Analysis of mRNA-miRNA-lncRNA differential expression in prediabetes/type 2 diabetes mellitus patients as potential players in insulin resistance.

Introduction: Type 2 diabetes mellitus (T2DM) is a major global health concern. It usually develops gradually and is frequently preceded by undetectable pre-diabetes mellitus (pre-DM) stage. The purpose of this study was to identify a novel set of seven candidate genes associated with the pathogenesis of insulin resistance (IR) and pre-DM, followed by their experimental validation in patients' serum samples. Methods: We used the bioinformatics tools and through a two-step process, we first identified and verified two mRNA candidate genes linked to insulin resistance molecular pathogenesis. Second, we identified a non-coding RNAs related to the selected mRNAs and implicated in the insulin resistance molecular pathways followed by pilot study for the RNA panel differential expression in 66 patients with T2DM, 49 individuals with prediabetes and 45 matched controls using real time PCR. Results: The levels of expression of TMEM173 and CHUK mRNAs, hsa-miR (-611, -5192, and -1976) miRNAs gradually increased from the healthy control group to the prediabetic group, reaching their maximum levels in the T2DM group (p <10-3), whereas the levels of expression of RP4-605O3.4 and AC074117.2 lncRNAs declined gradually from the healthy control group to the prediabetic group, reaching their lowest levels in the T2DM group (p <10-3). TMEM173, CHUK mRNAs, hsa_miR (-611 & -1976) and RP4-605O3.4 lncRNA were useful in distinguishing insulin resistant from insulin sensitive groups. miR_611 together with RP4-605O3.4 exhibited significant difference in good versus poor glycemic control groups. Discussion: The presented study provides an insight about this RNA based STING/NOD/IR associated panel that could be used for PreDM-T2DM diagnosis and also as a therapeutic target based on the differences of its expression level in the pre-DM and T2DM stages.

Identification of a Multi-Messenger RNA Signature as Type 2 Diabetes Mellitus Candidate Genes Involved in Crosstalk between Inflammation and Insulin Resistance.

Type 2 Diabetes Mellitus (T2DM) is a metabolic disease associated with inflammation widening the scope of immune-metabolism, linking the inflammation to insulin resistance and beta cell dysfunction. New potential and prognostic biomarkers are urgently required to identify individuals at high risk of ß-cell dysfunction and pre-DM. The DNA-sensing stimulator of interferon genes (STING) is an important component of innate immune signaling that governs inflammation-mediated T2DM. NOD-like receptor (NLR) reduces STING-dependent innate immune activation in response to cyclic di-GMP and DNA viruses by impeding STING-TBK1 interaction. We proposed exploring novel blood-based mRNA signatures that are selective for components related to inflammatory, immune, and metabolic stress which may reveal the landscape of T2DM progression for diagnosing or treating patients in the pre-DM state. In this study, we used microarray data set to identify a group of differentially expressed mRNAs related to the cGAS/STING, NODlike receptor pathways (NLR) and T2DM. Then, we comparatively analyzed six mRNAs expression levels in healthy individuals, prediabetes (pre-DM) and T2DM patients by real-time PCR. The expressions of ZBP1, DDX58, NFKB1 and CHUK were significantly higher in the pre-DM group compared to either healthy control or T2DM patients. The expression of ZBP1 and NFKB1 mRNA could discriminate between good versus poor glycemic control groups. HSPA1B mRNA showed a significant difference in its expression regarding the insulin resistance. Linear regression analysis revealed that LDLc, HSPA1B and NFKB1 were significant variables for the prediction of pre-DM from the healthy control. Our study shed light on a new finding that addresses the role of ZBP1 and HSPA1B in the early prediction and progression of T2DM.

Identifying SARS-CoV-2 Lineage Mutation Hallmarks and Correlating Them With Clinical Outcomes in Egypt: A Pilot Study.

The SARS-CoV-2 pandemic has led to over 4.9 million deaths as of October 2021. One of the main challenges of creating vaccines, treatment, or diagnostic tools for the virus is its mutations and emerging variants. A couple of variants were declared as more virulent and infectious than others. Some approaches were used as nomenclature for SARS-CoV-2 variants and lineages. One of the most used is the Pangolin nomenclature. In our study, we enrolled 35 confirmed SARS-CoV-2 patients and sequenced the viral RNA in their samples. We also aimed to highlight the hallmark mutations in the most frequent lineage. We identified a seven-mutation signature for the SARS-CoV-2 C36 lineage, detected in 56 countries and an emerging lineage in Egypt. In addition, we identified one mutation which was highly negatively correlated with the lineage. On the other hand, we found no significant correlation between our clinical outcomes and the C36 lineage. In conclusion, the C36 lineage is an emerging SARS-CoV-2 variant that needs more investigation regarding its clinical outcomes compared to other strains. Our study paves the way for easier diagnosis of variants of concern using mutation signatures.

In Silico Identification and Clinical Validation of a Novel Long Non-Coding RNA/mRNA/miRNA Molecular Network for Potential Biomarkers for Discriminating SARS CoV-2 Infection Severity.

(1) Background: The coronavirus (COVID-19) pandemic is still a major global health problem, despite the development of several vaccines and diagnostic assays. Moreover, the broad symptoms, from none to severe pneumonia, and the various responses to vaccines and the assays, make infection control challenging. Therefore, there is an urgent need to develop non-invasive biomarkers to quickly determine the infection severity. Circulating RNAs have been proven to be potential biomarkers for a variety of diseases, including infectious ones. This study aimed to develop a genetic network related to cytokines, with clinical validation for early infection severity prediction. (2) Methods: Extensive analyses of in silico data have established a novel IL11RA molecular network (IL11RNA mRNA, LncRNAs RP11-773H22.4 and hsa-miR-4257). We used different databases to confirm its validity. The differential expression within the retrieved network was clinically validated using quantitative RT-PCR, along with routine assessment diagnostic markers (CRP, LDH, D-dimmer, procalcitonin, Ferritin), in100 infected subjects (mild and severe cases) and 100 healthy volunteers. (3) Results: IL11RNA mRNA and LncRNA RP11-773H22.4, and the IL11RA protein, were significantly upregulated, and there was concomitant downregulation of hsa-miR-4257, in infected patients, compared to the healthy controls, in concordance with the infection severity. (4) Conclusion: The in-silico data and clinical validation led to the identification of a potential RNA/protein signature network for novel predictive biomarkers, which is in agreement with ferritin and procalcitonin for determination of COVID-19 severity.

ABHD4-Regulating RNA Panel: Novel Biomarkers in Acute Coronary Syndrome Diagnosis.

BACKGROUND: Acute coronary syndrome (ACS) is a major cause of death all over the world. STEMI represents a type of myocardial infarction with acute ST elevation. We aimed to assess the predictive power of potential RNA panel expression in acute coronary syndrome. METHOD: We used in silico data analysis to retrieve RNAs related to glycerophospholipid metabolism dysregulation and specific to ACS that results in the selection of Alpha/Beta hydrolase fold domain4 (ABHD4) mRNA and its epigenetic regulators (Foxf1 adjacent noncoding developmental regulatory RNA (FENDRR) lncRNA, miRNA-221, and miRNA-197). We assessed the expression of the serum RNA panel in 68 patients with ACS, 21 patients with chest pain due to non-cardiac causes, and 21 healthy volunteers by quantitative real-time polymerase chain reaction. RESULTS: The study data showed significant down regulation in the expression of the serum levels of FENDRR lncRNA and miRNA-221-3p by 120-fold and 22-fold in Unstable angina (UA) in comparison with healthy volunteers, and by 8.6-fold and 2-fold in ST segment elevation myocardial infarction (STEMI) patients versus UA; concomitant upregulation in the expression of ABHD4 mRNA and miRNA-197-5p by 444-fold and 10-fold in UA compared with healthy volunteers, and by 1.54-fold and 4.5-fold in STEMI versus unstable angina. Performance characteristics analysis showed that the ABHD4-regulating RNA panel were potential biomarkers for prediction of ACS. Moreover, there was a significant association between the 2 miRNAs and ABHD4 mRNA and the regulating FENDRR lncRNA. CONCLUSION: Collectively, ABHD4 mRNA regulating RNA panel based on putative interactions seems to be novel non-invasive biomarkers that could detect ACS early and stratify severity of the condition that could improve health outcome.

Promoting Effect of L-tyrosine Supplement on New Zealand Rabbit Bucks' Performance and Reproduction Through Upregulation of Steroidogenic Markers.

Delayed puberty and lower fertility are among the most challenging concerns in rabbit development during the summer season. This study was, therefore, aimed at enhancing male NZ rabbits' performance by using L-tyrosine. Thirty male, New Zealand rabbits, were employed for this purpose at the age of 60 days. Rabbits were divided accidentally into two groups: a control group and another treated with L-tyrosine (100 mg/kg body weight). After 4 weeks, three bucks of each group were assassinated. A comparable oral dose of L-tyrosine was administered to half of the treated group left untreated during the second half. Weekly blood samples were assembled from each group for testosterone, T3, and T4 hormone testing. The results showed that body weight and serum testosterone, T3, and T4 increased exponentially with increasing age in both groups. L-tyrosine contributed to another vital rise in dose-dependence than control, in bodyweight, GSI, and testosterone, T3, and T4. At the end of the third month, tests fell in the scrotum, compared to 2 weeks before in the L-tyrosine group. In the middle of the fourth month, the semen evaluations were first carried out for the L-tyrosine group and 1 month after for the control group. L-tyrosine has contributed to a substantial upsurge in semen quality and motility, and abnormalities have reduced dramatically (P < 0.01). The L-tyrosine-treated group showed significantly increased mRNA expression of steroidogenesis markers STAR, CYP11A1, and 3B-HSD. Besides, free sperm in the seminiferous tubular lumen was discovered at the end of the third month. Nevertheless, it achieves only in control of the spermatocyte stage. The research suggests that L-tyrosine supplements promote puberty and improve male New Zealand rabbit fertility during high-temperature periods in the year.

Deleterious impacts of heat stress on steroidogenesis markers, immunity status and ovarian tissue of Nile tilapia (Oreochromis niloticus).

The water temperature of aquacultures is a primary factor of fish welfare, reproductive patterns, and immunity. To elucidate the molecular and biological processes of the temperature modulation of reproduction and immunity, female Nile tilapia (190 ± 10g) were allocated into five groups following acclimatization (150 females, three replicates, each n = 10). Each group was subjected to various temperatures (28 °C, 30 °C, 32 °C, 34 °C, and 37 °C), the group at 28 °C representing the control. Their serum levels of estradiol, cortisol, and vitellogenin were measured as well as serum triiodothyronine (T3) hormone, thyroxine (T4) hormone, and non-specific immunity (phagocytic and lysozyme activity). In addition, steroidogenic acute regulatory protein (STAR), vitellogenin gene receptor, and heat shock protein 70 (HSP70) gene expression were evaluated. The serum levels of estradiol, cortisol, and vitellogenin markedly declined (P < 0.05) in fish group at higher temperatures. In addition to T3, T4 was significantly affected (P < 0.05) in the control group. The expressions of the STAR gene (steroidogenesis) and vitellogenin receptors were also considerably down-regulated. The histopathological photomicrograph of fish subjected to high water temperature revealed injuries in ovary tissues, demonstrating its harmful effects. The experimental results verified the possible role of water temperature as a main stressor on Nile tilapia' physiology through modulation of steroidogenesis-related gene expression and immunity.

lncRNA- RP11-156p1.3, novel diagnostic and therapeutic targeting via CRISPR/Cas9 editing in hepatocellular carcinoma.

We aim to characterize the expression of RNA panel in HCC. We assessed the expression of HCC-associated mRNA, miRNA and lncRNA network by real time PCR in sera and tissue samples. In a proof-of-principle approach, CRISPR cas9 mediated knock out for lncRNA- RP11-156p1.3 was performed in HEPG2 cell line to validate the role of the chosen RNA in HCC pathogenesis. The differential expression of RFTN1 mRNA, lncRNA- RP11-156p1.3 and miRNA-4764-5p was statistically different among the studied groups. After CRISPR cas9 mediated knockout of lncRNA- RP11-156p1.3 in HEPG2 cells, there was significant decrease in cell count and viability with reversal of the expression of the chosen RNAs. The chosen RNAs play a significant role in HCC pathogenesis and may be potential diagnostic and therapeutic targets.

Characterization of type IV antifreeze gene in Nile tilapia (Oreochromis niloticus) and influence of cold and hot weather on its expression and some immune-related genes.

The aim of this work is to study the effect of the thermal stress of ambient temperature during winter and summer on the expression of type IV antifreeze gene (ANF IV) in different tissues of Nile tilapia (Oreochromis niloticus) as well as some immune-related genes. At first, genomic ANF IV gene was characterized from one fish; 124 amino acids were identified with 92.7% similarity with that on the gene bank. Expression of ANF IV and immune-related genes were done twice, once at the end of December (winter sample, temperature 14 °C) and the other at August (summer sample, temperature 36 °C). Assessment of ANF IV gene expression in different organs of fish was done; splenic mRNA was used for assessment of immune-related gene transcripts (CXCl2 chemokine, cc-chemokine, INF-3A, and MHC IIß). Winter expression analysis of AFP IV in O. niloticus revealed significant upregulation of mRNA transcript levels in the intestine, gills, skin, spleen, liver, and brain with 324.03-, 170.06-, 107.63-, 97.61-, 94.35-, and 27.85-folds, respectively. Furthermore, upregulation in the gene was observed in some organs during summer: in the liver, gills, skin, intestine, and brain with lower levels compared with winter. The level of expression of immune-related genes in winter is significantly higher than summer in all assessed genes. Cc-chemokine gene expression was the most affected in both winter and summer. Variable expression profile of ANF IV in different organs and in different seasons together with its amino acid similarity of N-terminal and C-terminal with apolipoprotein (lipid binder) and form of high-density lipoprotein (HDL) suggests a different role for this protein which may be related to lipid metabolism.

Alleviation by gamma amino butyric acid supplementation of chronic heat stress-induced degenerative changes in jejunum in commercial broiler chickens.

High ambient temperature adversely influences poultry production. In the present study, gamma amino butyric acid (GABA) supplementation was used to alleviate the adverse changes due to heat stress (HS) in a broiler chicken strain (Ross 308). At 21 days of age, the birds were divided into four groups of 13. Two groups were housed under normal room temperature, one group was given orally 0.2 ml 0.9% physiological saline (CN) daily, the other group received 0.2 ml of 0.5% GABA solution orally (GN). A third group was exposed to environmental HS (33 ± 1 °C lasting for 2 weeks) + physiological saline (CH) and a fourth group was exposed to HS + GABA supplementation (GH). GABA supplementation during HS significantly reduced the birds' increased body temperature (p <.0001) and increased their body weight gain (p <.0001). This effect was associated with increases in the heat stress-induced reductions in jejunal villus length, crypt depth and mucous membrane thickness, and decreases in the vascular changes occurred due to HS. Additionally, GABA supplementation significantly modulated HS-induced changes in glucose facilitated transporter 2 (GLUT2), peptide transporter 1 (PEPT1) and heat shock protein 70 (HSP70) mRNA expression in the jejunal mucosa (p < .0001). GABA supplementation also significantly elevated the triiodothyronine (T3) hormone level and hemoglobin levels and decreased the heterophil-lymphocyte ratio (H/L ratio) (p <.0001). Furthermore, it induced higher hepatic glutathione peroxidase enzyme (GSH-Px) activities and decreased the malondialdehyde dehydrogenase (MDA) content. These results indicate that GABA supplementation during HS may be used to alleviate HS-related changes in broiler chickens.

Modulatory effect of different doses of ß-1,3/1,6-glucan on the expression of antioxidant, inflammatory, stress and immune-related genes of Oreochromis niloticus challenged with Streptococcus iniae.

ß-glucans are widely-known immunostimulants that are profusely used in aquaculture industry. The present study was conducted to evaluate the effect of different in-feed doses of ß-1,3/1,6-glucans on the expression of antioxidant and stress-related genes (GST, HSP-70, Vtg), inflammation related genes (Il-8, TNFα, CXC-chemokine and CAS) and adaptive immune-related genes (MHC-IIß, TLR-7, IgM-H, and Mx) of Oreochromis niloticus challenged and non-challenged with Streptococcus iniae. Six experimental groups were established: non-challenged control (non-supplemented diet), challenged control (non-supplemented diet), non-challenged supplemented with 0.1% ß-glucan, challenged supplemented with 0.1% ß-glucan, non-challenged supplemented with 0.2% ß-glucan and challenged supplemented with 0.2% ß-glucan. Fish were fed with ß-glucan for 21 days prior challenge and then sampled after 1, 3 and 7 days post-challenge. In non-challenged group, variable effects of the two doses of ß-Glucans on the expression of the studied genes were observed; 0.1% induced higher expression of HSP70, CXC chemokine, MHC-IIß and MX genes. Meanwhile, 0.2% induced better effect on the expression of Vtg, TNF-α, CAS and IgM-H, and almost equal effects of both doses on GST and IL8. However, with the challenged group, 0.2% ß-Glucans showed better effect than 0.1% at day one post challenge through significant up-regulation of GST, HSP, IL8, TNF-α, CXC, and MHC-IIß, meanwhile, the effect of 0.1% was only on the expression of HSP70, MHC-IIß, and TLR7 at day 3 post challenge. No stimulatory role for both doses of ß-Glucans on the expression of almost all genes at day 7 post-challenge. We conclude that both doses of ß-glucan can modulate the antioxidant, inflammation, stress and immune-related genes in Nile tilapia, moreover, 0.2% ß-Glucans showed better protective effect with Streptococcus iniae challange.

Modulatory Effect of Monochromatic Blue Light on Heat Stress Response in Commercial Broilers.

In a novel approach, monochromatic blue light was used to investigate its modulatory effect on heat stress biomarkers in two commercial broiler strains (Ross 308 and Cobb 500). At 21 days old, birds were divided into four groups including one group housed in white light, a second group exposed to blue light, a 3rd group exposed to white light + heat stress, and a 4th group exposed to blue light + heat stress. Heat treatment at 33°C lasted for five h for four successive days. Exposure to blue light during heat stress reduced MDA concentration and enhanced SOD and CAT enzyme activities as well as modulated their gene expression. Blue light also reduced the degenerative changes that occurred in the liver tissue as a result of heat stress. It regulated, though variably, liver HSP70, HSP90, HSF1, and HSF3 gene expression among Ross and Cobb chickens. Moreover, the Cobb strain showed better performance than Ross manifested by a significant reduction of rectal temperature in the case of H + B. Furthermore, a significant linear relationship was found between the lowered rectal temperature and the expression of all HSP genes. Generally, the performance of both strains by most assessed parameters under heat stress is improved when using blue light.

Assessment of complex water pollution with heavy metals and Pyrethroid pesticides on transcript levels of metallothionein and immune related genes.

Alteration of immunological function of an aquatic organism can be used as an indicator for evaluating the direct effect of exposure to pollutants. The aim of this work is to assess the impact of complex water pollution with special reference to Pyrethroid pesticides and heavy metals on mRNA transcript levels of Metallothionine and some immune related genes of Nile tilapia (Oreochromas Niloticus). Residues of six heavy metals and six Pyrethroid were assessed in water as well as fish tissues at three different sites of Lake Burullus, located at Northern Egypt. Variations of water physicochemical properties associated with different levels of heavy metals at the three different sections were recorded. Tissue residues of Fe, Mn and Zn, Cu, Ni exceed water levels in contrast to elevated water level of Pb. All assessed Pyrethroids are detected in fish tissue samples with higher concentration (3-42 folds) than that found in water samples especially Cypermethrin. Significant down-regulation of expression levels of metallothionein (MT) at the three sections of the lake was observed. The expression of immune related genes (IgM) and inflammatory cytokines (TNF, IL.8 and IL.1) were affected. IgM and TNF were significantly down-regulated at eastern and western section of the lake; meanwhile the expression of IL8 is down regulated at the three sections of the lack. IL1 was significantly up-regulated at eastern and middle sections. We conclude that, variable gene expression of MT and immune-related genes at the three sections of the lack impose different response to complex water pollution in relation to variable aquatic environment.

Vitamin C Modulates the Immunotoxic Effect of 17α-Methyltestosterone in Nile Tilapia.

The synthetic androgen 17α-methyltestosterone (MT) is profusely used and practically needed in the production of all-male Nile tilapia fry; however, such androgenic hormones badly disrupt the immune system. This study aimed to alleviate or counteract the immunotoxic effect of MT using vitamin C (ascorbic acid or vit C). Our results show that the highest phagocytic activity (PA), phagocytic index (PI), and lysozyme activity were detected in the vit C group and the MT plus vit C group. Furthermore, PA and PI were significantly suppressed, but lysozyme activity was stronger in the MT group than in the control. No differences were detected in the differential leukocyte count among the studied groups. Moreover, vit C obviously reduced the upregulated expression level of the innate immune-related genes, interleukin 1ß (il1ß), interleukin 8 (il8), tumor necrosis factor α (tnfα), CC-chemokine, Toll-like receptor 7 (tlr7), immunoglobulin M (IgM) heavy chain, and cellular apoptosis susceptibility (cas) induced by MT, excluding tnfα in the liver and CC-chemokine and tlr7 in the kidney. The micronucleus frequency was found to significantly improve in the vit C plus MT group in comparison to that in the MT group. Normal histoarchitecture of the liver, kidney, and spleen was observed in all the groups, except for the frequently observed melanomacrophage centers in the spleen and kidney of the fish that were treated with vit C and vit C plus MT. More importantly, our findings demonstrate that the upregulation of immune-related genes is not necessarily a sign of a stimulated or enhanced immune system.

Impaired antioxidant gene expression by pesticide residues and its relation with other cellular biomarkers in Nile Tilapia (Oreochromis niloticus) from Lake Burullus.

Organochlorines and Organophosphorus are the most commonly used pesticides. These pesticides constitute a considerable contaminating threat due to their excessive agricultural usage which in turn contaminates the aquatic system through agricultural drainage. The aim of this study was to evaluate water and tissue residues of both pesticides in O. niloticus obtained from three different sections in Lake Burullus, Egypt. Assessment of relative change in mRNA levels of GST and Vtg (oxidative stress indicator) was done and its relation with other cellular biomarkers including apoptosis, which is assessed by Cellular apoptosis susceptibility transcript level (CAS), comet assay and micronucleus assays (genotoxicity indicator). Pesticide residue levels in water are fluctuating. In fish tissues, most residues were higher than those found in water and were associated with down regulation of hepatic GST gene and Vtg expression. CAS gene involved in apoptosis, its transcript is down regulated in middle and western sections of the lake with higher pesticide residues. Different degrees of DNA damages in O. niloticus' liver cells were demonstrated by comet assay. Significant increase in the micronucleated cells in the three sections of the lake was observed; the western section fish showed the highest number. Persistent exposures of fish to pesticide caused impairment of antioxidant gene expression. This negatively affects apoptosis associated with damaging DNA and chromosome fragments.

The extent to which immunity, apoptosis and detoxification gene expression interact with 17 alpha-methyltestosterone.

Innate immunity is the first line of defence against invasion by foreign pathogens. One widely used synthetic androgen for the production of all-male fish, particularly commercially valuable Nile tilapia, Oreochromis niloticus, is 17 alpha-methyltestosterone (MT). The present study investigates the effect of MT on innate immunity, cellular apoptosis and detoxification and the mortality rate, during and after the feeding of fry with 0-, 40-and 60-mg MT/kg. Expression analysis was completed on interleukin 1 beta (il1ß), interleukin 8 (il8), tumour necrosis factor alpha (tnfα), CXC2- and CC-chemokines, interferon (ifn), myxovirus resistance (mx), toll-like receptor 7 (tlr7), immunoglobulin M heavy chain (IgM heavy chain), vitellogenin (vtg), cellular apoptosis susceptibility (cas) and glutathione S-transferase α1 (gstα1). Expression analysis revealed that MT had a significant impact on these genes, and this impact varied from induction to repression during and after the treatment. Linear regression analysis showed a significant association between the majority of the tested gene transcript levels and mortality rates on the 7th and 21st days of hormonal treatment and 2 weeks following hormonal cessation. The results are thoroughly discussed in this article. This is the first report concerning the hazardous effect of MT on a series of genes involved in immunity, apoptosis and detoxification in the Nile tilapia fry.

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor.

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low µM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low µM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.