The Influence of Plant Extracts and Phytoconstituents on Antioxidant Enzymes Activity and Gene Expression in the Prevention and Treatment of Impaired Glucose Homeostasis and Diabetes Complications.

Diabetes is a complex metabolic disorder resulting either from insulin resistance or an impaired insulin secretion. Prolonged elevated blood glucose concentration, the key clinical sign of diabetes, initiates an enhancement of reactive oxygen species derived from glucose autoxidation and glycosylation of proteins. Consequently, chronic oxidative stress overwhelms cellular endogenous antioxidant defenses and leads to the acute and long-standing structural and functional changes of macromolecules resulting in impaired cellular functioning, cell death and organ dysfunction. The oxidative stress provoked chain of pathological events over time cause diabetic complications such as nephropathy, peripheral neuropathy, cardiomyopathy, retinopathy, hypertension, and liver disease. Under diabetic conditions, accompanying genome/epigenome and metabolite markers alterations may also affect glucose homeostasis, pancreatic ß-cells, muscle, liver, and adipose tissue. By providing deeper genetic/epigenetic insight of direct or indirect dietary effects, nutrigenomics offers a promising opportunity to improve the quality of life of diabetic patients. Natural plant extracts, or their naturally occurring compounds, were shown to be very proficient in the prevention and treatment of different pathologies associated with oxidative stress including diabetes and its complications. Considering that food intake is one of the crucial components in diabetes' prevalence, progression and complications, this review summarizes the effect of the major plant secondary metabolite and phytoconstituents on the antioxidant enzymes activity and gene expression under diabetic conditions.

The association of tumor necrosis factor alpha, lymphotoxin alpha, tumor necrosis factor receptor 1 and tumor necrosis factor receptor 2 gene polymorphisms and serum levels with periodontitis and type 2 diabetes in Serbian population.

OBJECTIVES: Aiming to show that periodontitis (PD) and type 2 diabetes (T2D) are bidirectionally related and potentially linked by inflammatory cytokines, we searched for association between -308 G/A Tumor necrosis factor-alpha (TNFα), +252A/G Lymphotoxin-alpha (LTα), +36A/G Tumor necrosis factor receptor 1 (TNFR1) and +676 T/G tumor necrosis factor receptor 2 (TNFR2) single nucleotide polymorphisms (SNPs) and: risk of PD or PD + T2D; periodontitis parameters in PD and PD + T2D; serum levels of cytokines/their receptors. Relationship between periodontal inflammation and serum cytokine/receptor levels was also assessed. DESIGN: Subjects were stratified as: 57 healthy controls (HC); 58 PD; 65 PD + T2D. Sociodemographic, environmental, behavioral and periodontal clinical data were recorded. SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism, while cytokines/receptors levels were quantified by enzyme-linked immunosorbent assay. Impact of periodontal inflammation was measured using periodontal inflamed surface area (PISA). RESULTS: TNFα AA genotype showed protective effect in T2D + PD compared to PD, even adjusted for behavioral/environmental factors (OR 0.18; 95 %CI 0.037-0.886; p = 0.035). LTα AG heterozygotes had increased risk of PD (OR 3.27; 95 %CI 1.35-7.96; p = 0.016), while TNFR2 TG genotype had protective effect (OR = 0.44; 95 %CI 0.954-0.9794; p = 0.043). TNFR1 AA was predictor of periodontal pocket depth and clinical attachment loss in PD. Correlation between TNFR2 concentration and PISA was negative in PD, positive in PD + T2D. CONCLUSIONS: None of the SNPs showed cross-susceptibility between PD and T2D. + 252A/G LTα and +676 T/G TNFR2 SNPs are associated with PD risk. Periodontal destruction in healthy individuals is influenced by TNFR1 genotype. Impact of periodontal on systemic inflammation is masked by T2D.

DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-ß isoforms, TGF-ß1 and TGF-ß2, and their combination. TGF-ß1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCR-based array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCR-based array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-ß1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

Centaurium erythraea extract improves survival and functionality of pancreatic beta-cells in diabetes through multiple routes of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Centaurium erythraea Rafn (CE) is used as a traditional medicinal plant in Serbia to treat different ailments due to its antidiabetic, antipyretic, antiflatulent and detoxification effects. AIM OF THE STUDY: Elucidation of the mechanisms that underlie the antioxidant and pro-survival effects of the CE extract (CEE) in beta-cells and pancreatic islets from streptozotocin (STZ)-treated diabetic rats. MATERIAL AND METHODS: Diabetes was induced in rats by multiple applications of low doses of STZ (40 mg/kg intraperitoneally (i.p.), for five consecutive days). CEE (100 mg/kg) was administered orally, in the pre-treated group for two weeks before diabetes induction, during the treatments with STZ and for four weeks after diabetes onset, and in the post-treatment group for four weeks after diabetes induction. The impact of CEE on diabetic islets was estimated by histological and immunohistochemical examination of the pancreas. Molecular mechanisms of the effects of CEE were also analyzed in insulinoma Rin-5F cells treated with STZ (12 mM) and CEE (0.25 mg/mL). Oxidative stress was evaluated by assessing the levels of DNA damage, lipid peroxidation, protein S-glutathionylation and enzymatic activities and expression of CAT, MnSOD, CuZnSOD, GPx and GR in beta-cells. The presence and activities of the redox-sensitive and islet-enriched regulatory proteins were also analyzed. RESULTS: Treatment with CEE ameliorated the insulin level and glycemic control in STZ-induced diabetic rats by improving the structural and functional properties of pancreatic islets through multiple routes of action. The disturbance of islet morphology and islet cell contents in diabetes was reduced by the CEE treatment and was associated with a protective effect of CEE on the levels of insulin, GLUT-2 and p-Akt in diabetic islets. The antioxidant effect of CEE on STZ-treated beta-cells was displayed as reduced DNA damage, lipid peroxidation, protein S-glutathionylation and alleviation of STZ-induced disruption in MnSOD, CuZnSOD and CAT enzyme activities. The oxidative stress-induced disturbance of the transcriptional regulation of CAT, MnSOD, CuZnSOD, GPx and GR enzymes in beta-cells was improved after the CEE treatment, and was observed as readjustment of the presence and activities of redox-sensitive NFκB-p65, FOXO3A, Sp1 and Nrf-2 transcription factors. The observed CEE-mediated induction of proliferative and pro-survival pathways and insulin expression/secretion after STZ-induced oxidative stress in beta-cells could be partially attributed to a fine-tuned modulation of the activities of pro-survival Akt, ERK and p38 kinases and islet-enriched Pdx-1 and MafA regulatory factors. CONCLUSIONS: The results of this study provide evidence that CEE improves the structural and functional properties of pancreatic beta-cells by correcting the endogenous antioxidant regulatory mechanisms and by promoting proliferative and pro-survival pathways in beta-cells.

Absence of PARP-1 affects Cxcl12 expression by increasing DNA demethylation.

Poly [ADP-ribose] polymerase 1 (PARP-1) has an inhibitory effect on C-X-C motif chemokine 12 gene (Cxcl12) transcription. We examined whether PARP-1 affects the epigenetic control of Cxcl12 expression by changing its DNA methylation pattern. We observed increased expression of Cxcl12 in PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-) in comparison to wild-type mouse embryonic fibroblasts (NIH3T3). In the Cxcl12 gene, a CpG island is present in the promoter, the 5' untranslated region (5' UTR), the first exon and in the first intron. The methylation state of Cxcl12 in each cell line was investigated by methylation-specific PCR (MSP) and high resolution melting analysis (HRM). Both methods revealed strong demethylation in PARP1-/- compared to NIH3T3 cells in all four DNA regions. Increased expression of the Ten-eleven translocation (Tet) genes in PARP1-/- cells indicated that TETs could be important factors in Cxcl12 demethylation in the absence of PARP-1, accounting for its increased expression. Our results showed that PARP-1 was a potential upstream player in (de)methylation events that modulated Cxcl12 expression.

Centaurium erythraea methanol extract protects red blood cells from oxidative damage in streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Centaurium erythraea Rafn (CE) is a traditional medicinal herb in Serbia with antidiabetic, digestive, antipyretic and antiflatulent effects AIM OF THE STUDY: To investigate the potential protective effects of the methanol extract of the aerial parts of CE against glyco-oxidative stress in red blood cells (RBCs) in rats with experimentally induced diabetes. MATERIAL AND METHODS: Diabetes was induced in Wistar rats by intraperitoneal (i.p.) injection of multiple low-dose streptozotocin (STZ) (40mg/kg, for five consecutive days), with the 1st day after the last STZ injection taken as the day of diabetes onset. The methanol extract of CE (100mg/kg) was administered orally and daily, two weeks before the first STZ injection, during the 5-day treatment with STZ, and for four weeks after the STZ injections (pre-treated group) or for four weeks after diabetes onset (post-treated group). The effect of CE extract administration on the redox status of RBCs was evaluated by assessing lipid peroxidation, the ratio of reduced/oxidized glutathione (GSH/GSSG), the level of S-glutathionylated proteins (GSSP) and the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in RBCs four weeks after diabetes onset. The major biochemical parameters of diabetes, protein glycation/glycosylation of erythrocytes and parameters which correlate with their aggregation and deformability were also evaluated. RESULTS: Daily application of CE extract to STZ-induced diabetic rats provided important antidiabetic effects, observed in both pre-treated and post-treated groups of diabetic rats as elevated serum insulin concentration, reduction of blood glucose and glycated hemoglobin concentrations and an improved lipid profile. Antioxidant effects of CE extract were detected in RBCs of diabetic rats and observed as decreased lipid peroxidation and ameliorated oxidative damage as a result of increased SOD, CAT and GR activities, an improved GSH/GSSG ratio and reduced GSSP levels. Moreover, the CE extract protected RBC proteins from hyperglycemia-induced damage by reducing non-enzymatic glycation and enzymatic glycosylation processes. CE extract was more effective when applied before diabetes induction (pre-treated group). CONCLUSIONS: The results of this study show that the Centaurium erythraea methanol extract protects RBCs in diabetic animals from oxidative damage. They provide additional support for the application of this traditionally used plant in diabetes management.

Relationship between serum tumor necrosis factor receptor-2 concentration and periodontal destruction in patients with type 2 diabetes: Cross-sectional study.

Introduction: The role of tumor necrosis factor-α (TNFα) is well documented in pathogenesis of chronic periodontitis (CP) and type 2 diabetes (T2D). Considering short half-life of TNFα, tumor necrosis factor receptor-2 (TNFR2) is used as prosperous surrogate marker of TNFα activity. Objective: The aim was to detect TNFR2 serum concentration and correlate it with periodontal destruction in patients with diagnosed T2D and nondiabetics. Methods: The study included 85 patients divided into three groups: T2D + CP (group T2D, n = 34); nondiabetics + CP (Group PD, n = 27); and healthy controls (group HC, n = 24). T2D was diagnosed according to WHO criteria (2013) and periodontitis was diagnosed using International Workshop for a Classification of Periodontal Diseases and Conditions criteria (1999). TNFR2 level was measured by enzyme-linked immunosorbent assay (ELISA). Results: There was no difference in TNFR2 level among the groups (Kruskal­Wallis, p = 0.482). Significant correlation (Pearson's correlation coefficient) was observed between clinical attachment loss (CAL) and TNFR2 concentration in PD group (rp = -0.460, p = 0.016). In T2D group, correlations were observed between TNFR2 concentration and CAL (rp = 0.363, p = 0.005) and periodontal inflamed surface area (PISA) (rp = 0.345, p = 0.046) and periodontal epithelial surface area (PESA) (rp = 0.578, p = 0.000). Conclusion: Higher concentration of TNFR2 was associated with higher CAL, PESA, and PISA scores in T2D group. Contrary to that, nondiabetics with higher values of CAL exhibited lower concentration of TNFR2, presenting potential protective effect on periodontal destruction. These results imply that diabetes may alter TNFR2 secretion originated from periodontium.

Biochemical indicators and biomarkers in chub (Squalius cephalus L.) from the Sava River.

Biochemical indicators and biomarkers were analyzed in the liver and gills of chub caught in three localities along the Sava River exposed to different environmental impacts. Sampling sites were: downstream from Zagreb (Zgd), downstream Sremska Mitrovica (SM) and downstream from Belgrade (Bgd). We observed that the relative amounts and levels of activity of Cu, Zn containing superoxide dismutase and glutathione in both the liver and gills, and the relative amounts of heat shock protein (HSP90) and metallothioneins in the gills were highest in the Zgd locality, suggesting a higher impact of metal pollution. The Zgd locality had higher concentrations of trace metals in the water, especially iron. In the SM and Bgd localities, higher relative levels of glutathione peroxidase and catalase were recorded (especially in SM) as compared to the Zgd locality, pointing to the presence of hydrogen peroxide and different classes of organic peroxides. Low water oxygen and high temperature levels in the Bgd locality suggesting different metabolic activity between examined locations. Our results suggest that different presence and concentrations of individual environmental factors (total environment) influence the way how fish establish homeostasis.