hemagglutinin-Bearing Influenza Viruses.

Although vaccines confer protection against influenza A viruses, antiviral treatment becomes the first line of defense during pandemics because there is insufficient time to produce vaccines. Current antiviral drugs are susceptible to drug resistance, and developing new antivirals is essential. We studied host defense peptides from the skin of the South Indian frog and demonstrated that one of these, which we named "urumin," is virucidal for H1 hemagglutinin-bearing human influenza A viruses. This peptide specifically targeted the conserved stalk region of H1 hemagglutinin and was effective against drug-resistant H1 influenza viruses. Using electron microscopy, we showed that this peptide physically destroyed influenza virions. It also protected naive mice from lethal influenza infection. Urumin represents a unique class of anti-influenza virucide that specifically targets the hemagglutinin stalk region, similar to targeting of antibodies induced by universal influenza vaccines. Urumin therefore has the potential to contribute to first-line anti-viral treatments during influenza outbreaks.

Nobiletin enhances mitochondrial function by regulating SIRT1/PGC-1α signaling in porcine oocytes during in vitro maturation.

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 µM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1α signaling pathway.

GRP78 acts as a cAMP/PKA signaling modulator through the MC4R pathway in porcine embryonic development.

Glucose-regulated protein 78 (GRP78) binds to and stabilizes melanocortin 4 receptor (MC4R), which activates protein kinase A (PKA) by regulating G proteins. GRP78 is primarily used as a marker for endoplasmic reticulum stress; however, its other functions have not been well studied. Therefore, in this study, we aimed to investigate the function of GRP78 during porcine embryonic development. The developmental quality of porcine embryos, expression of cell cycle proteins, and function of mitochondria were evaluated by inhibiting the function of GRP78. Porcine oocytes were activated to undergo parthenogenesis, and blastocysts were obtained after 7 days of in vitro culture. GRP78 function was inhibited by adding 20 µM HA15 to the in vitro culture medium. The inhibition in GRP78 function led to a decrease in G proteins release, which subsequently downregulated the cyclic adenosine monophosphate (cAMP)/PKA pathway. Ultimately, inhibition of GRP78 function induced the inhibition of CDK1 and cyclin B expression and disruption of the cell cycle. In addition, inhibition of GRP78 function regulated DRP1 and SIRT1 expression, resulting in mitochondrial dysfunction. This study provides new insights into the role of GRP78 in porcine embryonic development, particularly its involvement in the regulation of the MC4R pathway and downstream cAMP/PKA signaling. The results suggest that the inhibition of GRP78 function in porcine embryos by HA15 treatment may have negative effects on embryo quality and development. This study also demonstrated that GRP78 plays a crucial role in the functioning of MC4R, which releases the G protein during porcine embryonic development.

Dendropanoxide Attenuates High Glucose-induced Oxidative Damage in NRK-52E Cells via AKT/mTOR Signaling Pathway.

Hyperglycemia is a potent risk factor for the development and progression of diabetes-induced nephropathy. Dendropanoxide (DPx) is a natural compound isolated from Dendropanax morbifera (Araliaceae) that exerts various biological effects. However, the role of DPx in hyperglycemia-induced renal tubular cell injury remains unclear. The present study explored the protective mechanism of DPx on high glucose (HG)-induced cytotoxicity in kidney tubular epithelial NRK-52E cells. The cells were cultured with normal glucose (5.6 mM), HG (30 mM), HG + metformin (10 µM), or HG + DPx (10 µM) for 48 h, and cell cycle and apoptosis were analyzed. Malondialdehyde (MDA), advanced glycation end products (AGEs), and reactive oxygen species (ROS) were measured. Protein-based nephrotoxicity biomarkers were measured in both the culture media and cell lysates. MDA and AGEs were significantly increased in NRK-52E cells cultured with HG, and these levels were markedly reduced by pretreatment with DPx or metformin. DPx significantly reduced the levels of kidney injury molecule-1 (KIM-1), pyruvate kinase M2 (PKM2), selenium-binding protein 1 (SBP1), or neutrophil gelatinase-associated lipocalin (NGAL) in NRK-52E cells cultured under HG conditions. Furthermore, treatment with DPx significantly increased antioxidant enzyme activity. DPx protects against HG-induced renal tubular cell damage, which may be mediated by its ability to inhibit oxidative stress through the protein kinase B/mammalian target of the rapamycin (AKT/mTOR) signaling pathway. These findings suggest that DPx can be used as a new drug for the treatment of high glucose-induced diabetic nephropathy.

Y-box binding protein 1 influences zygotic genome activation by regulating N6-methyladenosine in porcine embryos.

Y-box binding protein 1 (YBX1) is a member of the family of DNA- and RNA-binding proteins that play crucial roles in multiple aspects, including RNA stabilization, translational repression, and transcriptional regulation; however, its roles in embryo development remain less known. In this study, to investigate the function of YBX1 and its mechanism of action in porcine embryo development, YBX1 was knocked down by microinjecting YBX1 siRNA at the one-cell stage. YBX1 is located in the cytoplasm during embryonic development. The mRNA level of YBX1 was increased from the four-cell stage to the blastocyst stage but was significantly decreased in YBX1 knockdown embryos compared with the control. Moreover, the percentage of blastocysts was decreased following YBX1 knockdown compared with the control. Defecting YBX1 expression increased maternal gene mRNA expression and decreased zygotic genome activation (ZGA) gene mRNA expression and histone modification owing to decreased levels of N6-methyladenosine (m6A) writer N6-adenosine-methyltransferase 70 kDa subunit (METTL3) and reader insulin-like growth factor 2 mRNA-binding protein (IGF2BP1). In addition, IGF2BP1 knockdown showed that YBX1 regulated the ZGA process through m6A modification. In conclusion, YBX1 is essential for early embryo development because it regulates the ZGA process.

High Temperature-Induced m6A Epigenetic Changes Affect Early Porcine Embryonic Developmental Competence in Pigs.

N6-methyladenosine (m6A), the most prevalent modification in eukaryotic messenger RNA (mRNA), plays a key role in various developmental processes in mammals. Three proteins that affect RNA m6A modification have been identified: methyltransferases, demethylases, and m6A-binding proteins, known as "writer," "eraser," and "reader" proteins, respectively. However, changes in the m6A modification when early porcine embryos are exposed to stress remain unclear. In this study, we exposed porcine oocytes to a high temperature (HT, 41°C) for 10 h, after which the mature oocytes were parthenogenetically activated and cultured for 7 days to the blastocyst stage. HT significantly decreased the rates of the first polar body extrusion and blastocyst formation. Further detection of m6A modification found that HT can lead to increased expression levels of "reader," YTHDF2, and "writer," METTL3, and decreased expression levels of "eraser," FTO, resulting in an increased level of m6A modification in the embryos. Additionally, heat shock protein 70 (HSP70) is upregulated under HT conditions. Our study demonstrated that HT exposure alters m6A modification levels, which further affects early porcine embryonic development.

The Protective Role of Glutathione on Zinc-Induced Neuron Death after Brain Injuries.

Glutathione (GSH) is necessary for maintaining physiological antioxidant function, which is responsible for maintaining free radicals derived from reactive oxygen species at low levels and is associated with improved cognitive performance after brain injury. GSH is produced by the linkage of tripeptides that consist of glutamic acid, cysteine, and glycine. The adequate supplementation of GSH has neuroprotective effects in several brain injuries such as cerebral ischemia, hypoglycemia, and traumatic brain injury. Brain injuries produce an excess of reactive oxygen species through complex biochemical cascades, which exacerbates primary neuronal damage. GSH concentrations are known to be closely correlated with the activities of certain genes such as excitatory amino acid carrier 1 (EAAC1), glutamate transporter-associated protein 3-18 (Gtrap3-18), and zinc transporter 3 (ZnT3). Following brain-injury-induced oxidative stress, EAAC1 function is negatively impacted, which then reduces cysteine absorption and impairs neuronal GSH synthesis. In these circumstances, vesicular zinc is also released into the synaptic cleft and then translocated into postsynaptic neurons. The excessive influx of zinc inhibits glutathione reductase, which inhibits GSH's antioxidant functions in neurons, resulting in neuronal damage and ultimately in the impairment of cognitive function. Therefore, in this review, we explore the overall relationship between zinc and GSH in terms of oxidative stress and neuronal cell death. Furthermore, we seek to understand how the modulation of zinc can rescue brain-insult-induced neuronal death after ischemia, hypoglycemia, and traumatic brain injury.

ZSCAN4 Regulates Zygotic Genome Activation and Telomere Elongation in Porcine Parthenogenetic Embryos.

Zinc finger and SCAN domain-containing 4 (ZSCAN4), a DNA-binding protein, maintains telomere length and plays a key role in critical aspects of mouse embryonic stem cells, including maintaining genomic stability and defying cellular senescence. However, the effect of ZSCAN4 in porcine parthenogenetic embryos remains unclear. To investigate the function of ZSCAN4 and the underlying mechanism in porcine embryo development, ZSCAN4 was knocked down via dsRNA injection in the one-cell stage. ZSCAN4 was highly expressed in the four- and five- to eight-cell stages in porcine embryos. The percentage of four-cell stage embryos, five- to eight-cell stage embryos, and blastocysts was lower in the ZSCAN4 knockdown group than in the control group. Notably, depletion of ZSCAN4 induced the protein expression of DNMT1 and 5-Methylcytosine (5mC, a methylated form of the DNA base cytosine) in the four-cell stage. The H3K27ac level and ZGA genes expression decreased following ZSCAN4 knockdown. Furthermore, ZSCAN4 knockdown led to DNA damage and shortened telomere compared with the control. Additionally, DNMT1-dsRNA was injected to reduce DNA hypermethylation in ZSCAN4 knockdown embryos. DNMT1 knockdown rescued telomere shortening and developmental defects caused by ZSCAN4 knockdown. In conclusion, ZSCAN4 is involved in the regulation of transcriptional activity and is essential for maintaining telomere length by regulating DNMT1 expression in porcine ZGA.

Pathophysiological Roles of Transient Receptor Potential (Trp) Channels and Zinc Toxicity in Brain Disease.

Maintaining the correct ionic gradient from extracellular to intracellular space via several membrane-bound transporters is critical for maintaining overall cellular homeostasis. One of these transporters is the transient receptor potential (TRP) channel family that consists of six putative transmembrane segments systemically expressed in mammalian tissues. Upon the activation of TRP channels by brain disease, several cations are translocated through TRP channels. Brain disease, especially ischemic stroke, epilepsy, and traumatic brain injury, triggers the dysregulation of ionic gradients and promotes the excessive release of neuro-transmitters and zinc. The divalent metal cation zinc is highly distributed in the brain and is specifically located in the pre-synaptic vesicles as free ions, usually existing in cytoplasm bound with metallothionein. Although adequate zinc is essential for regulating diverse physiological functions, the brain-disease-induced excessive release and translocation of zinc causes cell damage, including oxidative stress, apoptotic cascades, and disturbances in energy metabolism. Therefore, the regulation of zinc homeostasis following brain disease is critical for the prevention of brain damage. In this review, we summarize recent experimental research findings regarding how TRP channels (mainly TRPC and TRPM) and zinc are regulated in animal brain-disease models of global cerebral ischemia, epilepsy, and traumatic brain injury. The blockade of zinc translocation via the inhibition of TRPC and TRPM channels using known channel antagonists, was shown to be neuroprotective in brain disease. The regulation of both zinc and TRP channels may serve as targets for treating and preventing neuronal death.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

Using intracellular metabolic profiling to identify novel biomarkers of cisplatin-induced acute kidney injury in NRK-52E cells.

The aim of this study was to investigate changes in the intracellular metabolism resulting from cisplatin (CDDP)-induced nephrotoxicity in normal kidney tubular epithelial NRK-52E cells. Cytotoxicity, cell cycle analysis, and apoptotic cell death were all evaluated in NRK-52E cells treated with CDDP. Subsequently, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to investigate cellular metabolic profiles. CDDP-induced nephrotoxicity was determined in vivo model. Cytotoxicity in the NRK-52E cells significantly rose following treatment with CDDP and these increases were found to be concentration-dependent. Both p53 and Bax protein expression was increased in CDDP-treated NRK-52E cells, correlating with enhanced cellular apoptosis. In addition, a number of metabolites were altered in both media and cell lysates in these cells. In cell lysates, citrate, creatinine, and acetate levels were dramatically reduced following treatment with 20 µM CDDP concentrations, while glutamate level was elevated. Lactate and acetate levels were significantly increased in culture media but citrate concentrations were reduced following high 20 µM CDDP concentrations incubation. In addition, excretion of clusterin, calbindin, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), selenium binding protein 1 (SBP1), and pyruvate kinase M2 (PKM2) into the culture media was significantly increased in CDDP-treated cells while expression of acetyl CoA synthetase 1 (AceCS1) was markedly reduced in these cells. These findings suggest that acetate-dependent metabolic pathway may be a reliable and useful biomarker for detecting CDDP-induced nephrotoxicity. Taken together, data demonstrate that the discovery of novel biomarkers by metabolite profiling in target cells may contribute to the detection of nephrotoxicity and new drug development.

Next-Generation Sequencing Results Vary Between Cultured and Uncultured Microbes.

Next-generation sequencing (NGS) technology has led to innovations in environmental metagenomics and investigations involving humans and microbes. However, it is necessary to analyze the components that will affect analysis of the method upon processing a large amount of information. In particular, the processing method after sample collection affects the NGS results, and it is necessary to check for inaccurate results. Here, we show that the microbial communities obtained from fingertip samples differ from those obtained from fingertips remaining on mobile phones and desks, when cultured or not for 24 h. We also confirmed changes in microbial communities in fingertip samples from desks incubated for 2, 4, 8, 16, and 24 h. Samples of prints from mobile phones that are considerably vulnerable to external factors were not analyzed. Ratios of Firmicutes and Bacillus were, respectively, increased in cultures at the phylum and species levels. Collectively, we identified bacterial species that can aid in determining whether a sample has been cultured. In addition, although microbial communities differed depending on sample types, we confirmed changes after culture for 4 and 8 h. However, since this study is a sample limited to three types, it is necessary to analyze other types of samples in the same way and check whether they are applicable to all types. This strategy can verify the suitability of samples for deriving informative results from cultured or uncultured bacterial communities.

Carvacrol Inhibits Expression of Transient Receptor Potential Melastatin 7 Channels and Alleviates Zinc Neurotoxicity Induced by Traumatic Brain Injury.

Carvacrol is a monoterpenoid phenol produced by aromatic plants such as oregano. Although the exact mechanism by which carvacrol acts has not yet been established, it appears to inhibit transient receptor potential melastatin 7 (TRPM7), which modulates the homeostasis of metal ions such as zinc and calcium. Several studies have demonstrated that carvacrol has protective effects against zinc neurotoxicity after ischemia and epilepsy. However, to date, no studies have investigated the effect of carvacrol on traumatic brain injury (TBI)-induced zinc neurotoxicity. In the present study, we investigated the therapeutic potential of carvacrol for the prevention of zinc-induced neuronal death after TBI. Rats were subjected to a controlled cortical impact, and carvacrol was injected at a dose of 50 mg/kg. Histological analysis was performed at 12 h, 24 h, and 7 days after TBI. We found that carvacrol reduced TBI-induced TRPM7 over-expression and free zinc accumulation. As a result, subsequent oxidative stress, dendritic damage, and neuronal degeneration were decreased. Moreover, carvacrol not only reduced microglial activation and delayed neuronal death but also improved neurological outcomes after TBI. Taken together, these findings suggest that carvacrol administration may have therapeutic potential after TBI by preventing neuronal death through the inhibition of TRPM7 expression and alleviation of zinc neurotoxicity.

Acid Sphingomyelinase Inhibitor, Imipramine, Reduces Hippocampal Neuronal Death after Traumatic Brain Injury.

Traumatic brain injury (TBI) broadly degrades the normal function of the brain after a bump, blow, or jolt to the head. TBI leads to the aggravation of pre-existing brain dysfunction and promotes neurotoxic cascades that involve processes such as oxidative stress, loss of dendritic arborization, and zinc accumulation. Acid sphingomyelinase (ASMase) is an enzyme that hydrolyzes sphingomyelin to ceramide in cells. Under normal conditions, ceramide plays an important role in various physiological functions, such as differentiation and apoptosis. However, under pathological conditions, excessive ceramide production is toxic and activates the neuronal-death pathway. Therefore, we hypothesized that the inhibition of ASMase activity by imipramine would reduce ceramide formation and thus prevent TBI-induced neuronal death. To test our hypothesis, an ASMase inhibitor, imipramine (10 mg/kg, i.p.), was administrated to rats immediately after TBI. Based on the results of this study, we confirmed that imipramine significantly reduced ceramide formation, dendritic loss, oxidative stress, and neuronal death in the TBI-imipramine group compared with the TBI-vehicle group. Additionally, we validated that imipramine prevented TBI-induced cognitive dysfunction and the modified neurological severity score. Consequently, we suggest that ASMase inhibition may be a promising therapeutic strategy to reduce hippocampal neuronal death after TBI.

EzMAP: Easy Microbiome Analysis Platform.

BACKGROUND: The rapid advances in next-generation sequencing technologies have revolutionized the microbiome research by greatly increasing our ability to understand diversity of microbes in a given sample. Over the past decade, several computational pipelines have been developed to efficiently process and annotate these microbiome data. However, most of these pipelines require an implementation of additional tools for downstream analyses as well as advanced programming skills. RESULTS: Here we introduce a user-friendly microbiome analysis platform, EzMAP (Easy Microbiome Analysis Platform), which was developed using Java Swings, Java Script and R programming language. EzMAP is a standalone package providing graphical user interface, enabling easy access to all the functionalities of QIIME2 (Quantitative Insights Into Microbial Ecology) as well as streamlined downstream analyses using QIIME2 output as input. This platform is designed to give users the detailed reports and the intermediate output files that are generated progressively. The users are allowed to download the features/OTU table (.biom;.tsv;.xls), representative sequences (.fasta) and phylogenetic tree (.nwk), taxonomy assignment file (optional). For downstream analyses, users are allowed to perform relative abundances (at all taxonomical levels), community comparison (alpha and beta diversity, core microbiome), differential abundances (DESeq2 and linear discriminant analysis) and functional prediction (PICRust, Tax4Fun and FunGuilds). Our case study using a published rice microbiome dataset demonstrates intuitive user interface and great accessibility of the EzMAP. CONCLUSIONS: This EzMAP allows users to consolidate the microbiome analysis processes from raw sequence processing to downstream analyses specific for individual projects. We believe that this will be an invaluable tool for the beginners in their microbiome data analysis. This platform is freely available at https://github.com/gnanibioinfo/EzMAP and will be continually updated for adoption of changes in methods and approaches.

Pharmacokinetic changes of clozapine and norclozapine in a rat model of non-alcoholic fatty liver disease induced by orotic acid.

Impaired in vitro oxidation of clozapine has been reported in steatotic rat liver due to downregulation of cytochrome P450 (CYP) 1A. Pharmacokinetic changes of clozapine and its major metabolite, norclozapine, were evaluated in a rat model of non-alcoholic fatty liver disease (NAFLD) induced by orotic acid. Significantly slower in vitro CLint for formation of norclozapine from clozapine was observed in NAFLD rats than in control rats as a result of the reduced protein expression and metabolic activity of CYP1A1/2. However, systemic exposures to clozapine in NAFLD rats were comparable to those in controls after intravenous (4 mg/kg) and oral (10 mg/kg) administration of clozapine. Of note, the AUC of the norclozapine and AUCnorclozapine/AUCclozapine ratio following intravenous and oral administration of clozapine rather increased significantly in NAFLD rats, as a result of the slowed subsequent metabolism of norclozapine via CYP1A1/2. Steady-state brain concentrations of both clozapine and norclozapine were significantly higher in NAFLD rats than those in control rats following intravenous infusion of clozapine. Increased systemic exposure to norclozapine and elevated brain concentrations of clozapine and norclozapine observed in NAFLD rats imply that further studies are warranted on the pharmacotherapy of clozapine in patients with pre-existing or drug-induced hepatic steatosis.

Effects of Transient Receptor Potential Cation 5 (TRPC5) Inhibitor, NU6027, on Hippocampal Neuronal Death after Traumatic Brain Injury.

Traumatic brain injury (TBI) can cause physical, cognitive, social, and behavioral changes that can lead to permanent disability or death. After primary brain injury, translocated free zinc can accumulate in neurons and lead to secondary events such as oxidative stress, inflammation, edema, swelling, and cognitive impairment. Under pathological conditions, such as ischemia and TBI, excessive zinc release, and accumulation occurs in neurons. Based on previous research, it hypothesized that calcium as well as zinc would be influx into the TRPC5 channel. Therefore, we hypothesized that the suppression of TRPC5 would prevent neuronal cell death by reducing the influx of zinc and calcium. To test our hypothesis, we used a TBI animal model. After the TBI, we immediately injected NU6027 (1 mg/kg, intraperitoneal), TRPC5 inhibitor, and then sacrificed animals 24 h later. We conducted Fluoro-Jade B (FJB) staining to confirm the presence of degenerating neurons in the hippocampal cornus ammonis 3 (CA3). After the TBI, the degenerating neuronal cell count was decreased in the NU6027-treated group compared with the vehicle-treated group. Our findings suggest that the suppression of TRPC5 can open a new therapeutic window for a reduction of the neuronal death that may occur after TBI.

An Inhibitor of the Sodium-Hydrogen Exchanger-1 (NHE-1), Amiloride, Reduced Zinc Accumulation and Hippocampal Neuronal Death after Ischemia.

Acidosis in the brain plays an important role in neuronal injury and is a common feature of several neurological diseases. It has been reported that the sodium-hydrogen exchanger-1 (NHE-1) is a key mediator of acidosis-induced neuronal injury. It modulates the concentration of intra- and extra-cellular sodium and hydrogen ions. During the ischemic state, excessive sodium ions enter neurons and inappropriately activate the sodium-calcium exchanger (NCX). Zinc can also enter neurons through voltage-gated calcium channels and NCX. Here, we tested the hypothesis that zinc enters the intracellular space through NCX and the subsequent zinc accumulation induces neuronal cell death after global cerebral ischemia (GCI). Thus, we conducted the present study to confirm whether inhibition of NHE-1 by amiloride attenuates zinc accumulation and subsequent hippocampus neuronal death following GCI. Mice were subjected to GCI by bilateral common carotid artery (BCCA) occlusion for 30 min, followed by restoration of blood flow and resuscitation. Amiloride (10 mg/kg, intraperitoneally (i.p.)) was immediately injected, which reduced zinc accumulation and neuronal death after GCI. Therefore, the present study demonstrates that amiloride attenuates GCI-induced neuronal injury, likely via the prevention of intracellular zinc accumulation. Consequently, we suggest that amiloride may have a high therapeutic potential for the prevention of GCI-induced neuronal death.

Transient Receptor Potential Melastatin 2 (TRPM2) Inhibition by Antioxidant, N-Acetyl-l-Cysteine, Reduces Global Cerebral Ischemia-Induced Neuronal Death.

A variety of pathogenic mechanisms, such as cytoplasmic calcium/zinc influx, reactive oxygen species production, and ionic imbalance, have been suggested to play a role in cerebral ischemia induced neurodegeneration. During the ischemic state that occurs after stroke or heart attack, it is observed that vesicular zinc can be released into the synaptic cleft, and then translocated into the cytoplasm via various cation channels. Transient receptor potential melastatin 2 (TRPM2) is highly distributed in the central nervous system and has high sensitivity to oxidative damage. Several previous studies have shown that TRPM2 channel activation contributes to neuroinflammation and neurodegeneration cascades. Therefore, we examined whether anti-oxidant treatment, such as with N-acetyl-l-cysteine (NAC), provides neuroprotection via regulation of TRPM2, following global cerebral ischemia (GCI). Experimental animals were then immediately injected with NAC (150 mg/kg/day) for 3 and 7 days, before sacrifice. We demonstrated that NAC administration reduced activation of GCI-induced neuronal death cascades, such as lipid peroxidation, microglia and astroglia activation, free zinc accumulation, and TRPM2 over-activation. Therefore, modulation of the TRPM2 channel can be a potential therapeutic target to prevent ischemia-induced neuronal death.

The Transient Receptor Potential Melastatin 7 (TRPM7) Inhibitors Suppress Seizure-Induced Neuron Death by Inhibiting Zinc Neurotoxicity.

Transient receptor potential melastatin 7 (TRPM7) is an ion channel that mediates monovalent cations out of cells, as well as the entry of divalent cations, such as zinc, magnesium, and calcium, into the cell. It has been reported that inhibitors of TRPM7 are neuroprotective in various neurological diseases. Previous studies in our lab suggested that seizure-induced neuronal death may be caused by the excessive release of vesicular zinc and the subsequent accumulation of zinc in the neurons. However, no studies have evaluated the effects of carvacrol and 2-aminoethoxydiphenyl borate (2-APB), both inhibitors of TRPM7, on the accumulation of intracellular zinc in dying neurons following seizure. Here, we investigated the therapeutic efficacy of carvacrol and 2-APB against pilocarpine-induced seizure. Carvacrol (50 mg/kg) was injected once per day for 3 or 7 days after seizure. 2-APB (2 mg/kg) was also injected once per day for 3 days after seizure. We found that inhibitors of TRPM7 reduced seizure-induced TRPM7 overexpression, intracellular zinc accumulation, and reactive oxygen species production. Moreover, there was a suppression of oxidative stress, glial activation, and the blood-brain barrier breakdown. In addition, inhibitors of TRPM7 remarkably decreased apoptotic neuron death following seizure. Taken together, the present study demonstrates that TRPM7-mediated zinc translocation is involved in neuron death after seizure. The present study suggests that inhibitors of TRPM7 may have high therapeutic potential to reduce seizure-induced neuron death.