Involvement of Ferroptosis in Diabetes-Induced Liver Pathology.

Cell death plays an important role in diabetes-induced liver dysfunction. Ferroptosis is a newly defined regulated cell death caused by iron-dependent lipid peroxidation. Our previous studies have shown that high glucose and streptozotocin (STZ) cause ß-cell death through ferroptosis and that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, improves ß-cell viability, islet morphology, and function. This study was aimed to examine in vivo the involvement of ferroptosis in diabetes-related pathological changes in the liver. For this purpose, male C57BL/6 mice, in which diabetes was induced with STZ (40 mg/kg/5 consecutive days), were treated with Fer-1 (1 mg/kg, from day 1-21 day). It was found that in diabetic mice Fer-1 improved serum levels of ALT and triglycerides and decreased liver fibrosis, hepatocytes size, and binucleation. This improvement was due to the Fer-1-induced attenuation of ferroptotic events in the liver of diabetic mice, such as accumulation of pro-oxidative parameters (iron, lipofuscin, 4-HNE), decrease in expression level/activity of antioxidative defense-related molecules (GPX4, Nrf2, xCT, GSH, GCL, HO-1, SOD), and HMGB1 translocation from nucleus into cytosol. We concluded that ferroptosis contributes to diabetes-related pathological changes in the liver and that the targeting of ferroptosis represents a promising approach in the management of diabetes-induced liver injury.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Investigation of metabolic properties and effects of 17ß-carboxamide glucocorticoids on human peripheral blood leukocytes.

The biological activity of three previously synthesized 17ß-carboxamide glucocorticoids (BG, BEG, and MPEA) was tested in vitro on mitogen stimulated and non-stimulated peripheral blood mononuclear cells (MNCs) and granulocytes from human healthy donors, and the results were compared to the conventional glucocorticoid dexamethasone. The tested 17ß-carboxamide glucocorticoids did not induce decreases in MNC viability and proliferation, while modulation of reactive oxygen species (ROS) synthesis in granulocytes was dependent on the cell donor. The obtained results indicate the possibility of avoidance of strong lymphocyte suppression, which is generally recognized during administration of conventional glucocorticoids. Furthermore, the metabolism of the tested derivatives was predicted in silico. The predicted metabolites were synthesized and the in silico results were confirmed by in vitro evaluation of the metabolism of BG, BEG, and MPEA in human serum and in cultures of peripheral blood MNCs. The results of the biological activity and metabolism evaluation and of previous in vivo evaluations of biological activity indicate the soft drug nature of BG, BEG, and MPEA. In order to be fully considered as soft glucocorticoids, further investigations on the toxicity and activity of the formed metabolites are required.

Expression patterns of mitochondrial OXPHOS components, mitofusin 1 and dynamin-related protein 1 are associated with human embryo fragmentation.

Developmental dysfunction in embryos, such as a lethal level of fragmentation, is assumed to be mitochondrial in origin. This study investigated the molecular basis of mitochondrial impairment in embryo fragmentation. Transcription patterns of factors that determine mitochondrial functionality: (i) components of the oxidative phosphorylation (OXPHOS) - complex I, cytochrome b, complex IV and ATP synthase; (ii) mitochondrial membrane potential (MMP); (iii) mitochondrial DNA (mtDNA) content and (iv) proteins involved in mitochondrial dynamics, mitofusin 1 (Mfn1) and dynamin related protein 1 (Drp1) were examined in six-cells Day 3 non-fragmented (control), low-fragmented (LF) and high-fragmented (HF) human embryos. Gene expression of mitochondria-encoded components of complex I and IV, cytochrome b and mtDNA were increased in HF embryos compared with control and LF embryos. In LF embryos, expression of these molecules was decreased compared with control and HF embryos. Both classes of fragmented embryos had decreased MMP compared with control. LF embryos had increased gene expression of Mfn1 accompanied by decreased expression of Drp1, while HF embryos had decreased Mfn1 expression but increased Drp1 expression. The study revealed that each improper transcriptional (in)activation of mitochondria-encoded components of the OXPHOS during early in vitro embryo development is associated with a decrease in MMP and with embryo fragmentation. The results also showed the importance of mitochondrial dynamics in fragmentation, at least in the extent of this process.

Two key temporally distinguishable molecular and cellular components of white adipose tissue browning during cold acclimation.

KEY POINTS: White to brown adipose tissue conversion and thermogenesis can be ignited by different conditions or agents and its sustainability over the long term is still unclear. Browning of rat retroperitoneal white adipose tissue (rpWAT) during cold acclimation involves two temporally apparent components: (1) a predominant non-selective browning of most adipocytes and an initial sharp but transient induction of uncoupling protein 1, peroxisome proliferator-activated receptor (PPAR) coactivator-1α, PPARγ and PPARα expression, and (2) the subsistence of relatively few thermogenically competent adipocytes after 45 days of cold acclimation. The different behaviours of two rpWAT beige/brown adipocyte subsets control temporal aspects of the browning process, and thus regulation of both components may influence body weight and the potential successfulness of anti-obesity therapies. ABSTRACT: Conversion of white into brown adipose tissue may have important implications in obesity resistance and treatment. Several browning agents or conditions ignite thermogenesis in white adipose tissue (WAT). To reveal the capacity of WAT to function in a brownish/burning mode over the long term, we investigated the progression of the rat retroperitoneal WAT (rpWAT) browning during 45 days of cold acclimation. During the early stages of cold acclimation, the majority of rpWAT adipocytes underwent multilocularization and thermogenic-profile induction, as demonstrated by the presence of a multitude of uncoupling protein 1 (UCP1)-immunopositive paucilocular adipocytes containing peroxisome proliferator-activated receptor (PPAR) coactivator-1α (PGC-1α) and PR domain-containing 16 (PRDM16) in their nuclei. After 45 days, all adipocytes remained PRDM16 immunopositive, but only a few multilocular adipocytes rich in mitochondria remained UCP1/PGC-1α immunopositive. Molecular evidence showed that thermogenic recruitment of rpWAT occurred following cold exposure, but returned to starting levels after cold acclimation. Compared with controls (22 ± 1 °C), levels of UCP1 mRNA increased in parallel with PPARγ (PPARα from days 1 to 7 and PGC-1α on day 1). Transcriptional recruitment of rpWAT was followed by an increase in UCP1 protein content (from days 1 to 21). Results clearly showed that most of the adipocytes within rpWAT underwent transient brown-fat-like thermogenic recruitment upon stimulation, but only a minority of cells retained a brown adipose tissue-like phenotype after the attainment of cold acclimation. Therefore, browning of WAT is dependent on both maintaining the thermogenic response and retaining enough brown-like thermogenically competent adipocytes in the long-term. Both aspects of browning could be important for long-term energy homeostasis and body-weight regulation.

Long-term dietary L-arginine supplementation increases endothelial nitric oxide synthase and vasoactive intestinal peptide immunoexpression in rat small intestine.

BACKGROUND AND AIMS: Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term L-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold. METHODS: Animals (six per group) acclimated to room temperature (22 ± 1 °C) and cold (4 ± 1 °C), respectively, were treated with 2.25% L-arginine, a substrate for NOSs, or with 0.01% N(ω)-nitro-L-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression. RESULTS: Long-term dietary L-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine. CONCLUSION: Our results demonstrate that long-term dietary L-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP.

Sulforaphane prevents diabetes-induced hepatic ferroptosis by activating Nrf2 signaling axis.

Recently, we characterized the ferroptotic phenotype in the liver of diabetic mice and revealed nuclear factor (erythroid-derived-2)-related factor 2 (Nrf2) inactivation as an integral part of hepatic injury. Here, we aim to investigate whether sulforaphane, an Nrf2 activator and antioxidant, prevents diabetes-induced hepatic ferroptosis and the mechanisms involved. Male C57BL/6 mice were divided into four groups: control (vehicle-treated), diabetic (streptozotocin-induced; 40 mg/kg, from Days 1 to 5), diabetic sulforaphane-treated (2.5 mg/kg from Days 1 to 42) and non-diabetic sulforaphane-treated group (2.5 mg/kg from Days 1 to 42). Results showed that diabetes-induced inactivation of Nrf2 and decreased expression of its downstream antiferroptotic molecules critical for antioxidative defense (catalase, superoxide dismutases, thioredoxin reductase), iron metabolism (ferritin heavy chain (FTH1), ferroportin 1), glutathione (GSH) synthesis (cystine-glutamate antiporter system, cystathionase, glutamate-cysteine ligase catalitic subunit, glutamate-cysteine ligase modifier subunit, glutathione synthetase), and GSH recycling - glutathione reductase (GR) were reversed/increased by sulforaphane treatment. In addition, we found that the ferroptotic phenotype in diabetic liver is associated with increased ferritinophagy and decreased FTH1 immunopositivity. The antiferroptotic effect of sulforaphane was further evidenced through the increased level of GSH, decreased accumulation of labile iron and lipid peroxides (4-hydroxy-2-nonenal, lipofuscin), decreased ferritinophagy and liver damage (decreased fibrosis, alanine aminotransferase, and aspartate aminotransferase). Finally, diabetes-induced increase in serum glucose and triglyceride level was significantly reduced by sulforaphane. Regardless of the fact that this study is limited by the use of one model of experimentally induced diabetes, the results obtained demonstrate for the first time that sulforaphane prevents diabetes-induced hepatic ferroptosis in vivo through the activation of Nrf2 signaling pathways. This nominates sulforaphane as a promising phytopharmaceutical for the prevention/alleviation of ferroptosis in diabetes-related pathologies.

Regulatory role of PGC-1α/PPAR signaling in skeletal muscle metabolic recruitment during cold acclimation.

This study examined the molecular basis of energy-related regulatory mechanisms underlying metabolic recruitment of skeletal muscle during cold acclimation and possible involvement of the l-arginine/nitric oxide-producing pathway. Rats exposed to cold (4±1°C) for periods of 1, 3, 7, 12, 21 and 45 days were divided into three groups: untreated, l-arginine treated and N(ω)-nitro-l-arginine methyl ester (l-NAME) treated. Compared with controls (22±1°C), there was an initial increase in the protein level of 5'-AMP-activated protein kinase α (day 1), followed by an increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and peroxisome proliferator-activated receptors (PPARs): PPARα and PPARγ from day 1 and PPARδ from day 7 of cold acclimation. Activation of the PGC-1α/PPAR transcription program was accompanied by increased protein expression of the key metabolic enzymes in ß-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation, with the exceptions in complex I (no changes) and ATP synthase (decreased at day 1). Cold did not affect hexokinase and GAPDH protein levels, but increased lactate dehydrogenase activity compared with controls (1-45 days). l-arginine sustained, accelerated and/or intensified cold-induced molecular remodeling throughout cold acclimation. l-NAME exerted phase-dependent effects: similar to l-arginine in early cold acclimation and opposite after prolonged cold exposure (from day 21). It seems that upregulation of the PGC-1α/PPAR transcription program early during cold acclimation triggers the molecular recruitment of skeletal muscle underlying the shift to more oxidative metabolism during prolonged cold acclimation. Our results suggest that nitric oxide has a role in maintaining the skeletal muscle oxidative phenotype in late cold acclimation but question its role early in cold acclimation.

Defining the ferroptotic phenotype of beta cells in type 1 diabetes and its inhibition as a potential antidiabetic strategy.

Introduction: Recently, the involvement of ferroptotic cell death in the reduction of ß-cell mass in diabetes has been demonstrated. To elucidate the mechanisms of ß-cell ferroptosis and potential antidiabetic effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in vivo, a mouse model of type 1 diabetes (T1D) was used. Methods: Animals were divided into three groups: control (vehicle-treated), diabetic (streptozotocin-treated, 40 mg/kg, from days 1-5), and diabetic treated with Fer-1 (1 mg/kg, from days 1-21). On day 22, glycemia and insulinemia were measured and pancreases were isolated for microscopic analyses. Results: Diabetes disturbed general parameters of ß-cell mass (islet size, ß-cell abundance and distribution) and health (insulin and PDX-1 expression), increased lipid peroxidation in islet cells, and phagocytic removal of iron-containing material. It also downregulated the main players of the antiferroptotic pathway - Nrf2, GPX4, and xCT. In contrast, Fer-1 ameliorated the signs of deterioration of ß-cell/islets, decreased lipid peroxidation, and reduced phagocytic activity, while upregulated expression of Nrf2 (and its nuclear translocation), GPX4, and xCT in ß-cell/islets. Discussion: Overall, our study confirms ferroptosis as an important mode of ß-cell death in T1D and suggests antiferroptotic agents as a promising strategy for the prevention and treatment of diabetes.

Interscapular brown adipose tissue metabolic reprogramming during cold acclimation: Interplay of HIF-1α and AMPKα.

BACKGROUND: Brown adipose tissue thermogenic program includes complex molecular and structural changes. However, energetic aspects of this process are poorly depicted. METHODS: We investigated time-dependent reprogramming of interscapular brown adipose tissue (IBAT) energy metabolism during cold-acclimation, as well as the effects of nitric oxide (()NO) on those changes. Rats were exposed to cold (4±1°C) for periods of 1, 3, 7, 12, 21, and 45days, and divided into three groups: control, treated with L-arginine, and treated with N(ω)-nitro-L-arginine methyl ester (L-NAME). RESULTS: In the early phase of cold-acclimation (up to 7days), the protein levels of all metabolic parameters and oxidative phosphorylation components were below the control. However, metabolic parameters and respiratory chain components entered a new homeostatic level in the late phase of cold-acclimation. These changes were accompanied with increased protein levels of phospho-AMP-dependent protein kinase-α (phospho-AMPKα) on the first day of cold-acclimation, and hypoxia-inducible factor-1α (HIF-1α) throughout early cold-acclimation. L-arginine positively affected protein expression of enzymes involved in glucose metabolism and ß-oxidation of fatty acids in the early phase of cold-acclimation, and oxidative phosphorylation components throughout cold-acclimation. In contrast, L-NAME had the opposite effects. CONCLUSION: Results suggest that IBAT structural remodeling is followed by energy metabolism reprogramming, which control might be orchestrated by the action of AMPKα and HIF-1α. Data also indicated the involvement of L-arginine-()NO in the regulation of IBAT metabolism. GENERAL SIGNIFICANCE: Results obtained in this study might be of great importance for elucidating regulatory pathways governing energy metabolism in both physiological and pathophysiological states.

Mitochondrial molecular basis of sevoflurane and propofol cardioprotection in patients undergoing aortic valve replacement with cardiopulmonary bypass.

BACKGROUND/AIMS: Study elucidates and compares the mitochondrial bioenergetic-related molecular basis of sevoflurane and propofol cardioprotection during aortic valve replacement surgery due to aortic valve stenosis. METHODS: Twenty-two patients were prospectively randomized in two groups regarding the anesthetic regime: sevoflurane and propofol. Hemodynamic parameters, biomarkers of cardiac injury and brain natriuretic peptide (BNP) were measured preoperatively and postoperatively. In tissue samples, taken from the interventricular septum, key mitochondrial molecules were determined by Western blot, real time PCR, as well as confocal microscopy and immunohisto- and immunocyto-chemical analysis. RESULTS: The protein levels of cytochrome c oxidase and ATP synthase were higher in sevoflurane than in propofol group. Nevertheless, cytochrome c protein content was higher in propofol than sevoflurane receiving patients. Propofol group also showed higher protein level of connexin 43 (Cx43) than sevoflurane group. Besides, immunogold analysis showed its mitochondrial localization. The mRNA level of mtDNA and uncoupling protein (UCP2) were higher in propofol than sevoflurane patients, as well. On the other hand, there were no significant differences between groups in hemodynamic assessment, intensive care unit length of stay, troponin I and BNP level. CONCLUSIONS: Our data indicate that sevoflurane and propofol lead to cardiac protection via different mitochondrially related molecular mechanisms. It appears that sevoflurane acts regulating cytochrome c oxidase and ATP synthase, while the effects of propofol occur through regulation of cytochrome c, Cx43, mtDNA transcription and UCP2.

Protein expression of ubiquitin in interscapular brown adipose tissue during acclimation of rats to cold: the impact of (∙)NO.

In this study, the effects of L-arginine-nitric-oxide ((∙)NO)-producing pathway on protein content of ubiquitin, as an important component of ubiquitin-proteasome system for protein removal, were investigated. We showed that L-arginine markedly decreased ubiquitin protein content in interscapular brown adipose tissue, both in thermogenic inactive (at room temperature) and thermogenic active (on cold) states; while in L-NAME-treated groups this effect was abolished. This result suggests that nitric oxide ((∙)NO), besides well established roles, is involved in this aspect of structure remodeling, as well.

Relation of CuZnSOD activity with renal insufficiency in hypertensive diabetic patients.

Diabetes and renal insufficiency are interrelated metabolic disorders closely associated with redox homeostasis disturbances. The aim of this study was to compare the activity of copper zinc superoxide dismutase (CuZnSOD) in the erythrocytes of hypertensive diabetic patients with or without renal insufficiency with normal healthy control subjects. In both groups of diabetic patients, blood glucose level and the content of glycosylated hemoglobin (HbA1c) were higher than in the control group. However, CuZnSOD activity was significantly higher than control only in hypertensive diabetic patients with renal insufficiency. Our results suggest that disturbances in superoxide homeostasis do correlate with long-term complication in diabetes, i.e. diabetic renal insufficiency and hypertension.

Extracellular Hemoglobin: Modulation of Cellular Functions and Pathophysiological Effects.

Hemoglobin is essential for maintaining cellular bioenergetic homeostasis through its ability to bind and transport oxygen to the tissues. Besides its ability to transport oxygen, hemoglobin within erythrocytes plays an important role in cellular signaling and modulation of the inflammatory response either directly by binding gas molecules (NO, CO, and CO2) or indirectly by acting as their source. Once hemoglobin reaches the extracellular environment, it acquires several secondary functions affecting surrounding cells and tissues. By modulating the cell functions, this macromolecule becomes involved in the etiology and pathophysiology of various diseases. The up-to-date results disclose the impact of extracellular hemoglobin on (i) redox status, (ii) inflammatory state of cells, (iii) proliferation and chemotaxis, (iv) mitochondrial dynamic, (v) chemoresistance and (vi) differentiation. This review pays special attention to applied biomedical research and the use of non-vertebrate and vertebrate extracellular hemoglobin as a promising candidate for hemoglobin-based oxygen carriers, as well as cell culture medium additive. Although recent experimental settings have some limitations, they provide additional insight into the modulatory activity of extracellular hemoglobin in various cellular microenvironments, such as stem or tumor cells niches.

Ferroptosis as a Novel Determinant of ß-Cell Death in Diabetic Conditions.

The main pathological hallmark of diabetes is the loss of functional ß-cells. Among several types of ß-cell death in diabetes, the involvement of ferroptosis remains elusive. Therefore, we investigated the potential of diabetes-mimicking factors: high glucose (HG), proinflammatory cytokines, hydrogen peroxide (H2O2), or diabetogenic agent streptozotocin (STZ) to induce ferroptosis of ß-cells in vitro. Furthermore, we tested the contribution of ferroptosis to injury of pancreatic islets in an STZ-induced in vivo diabetic model. All in vitro treatments increased loss of Rin-5F cells along with the accumulation of reactive oxygen species, lipid peroxides and iron, inactivation of NF-E2-related factor 2 (Nrf2), and decrease in glutathione peroxidase 4 expression and mitochondrial membrane potential (MMP). Ferrostatin 1 (Fer-1), ferroptosis inhibitor, diminished the above-stated effects and rescued cells from death in case of HG, STZ, and H2O2 treatments, while failed to increase MMP and to attenuate cell death after the cytokines' treatment. Moreover, Fer-1 protected pancreatic islets from STZ-induced injury in diabetic in vivo model, since it decreased infiltration of macrophages and accumulation of lipid peroxides and increased the population of insulin-positive cells. Such results revealed differences between diabetogenic stimuli in determining the destiny of ß-cells, emerging HG, H2O2, and STZ, but not cytokines, as contributing factors to ferroptosis and shed new light on an antidiabetic strategy based on Nrf2 activation. Thus, targeting ferroptosis in diabetes might be a promising new approach for preservation of the ß-cell population. Our results obtained from in vivo study strongly justify this approach.

Adipokine signatures of subcutaneous and visceral abdominal fat in normal-weight and obese women with different metabolic profiles.

INTRODUCTION: Metabolic syndrome arises from abnormal adipose function accompanied by insulin resistance. As early factors reflecting/impacting lipid storage dysfunction of adipose tissues, we sought to determine adipokine levels in subcutaneous and visceral adipose tissues (SAT and VAT). MATERIAL AND METHODS: Gene and protein expression levels of leptin, adiponectin, and resistin were analysed in SAT and VAT of normal-weight and overweight/obese women, subclassified according to insulin resistance index, triglyceride, total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels into metabolically healthy and "at risk" groups. RESULTS: Compared with normal-weight women, obese women had higher serum leptin levels (p < 0.05), as well as increased leptin gene and protein expression in VAT. Conversely, expression levels of leptin were lower in SAT of obese women, and minor in the SAT of "at risk" groups of women, compared with weight-matched healthy groups. In addition, lower adiponectin levels were detected in SAT of metabolically healthy obese women (p < 0.01), and lower in SAT and VAT (p < 0.05) of "at risk" obese women compared to healthy, obese women. Significant differences in resistin levels were only observed in obese women; resistin gene expression was higher in VAT and SAT of obese, compared to normal-weight women. However, higher gene expression was not consistent with protein expression of resistin. CONCLUSIONS: Low adiponectin in both examined adipose tissues and inappropriate leptin expression levels in SAT appear to be important characteristics of obesity-related metabolic syndrome. Intriguingly, this adipokine dysregulation is primary seen in SAT, suggesting that endocrine dysfunction in this abdominal depot may be an early risk sign of metabolic syndrome.

Insight into the Biological Activity of Hennosides-Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead.

Henna is the current name of the dye prepared from the dry leaf powder of Lawsonia inermis (Lythraceae). Several studies have focused on the chemistry and pharmacology of the henna dyeing active compound, lawsone, obtained from the main constituents of leaves, hennosides, during the processing of plant material. However, knowledge regarding the biological activity of hennosides is largely lacking. In this paper, the redox activity of three hennoside isomers is reported. The pro-oxidative activity was confirmed by their ability to induce mild lysis of erythrocytes and to increase the level of methemoglobin at the concentration ≥ 500 µg/mL. The antioxidant activity of hennosides (concentration ≥100 µg/mL) was determined by FRAP and ABTS assays. At concentration of 500 µg/mL, antioxidant activity of hennoside isomers was equivalent to 0.46 ± 0.08, 0.62 ± 0.28 and 0.35 ± 0.03 mM FeSO4 × 7H2O, and 0.15 ± 0.01, 0.30 ± 0.01 and 0.09 ± 0.01 mM Trolox. Hennosides at 100 µg/mL concentration did not influence viability of human breast cancer cell lines MDA231 and MCF-7 and primary human peripheral blood and periodontal ligament-mesenchymal stem cells, but produced a modest increase in concentration of antioxidants in the cell culture supernatants. The evidenced antioxidant and pro-oxidant activities indicate their potential to act as redox balance regulator, which opens up the possibility of using hennosides in commercial phytomedicines.

Reactive oxygen, nitrogen, and sulfur species in human male fertility. A crossroad of cellular signaling and pathology.

Infertility is a significant global health problem that currently affects one of six couples in reproductive age. The quality of male reproductive cells dramatically decreased over the last years and almost every aspect of modern life additionally worsen sperm functional parameters that consequently markedly increase male infertility. This clearly points out the importance of finding a new approach to treat male infertility. Redox signaling mediated by reactive oxygen, nitrogen and sulfur species (ROS, RNS, and RSS respectively), has appeared important for sperm reproductive function. Present review summarizes the current knowledge of ROS, RNS, and RSS in male reproductive biology and identifies potential targets for development of novel pharmacological and therapeutic approaches for male infertility by targeted therapeutic modulation of redox signaling.

Native bovine hemoglobin reduces differentiation capacity of mesenchymal stromal cells in vitro.

We have tested in vitro effects of hemoglobin from bovine slaughterhouse blood (BHb) on stromal cells of mesodermal origin, with an aim to explore its use as a component of cell culture media. Human peripheral blood mesenchymal stromal cells (PB-MSCs) and three mouse cell lines (ATDC5, MC3T3-E1 and 3T3-L1) were employed to study BHb effects on their growth and migration. The cells multilineage differentiation capacity in the presence of BHb was evaluated after induced differentiation, by histochemical staining and by RT-PCR analysis of the expression of genes specific for chondrogenic, adipogenic and osteogenic lineages. The effects of BHb on the cell proliferation and motility were dependent on both, cell type and BHb concentration (0.1 µM, 1 µM and 10 µM). In the lowest concentration (0.1 µM) BHb showed the least prominent effect on the cell proliferation and migration. In this concentration BHb reduced the differentiation capacity of all tested cells and its effect was dependent of composition of induction medium and the culture period. Obtained data suggest that BHb has the potential to be used as a component of cell culture media through maintaining proliferation and reducing differentiation capacity of mesenchymal stromal cells.

Evaluation of the antioxidative enzymes in the seminal plasma of infertile men: Contribution to classic semen quality analysis.

Protein expression/activity of antioxidative defense enzymes (AD) in seminal plasma of fertile men might be used as biomarkers of male fertility status. To test this concept, the present study examined the semen parameters of males among 14 normal idiopathic (normozoospermia) and 84 subnormal (teratozoospermia, oligoteratozoospermia, oligoasthenoteratozoospermia) infertile individuals\. We investigated levels of protein expression/activity of Cu, Zn superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GSH-Px), their association with functional sperm parameters, as well as their potential to serve as biomarkers of specific sperm pathologies. Although the activity of CuZnSOD and protein expression of catalase were significantly correlated with several sperm parameters, underlying their potential role in etiology of various sperm abnormalities, investigation of their potential usefulness as a biomarker of semen quality showed that these AD enzymes could not distinguish subtle differences between various sperm pathologies. In contrast, GSH-Px activity was decreased in all groups with sperm pathologies and was a very good indicator of aberrations in functional sperm parameters, explaining up to 94.6% of infertility cases where functional sperm parameters were affected. Therefore, assessment of GSH-Px activity showed the potential to discriminate between infertile males with normal and subnormal semen characteristics and may prove useful in the evaluation of male (in)fertility. Abbreviations: AD: antioxidative defense; Cu, Zn SOD: copper, zinc superoxide dismutase; GSH-Px: glutathione peroxidase; MnSOD: manganese superoxide dismutase; NS: normospermia; OATS: oligoasthenoteratozoospermia; OTS: oligoteratozoospermia; ROC: receiver operating characteristic; ROS: reactive oxygen species; TS: teratozoospermia; WHO: world health organization.