Liposome Encapsulation Enhances the Antidiabetic Efficacy of Silibinin.

Silibinin has considerable therapeutic potential for the treatment of diabetes through anti-inflammatory, antioxidant, and immunomodulatory properties. However, the therapeutic application of silibinin is quite limited due to its poor bioavailability. In the present study, an attempt was made to improve the antidiabetic efficacy of silibinin by its encapsulation in liposomal vesicles. The liposomes with a high encapsulation efficiency of silibinin (96%) and a zeta potential of -26.2 ± 0.6 mV were developed and studied using nicotinamide/streptozotocin-induced diabetic rats. Administration of silibinin-loaded liposomes to diabetic rats lowered glucose levels, increased insulin levels, and improved pancreatic islet architecture. The anti-inflammatory effect of silibinin-loaded liposomes was demonstrated by a decrease in serum C-reactive protein (CRP) levels and a reduced deposition of collagen fibers in the islets of diabetic rats. Furthermore, silibinin-loaded liposomes were more efficient in lowering glucose, alanine transaminase, triglyceride, and creatinine levels in diabetic rats than pure silibinin. In addition, silibinin-loaded liposomes had a significantly better effect on beta-cell mass and Glut2 glucose receptor distribution in diabetic islets than pure silibinin. The present results clearly show that liposome encapsulation of silibinin enhances its antidiabetic efficacy, which may contribute to the therapeutic benefit of silibinin in the treatment of diabetes and its complications.

Synchrotron-based FTIR microspectroscopy reveals DNA methylation profile in DNA-HALO structure.

Fourier transform infrared (FTIR) spectroscopy is a rapid, non-destructive and label-free technique for identifying subtle changes in all bio-macromolecules, and has been used as a method of choice for studying DNA conformation, secondary DNA structure transition and DNA damage. In addition, the specific level of chromatin complexity is introduced via epigenetic modifications forcing the technological upgrade in the analysis of such an intricacy. As the most studied epigenetic mechanism, DNA methylation is a major regulator of transcriptional activity, involved in the suppression of a broad spectrum of genes and its deregulation is involved in all non-communicable diseases. The present study was designed to explore the use of synchrotron-based FTIR analysis to monitor the subtle changes in molecule bases regarding the DNA methylation status of cytosine in the whole genome. In order to reveal the conformation-related best sample for FTIR-based DNA methylation analysis in situ, we used methodology for nuclear HALO preparations and slightly modified it to isolated DNA in HALO formations. Nuclear DNA-HALOs represent samples with preserved higher-order chromatin structure liberated of any protein residues that are closer to native DNA conformation than genomic DNA (gDNA) isolated by the standard batch procedure. Using FTIR spectroscopy we analyzed the DNA methylation profile of isolated gDNA and compared it with the DNA-HALOs. This study demonstrated the potential of FTIR microspectroscopy to detect DNA methylation marks in analyzed DNA-HALO specimens more precisely in comparison with classical DNA extraction procedures that yield unstructured whole genomic DNA. In addition, we used different cell types to assess their global DNA methylation profile, as well as defined specific infrared peaks that can be used for screening DNA methylation.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Introduction: Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. Results: In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. Conclusion: In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile-The Example of SRXN1 Gene.

The role of catechins in the epigenetic regulation of gene expression has been widely studied; however, if and how this phenomenon relates to the redox properties of these polyphenols remains unknown. Our earlier study demonstrated that exposure of the human colon adenocarcinoma HT29 cell line to these antioxidants affects the expression of redox-related genes. In particular, treatment with (-)-epigallocatechin (EGC) downregulated transcription of gene encoding sulfiredoxin-1 (SRXN1), the peroxidase involved in the protection of cells against hydrogen peroxide-induced oxidative stress. The aim of this study was to investigate whether the observed SRXN1 downregulation was accompanied by changes in the DNA methylation level of its promoter and, if so, whether it was correlated with the redox properties of catechins. The impact on DNA methylation profile in HT29 cells treated with different concentrations of five catechins, varying in chemical structures and standard reduction potentials as well as susceptibility to oxidation, was monitored by a methylation-sensitive high-resolution melting technique employing the SRXN1 promoter region as a model target. We demonstrated that catechins, indeed, are able to modulate DNA methylation of the SRXN1 gene in a redox-related manner. The nonlinear method in the statistical analysis made it possible to fish out two parameters (charge transfer in oxidation process Qox and time of electron transfer t), whose strong interactions correlated with observed modulation of DNA methylation by catechins. Based on these findings, we present a proof-of-concept that DNA methylation, which limits SRXN1 expression and thus restricts the multidirectional antioxidant action of SRXN1, may represent a mechanism protecting cells against reductive stress caused by particularly fast-reacting reductants such as EGC and (-)-epicatechin gallate (ECG) in our study.

Oxidative stress-mediated beta cell death and dysfunction as a target for diabetes management.

The biggest drawback of a current diabetes therapy is the treatment of the consequences not the cause of the disease. Regardless of the diabetes type, preservation and recovery of functional pancreatic beta cells stands as the biggest challenge in the treatment of diabetes. Free radicals and oxidative stress are among the major mediators of autoimmune destruction of beta cells in type 1 diabetes (T1D) or beta cell malfunction and death provoked by glucotoxicity and insulin resistance in type 2 diabetes (T2D). Additionally, oxidative stress reduces functionality of beta cells in T2D by stimulating their de-/trans-differentiation through the loss of transcription factors critical for beta cell development, maturity and regeneration. This review summarizes up to date clarified redox-related mechanisms involved in regulating beta cell identity and death, underlining similarities and differences between T1D and T2D. The protective effects of natural antioxidants on the oxidative stress-induced beta cell failure were also discussed. Considering that oxidative stress affects epigenetic regulatory mechanisms involved in the regulation of pancreatic beta cell survival and insulin secretion, this review highlighted huge potential of epigenetic therapy. Special attention was paid on application of the state-of-the-art CRISPR/Cas9 technology, based on targeted epigenome editing with the purpose of changing the differentiation state of different cell types, making them insulin-producing with ability to attenuate diabetes. Clarification of the above-mentioned mechanisms could provide better insight into diabetes etiology and pathogenesis, which would allow development of novel, potentially more efficient therapeutic strategies for the prevention or reversion of beta cell loss.

TET-mediated DNA hydroxymethylation is negatively influenced by the PARP-dependent PARylation.

BACKGROUND: Poly(ADP-ribosyl)ation (PARylation), a posttranslational modification introduced by PARP-1 and PARP-2, has first been implicated in DNA demethylation due to its role in base excision repair. Recent evidence indicates a direct influence of PARP-dependent PARylation on TET enzymes which catalyse hydroxymethylation of DNA-the first step in DNA demethylation. However, the exact nature of influence that PARylation exerts on TET activity is still ambiguous. In our recent study, we have observed a negative influence of PARP-1 on local TET-mediated DNA demethylation of a single gene and in this study, we further explore PARP-TET interplay. RESULTS: Expanding on our previous work, we show that both TET1 and TET2 can be in vitro PARylated by PARP-1 and PARP-2 enzymes and that TET1 PARylation negatively affects the TET1 catalytic activity in vitro. Furthermore, we show that PARylation inhibits TET-mediated DNA demethylation at the global genome level in cellulo. CONCLUSIONS: According to our findings, PARP inhibition can positively influence TET activity and therefore affect global levels of DNA methylation and hydroxymethylation. This gives a strong rationale for future examination of PARP inhibitors' potential use in the therapy of cancers characterised by loss of 5-hydroxymethylcytosine.

α-Lipoic Acid Increases Collagen Synthesis and Deposition in Nondiabetic and Diabetic Rat Kidneys.

α-Lipoic acid (ALA) is widely used as a nutritional supplement and therapeutic agent in diabetes management. Well-established antioxidant and hypoglycemic effects of ALA were considered to be particularly important in combating diabetic complications including renal injury. The present study evaluated the potential of ALA to affect profibrotic events in kidney that could alter its structure and functioning. ALA was administered intraperitoneally (10 mg/kg) to nondiabetic and streptozotocin-induced diabetic male Wistar rats for 4 and 8 weeks. The effects of ALA were assessed starting from structural/morphological alterations through changes that characterize profibrotic processes, to regulation of collagen gene expression in kidney. Here, we demonstrated that ALA improved systemic glucose and urea level, reduced formation of renal advanced glycation end products (AGEs), and maintained renal structural integrity in diabetic rats. However, profibrotic events provoked in diabetes were not alleviated by ALA since collagen synthesis/deposition and expression of transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) remained elevated in ALA-treated diabetic rats, especially after 8 weeks of diabetes onset. Moreover, 8 weeks treatment of nondiabetic rats with ALA led to the development of profibrotic features reflected in increased collagen synthesis/deposition. Besides the TGF-ß1 downstream signaling, the additional mechanism underlying the upregulation of collagen IV in nondiabetic rats treated with ALA involves decreased DNA methylation of its promoter that could arise from increased Tet1 expression. These findings emphasize the therapeutic caution in the use of ALA, especially in patients with renal diabetic complication.

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.

Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA-Implications for Epithelial-Mesenchymal Transition.

Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

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.

Association of CXCL12 gene promoter methylation with periodontitis in patients with diabetes mellitus type 2.

OBJECTIVES: CXCL12 is widely expressed, constitutive chemokine involved in tissue repair and regeneration, while the extent of its expression is important in various chronic inflammatory conditions. Involvement of DNA methylation in CXCL12 gene suppression (CXCL12) has been shown in malignancy and some autoimmune diseases. The aim of this study was to investigate whether the alterations in DNA methylation of CXCL12 are also involved in progression of periodontitis in combination with diabetes, as these chronic inflammatory conditions are strongly interrelated. DESIGN: Study included 72 subjects divided in three groups: healthy control (C, n=21), periodontitis (P, n=29) and diabetes/periodontitis group (D/P, n=22). DNA extracted from epithelial cells obtained by sterile cotton swabs from buccal mucosa was subjected to methylation specific polymerase chain reaction (MSP) to obtain DNA methylation pattern of CXCL12 promoter. RESULTS: CXCL12 promoter was predominantly unmethylated in all groups. However, increase in the frequency of the methylated form and increase in percent of methylation of CXCL12 promoter in periodontitis and diabetes/periodontitis group compared to control group were found, although without statistical significance. However, statistically significant increase in Tm of MSP products in diabetes/periodontitis group was observed. Correlation analysis revealed statistically significant relationship between the extent of DNA methylation of the CXCL12 promoter and periodontal parameters, as well as between DNA methylation of CXCL12 and glycosylated hemoglobin. CONCLUSION: Presented results suggest that chronic inflammation contributes to the change of CXCL12 DNA methylation in buccal cells and that DNA methylation profile of CXCL12 promoter plays important role in development and progression of periodontal disease.