Anti-Inflammatory Effects of Melatonin in Rats with Induced Type 2 Diabetes Mellitus.

INTRODUCTION: Insulin resistance is associated with a pro-inflammatory state increasing the risk for complications in patients with type 2 diabetes mellitus (T2DM). In addition to its chronobiotic effects, the pineal hormone melatonin is known to exert anti-inflammatory and antioxidant effects. Melatonin was also suggested to affect insulin secretion. The aim of this study was therefore to investigate the effect of melatonin on inflammation in diabetic rats and to study the possible involvement of the melatonin receptor, MT2. MATERIALS AND METHODS: Male Sprague Dawley rats were randomly divided into four experimental groups (n = 10 per group): (1) control, (2) streptozotocin/nicotinamide induced diabetes type 2 (T2DM), (3) T2DM treated with melatonin (500 µg/kg/day), and (4) T2DM treated with melatonin (500 µg/kg/day for 6 weeks) and the selective MT2 receptor antagonist luzindole (0.25 g/kg/day for 6 weeks). Blood samples were taken for biochemical parameters and various tissue samples (liver, adipose tissue, brain) were removed for immunohistochemistry (IHC), Western blot (WB), and Q-PCR analyses, respectively. RESULTS: Melatonin significantly reduced increased blood levels of liver transaminases (AST, ALT), blood urea nitrogen (BUN), triglyceride, very low-density lipoprotein (VLDL), and cholesterol in diabetic rats with luzindole treatment partly reversing this effect regarding the lipids. Furthermore, the liver and adipose tissues of T2DM rats treated with melatonin showed lower expression of the inflammatory markers IL-1ß, IL-6, TNF-α, and NF-κB as compared to the T2DM group without melatonin. The results also showed that the MT2 receptor is at least partly involved in the protective effects of melatonin. CONCLUSIONS: Our results suggest that melatonin exerts relevant anti-inflammatory effects on various tissues in type 2 diabetic rats.

Ellagic Acid Enhances the Antitumor Efficacy of Bevacizumab in an In Vitro Glioblastoma Model.

BACKGROUND: The anticarcinogenic effect of ellagic acid (EA), a natural phenol of fruits and vegetables, has been investigated in several types of tumors. The combined effect of EA with bevacizumab (BEV), a common drug used in treatment of recurrent glioma, on glioblastoma has not been reported. This study observed the combined effect of EA with BEV on the expression profile of the C6 glioma cell line. METHODS: Rat C6 glioma cells were treated with EA at 100 µmol/L concentration in combination with BEV at 100 ng/mL concentration for 24, 48, and 72 hours. Cell proliferation was detected by 5-bromo-2'-deoxyuridine immunohistochemistry, and p53 and caspase-3 protein levels were determined by immunohistochemistry and assessed by the H-Score. Expression profiles for P-glycoprotein (MDR1), O6-methylguanine DNA methyltransferase (MGMT), caspase-3, and p53 related proteins were detected by reverse transcriptase polymerase chain reaction after EA treatment with or without BEV. RESULTS: EA combined with BEV conspicuously reduced the cell viability of C6 glioma cells for all incubation times. EA significantly downregulated expression of MGMT regardless of combination with BEV even in the early hours after treatment. Combined EA and BEV reduced MDR1 expression only at 72 hours. EA affected the apoptotic proteins of p53 and caspase-3 at protein level in a time-dependent manner, but not at gene level. CONCLUSIONS: This study suggests successful antiproliferative efficacy of EA combined with BEV, probably through inhibition of MGMT expression and time-dependent inhibition of MDR1. EA combined with BEV may be an alternative treatment for drug-resistant gliomas.

Administration of Selenium Decreases Lipid Peroxidation and Increases Vascular Endothelial Growth Factor in Streptozotocin Induced Diabetes Mellitus.

OBJECTIVES: The imbalance in oxidant/antioxidant status plays a pivotal role in diabetes mellitus (DM). Selenium is a integral component of the antioxidant enzyme glutathione peroxidase. Se treatment induces angiogenesis and improves endothelial function through increased expression of vascular endothelial growth factor (VEGF). The aim of this study is to investigate the effect of selenium on oxidative stress, VEGF, and endothelin 1 (ET1) in a DM rat model. MATERIALS AND METHODS: We performed an experimental animal study with 64 adult male Wistar-Albino rats. Rats were divided into the following groups (n=8): control (C)7, C21, C+sodium selenite (Se)7, and C+Se21 (control rats), and DM7, DM21, DM+Se7, and DM+Se21 (diabetic rats). Diabetes was induced by 2-deoxy-2-(3-methyl-3-nitrosoureido)- D-glucopyranose [streptozotocin (STZ)]. Three weeks after STZ, DM+Se7 rats received intraperitoneal (i.p.) injections of 0.4 mg/kg Se for 7 days. The DM+Se21 rats received these injections for 21 days. The same dose/duration of Se was administered to the C+Se7 and C+Se21 groups. The remaining rats (C7, C21, DM7, DM21) received physiologic saline injections for 7 or 21 days. Ferric reducing antioxidant power (FRAP), malondialdehyde (MDA), advanced oxidation protein products (AOPP), and endothelial function markers (VEGF and ET1) in plasma samples were measured. RESULTS: Diabetic rats (DM7 and DM21) had significantly increased plasma FRAP (P=0.002, P=0.001), AOPP (P=0.024, P=0.01), MDA (P=0.004, P=0.001), and ET1 (P=0.028, P=0.003) levels compared with C7 and C21 control rats. VEGF (P=0.02, P=0.01) significantly decreased in DM7 and DM21 diabetic rats compared with their controls (C7, C21). Se administration reversed the increased MDA and decreased VEGF levels, and lowered plasma glucose levels in the DM+Se7 and DM+Se21 diabetic groups compared with diabetic rats (DM7, DM21). We observed positive correlations between FRAP-AOPP (r=0.460), FRAP-ET1 (r=0.510), AOPP-MDA (r=0.270), and AOPP-ET1 (r=0.407), and a negative correlation between MDA-VEGF (r=-0.314). CONCLUSIONS: We observed accentuated oxidative stress and impaired endothelial function in diabetes. Se treatment reduced lipid peroxidation and hyperglycemia. Se probably improved endothelial dysfunction in diabetic rats because of the increased VEGF levels.

Impact of Neuro-Behçet Disease Immunoglobulin G on Neuronal Apoptosis.

INTRODUCTION: Parenchymal neuro-Behçet disease (NBD) is encountered in 5%-15% of Behçet disease (BD) patients and is characterized by inflammation of the brainstem and diencephalon structures. Neuronal apoptosis has been shown to participate in neuronal cell loss. Anti-neuronal antibodies have been identified in NBD patients. However, the pathogenic properties of these antibodies have not been studied. METHODS: To delineate the potential pathogenic activity of serum antibodies on neurons, pooled sera from seven NBD patients and seven healthy controls were divided into purified immunoglobulin G (IgG) and IgG-depleted serum fractions, and each fraction was administered to cultured SH-SY5Y neuroblastoma cells. Cell death was evaluated with a toxicity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Moreover, expression levels of several apoptosis markers were evaluated with real time polymerase chain reaction (PCR). RESULTS: Administration of NBD IgG to cultured SH-SY5Y cells induced significantly increased cell death and apoptosis compared with other treatments. NBD IgG also enhanced the mRNA expression levels of major apoptosis and cell survival pathway factors. CONCLUSION: Our results suggest that IgGs isolated from the sera of NBD patients have a neurotoxic activity that is presumably mediated by apoptotic mechanisms.

Arg25Pro (c.915G>C) polymorphism of transforming growth factor ß1 gene suggests an association with increased risk for Hashimoto's thyroiditis.

BACKGROUND: The etiopathogenesis of Hashimoto's thyroiditis (HT) - has not been clearly elucidated although the role of chronic inflammation, endothelial dysfunction, and imbalance between pro- and anti-inflammatory cytokines has been established. Transforming growth factor ß1 (TGFß1) is required to maintain immune homeostasis, and is implicated in lymphocyte infiltration, production of autoantibodies and thyrocyte destruction seen in patients with HT. AIM: The aim of the present study was to investigate the possible association of Leu10Pro (c.869T>C) and Arg25Pro (c.915G>C) single nucleotide polymorphisms (SNPs) of TGFß1 gene with the occurrence of HT. METHODS: We analyzed the genotype and allele frequencies of polymorphisms at codon 10 and 25 in 178 patients who had been diagnosed as having HT and 197 healthy controls using PCR-restriction fragment length polymorphism (RFLP). RESULTS: There was no notable risk for HT afflicted by Leu10Pro (c.869T>C) polymorphism of TGFß1 gene. However, there was a significant increase of Arg25Pro (c.915G>C) C allele frequency in patients with HT compared with healthy controls (p=0.003, OR=1.87, 95% CI=1.23-2.84). Moreover, heterozygous (CG) subjects had a 2.53-fold increased risk for developing HT with respect to wild (GG) homozygotes (p<0.001, 95% CI=1.57-4.05). TSH levels in CG heterozygous patients were increased in comparison with wild homozygotes (p=0.006). CONCLUSION: This study indicates that the Arg25Pro (c.915G>C) polymorphism of TGFß1 gene may be related to increased risk for HT.

Tumor necrosis factor alpha (-308), interleukin-6 (-174) and interleukin-10 (-1082) gene polymorphisms in polycystic ovary syndrome.

OBJECTIVE: The imbalance between pro- and anti-inflammatory cytokines and polymorphism of cytokine genes may play a role in the etiology of the polycystic ovary syndrome (PCOS). The aim of this study was to investigate the association of polymorphisms of TNFalpha, IL-6 and IL-10 genes with the occurrence and the clinical/laboratory characteristics of PCOS in the Turkish population. STUDY DESIGN: Single nucleotide polymorphisms (SNPs) of TNFalpha (-308 G/A), IL-6 (-174 G/C), IL-10 (-1082 G/A) genes in DNA from peripheral blood leukocytes of 97 PCOS patients and 95 healthy control women were investigated. RESULTS: There is a tendency toward lower frequency of the IL-6 CC genotype and C allele among PCOS women compared with healthy controls although the difference did not reach a significant level. No notable differences were observed in allele or genotype frequencies for TNFalpha and IL-10 genes between groups. The concomitant presence of wild homozygous TNFalpha genotype together with mutant IL-6 C allele has a protective effect against PCOS with an OR=0.45 (95% CI=0.23-0.86). While TNFalpha (-308) and IL-10 (-1082) genotypes did not influence clinical/laboratory parameters in PCOS, IL-6 (-174) CC or pooled CG+CC genotypes have lower glucose, insulin, HOMA, cholesterol, triglyceride, and LDL-C, and higher GIR and HDL-C values than GG genotypes. CONCLUSIONS: We suggest that the IL-6 promoter region polymorphism may be related to occurrence and metabolic abnormalities seen in PCOS in the Turkish population. However, more studies with larger sample size are necessary to support our findings in other populations before any statement can be made about the relationship between PCOS and cytokine polymorphism.

Tumor necrosis factor alpha, interleukin-6 and interleukin-10 polymorphisms in preeclampsia.

AIM: Preeclampsia (PE) is one of the most serious disorders of pregnancy. The imbalance between pro- and anti-inflammatory cytokines may play a role in its etiology. The aim of the present study was to investigate whether cytokine gene polymorphism is associated with PE, and to evaluate the relationship between genotypes and clinical/laboratory manifestation of PE. METHODS: We investigated single nucleotide polymorphisms of tumor necrosis factor (TNF)alpha(-308 G/A), interleukin (IL)-6 (-174 G/C), IL-10 (-1082 G/A) genes in DNA from peripheral blood leukocytes of 101 PE patients and 95 healthy control women. RESULTS: In PE, there was a significant increase of the IL-10 (-1082) A allele frequency (P = 0.04). No significant differences were found in genotypes or allele frequencies of TNFalpha(-308) and IL-6 (-174) genes between PE women and controls. While TNFalpha(-308) and IL-6 (-174) genotypes did not influence clinical/laboratory parameters in PE, IL-10 (-1082) A allele carrying genotypes (AG + AA) were associated with higher glucose and lower HDL-cholesterol levels. CONCLUSION: Because women with IL-10 (-1082) AA genotype have 3.38-fold increased risk of developing PE according to GG genotype (95% CI 1.21-9.4, P = 0.01), we suggest that IL-10 (-1082) variant A allele is associated with an increased risk of preeclampsia, which is independent from its metabolic effects.

Genetic polymorphisms in DNA repair gene APE1, XRCC1 and XPD and the risk of pre-eclampsia.

OBJECTIVE: Oxidative stress has been postulated as a major contributor to placental hypoperfusion and ischemia in pre-eclampsia (PE). Reactive oxygen species (ROS) generated during placental ischemia can cause oxidative damage to nucleic acids. Base excision repair (BER) and nucleotide excision repair (NER) are major pathways responsible for removing the oxidative DNA damage. Polymorphisms in DNA repair genes may be associated with differences in the repair efficiency of DNA damage. STUDY DESIGN: In order to investigate the possible association between DNA repair genes and PE susceptibility, we analyzed genotype and allele distributions of APE1-148, XRCC1-194, XRCC1-399 and XPD-751 genes in 101 patients with PE and 107 healthy women. Differences in genotype distributions and allele frequencies in the cases and the controls were compared for statistical significance using the chi(2)-test. Haplotype frequencies were estimated using a contingency chi(2)-test. One-way ANOVA and Mann-Whitney U-test were used for the statistics of the clinical and biochemical parameters. RESULTS: No significant association between PE and the variant alleles of APE1 codon 148 (OR: 0.77, 95% CI=0.51-1.15), XRCC1 codon 194 (OR: 0.64, 95% CI=0.30-1.37), XRCC1 codon 399 (OR: 1.16, 95% CI=0.78-1.74) and XPD codon 751 (OR: 1.21, 95% CI=0.81-1.80) was observed. Results of our haplotype analysis demonstrated that there is a high linkage disequilibrium (D': 1.0, r(2)=0.042) between the haplotypes of XRCC1 codon 194 and codon 399 markers. CONCLUSIONS: These preliminary results suggest that the polymorphic variants of APE1-148, XRCC1-194, XRCC1-399, and XPD-751 genes are not significant risk factors for PE development.

The relationship between transforming growth factor-beta1, vascular endothelial growth factor, nitric oxide and Hashimoto's thyroiditis.

BACKGROUND: Hashimoto's thyroiditis (HT) is one of the autoimmune disorders of the thyroid gland. The pathogenesis of HT has not been clearly understood. This study was designed to investigate plasma transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and nitrate/nitrite (NOx - two end products of nitric oxide [NO] metabolism) in HT. METHODS: Forty patients diagnosed HT and 40 age- and sex-matched healthy controls were included in the study. TGF-beta1 and VEGF levels were measured by ELISA, NOx levels were measured spectrophotometrically. RESULTS: Plasma TGF-beta1 and VEGF were decreased, and NOx increased in HT patients in comparison with controls. There was a significant correlation between TGF-beta1 and VEGF, and weak but significant correlation between TGF-beta1 and NOx in HT. CONCLUSION: This study indicates that TGF-beta1, VEGF and NO probably have a role in the pathogenesis of Hashimoto's thyroiditis, and development of autoimmunity. Clearly, further studies are necessary to establish the exact mechanism of TGF-beta1, VEGF and NO interaction in Hashimoto's thyroiditis.

Acute-phase reactans in Hashimoto thyroiditis.

Hashimoto thyroiditis (HT) is one of the autoimmune diseases of the thyroid gland. It is characterized by diffuse lymphocytic infiltration of the thyroid gland and the elevated levels of the serum anti-thyroid antibodies. Recently Hashimoto thyroiditis has been proposed as a systemic disorder. Acute-phase reactans have been implicated for their involvement as pro-inflammatory molecules in various inflammatory diseases. The aim of the present study was to examine the blood concentrations of acute-phase proteins, namely C-reactive protein (CRP), serum amyloid A (SAA) and fibrinogen in the patients with Hashimoto thyroiditis. Two groups were enrolled: study group consisting from euthyroid patients with HT (n=30), and healthy control subjects (n=30). Blood samples were obtained from all the patients and control subjects and were analyzed for serum CRP, SAA, fibrinogen, and ESR. Mean ESR was found to be higher in HT patients than control group (p=0.024). The mean fibrinogen and SAA values in HT were also significantly higher than that of the control group (p=0.03, p=0.002). We therefore suggest that a low-grade systemic inflammation may exist in HT patients even if they were euthyroid.