Ivana Nikolic

Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity

The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia officinalis extracts against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MS(n)). For the quantitative estimation of triterpenic acids in the crude extracts an NMR based methodology was used and compared with the HPLC measurements, both applied for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-α release from macrophages therefore enhancing their cytocidal action. The rosemary extract developed more pronounced antioxidant, cytotoxic and immunomodifying activities, probably due to the presence of betulinic acid and a higher concentration of carnosic acid in its phytochemical profile.

Protective role of IL-33/ST2 axis in Con A-induced hepatitis

BACKGROUND & AIMS We used Concanavalin A-induced liver injury to study the role of Interleukin 33 and its receptor ST2 in the induction of inflammatory pathology and hepatocellular damage. METHODS We tested susceptibility to Concanavalin A induced hepatitis in ST2 deficient and wild type BALB/c mice and analyzed the effects of single injection of Interleukin 33 as evaluated by liver enzyme test, quantitative histology, mononuclear cell infiltration, cytokine production, intracellular staining of immune cells, and markers of apoptosis in the liver. RESULTS ST2 deficient mice developed significantly more severe hepatitis and had significantly higher number of mononuclear cells in the liver, CD4+ and CD8+ T cells, NKp46+ and CD3+NKp46+ cells, and F4/80+ macrophages. The level of pro-inflammatory cytokines in the sera and number of TNF alpha, IFN gamma, and IL-17 producing cells was higher in ST2 deficient mice. In contrast, number of CD4+Foxp3+ cells was statistically higher in wild type mice. Additionally, treatment of wild type mice with single (1 μg) injection of Interleukin 33 led to attenuation of the liver injury and milder infiltration of mononuclear cells, increase in total number of liver CD4+Foxp3+ cells and IL-4 producing CD4+ T cells. Interleukin 33 also suppressed the activation of caspase 3, prevented the expression of BAX, and enhanced the expression of antiapoptotic Bcl-2 in the liver. CONCLUSIONS We concluded that Interleukin 33/ST2 axis downregulated Concanavalin A-induced liver injury and should be evaluated as potential target in fulminant hepatitis in humans.

Galectin-3 Deficiency Accelerates High-Fat Diet Induced Obesity and Amplifies Inflammation in Adipose Tissue and Pancreatic Islets

Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet–induced obesity and diabetes. Obese LGALS3−/− mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3−/− mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c+CD11b+ macrophages and decreased CD4+CD25+FoxP3+ regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1β (IL-1β) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3−/− animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3−/− peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1–dependent IL-1β production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3−/− macrophages with NLRP3 small interfering RNA attenuated IL-1β production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes.

Pharmacological application of carbon monoxide ameliorates islet-directed autoimmunity in mice via anti-inflammatory and anti-apoptotic effects

Aims/hypothesisRecent studies have identified carbon monoxide (CO) as a potential therapeutic molecule for the treatment of autoimmune diseases owing to its anti-inflammatory and anti-apoptotic properties. We explored the efficacy and the mechanisms of action of the CO-releasing molecule (CORM)-A1 in preclinical models of type 1 diabetes.MethodsThe impact of CORM-A1 on diabetes development was evaluated in models of spontaneous diabetes in NOD mice and in diabetes induced in C57BL/6 mice by multiple low-dose streptozotocin (MLDS). Ex vivo analysis was performed to determine the impact of CORM-A1 both on T helper (Th) cell and macrophage differentiation and on their production of soluble mediators in peripheral tissues and in infiltrates of pancreatic islets. The potential effect of CORM-A1 on cytokine-induced apoptosis in pancreatic islets or beta cells was evaluated in vitro.ResultsCORM-A1 conferred protection from diabetes in MLDS-induced mice and reduced diabetes incidence in NOD mice as confirmed by preserved insulin secretion and improved histological signs of the disease. In MLDS-challenged mice, CORM-A1 attenuated Th1, Th17, and M1 macrophage response and facilitated Th2 cell differentiation. In addition, CORM-A1 treatment in NOD mice upregulated the regulatory arm of the immune response (M2 macrophages and FoxP3+ regulatory T cells). Importantly, CORM-A1 interfered with in vitro cytokine-induced beta cell apoptosis through the reduction of cytochrome c and caspase 3 levels.Conclusions/interpretationThe ability of CORM-A1 to protect mice from developing type 1 diabetes provides a valuable proof of concept for the potential exploitation of controlled CO delivery in clinical settings for the treatment of autoimmune diabetes.

Galectin-3 deficiency protects pancreatic islet cells from cytokine-triggered apoptosis in vitro†‡

Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin‐3 deficient (LGALS3−/−) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin‐3 and to delineate the underlying mechanism. Deficiency of galectin‐3, either hereditary or induced through application of chemical inhibitors, β‐lactose or TD139, supported survival and function of islet beta cells compromised by TNF‐α + IFN‐γ + IL‐1β stimulus. Similarly, inhibition of galectin‐3 by β‐lactose or TD139 reduced cytokine‐triggered apoptosis of beta cells, leading to conclusion that endogenous galectin‐3 propagates beta apoptosis in the presence of an inflammatory milieu. Exploring apoptosis‐related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin‐3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase‐9, Apaf, SMAC, caspase‐3, and AIF. In contrast, anti‐apoptotic molecules Bcl‐2 and Bcl‐XL were up‐regulated in LGALS3−/− islet cells when compared to wild‐type (WT) counterparts (C57BL/6), resulting in increased ratio of anti‐apoptotic versus pro‐apoptotic molecules. However, Fas‐triggered apoptotic pathway as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) was not influenced by LGALS‐3 deletion. All together, these results point to an important role of endogenous galectin‐3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin‐3. J. Cell. Physiol. 228: 1568–1576, 2013.

Macrophage migration inhibitory factor deficiency protects pancreatic islets from cytokine-induced apoptosis in vitro

During pathogenesis of diabetes, pancreatic islets are exposed to high levels of cytokines and other inflammatory mediators that induce deterioration of insulin‐producing beta cells. Macrophage migration inhibitory factor (MIF) plays a key role in the onset and development of several immunoinflammatory diseases and also controls apoptotic cell death. Because the occurrence of apoptosis plays a pathogenetic role in beta cell death during type 1 diabetes development and MIF is expressed in beta cells, we explored the influence of MIF deficiency on cytokine‐induced apoptosis in pancreatic islets. The results indicated clearly that elevated MIF secretion preceded C57BL/6 pancreatic islets death induced by interferon (IFN)‐γ + tumour necrosis factor (TNF)‐α + interleukin (IL)‐1β. Consequently, MIF‐deficient [MIF‐knock‐out (KO)] pancreatic islets or islet cells showed significant resistance to cytokine‐induced death than those isolated from C57BL/6 mice. Furthermore, upon exposure to cytokines pancreatic islets from MIF‐KO mice maintained normal insulin expression and produced less cyclooxygenase‐2 (COX‐2) than those from wild‐type C57BL6 mice. The final outcome of cytokine‐induced islet apoptosis in islets from wild‐type mice was the activation of mitochondrial membrane pore‐forming protein Bcl‐2‐associated X protein and effector caspase 3. In contrast, these apoptotic mediators remained at normal levels in islets from MIF‐KO mice suggesting that MIF absence prevented initiation of the mitochondrial apoptotic pathway. Additionally, the protection from apoptosis was also mediated by up‐regulation of prosurvival kinase extracellular‐regulated kinase 1/2 in MIF‐KO islets. These data indicate that MIF is involved in the propagation of pancreatic islets apoptosis probably via nuclear factor‐κB and mitochondria‐related proteins.

cDNA cloning, sequence comparison, and developmental expression of Xenopus rac1

The Rho family of small GTP-binding proteins are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. Here, we describe the temporal and spatial patterns of expression of the Rho family member, rac, during the development of the amphibian, Xenopus laevis. We also present the deduced amino acid sequence of Xenopus rac (Xrac). At the amino acid level, Xrac is highly conserved relative to previously characterized rac homologs, and is nearly identical to human rac1. RNase protection assays and Western blot analysis indicate that Xrac mRNA and protein are present from fertilization through tailbud stages of development. Whole-mount in situ hybridizations show that Xrac transcripts are especially abundant in cells of the involuting marginal zone, and later, in the cranial neural crest, the developing central nervous system, and in the somites. The remarkable degree of evolutionary conservation observed in the Xrac primary structure together with its high level of expression in cells and structures critical to morphogenesis suggest a functionally important role for this Rho family member in early vertebrate development.

Regulation of Xenopus embryonic cell adhesion by the small GTPase, rac

TGF-beta family signalling pathways are important for germ layer formation and gastrulation in vertebrate embryos and have been studied extensively using embryos of Xenopus laevis. Activin causes changes in cell movements and cell adhesion in Xenopus animal caps and dispersed animal cap cells. Rho family GTPases, including rac, mediate growth factor-induced changes in the actin cytoskeleton, and consequently, in cell adhesion and motility, in a number of different cell types. Ectopic expression of mutant rac isoforms in Xenopus embryos was combined with animal cap adhesion assays and a biochemical assay for rac activity to investigate the role of rac in activin-induced changes in cell adhesion. The results indicate that (1) the perturbation of rac signalling disrupts embryonic cell-cell adhesion, (2) that rac activity is required for activin-induced changes in cell adhesive behavior on fibronectin, and (3) that activin increases endogenous rac activity in animal cap explants.

Methanolic extract of Origanum vulgare ameliorates type 1 diabetes through antioxidant, anti-inflammatory and anti-apoptotic activity

Type 1 diabetes (T1D), an autoimmune inflammatory disorder, develops as a consequence of pancreatic β-cell destruction and results in hyperglycaemia. Since current T1D therapy mainly involves insulin replacement, the aim of the present study was to evaluate the therapeutic potential of Origanum vulgare L. ssp. hirtum (Greek oregano) leaf extract rich in biophenols for the treatment of T1D. The phytochemical profile of methanolic oregano extract (MOE) and aqueous oregano extract (AOE) was determined by liquid chromatography/electrospray ion-trap tandem MS (LC/DAD/ESI-MSn), while their main compounds were quantified by HPLC with diode array detection. After establishing their potent in vitro antioxidant activity, the extracts were administered to C57BL/6 mice treated with multiple low doses of streptozotocin for diabetes induction. While prophylactic AOE therapy had no impact on diabetes induction, MOE reduced diabetes incidence and preserved normal insulin secretion. In addition, MOE scavenged reactive oxygen and nitrogen species and, therefore, alleviated the need for the up-regulation of antioxidant enzymes. MOE treatment specifically attenuated the pro-inflammatory response mediated by T helper 17 cells and enhanced anti-inflammatory T helper 2 and T regulatory cells through the impact on specific signalling pathways and transcription factors. Importantly, MOE preserved β-cells from in vitro apoptosis via blockade of caspase 3. Finally, rosmarinic acid, a predominant compound in MOE, exhibited only partial protection from diabetes induction. In conclusion, acting as an antioxidant, immunomodulator and in an anti-apoptotic manner, MOE protected mice from diabetes development. Seemingly, there is more than one compound responsible for the beneficial effect of MOE.

Carbon Monoxide–Releasing Molecule‐A1 Inhibits Th1/Th17 and Stimulates Th2 Differentiation In vitro

Carbon monoxide (CO) is endogenously produced by haeme oxygenase‐1 and has profound effects on intracellular signalling processes, generating anti‐inflammatory, antiproliferative and antiapoptotic effects. A boron‐containing compound CORM‐A1 is capable of releasing CO in such a way to mimic physiological functions of haeme oxygenase‐1. Considering the importance of Th1/Th17 versus Th2 balance in the final outcome of immune and inflammatory responses in this study we focused on immune‐modulatory effects of CORM‐A1 on murine lymph node–derived T cells in vitro and its influence on T‐cell proliferation, activation and differentiation. Anti‐CD3/CD28 antibody‐triggered lymph node cells proliferation remained unaffected after 24‐hour CORM‐A1 treatment, as well as the expression of the early activation marker CD25. However, CORM‐A1 successfully reduced the secretion of the two representative pro‐inflammatory cytokines, IFN‐γ and IL‐17, while the secretion of anti‐inflammatory cytokine IL‐4 remained unchanged. Furthermore, CORM‐A1 efficiently reduced the percentage of CD4+IFN‐γ+ and CD4+IL‐17+ cells, whereas CD4+IL‐4+ cell population increased after treatment. Also, CORM‐A1 significantly reduced expression of transcription factor RORγT, necessary for Th17 development, but the expression of Th1‐related and Th2‐related transcription factors (T‐bet and GATA‐3, respectively) remained unchanged. In conclusion, our findings indicate that CO has anti‐inflammatory role through the regulation of balance between pro‐inflammatory Th1/Th17 and anti‐inflammatory Th2 cells. Observed immunomodulatory effects of CORM‐A1 could be useful for developing novel therapeutic approaches in managing Th1/Th17‐mediated immune disorders.