Effects of transcranial direct current stimulation on the glutamatergic pathway in the male rat hippocampus after experimental focal cerebral ischemia.

This study aimed to assess the focal cerebral ischemia-induced changes in learning and memory together with glutamatergic pathway in rats and the effects of treatment of the animals with transcranial Direct Current Stimulation (tDCS). One hundred male rats were divided into five groups as sham, tDCS, Ischemia/Reperfusion (IR), IR + tDCS, and IR + E-tDCS groups. Learning, memory, and locomotor activity functions were evaluated by behavioral experiments in rats. Glutamate and glutamine levels, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR1), N-Methyl-D-Aspartate receptors (NMDAR1 and NMDAR2A), vesicular glutamate transporter-1 (VGLUT-1), and excitatory amino acid transporters (EAAT1-3) mRNA expressions in hippocampus tissues were measured. Ischemic areas were analyzed by TTC staining. The increase was observed in IR + tDCS, and IR + E-tDCS groups compared to the IR group while a significant decrease was observed in IR group compared to the sham in the locomotor activity, learning, and memory tests. While glutamate and glutamine levels, AMPAR1, NMDAR1, NMDAR2A, VGLUT1, and EAAT1 mRNA expressions were significantly higher in IR group compared to the sham group, it was found to be significantly lower in IR + tDCS and IR + E-tDCS groups compared to the IR group. EAAT2 and EAAT3 mRNA expressions were significantly higher in IR + tDCS and IR + E-tDCS groups compared to the IR group. Ischemic areas were significantly decreased in IR + tDCS and IR + E-tDCS groups compared to the IR group. Current results suggest that tDCS application after ischemia improves learning and memory disorders and these effects of tDCS may be provided through transporters that regulate glutamate levels.

Effects of aurantiamide on a rat model of renovascular arterial hypertension.

Asperglaucide (ASP) is an aurantiamide, an effective constituent of purslane (Portulaca oleracea L.), a safe to eat greenery. Effects of ASP on endothelial function, endothelial nitric oxide synthase (eNOS) expression, vascular fluidity, renal and vascular reactive oxygen, and nitrogen species (ROS/RNS) production was examined in the two-kidney one-clip (2 K-1C) rat model of renovascular arterial hypertension. ASP toxicity, dose dependent eNOS gene expression and protein levels were also analyzed in human umbilical vein endothelial cells (HUVEC). The 2 K-1C model of hypertension was created via surgery and mean blood pressure (MBP) was measured by tail-cuff method during four weeks of ASP treatment. Erythrocyte deformability was monitored by rotational ektacytometry, while vascular constrictor and dilator responses were determined in organ baths. eNOS gene expression and protein levels were assessed in thoracic aorta and HUVEC. MBP was significantly decreased in hypertensive rats treated with ASP. Endothelium dependent vascular dilator and constrictor responses were also considerably improved following ASP treatment. There was a notable increase in red blood cell deformability in hypertensive rats treated with ASP as compared to hypertensive rats alone. A significant increase was observed in eNOS gene expression and protein levels in both normotensive and hypertensive rats treated with ASP. Treatment of HUVEC with 3 µM ASP notably increased eNOS mRNA and protein levels. In conclusion, ASP lowered blood pressure, improved endothelium-mediated relaxation, decreased renovascular ROS/RNS production in hypertensive rats. ASP also increased eNOS protein expression in aorta and HUVEC at nontoxic doses. ASP may have future potential as an anti-hypertensive agent.

Inflammatory signal transduction pathways induced by prilocaine toxicity in cultured ARPE-19 cells.

Prilocaine (PRL) is a common local anesthetic. Despite the successful use of regional anesthesia for intraocular surgery, there are associated side effects that may affect the retina in case of accidental intravitreal injection. This study examined the signal transduction pathways activated by PRL toxicity and determined the protective role of nitric oxide synthase-2 (NOS2) inhibition in cultured human-derived retinal pigment epithelial cells (ARPE-19). Toxicity analysis was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to detect the toxic dose of PRL and protective effectiveness of asperglaucide (ASP), an NOS2 inhibitor. Nuclear factor kappa B p65 (NF-κB p65), phosphorylated NF-κB p65, phospho-protein kinase B (AKT), NOS2, nitrotyrosine, and cleaved caspase-3 protein levels were evaluated by immunofluorescence staining and/or western blot analysis. Interleukin-6 (IL-6) and nitrated protein levels were quantified using an immunoassay, whereas caspase-3 activity and nitrite/nitrate levels were measured using a fluorometric method. A significant increase in NF-κB p65, and phosphorylated NF-κB p65 and AKT levels due to PRL toxicity was observed. Similarly, IL-6, NOS2, nitrite/nitrate, and nitrotyrosine levels were significantly higher in PRL-treated cells than in control cells. Application of ASP to PRL-treated cells reduced NF-κB p65, and phosphorylated NF-κB p65 and AKT to basal levels. IL-6, NOS2, nitrite/nitrate, and nitrotyrosine levels also considerably decreased following ASP treatment in cells experiencing PRL-induced toxicity. Moreover, the caspase-3-dependent apoptotic pathway was not activated. Our results indicate that ASP could ameliorate PRL-induced activation of NF-κB p65 that led to inflammation in cultured ARPE-19 cells.

Sphingolipidomic profile and HDL subfractions in obese dyslipidemic type 2 diabetic patients.

PURPOSE: The aim of the study was to investigate changes in serum sphingolipid levels and high density lipoprotein (HDL) subtypes with relation to low-density lipoprotein cholesterol (LDL-C), non-HDL-C and triglyceride (TG) levels in type 2 diabetes mellitus (T2DM) patients. METHODS: Blood was obtained from 60 patients with T2DM. Levels of sphingosine-1-phosphate (S1P), C16-C24 sphingomyelins (SMs), C16-C24 ceramides (CERs), and C16 CER-1 P were determined by LC-MS/MS. Serum concentrations of cholesterol ester transfer protein (CETP), lecithin-cholesterol acyltransferase (LCAT) and apolipoprotein A-1 (apoA-I) were analyzed by enzyme-linked immunosorbent assay (ELISA). HDL subfraction analysis was performed by Disc polyacrylamide gel electrophoresis. RESULTS: C16 SM, C24 SM, C24-C16 CER and C16 CER-1 P levels were significantly increased in T2DM patients with LDL-C above 160 mg/dL, compared to those with LDL-C below 100 mg/dL. A significant correlation was observed between C24:C16 SM, C24:C16 CER ratios and LDL-C, non HDL-C levels. Higher serum levels of C24 SM, C24-C18 CER and C24:C16 SM ratio was seen in obese T2DM patients (BMI>30) compared to those with BMI 27-30. Patients with fasting TG levels below 150 mg/dL had significantly increased HDL-large and significantly decreased HDL-small fractions compared to those with fasting TG levels above 150 mg/dL. CONCLUSION: Obese dyslipidemic T2DM patients had increased levels of serum sphingomyelins, ceramides and HDL-small fractions. The ratio of serum C24:C16 SM, C24:C16 CER and long chain CER levels may be used as diagnostic and prognostic indicators of dyslipidemia in T2DM.

Endoplasmic-reticulum-stress-induced lipotoxicity in human kidney epithelial cells.

Accumulation of lipids and their intermediary metabolites under endoplasmic reticulum (ER) stress instigates metabolic failure, described as lipotoxicity, in the kidney. This study aimed to determine ER-stress-related sphingolipid and polyunsaturated fatty acid (PUFA) changes in human kidney cells. Tunicamycin (TM) was employed to induce ER stress and an ER stress inhibitor, tauroursodeoxycholic acid (TUDCA), was given to minimize cytotoxicity. Cell viability was determined by MTT assay. Sphingomyelin (SM), ceramide (CER), and PUFA levels were measured by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78), cleaved caspase-3 and cyclooxygenase-1 (COX-1) levels were assessed by immunofluorescence. Cytosolic phospholipase A2 (cPLA2), total COX, and prostaglandin E2 (PGE2) were measured to evaluate changes in enzyme activity. Decreased cell viability was observed in TM treated cells. Administration of TUDCA following TM treatment significantly increased cell viability compared to TM treatment alone. Tunicamycin-induced ER stress was confirmed by significantly increased protein levels of GRP78. A significant increase was observed in C18-C24 CERs and caspase-3 activity, while a significant decrease occurred in sphingosine-1-phosphate (S1P) and cPLA2 activity in cells treated with TM versus controls. The decrease in cPLA2 activity was accompanied by significantly increased PUFA levels in TM treated cells. TUDCA treatment in conjunction with TM significantly decreased ER stress, C18-C24 CERs, caspase 3 activity, and increased S1P levels. Results show the buildup of long chain CERs and PUFAs in kidney cells undergoing ER stress alongside increased apoptotic activity. TUDCA administration, along with TM treatment alleviated the buildup of CERs and TM-induced apoptotic activity in kidney epithelial cells.

Vortioxetine ameliorates motor and cognitive impairments in the rotenone-induced Parkinson's disease via targeting TLR-2 mediated neuroinflammation.

Parkinson's disease (PD) is characterized by motor and non-motor symptoms associated with dopaminergic and non-dopaminergic injury. Vortioxetine is a multimodal serotonergic antidepressant with potential procognitive effects. This study aimed to explore the effects of vortioxetine on motor functions, spatial learning and memory, and depression-like behavior in the rotenone-induced rat model of PD. Male Sprague-Dawley rats were daily administered with the rotenone (2 mg kg-1, s.c.) and/or vortioxetine (10 mg kg-1, s.c.) for 28 days. Motor functions (rotarod, catalepsy, open-field), depression-like behaviors (sucrose preference test), anxiety (elevated plus maze), and spatial learning and memory abilities (novel object recognition and Morris water maze) were evaluated in behavioral tests. Then immunohistochemical, neurochemical, and biochemical analysis on specific brain areas were performed. Vortioxetine treatment markedly reduced rotenone-induced neurodegeneration, improved motor and cognitive dysfunction, decreased depression-like behaviors without affecting anxiety-like parameters. Vortioxetine also restored the impaired inflammatory response and affected neurotransmitter levels in brain tissues. Interestingly, vortioxetine was thought to trigger a sort of dysfunction in basal ganglia as evidenced by increased Toll-like receptor-2 (TLR-2) and decreased TH immunoreactivity only in substantia nigra tissue of PD rats compared to the control group. The present study indicates that vortioxetine has beneficial effects on motor dysfunction as well as cognitive impairment associated with neurodegeneration in the rotenone-induced PD model. Possible mechanisms underlying these beneficial effects cover TLR-2 inhibition and neurochemical restoration of vortioxetine.

Diclofenac down-regulates COX-2 induced expression of CD44 and ICAM-1 in human HT29 colorectal cancer cells.

Cyclooxygenase-2 (COX-2) is expressed in a variety of human colorectal cancer cells and can contribute to carcinogenesis. This study aimed to investigate the effect of diclofenac (DCF), a selective COX-2 inhibitor, on cell adhesion molecules and apoptosis in human colon adenocarcinoma cells. Levels of homing cell adhesion molecule (H-CAM, CD44), intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule-1 (VCAM-1, CD106), and epithelial cell adhesion molecule (EpCAM, CD326) were evaluated in cancer cells overexpressing (HT29) or not expressing (HCT116) COX-2. Cell viability was determined by MTT assay, COX-2 protein levels and activity were assessed by immunofluorescence and fluorometric analysis, respectively. Endogenous levels of polyunsaturated fatty acids (PUFAs) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) while expression of cell adhesion molecules was analyzed by flow cytometry. Annexin V-FITC/propidium iodide-labelling and fluorometric caspase-3 activity measurements were carried out to determine apoptosis. Flow cytometry analysis revealed that the percentage of CD44 and ICAM-1 staining in HCT116 cells was significantly lower compared to HT29 cells. In HT29 cells, phorbol 12-myristate 13-acetate (PMA) induced COX-2 expression and increased CD44 and ICAM-1 levels were down-regulated by diclofenac. Stimulation of COX-2 activity in HT29 cells via PMA significantly decreased diclofenac associated increase in PUFA levels. Treatment with both diclofenac and PMA significantly increased the number of apoptotic cells and caspase-3 activity in colon adenocarcinoma cells compared to control groups. In conclusion, diclofenac's effect to retard colorectal tumor growth and metastasis occurs in COX-2 overexpressing colon cancer cells by increased apoptosis and decreased expression of CD44 and ICAM-1.

Antiproliferative Effects of Thymoquinone in MCF-7 Breast and HepG2 Liver Cancer Cells: Possible Role of Ceramide and ER Stress.

We aimed to investigate the impact of thymoquinone (TQ), on sphingolipid metabolites, ER stress and apoptotic pathways in MCF-7 and HepG2 cancer cells. Antiproliferative effect was exerted in cancer cells via TQ incubation at different doses and durations. Cell viability was measured by MTT assay. Levels of sphingosine-1-phosphate (S1P), C16-C24 sphingomyelins (SM) and C16-C24 ceramides (CER) were determined by LC-MS/MS. Neutral sphingomyelinase (N-SMase) enzyme activity was measured by colorimetric assay and ceramide-1-phosphate (C1P) levels were determined by immunoassay. Nuclear factor kappa-b subunit 1 (NFκB1) and glucose-regulated protein 78-kd (GRP78) gene expressions were evaluated by quantitative PCR analysis, while NF-κB p65, GRP 78 and cleaved caspase-3 protein levels were assesed by immunofluorescence and western blot analysis. Incubation with TQ significantly decreased cell viability, S1P, C1P, NF-κB1 mRNA and NF-κB p65 protein levels in cancer cells compared to controls. A significant increase was observed in N-SMase activity, cellular levels of C16-C24 CERs and cleaved caspase-3 levels in cancer cells treated with TQ. GRP78 mRNA and protein levels also increased in cancer cells treated with TQ. In conclusion, TQ-induced ceramide accumulation and ER stress in conjunction with decreased S1P, C1P and NF-κB mediated cell survival may promote cancer cell death by triggering apoptosis.

Increased PUFA levels in kidney epithelial cells in the course of diclofenac toxicity.

This study evaluated polyunsaturated fatty acids (PUFAs) in human kidney epithelial cells exposed to diclofenac (DCL) toxicity. Kidney cells were treated with DCL to induce cytotoxicity and thymoquinone (TQ) was administered to decrease cytotoxic effects. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were determined by liquid chromatography coupled with tandem mass spectrometry. Cytosolic phospholipase A2 (cPLA2), cyclooxygenase 1 (COX-1) and prostaglandin E2 (PGE2) were measured to evaluate changes in enzyme activity. Immunofluorescence staining and western blot analysis was performed to determine protein levels of COX- 1. Renal cell toxicity was accomplished by DCL and was alleviated by TQ treatment. Diclofenac significantly increased all measured PUFAs while pretreatment with TQ decreased PUFA levels in DCL treated cells. Cytosolic PLA2 and total COX activity was significantly decreased in DCL treated cells. Immunofluorescence staining and western blot analysis confirmed significantly decreased COX-1 levels in DCL and DCL + TQ treated groups. The results of this study reveal that DCL treatment is associated with accumulation of PUFAs in kidney cells. We suggest that PUFA accumulation in DCL toxicity might be a consequence of both cPLA2 and COX-1 inhibition. Thymoquinone administration, along with DCL treatment alleviated the buildup of PUFAs and DCL-induced cell death in kidney cells.

Effect of ER stress on sphingolipid levels and apoptotic pathways in retinal pigment epithelial cells.

BACKGROUND: We aimed to determine sphingolipid levels and examine apoptotic pathways in human retinal pigment epithelial cells (ARPE-19) undergoing endoplasmic reticulum (ER) stress. METHODS: Cells were treated with tunicamycin (TM) to induce ER stress and tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, was administered to decrease cytotoxicity. Cell viability was measured by MTT assay. Levels of C16-C24 sphingomyelins (SM) and C16-C24 ceramides (CERs) were determined by LC-MS/MS. Glucose-regulated protein 78-kd (GRP78) and nuclear factor kappa-b subunit 1 (NFκB1) gene expressions were evaluated by quantitative PCR analysis, while GRP 78, NF-κB p65, cleaved caspase-3 and caspase-12 protein levels were assesed by immunofluorescence. Ceramide-1-phosphate (C1P) levels were determined by immunoassay, while caspase -3 and -12 activity in cell lysates were measured via a fluorometric method. RESULTS: Induction of ER stress in TM treated groups were confirmed by significantly increased mRNA and protein levels of GRP78. TM significantly decreased cell viability compared to controls. Treatment with TUDCA along with TM significantly increased cell viability compared to the TM group. A significant increase was observed in C22-C24 CERs, C1P, caspase-3, caspase-12, NFκB1 mRNA and NF-κB p65 protein levels in cells treated with TM compared to controls. Administration of TUDCA lead to a partial decrease in GRP78 expression, NFκB1 mRNA, NF-κB p65 protein, C22-C24 CERs and C1P levels along with a decrease in caspase-3 and -12 activity. CONCLUSIONS: The results of this study reveal the presence of increased long chain CERs, C1P and apoptotic markers in retinal cells undergoing ER stress.