Antiproliferative Effect of 7-Ketositosterol in Breast and Liver Cancer Cells: Possible Impact on Ceramide, Extracellular Signal-Regulated Kinases, and Nuclear Factor Kappa B Signaling Pathways.

Background: This study aimed to examine the effect of 7-Ketositosterol (7-KSS), on sphingomyelin/ceramide metabolites and apoptosis in human breast MCF-7 and human liver HepG2 cancer cells. Methods: Anti-proliferative effects of 7-KSS treatment were assessed at different concentrations and periods. Cell viability was assessed through MTT analysis, whereas the levels of sphingosine-1-phosphate (S1P), sphingomyelins (SMs), and ceramides (CERs) were measured using LC-MS/MS. Phosphorylated 44/42 ERK1/2 and NF-κB p65 (Ser536) protein levels were measured by Western blot analysis and immunofluorescence staining. Apoptosis was evaluated by TUNEL staining and flow cytometric assessment of annexin-V and propidium iodide (PI) labeling. Results: Treatment with 7-KSS significantly decreased cell survival and S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels in cancer cells compared to controls. A substantial rise was detected in intracellular amounts of C16-C24 CERs and apoptosis in cancer cells incubated with 7-KSS. Conclusions: 7-KSS stimulated ceramide accumulation and apoptosis while decreasing cell proliferation via downregulating S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels.

Protective Effects of Adropin in Experimental Subarachnoid Hemorrhage.

PURPOSE: We aimed to investigate early effects of exogenously administered adropin (AD) on neurological function, endothelial nitric oxide synthase (eNOS) expression, nitrite/nitrate levels, oxidative stress, and apoptosis in subarachnoid hemorrhage (SAH). METHODS: Following intracerebroventricular AD administration (10 µg/5 µl at a rate of 1 µl/min) SAH model was carried out in Sprague-Dawley rats by injection of autologous blood into the prechiasmatic cistern. The effects of AD were assessed 24 h following SAH. The modified Garcia score was employed to evaluate functional insufficiencies. Adropin and caspase-3 proteins were measured by ELISA, while nitrite/nitrate levels, total antioxidant capacity (TAC) and reactive oxygen/nitrogen species (ROS/RNS) were assayed by standard kits. eNOS expression and apoptotic neurons were detected by immunohistochemical analysis. RESULTS: The SAH group performed notably lower on the modified Garcia score compared to sham and SAH + AD groups. Adropin administration increased brain eNOS expression, nitrite/nitrate and AD levels compared to SHAM and SAH groups. SAH produced enhanced ROS/RNS generation and reduced antioxidant capacity in the brain. Adropin boosted brain TAC and diminished ROS/RNS production in SAH rats and no considerable change amongst SHAM and SAH + AD groups were detected. Apoptotic cells were notably increased in intensity and number after SAH and were reduced by AD administration. CONCLUSIONS: Adropin increases eNOS expression and reduces neurobehavioral deficits, oxidative stress, and apoptotic cell death in SAH model. Presented results indicate that AD provides protection in early brain injury associated with SAH.

Nasal application of kisspeptin-54 mitigates motor deficits by reducing nigrostriatal dopamine loss in hemiparkinsonian rats.

Parkinson's Disease is a progressive neurodegenerative disorder characterized by motor symptoms resulting from the loss of nigrostriatal dopaminergic neurons. Kisspeptins (KPs) are a family of neuropeptides that are encoded by the Kiss-1 gene, which exert their physiological effects through interaction with the GPR54 receptor. In the current investigation, we investigated the prospective protective effects of central KP-54 treatments on nigrostriatal dopaminergic neurons and consequent motor performance correlates in 6-hydroxydopamine (6-OHDA)-lesioned rats. Male adult Sprague Dawley rats underwent stereotaxic injection of 6-OHDA into the right medial forebrain bundle to induce hemiparkinsonism. Following surgery, rats received chronic central treatments of nasal or intracerebroventricular KP-54 (logarithmically increasing doses) for seven consecutive days. Motor performance was evaluated seven days post-surgery utilizing the open field test and catalepsy test. The levels of dopamine in the striatum were determined with mass spectrometry. Immunohistochemical analysis was conducted to assess the immunoreactivities of tyrosine hydroxylase (TH) and the GPR54 in the substantia nigra. The dose-response curve revealed a median effective dose value of ≈3 nmol/kg for both central injections. Due to its non-invasive and effective nature, nasal administration was utilized in the second phase of our study. Chronic administration of KP-54 (3nmol/kg, nasally) significantly protected 6-OHDA-induced motor deficits. Nasal KP-54 attenuated the loss of nigrostriatal dopaminergic neurons induced by 6-OHDA. Additionally, significant correlations were observed between motor performance and nigrostriatal dopamine levels. Immunohistochemical analysis demonstrated the localization of the GPR54 within TH-positive nigral cells. These findings suggest the potential efficacy of central KP-54 on motor impairments in hemiparkinsonism.

Decreased plasma levels of sphingolipids and total cholesterol in adult cystic fibrosis patients.

BACKGROUND: Sphingolipid species in the lung epithelium have a critical role for continuity of membrane structure, vesicular transport, and cell survival. Sphingolipid species were reported to have a role in the inflammatory etiology of cystic fibrosis by previous work. The aim of the study was to investigate the levels of plasma sphingomyelin and ceramide in adult cystic fibrosis (CF) patients and compared with healthy controls. MATERIALS AND METHODS: Blood samples were obtained from CF patients at exacerbation (n = 15), discharge (n = 13) and stable periods (n = 11). Healthy individuals (n = 15) of similar age served as control. Levels of C16-C24 sphingomyelin and C16-C24 ceramide were measured in the plasma by LC-MS/MS. Also, cholesterol and triglyceride levels were determined in plasma samples of the patients at stable period. RESULTS: All measured sphingomyelin and ceramide levels in all periods of CF patients were significantly lower than healthy controls except C16 sphingomyelin level in the stable period. However, plasma Cer and SM levels among exacerbation, discharge, and stable periods of CF were not different. CF patients had significantly lower cholesterol levels compared to healthy individuals. We found significant correlation of cholesterol with C16 sphingomyelin. CONCLUSION: We observed lower plasma Cer and SM levels in adult CF patients at exacerbation, discharge, and stable periods compared to healthy controls. We didn't find any significant difference between patient Cer and SM levels among these three periods. Our limited number of patients might have resulted with this statistical insignificance. However, percentage of SM16 levels were increased at discharge compared to exacerbation levels, while percentage of Cer16 and Cer 20 decreased at stable compared to exacerbation. Inclusion of a larger number of CF patients in such a follow up study may better demonstrate any possible difference between exacerbation, discharge, and stable periods.

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.

Relationship between serum Betatrophin, GPIHBP1, and LDL subfractions in patients with gestational diabetes mellitus.

OBJECTIVES: Gestational diabetes mellitus (GDM) leads to changes in the lipid metabolism. In this study, we aimed to compare serum levels of LDL subfractions, betatrophin, and glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) between patients with GDM and healthy pregnant women. DESIGN AND METHODS: We designed a prospective case-control study with 41 pregnant women. Subjects were divided into two groups: GDM and control. Betatrophin and GPIHBP1 levels were measured by ELISA method. Lipoprint LDL subfraction kit was used to perform LDL subfraction analysis electrophoretically. RESULTS: Serum levels of LDL6 subfraction, betatrophin, and GPIHBP1 were found to be higher in GDM group compared to the controls (p < 0.001). The mean LDL size were also found larger in GDM group. A positive correlation was found between betatrophin and GPIHBP1 levels (rho = 0.96, p < 0.001). CONCLUSIONS: Our findings suggest that betatrophin, and GPIHBP1 levels were found to be increased in GDM. This maybe the result of adaptive mechanisms in response to insulin resistance, but also this relationship should be evaluated for their effects on impaired lipid metabolism and lipoprotein lipase metabolism. There is a need for further prospective studies with larger samples to fully elucidate the mechanisms of this relationship both in pregnant patients and the other patient groups.

Resveratrol in Neurodegeneration, in Neurodegenerative Diseases, and in the Redox Biology of the Mitochondria.

Neurodegeneration is a process leading to the progressive loss of structure and functions of neurons. Many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease have shown many common points at the subcellular level. Neurons are metabolically active cells and need a high amount of energy. Mitochondria are known as the energy synthesis center for cells, involved in the synthesis of adenosine triphosphate by oxidative phosphorylation. Rather than just being an energy synthesis center, it has critical importance for many cellular functions such as calcium homeostasis, cell proliferation, cell growth, and apoptosis. In the process of mitochondrial dysfunction, cellular functions are disrupted and cells enter the apoptotic or necrotic pathway. Resveratrol (trans-3,5,4-trihydoxystilbene), a plant-derived polyphenol found in the seed of grapes, berries, peanuts, and wine, has many biological effects such as inhibition of lipid peroxidation, scavenging of free radicals, changes in eicosanoid synthesis, inhibition of platelet aggregation, anti-inflammatory and anticancer activity, and regulation of lipid metabolism. Through the reviewed literature, the current study investigated the protective role of resveratrol in neurodegenerative diseases. Studies show that resveratrol moderates mitochondrial function, redox status, and cellular dynamics in both in vivo and in vitro experimental models of neurodegeneration. Resveratrol suppresses reactive oxygen species production by reducing the activity of complex III due to its competition effect with coenzyme Q. In the present work, we discussed the protective effects of resveratrol on neurodegeneration, neurodegenerative diseases, and the redox biology of the mitochondria.

Adropin increases with swimming exercise and exerts a protective effect on the brain of aged rats.

Adropin is a protein in the brain that decreases with age. Exercise has a protective effect on the endothelium by increasing the level of adropin in circulation. In this study, whether adropin, whose level in the brain decreases with age, may increase with swimming exercise, and exhibit a protective effect was investigated. Young and aged male Sprague Dawley rats were submitted to 1 h of swimming exercise every day for 8 weeks. Motor activity parameters were recorded at the end of the exercise or waiting periods before the animals were euthanized. Increased motor functions were observed in only the young rats that exercised regularly. Adropin levels in the plasma, and the adropin and VEGFR2 immunoreactivities and p-Akt (Ser473) levels in the frontal cortex were significantly increased in the aged rats that exercised regularly. It was also observed that the BAX/Bcl2 ratio and ROS-RNS levels decreased, while the TAC levels increased in the aged rats that exercised regularly. The results of the study indicated that low-moderate chronic swimming exercise had protective effects by increasing the level of adropin in the frontal cortex tissues of the aged rats. Adropin is thought to achieve this effect by increasing the VEGFR2 expression level and causing Akt (Ser473) phosphorylation. These results indicated that an exercise-mediated increase in endogenous adropin may be effective in preventing the destructive effects of aging on the brain.

Effects of biotin deficiency on short term memory: The role of glutamate, glutamic acid, dopamine and protein kinase A.

Insufficient dietary biotin intake, biotinidase deficiency, drug-biotin interactions can cause biotin deficiency which may result in central nervous system dysfunctions. We hypothesized that biotin deficiency could disrupt learning and memory functions by altering glutamate, glutamine, dopamine levels and protein kinase A (PKA) activity in the hippocampus. Sixteen female and 4 male Wistar rats were mated and females were separated into 4 groups. Three pups were selected from each mother and a total of 48 pups were divided into the following experimental groups. NN group, normal diet in the prenatal and postnatal period. NB group, normal diet in the prenatal and a biotin-deficient diet in the postnatal period. BN group: biotin-deficient diet in the prenatal and a normal diet in the postnatal period, BB group: biotin-deficient diet in both the prenatal and postnatal period. Open Field, Y-Maze, Object Location, and Novel Object Recognition Tests were performed in all groups and rats were sacrificed. Glutamine, glutamate, dopamine levels and PKA activity were analyzed in the hippocampi. In the open field test, distance and velocity values of NB, BN and BB groups were decreased with respect to the NN group. Learning and memory functions of NB, BN and BB groups were found to be impaired in behavioral tests. Dopamine levels and PKA activity were also decreased in all rat pups fed with a biotin deficient diet. In conclusion, we demonstrated that biotin deficiency deteriorates short-term memory and locomotor activity. This impairment may relate to decreased dopamine levels and PKA activity in the hippocampus.

The influence of syringic acid treatment on total dopamine levels of the hippocampus and on cognitive behavioral skills.

BACKGROUND: Natural polyphenols have been investigated and are claimed to be mediators of the relationship between dopamine (DA) and memory. Therefore, we aimed to measure and evaluate the effect of syringic acid (SA) on DA expression by behavioral tests related to short-term and recognition memory in Wistar rats. METHODS: Rats were randomly assigned to control (0.5 cc corn oil, n = 10), SA (25 mg/kg/day, o.g, n = 10), Deltamethrin (DTM) (1.28 mg/kg/day o.g, n = 10) and DTM (1.28 mg/kg/day o.g, n = 10) + SA (25 mg/kg/day) groups. The Y-maze and Novel Object Recognition (NOR) tests were performed to assess cognitive and behavioral functions in the rats. Dopamine levels in the hippocampus were measured by mass spectrometry. RESULTS: Syringic acid significantly increased DA (5.45 ± 1.06 ng/ml, p = 0.0026, p < 0.05) compared with the other groups. SA increased the percent alternation (34.85 ± 0.72%, p < 0.05), time spent in the novel arm (2.88 ± 0.18 min, p < 0.05), and frequency of novel arm entries (44.91 ± 2.28%, p < 0.05), of the rats after the Y-maze test. The SA elevated the discrimination index (70.42 ± 3.59%, p < 0.001), and exploration time (30.44 ± 1.8 sec, p < 0.05) in the NOR test, and increased the short term and recognition memory in behavioral tests. CONCLUSION: Our findings support the hypothesis that SA-induced DA levels of the hippocampus may facilitate recognition and short-term memory in Wistar rats through the activation of dopaminergic receptors or pathways during the learning process, and that this can be seen in the cognitive behavior of SA-treated rats.

Effects of adropin on learning and memory in rats tested in the Morris water maze.

Adropin is a secreted peptide, which is composed of 43 amino acids and shows an effective role in regulating energy metabolism and insulin resistance. Motor coordination and locomotor activity were improved by adropin in the cerebellum. However, it is not known whether adropin administration has an effect on spatial learning and memory. In this study, we investigated the effect of adropin on spatial learning and memory and characterized the biochemical properties of adropin in the hippocampus. Thirty male Sprague-Dawley rats were randomly divided into two groups as control and adropin groups. The control group received 0.9% NaCl intracerebroventricular for 6 days, while the adropin groups received 1 nmol of adropin dissolved in 0.9% NaCl (for 6 days). The Morris water maze, Y maze, and object location recognition tests were performed to evaluate learning and memory. Also, the locomotor activity tests were measured to assess the motor function. The expression of Akt, phospho-Akt, CREB, phospho-CREB, Erk1/2, phospho-Erk1/2, glycogen synthase kinase 3 ß (GSK3ß), phospho-GSK3ß, brain-derived neurotrophic factor (BDNF), and N-methyl-d-aspartate receptor NR2B subunit were determined in the hippocampal tissues by using western blot. Behavior tests showed that adropin significantly increase spatial memory performance. Meanwhile, the western blot analyses revealed that the phosphorylated form of the Akt and CREB were enhanced with adropin administration in the hippocampus. Also, the expression of BDNF showed an enhancement in adropin group in comparison to the control group. In conclusion, we have shown for the first time that adropin exerts its enhancing effect on spatial memory capacity through Akt/CREB/BDNF signaling pathways.

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.

Serum Levels of S100ß, Neuron-Specific Enolase, Glial Fibrillary Acidic Protein in Kidney Transplant Recipients and Donors: A Prospective Cohort Study.

BACKGROUND: The aim of this study was to evaluate changes in serum levels of S100ß, neuron-specific enolase, glial fibrillary acidic protein in living donors and recipients after kidney transplantation. METHODS: We enrolled 56 patients into the study. Of these, 27 underwent donor nephrectomy (group D), and the remaining 29 underwent kidney transplantation (recipient, group R). Neuromarkers were measured in samples obtained before the procedure, on postoperative day 7, and at 1 month postoperatively. RESULTS: Postoperative kidney functions were impaired in patients who underwent living donor nephrectomy compared with their preoperative levels (P < .001), although no significant difference was observed in their neuromarkers. The postoperative delirium rating scale was also impaired after living donor nephrectomy compared with preoperative levels (P < .05). Postoperative kidney functions were improved (P < .001), and a progressive decrease in neuromarker levels (P < .05) was observed in kidney transplant recipients compared with their preoperative levels. Linear regression analysis showed a significant correlation between neuron-specific enolase, glial fibrillary acidic protein levels and kidney functions in recipients. CONCLUSION: The present study demonstrated that neuron-specific enolase and glial fibrillary acidic protein levels decrease in kidney transplant recipients and do not change in donors. This result indicated that there is no evidence of neurotoxicity in either recipients and donors in kidney transplantation.

Protective mechanism of Syringic acid in an experimental model of Parkinson's disease.

6-Hydroxydopamine (6-OHDA) is a widely used chemical to model Parkinson's disease (PD) in rats. Syringic acid (SA) is a polyphenolic compound which has antioxidant and anti-inflammatory properties. The present study aimed to evaluate the neuroprotective role of SA in a rat model of 6-OHDA-induced PD. Parkinson's disease was created by injection of 6-OHDA into the medial forebrain bundle via stereotaxic surgery. Syringic acid was administered daily by oral gavage, before or after surgery. All groups were tested for locomotor activity, rotarod performance and catatony. Dopamine levels in SN were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). The immunoreactivities for tyrosine hydroxylase (TH) and inducible nitric oxide synthase (iNOS) were detected by immunohistochemistry in frozen substantia nigra (SN) sections. Nitrite/nitrate levels, iNOS protein, total oxidant (TOS) and total antioxidant (TAS) status were assayed in SN tissue by standard kits. Motor dysfunction, impaired nigral dopamine release, increased iNOS expression and elevated nitrite/nitrate levels induced by 6-OHDA were significantly restored by SA treatment. Syringic acid significantly improved the loss of nigral TH-positive cells, while increasing TAS capacity and reducing TOS capacity in SN of PD rats. These data conclude that SA is a potential therapeutic agent for the treatment of 6-OHDA-induced rat model of PD. Syringic acid reduced the progression of PD via its neuroprotective, antioxidant and anti-inflammatory effects.

Alleviation of prilocaine-induced epileptiform activity and cardiotoxicity by thymoquinone.

PURPOSE: This study investigated whether thymoquinone (TQ) could alleviate central nervous system (CNS) and cardiovascular toxicity of prilocaine, a commonly used local anesthetic. METHODS: Rats were randomized to the following groups: control, prilocaine treated, TQ treated and prilocaine + TQ treated. Electroencephalography and electrocardiography electrodes were placed and trachea was intubated. Mechanical ventilation was initiated, right femoral artery was cannulated for continuous blood pressure measurements and blood-gas sampling while the left femoral vein was cannulated for prilocaine infusion. Markers of myocardial injury, reactive oxygen/nitrogen species (ROS/RNS) generation and total antioxidant capacity (TAC) were assayed by standard kits. Aquaporin-4 (AQP4), nuclear factor(NF)κB-p65 and -p50 subunit in brain tissue were evaluated by histological scoring. RESULTS: Blood pH and partial oxygen pressure, was significantly decreased after prilocaine infusion. The decrease in blood pH was alleviated in the prilocaine + TQ treated group. Prilocaine produced seizure activity, cardiac arrhythmia and asystole at significantly lower doses compared to prilocaine + TQ treated rats. Thymoquinone administration attenuated levels of myocardial injury induced by prilocaine. Prilocaine treatment caused increased ROS/RNS formation and decreased TAC in heart and brain tissue. Thymoquinone increased heart and brain TAC and decreased ROS/RNS formation in prilocaine treated rats. AQP4, NFκB-p65 and NFκB-p50 expressions were increased in cerebellum, cerebral cortex, choroid plexus and thalamic nucleus in prilocaine treated rats. Thymoquinone, decreased the expression of AQP4, NFκB-p65 and NFκB-p50 in brain tissue in prilocaine + TQ treated rats. CONCLUSION: Results indicate that TQ could ameliorate prilocaine-induced CNS and cardiovascular toxicity.

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.

Evaluation of Preoperative and Postoperative S100ß and NSE Levels in Liver Transplantation and Right Lobe Living-Donor Hepatectomy: A Prospective Cohort Study.

BACKGROUND AND AIMS: This study aimed to evaluate plasma neuron-specific enolase (NSE) and S100ß levels in orthotopic liver transplantation. MATERIALS AND METHODS: A total of 56 patients who underwent orthotopic liver transplantation were divided into 3 groups. Healthy donors (group D), end-stage liver failure (ESLF) patients (recipient, group R), and ESLF patients diagnosed with hepatic encephalopathy (HE, group HE). Prognosis, preoperative routine laboratory findings, serum NSE, and S100ß in samples obtained preoperation and first and sixth months postoperation were analyzed. RESULTS: Serum NSE and S100ß levels were significantly higher in ESLF patients compared to healthy donors, particularly during the preoperative period. There was a significant decrease in serum NSE and S100ß in ESLF patients during the postoperative measurement periods compared to preoperative levels. Serum NSE and S100ß levels measured at 3 different time points showed no significant difference between ESLF patients and ESLF patients with HE. However, the recent Model of End-Stage Liver Disease (MELD) and Child-Turcotte-Pugh (CTP) scores showed a significant correlation with serum NSE and S100ß in ESLF patients diagnosed with HE. Serum NSE and S100ß levels in healthy donors significantly increased within the first month following hepatectomy and decreased in the sixth month following surgery. CONCLUSION: Although serum NSE and S100ß levels significantly decreased with improved liver function in recipients following liver transplantation, there was no complete recovery within 6 months after surgery. The increase in serum levels of NSE and S100ß in donors measured following hepatectomy was detected to remain slightly higher in the sixth postoperative months.

Plasma Ceramides and Sphingomyelins of Pediatric Patients Increase in Primary Ciliary Dyskinesia but Decrease in Cystic Fibrosis.

We investigated plasma sphingomyelin (CerPCho) and ceramide (Cer) levels in pediatric patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD). Plasma samples were obtained from CF (n = 19) and PCD (n = 7) patients at exacerbation, discharge, and stable periods. Healthy children (n = 17) of similar age served as control. Levels of 16-24 CerPCho and 16-24 Cer were measured by LC-MS/MS. Concentrations of all CerPCho and Cer species measured at exacerbation were significantly lower in patients with CF than PCD. 16, 18, 24 CerPCho, and 22, 24 Cer in exacerbation; 18, 24 CerPCho, and 18, 20, 22, 24 Cer at discharge; 18, 24 CerPCho and 24 Cer at stable period were significantly lower in CF patients than healthy children (p < 0.001 and p < 0.05). All CerPCho and Cer levels of PCD patients were significantly higher except 24 CerPCho and 24 Cer during exacerbation, 24 CerPCho at discharge, and 18, 22 CerPCho levels at stable period (p < 0.001 and p < 0.05) compared with healthy children. There was no significant difference among exacerbation, discharge, and stable periods in each group for Cer and CerPCho levels. This is the first study measuring plasma Cer and CerPCho levels in PCD and third study in CF patients. The dramatic difference in plasma levels of most CerPCho and Cer species found between two diseases suggest that cilia pathology in PCD and CFTR mutation in CF seem to alter sphingolipid metabolism possibly in opposite directions.

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.