Evaluation of the Antidiabetic Potential of Xanthone-Rich Extracts from Gentiana dinarica and Gentiana utriculosa.

Despite the existence of various therapeutic approaches, diabetes mellitus and its complications have been an increasing burden of mortality and disability globally. Hence, it is necessary to evaluate the efficacy and safety of medicinal plants to support existing drugs in treating diabetes. Xanthones, the main secondary metabolites found in Gentiana dinarica and Gentiana utriculosa, display various biological activities. In in vitro cultured and particularly in genetically transformed G. dinarica and G. utriculosa roots, there is a higher content of xanthones. The aim of this study was to investigate and compare antidiabetic properties of secondary metabolites (extracts) prepared from these two Gentiana species, cultured in vitro and genetically transformed with those collected from nature. We compare HPLC secondary metabolite profiles and the content of the main extract compounds of G. dinarica and G. utriculosa methanol extracts with their ability to scavenge DPPH free radicals and inhibit intestinal α-glucosidase in vitro. Anti-hyperglycemic activity of selected extracts was tested further in vivo on glucose-loaded Wistar rats. Our findings reveal that the most prominent radical scavenging potential and potential to control the rise in glucose level, detected in xanthone-rich extracts, were in direct correlation with an accumulation of xanthones norswertianin and norswertianin-1-O-primeveroside in G. dinarica and decussatin and decussatin-1-O-primeveroside in G. utriculosa.

Beneficial Effects of Probiotic Lactobacillus paraplantarum BGCG11 on Pancreatic and Duodenum Function in Diabetic Rats.

Diabetes mellitus, as a chronic metabolic disorder, significantly impacts the pancreas and among other organs, affects duodenal function. Emerging evidence suggests that probiotics can exert beneficial effects on gut health and metabolism. In our previous research, we evaluated the probiotic Lactobacillus paraplantarum BGCG11 primarily for its protective properties against diabetic rats' damaged liver and kidneys. In this work, we further examined the effects of probiotic strain BGCG11 on the function of the duodenum and pancreas in diabetic rats. We explored the potential mechanisms underlying the probiotic's effects, focusing on general indicators of diabetes, the architecture and morphology of pancreatic islets, duodenal integrity (measuring the transfer of fluid and serum zonulin level), and the modulation of gut microbiota composition. Our findings reveal the protective and regulatory roles of L. paraplantarum BGCG11 in mitigating diabetes-induced pancreatic and duodenal dysfunction regardless of its application time (pre- or post-treatment), highlighting its therapeutic potential in managing diabetes-related gastrointestinal complications.

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.

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.

CXCL12 protects pancreatic ß-cells from oxidative stress by a Nrf2-induced increase in catalase expression and activity.

Due to intrinsically low levels of antioxidant enzyme expression and activity, insulin producing pancreatic ß-cells are particularly susceptible to free radical attack. In diabetes mellitus, which is accompanied by high levels of oxidative stress, this feature of ß-cells significantly contributes to their damage and dysfunction. In light of the documented pro-survival effect of chemokine C-X-C Ligand 12 (CXCL12) on pancreatic ß-cells, we examined its potential role in antioxidant protection. We report that CXCL12 overexpression enhanced the resistance of rat insulinoma (Rin-5F) and primary pancreatic islet cells to hydrogen peroxide (H2O2). CXCL12 lowered the levels of DNA damage and lipid peroxidation and preserved insulin expression. This effect was mediated through an increase in catalase (CAT) activity. By activating downstream p38, Akt and ERK kinases, CXCL12 facilitated Nrf2 nuclear translocation and enhanced its binding to the CAT gene promoter, inducing constitutive CAT expression and activity that was essential for protecting ß-cells from H2O2.

Identification of transcription factors involved in the transcriptional regulation of the CXCL12 gene in rat pancreatic insulinoma Rin-5F cell line.

Diabetes is characterized by a deficit in the number of functional pancreatic ß-cells. Understanding the mechanisms that stimulate neogenesis of ß-cells should contribute to improved maintenance of ß-cell mass. Chemokine CXCL12 has recently become established as a novel ß-cell growth factor, however the mechanisms controlling its expression require clarification. We investigated the proteins involved in the transcriptional regulation of the rat ß-cell CXCL12 gene (Cxcl12). Using the electrophoretic mobility shift assay and chromatin immunoprecipitation, we established the in vitro and in vivo binding of C/EBPß, C/EBPα, STAT3, p53, FOXO3a, and HMG I/Y to the Cxcl12 promoter. Co-immunoprecipitation experiments revealed protein-protein interactions between YY1 and PARP-1, FOXO3a and PARP-1, Sp1 and PARP-1, p53 and PARP-1, C/EBPß and PARP-1, YY1 and p53, YY1 and FOXO3a, p53 and FOXO3a, Sp1 and FOXO3a, C/EBPß and FOXO3a, C/EBPα and FOXO3a, Sp1 and STAT3. Our data lay the foundation for research into the interplay of signaling pathways that determine the ß-cell Cxcl12 expression profile.

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.

Decreased O-GlcNAcylation of the key proteins in kinase and redox signalling pathways is a novel mechanism of the beneficial effect of α-lipoic acid in diabetic liver.

The present study aimed to investigate the effects of the treatment with a-lipoic acid (LA), a naturally occurring compound possessing antioxidant activity, on liver oxidant stress in a rat model of streptozotocin (STZ)-induced diabetes by examining potential mechanistic points that influence changes in the expression of antioxidant enzymes such as catalase (CAT) and CuZn/Mn superoxide dismutase(s) (SOD). LA was administered for 4 weeks by daily intraperitoneal injections (10 mg/kg) to STZ-induced diabetic rats, starting from the last STZ treatment. LA administration practically normalised the activities of the indicators of hepatocellular injury, alanine and aspartate aminotransferases, and lowered oxidative stress, as observed by the thiobarbituric acid-reactive substance assay, restored the reduced glutathione:glutathione disulphide ratio and increased the protein sulfhydryl group content. The lower level of DNA damage detected by the comet assay revealed that LA reduced cytotoxic signalling, exerting a hepatoprotective effect. The LA-treated diabetic rats displayed restored specific enzymatic activities of CAT, CuZnSOD and MnSOD. Quantitative real-time PCR analysis showed that LA restored CAT gene expression to its physiological level and increased CuZnSOD gene expression, but the gene expression of MnSOD remained at the diabetic level. Although the amounts of CAT and CuZnSOD protein expression returned to the control levels, the protein expression of MnSOD was elevated. These results suggested that LA administration affected CAT and CuZnSOD expression mainly at the transcriptional level, and MnSOD expression at the post-transcriptional level. The observed LA-promoted decrease in the O-GlcNAcylation of extracellular signal-regulated kinase, protein 38 kinase, NF-kB, CCAAT/enhancer-binding protein and the antioxidative enzymes themselves in diabetic rats suggests that the regulatory mechanisms that supported the changes in antioxidative enzyme expression were also influenced by post-translational mechanisms.

Alpha-lipoic acid upregulates antioxidant enzyme gene expression and enzymatic activity in diabetic rat kidneys through an O-GlcNAc-dependent mechanism.

PURPOSE: The combined hyperglycemia lowering and antioxidant actions of α-lipoic acid (LA) contribute to its usefulness in preventing renal injury and other diabetic complications. The precise mechanisms by which LA alters diabetic oxidative renal injury are not known. We hypothesized that LA through its hypoglycemic effect lowers O-GlcNAcylation which influences the expression and activities of antioxidant enzymes which assume important roles in preventing diabetes-induced oxidative renal injury. METHODS: An experimental model of diabetes was induced in rats by the administration of 40 mg/kg streptozotocin (STZ) intraperitoneally (i.p.) for five consecutive days. LA was applied at a dose of 10 mg/kg i.p. for 4 weeks, starting from the last day of STZ administration. RESULTS: An improved glycemic status of LA-treated diabetic rats was accompanied by a significant suppression of oxidative stress and a reduction of oxidative damage of lipids, proteins and DNA. LA treatment normalized CuZn-superoxide dismutase (SOD) and catalase activities in renal tissue of diabetic rats. These changes were allied with upregulated gene expression and lower levels of O-GlcNA glycosylation. The accompanying increase in MnSOD activity was only linked with upregulated gene expression. The observed antioxidant enzyme gene regulation was accompanied by nuclear translocation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), enhanced expression of heat shock proteins (HSPs) and by reduction in O-GlcNAcylation of HSP90, HSP70, and extracellular regulated kinase and p38. CONCLUSION: α-Lipoic acid administration activates a coordinated cytoprotective response against diabetes-induced oxidative injury in kidney tissue through an O-GlcNAc-dependent mechanism.

Methanol extract from the stem of Cotinus coggygria Scop., and its major bioactive phytochemical constituent myricetin modulate pyrogallol-induced DNA damage and liver injury.

The present study was undertaken to investigate the hepatoprotective effect of the methanol extract of Cotinus coggygria Scop. in rats exposed to the hepatotoxic compound pyrogallol. Assessed with the alkaline version of the comet assay, 1000 and 2000mg/kg body weight (bw) of the extract showed a low level of genotoxicity, while 500mg/kg bw of the extract showed no genotoxic potential. Quantitative HPLC analysis of phenolic acids and flavonoids in the methanol extract of C. coggygria showed that myricetin was a major component. To test the hepatoprotective effect, a non-genotoxic dose of the C. coggygria extract and an equivalent amount of synthetic myricetin, as present in the extract, were applied either 2 or 12h prior to administration of 100mg/kg bw of pyrogallol. The extract and myricetin promoted restoration of hepatic function by significantly reducing pyrogallol-induced elevation in the serum enzymes AST, ALT, ALP and in total bilirubin. As measured by the decrease in total score and tail moment, the DNA damage in liver was also reduced by the extract and by myricetin. Our results suggest that pro-surviving Akt activity and STAT3 protein expression play important roles in decreasing DNA damage and in mediating hepatic protection by the extract. These results suggest that myricetin, as a major component in the extract, is responsible for the antigenotoxic and hepatoprotective properties of the methanol extract of C. coggygria against pyrogallol-induced toxicity.

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.

The protective effect of a mix of Lactarius deterrimus and Castanea sativa extracts on streptozotocin-induced oxidative stress and pancreatic ß-cell death.

Pancreatic ß-cell death or dysfunction mediated by oxidative stress underlies the development and progression of diabetes mellitus. In the present study, we tested extracts from the edible mushroom Lactarius deterrimus and the chestnut Castanea sativa, as well as their mixture (MIX Ld/Cs), for potential beneficial effects on streptozotocin (STZ)-induced pancreatic ß-cell death. Analysis of chelating effects, reducing power and radical-scavenging assays revealed strong antioxidant effects of the C. sativa extract and MIX Ld/Cs, while the L. deterrimus extract displayed a weak to moderate effect. The antioxidative effect of the chestnut extract corresponds with the high content of phenolics and flavonoids identified by HPLC analysis. In contrast, the mushroom extract contains relatively small amounts of phenols and flavonoids. However, both extracts, and especially their combination MIX Ld/Cs, increased cell viability after the STZ treatment as a result of a significant reduction of DNA damage and improved redox status. The chestnut extract and MIX Ld/Cs significantly lowered the STZ-induced increases in superoxide dismutase and catalase activities, while the mushroom extract had no impact on the activities of these antioxidant enzymes. However, the L. deterrimus extract exhibited good NO-scavenging activity. Different mechanisms that underlie antioxidant effects of the mushroom and chestnut extracts were discussed. When combined as in the MIX Ld/Cs, the extracts exhibited diverse but synergistic actions that ultimately exerted beneficial and protective effects against STZ-induced pancreatic ß-cell death.

STAT3/NF-κB interactions determine the level of haptoglobin expression in male rats exposed to dietary restriction and/or acute phase stimuli.

Haptoglobin is a constitutively expressed protein which is predominantly synthesized in the liver. During the acute-phase (AP) response haptoglobin is upregulated along with other AP proteins. Its upregulation during the AP response is mediated by cis-trans interactions between the hormone-responsive element (HRE) residing in the haptoglobin gene and inducible transcription factors STAT3 and C/EBP ß. In male rats that have been subjected to chronic 50% dietary restriction (DR), the basal haptoglobin serum level is decreased. The aim of this study was to characterize the trans-acting factor(s) responsible for the reduction of haptoglobin expression in male rats subjected to 50% DR for 6 weeks. Protein-DNA interactions between C/EBP and STAT families of transcription factors and the HRE region of the haptoglobin gene were examined in livers of male rats subjected to DR, as well as during the AP response that was induced by turpentine administration. In DR rats, we observed associations between the HRE and C/EBPα/ß, STAT5b and NF-κB p50, and the absence of interactions between STAT3 and NF-kB p65. Subsequent induction of the AP response in DR rats by turpentine administration elicited a normal, almost 2-fold increase in the serum haptoglobin level that was accompanied by HRE-binding of C/EBPß, STAT3/5b and NF-kB p65/p50, and the establishment of interaction between STAT3 and NF-κB p65. These results suggest that STAT3 and NF-κB p65 crosstalk plays a central role while C/EBPß acquires an accessory role in establishing the level of haptoglobin gene expression in male rats exposed to DR and AP stimuli.

The usefulness of myeloperoxidase in prediction of in-hospital mortality in patients with ST-segment elevation myocardial infarction treated by primary percutaneous coronary intervention.

BACKGROUND: The predictive value of myeloperoxidase (MPO) in ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (PCI) has not been established. The aim of the present study was to investigate MPO as a predictor of in-hospital mortality in STEMI patients treated by primary PCI. METHODS: Study population consisted of 189 STEMI patients having undergone primary PCI. Plasma MPO level was measured 24 hours after symptom onset using chemiluminescent microparticle immunoassay (Abbott Diagnostics, Germany). The Receiver Operating Characteristic analysis was performed to identify the most useful MPO cut-off level for the prediction of in-hospital mortality. The patients were divided into two groups according to the cut-off MPO level: high MPO group (> or = 840 pmol/L, n = 65) and low MPO group (< 840 pmol/L, n = 124). RESULTS: The high MPO group had significantly more frequent anterior wall infarctions (p < 0.001) and Killip class > 1 on admission (p = 0.013) as well as lower left ventricular ejection fraction (LVEF) (p = 0.011) and higher B-type natriuretic peptide (BNP) (p = 0.029) than the low MPO group. The incidence of in-hospital mortality was 5.8% and was significantly higher in the high MPO group (13.8%) than in the low MPO group (1.6%) (p = 0.001). Multiple logistic regression analysis identified the plasma MPO level as an independent predictor of in-hospital mortality (OR 3.88, 95% CI 1.13 - 13.34, p = 0.031). CONCLUSIONS: Plasma MPO level independently predicts in-hospital mortality in STEMI patients treated by primary PCI.

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.

Extract of the plant Cotinus coggygria Scop. attenuates pyrogallol-induced hepatic oxidative stress in Wistar rats.

To examine the protective potential of the Cotinus coggygria Scop. methanol extract, Wistar rats were treated with the hepatotoxic compound pyrogallol, which possesses a potent ability to generate free radicals and induce oxidative stress. The ability of the extract to counteract the oxidative stress was examined in rats that were injected with the extract intraperitoneally (500 mg·(kg body weight)(-1)) either 2 or 12 h before the pyrogallol treatment. The extract possesses a reducing activity in vitro and an ability to chelate the ferrous ion both in vivo and in vitro. Application of the extract prior to pyrogallol treatment led to a decrease in the levels of thiobarbituric acid-reactive substances, aspartate aminotransferase, and alanine aminotransferase, increased activities of antioxidant enzymes and attenuation of DNA damage, as well as increased Akt activity and inhibition of NF-κB protein expression. Treatment with the extract 12 h prior to pyrogallol administration was more effective in suppressing pyrogallol-induced oxidative damage than the 2 h pretreatment. Extract administration promoted an increase in acute phase reactants haptoglobin and α(2)-macroglobulin that was short of a full-fledged acute phase response. Administration of the extract considerably improved the markers of oxidative stress, thus revealing a potential hepatoprotective activity. Our results suggest that Akt activation, NF-κB inhibition, and induction of the acute phase play important roles in mediating hepatic protection by the extract. The greater effectiveness of the 12 h pretreatment with extract points to the important role that preconditioning assumes in improving resistance to subsequent exposure to oxidative stress.

Administration of rat acute-phase protein α(2)-macroglobulin before total-body irradiation initiates cytoprotective mechanisms in the liver.

Previously, we showed that administration of the acute-phase protein α(2)-macroglobulin (α(2)M) to rats before total-body irradiation with 6.7 Gy (LD(50/30)) of X-rays provides the same level of radioprotection as amifostine. Here, we compare the cytoprotective effects of α(2)M and amifostine on rat liver. The potential of the liver to replenish cells destroyed by ionizing radiation was assessed by immunoblot analysis with antibody to proliferating cell nuclear antigen (PCNA). After irradiation, in unprotected rats PCNA decreased 6-fold from the basal level. In rats pretreated with either α(2)M or amifostine, PCNA was increased throughout a 4 week follow-up period, indicating that hepatocyte proliferation was unaffected. Since PCNA is an important component of the repair machinery, its increased expression was accompanied by significantly lower DNA damage in α(2)M- and amifostine-treated rats. At 2 weeks after irradiation, the Comet assay revealed a 15-fold increase in DNA damage in unprotected rats, while in α(2)M- and amifostine-treated rats we observed 3- and 4-fold rise in damage, respectively. The improved protection to DNA damage was supported by elevated activity of the antioxidant systems. Compared to untreated rats, pretreatments with α(2)M and amifostine led to similar increases in levels of the inflammatory cytokine IL-6 and the redox-sensitive transcription factor NFκB, promoting upregulation of MnSOD, the major component of the cell's antioxidant axis, and subsequent increases in Mn/CuZnSOD and catalase enzymatic activities. The results show that α(2)M induces protein factors whose interplay underlies radioprotection and support the idea that α(2)M is the central effector of natural radioprotection in the rat.

In vitro antioxidant activity of selected 4-hydroxy-chromene-2-one derivatives-SAR, QSAR and DFT studies.

The series of fifteen synthesized 4-hydroxycoumarin derivatives was subjected to antioxidant activity evaluation in vitro, through total antioxidant capacity, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), hydroxyl radical, lipid peroxide scavenging and chelating activity. The highest activity was detected during the radicals scavenging, with 2b, 6b, 2c, and 4c noticed as the most active. The antioxidant activity was further quantified by the quantitative structure-activity relationships (QSAR) studies. For this purpose, the structures were optimized using Paramethric Method 6 (PM6) semi-empirical and Density Functional Theory (DFT) B3LYP methods. Bond dissociation enthalpies of coumarin 4-OH, Natural Bond Orbital (NBO) gained hybridization of the oxygen, acidity of the hydrogen atom and various molecular descriptors obtained, were correlated with biological activity, after which we designed 20 new antioxidant structures, using the most favorable structural motifs, with much improved predicted activity in vitro.

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.