Radical Oxygen Species Generation Research Articles

Elucidating the Mechanism of Electrochemical Inactivation of Direct Electron Transfer Type Glucose Dehydrogenase Enzyme-Employing Biosensor

Introduction: Diabetes management has evolved through the introduction of self-monitoring blood glucose (SMBG) and continuous glucose monitoring (CGM) systems, enabling real-time patient feedback.[1] Of these, CGM systems, capable of prolonged, continuous monitoring, hinge upon the dependable performance of the enzymes to maintain precise measurements and long-term sensing capabilities. Current CGM systems utilize fungi-derived glucose oxidase (GOx) to catalyze the reaction with glucose. This enzyme is able to record glucose values within the physiological range with high accuracy; however, this enzyme also creates hydrogen peroxide during continuous operation leading to self-inactivation via radical oxygen species(ROS) formation. Our group has been involved in the characterization and functional engineering of a FAD-dependent glucose dehydrogenase enzyme derived from Burkholderia cepacia (BcGDH) which is capable of direct electron transfer (DET) with an electrode.[2, 3] By eliminating the need for hydrogen peroxide or toxic external mediators—as are common with 1st and 2nd generation CGM sensors, respectively—we are able to simplify our sensing platform and create the ideal sensing modality. Additionally, we have improved the thermal stability of BcGDH by introducing inter-subunit disulfide bonds which has greatly improved the in vitro half-life of our enzyme.[4] Although we have been able to engineer our BcGDH to have a much greater half-life in vitro than the wild type BcGDH or GOx, we observe uncharacteristic inactivation during continuous applied potential electrochemistry experiments. This study focuses on the elucidation of the electrochemical inactivation mechanism of a DET-type BcGDH enzymatic sensor. Materials & Methods: BcGDH engineered enzyme, from the introduction of inter-subunit disulfide bonds was recombinantly produced and purified. A ϕ=2mm gold disk electrode was polished and electrochemically cleaned before functionalized with dithiobis(succinimidyl hexanoate) to form a self-assembled monolayer(SAM). The purified BcGDH mutant was then immobilized to the SAM. Variations in potentials (none, 100mV or 400mV vs Ag/AgCl), glucose concentration (none or 20mM), and oxygen (argon purged or ambient oxygen) were used for the incubation conditions. Every three hours (0 to 9 hours) calibration curves were investigated unilaterally at 45°C, 400mV vs Ag/AgCl applied potential, ambient oxygen, and varying concentrations from 0 to 20mM glucose. Results & Discussion: The disulfide bond stabilized BcGDH engineered enzyme was stable at more than 80°C confirming its thermal stability and extended half-life at supraphysiological temperatures. However, when utilized in a sensor format at 45°C, engineered enzyme was inactivated much more rapidly than expected. The largest rate of inactivation was revealed to be due to application of a high over-potential (+400mV vs Ag/AgCl) coupled with hyper-physiological glucose levels with ambient oxygen showing a sensor half-life of 2.89±0.74 hours at 45°C. The impact of oxygen-purging the system by utilizing argon gas was shown to improve the sensor half-life to 35.04±6.17 hours at 45°C. Additionally, without the high over-potential, the hyper-physiological glucose concentrations had no detriment to the sensor and no sensor inactivation was observed. . The high over-potential combined with hyper-physiological glucose and ambient oxygen were assumed to produce radical oxygen species (ROS) which are responsible for the inactivation of the enzyme. Conclusions: We demonstrate the ability to use this DET-type GDH electrochemical sensor to investigate the factors which accelerate its inactivation. While hyper-physiological glucose levels do not significantly inactivate the enzyme-sensor, the high over-potential incubation inactivates the sensor much faster—likely due to the formation of ROS from generation of H2O2 at the cathode. By removing oxygen from the system via an Argon purge, the conversion of oxygen to hydrogen peroxide is thwarted. In the future, studies should be done to mitigate the ROS generation as glucose levels cannot be controlled. This strategy may be done by applying a lower over-potential to the sensor would minimize the formation of ROS even in the presence of hyper-physiological glucose.

Perspectives for Photochemical Leaching Processes of Chalcopyrite: A Solar Radical-Leaching Process

This paper review presents a comparison between conventional leaching and advanced photochemical leaching processes and their potential for use in chalcopyrite leaching. Likewise, it presents an analysis of the differences between the advanced leaching processes, photoleaching and radical-leaching, indicating that the photochemical mechanisms (photooxidation/photoreduction and generation of radical oxygen species (ROS) and radical sulfur species (RSS)) would improve the oxidative dissolution of chalcopyrite, taking advantage of the high oxidizing power of free radicals. Initial experimental results of solar-assisted radical-leaching on chalcopyrite are presented, demonstrating that sulfate radicals (SO4−) allow copper to be leached at a rate 4.7 times higher than in the absence of radicals and sunlight. With these results, a radical-leaching process is presented for the first time, with a perspective toward the future development of a new hydrometallurgical route: solar-assisted radical-leaching.

Chitosan-Biotin-Conjugated pH-Responsive Ru(II) Glucose Nanogel: A Dual Pathway of Targeting Cancer Cells and Self-Drug Delivery.

The current study paves the way for improved chemotherapy by creating pH-responsive nanogels (NGs) (GC1 and GC2) loaded with synthetic ruthenium(II) arene complexes to increase biological potency. NGs are fabricated by the conjugation of chitosan (CTS)-biotin biopolymers that selectively target the cancer cells as CTS has the pH-responsive property, which helps in releasing the drug in cancer cells having pH ∼ 5.5, and biotin provides the way to target the cancer cells selectively due to the overexpression of integrin. The synthesized compounds and NGs were thoroughly characterized using various spectroscopic and analytical techniques such as NMR, electrospray ionization-mass spectrometry, Fourier transform infrared, UV-vis, scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, rheology, Brunauer-Emmett-Teller, and others. NGs displayed exceptional increased efficacy toward cancerous cells with IC50 values ranging from 7.50 to 18.86 μM via induced apoptosis in three human cancer cell lines. Apart from its potency, NGs were found to be highly selective toward cancer cells. Moreover, based on the results of immunoblot analysis, it was observed that the synthesized compounds exhibit a significant increase in the expression of cleaved caspase-3 and a decrease in the expression of the antiapoptotic protein BCL-XL. Interestingly, the complexes were discovered to have the additional capability of catalyzing the conversion of NADH to NAD+, leading to the generation of radical oxygen species within the cells. Additionally, it was discovered that NG-induced apoptosis depends on ROS production and DNA binding. A narrower range of LD50 values (1185.93 and 823.03 μM) was seen after administering NGs to zebrafish embryos in vivo. The results support the use of drug-loaded NGs as potential chemotherapeutic and chemopreventive agents for human cancer cells.

Maternal Oxygen Administration during Labor: A Controversial Practice.

Oxygen administration to the mother is commonly performed during labor, especially in the case of a non-reassuring fetal heart rate, aiming to increase oxygen diffusion through the placenta to fetal tissues. The benefits and potential risks are controversial, especially when the mother is not hypoxemic. Its impact on placental gas exchange and the fetal acid-base equilibrium is not fully understood and it probably affects the sensible placental oxygen equilibrium causing a time-dependent vasoconstriction of umbilical and placental vessels. Hyperoxia might also cause the generation of radical oxygen species, raising concerns for the developing fetal cells. Moreover, this practice affects the maternal cardiovascular system, causing alterations of the cardiac index, heart rate and vascular resistance, and unclear effects on uterine blood flow. In conclusion, there is no evidence that maternal oxygen administration can provide any benefit in the case of a non-reassuring fetal heart rate pattern, while possible collateral effects warn of its utilization. Oxygen administration during labor should be reserved for cases of maternal hypoxia.

Chrysin and its nanoliposome ameliorated non-alcoholic steatohepatitis via inhibiting TLR4 signalling pathway.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease histologically characterized by liver steatosis, hepatocellular injury, inflammation and fibrosis, resulting in cirrhosis and hepatocellular carcinoma, but effective measures and obvious pathogenesis for NASH remain elusive. Chrysin (CH) has been reported to have anti-inflammatory effects but shows lower bioavailability. In this study, a chrysin nanoliposome (CH-NL) was first prepared and characterized. Then, we used the methionine-choline-deficient (MCD) diet to induce a mouse model of NASH. Finally, the effects of CH and CH-NL on NASH were evaluated in the liver of NASH mice. The results showed that CH or CH-NL significantly reduced the accumulation of lipids in hepatocytes, alleviated liver injury, decreased the generation of radical oxygen species, and attenuated the accumulation of collagen fibre in the liver of NASH mice. In addition, CH and its nano-liposomes markedly inhibited the production of inflammatory cytokines and inflammatory cell infiltration in the liver of NASH mice. Further studies found that CH-NL and CH-NL downregulated the MCD diet-induced activation of Toll-like receptor 4 (TLR4) signalling pathway in the liver of mice. CH and its nanoliposome alleviated MCD diet-induced NASH in mice, which might be through inhibiting TLR4 signalling pathway.

Multimodal analysis of granulocytes, monocytes, and platelets in patients with cystic fibrosis before and after Elexacaftor-Tezacaftor-Ivacaftor treatment.

Cystic fibrosis (CF) is a monogenetic disease caused by an impairment of the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organs and is associated with acute and chronic inflammation. In 2020, Elexacaftor-Tezacaftor-Ivacaftor (ETI) was approved to enhance and restore the remaining CFTR functionality. This study investigates cellular innate immunity, with a focus on neutrophil activation and phenotype, comparing healthy volunteers with patients with CF before (T1, n = 13) and after six months (T2, n = 11) of ETI treatment. ETI treatment reduced sweat chloride (T1: 95 mmol/l (83|108) vs. T2: 32 mmol/l (25|62), p < 0.01, median, first|third quartile) and significantly improved pulmonal function (FEV1 T1: 2.66 l (1.92|3.04) vs. T2: 3.69 l (3.00|4.03), p < 0.01). Moreover, there was a significant decrease in the biomarker human epididymis protein 4 (T1: 6.2 ng/ml (4.6|6.3) vs. T2: 3.0 ng/ml (2.2|3.7), p < 0.01) and a small but significant decrease in matrix metallopeptidase 9 (T1: 45.5 ng/ml (32.5|140.1) vs. T2: 28.2 ng/ml (18.2|33.6), p < 0.05). Neutrophil phenotype (CD10, CD11b, CD62L, and CD66b) and function (radical oxygen species generation, chemotactic and phagocytic activity) remained largely unaffected by ETI treatment. Likewise, monocyte phenotype and markers of platelet activation were similar at T1 and T2. In summary, the present study confirmed a positive impact on patients with CF after ETI treatment. However, neither beneficial nor harmful effects of ETI treatment on cellular innate immunity could be detected, possibly due to the study population consisting of patients with well-controlled CF.

Metal-Polymer Nanoconjugates Application in Cancer Imaging and Therapy.

Metallic-based nanoparticles present a unique set of physicochemical properties that support their application in different fields, such as electronics, medical diagnostics, and therapeutics. Particularly, in cancer therapy, the plasmonic resonance, magnetic behavior, X-ray attenuation, and radical oxygen species generation capacity displayed by metallic nanoparticles make them highly promising theragnostic solutions. Nevertheless, metallic-based nanoparticles are often associated with some toxicological issues, lack of colloidal stability, and establishment of off-target interactions. Therefore, researchers have been exploiting the combination of metallic nanoparticles with other materials, inorganic (e.g., silica) and/or organic (e.g., polymers). In terms of biological performance, metal-polymer conjugation can be advantageous for improving biocompatibility, colloidal stability, and tumor specificity. In this review, the application of metallic-polymer nanoconjugates/nanohybrids as a multifunctional all-in-one solution for cancer therapy will be summarized, focusing on the physicochemical properties that make metallic nanomaterials capable of acting as imaging and/or therapeutic agents. Then, an overview of the main advantages of metal-polymer conjugation as well as the most common structural arrangements will be provided. Moreover, the application of metallic-polymer nanoconjugates/nanohybrids made of gold, iron, copper, and other metals in cancer therapy will be discussed, in addition to an outlook of the current solution in clinical trials.

Controllable synthesis of ultrasmall copper nanoparticles decorated fullerenol composite for antibacterial application and wound healing under visible light

Developing an efficient antimicrobial agent is a key concern of present scientific community. Fullerene has been proved antibacterial by means of photodynamic therapy (PDT), but the severe hydrophobicity strongly hinders its application in biomedical fields. To solve this problem, a highly stable and water-soluble composite of which spherical fullerenol (hydroxylated fullerene) encapped by ultrasmall (5 nm sized) copper nanoparticles (CuNPs@fullerenol) was simply prepared by the combination of liquid-liquid interface precipitation (LLIP) and photochemical reduction strategy. Under the irradiation of 660 nm light for 15 min, 98.1 % E. coli and 97.6 % S. aureus treated by CuNPs@fullerenol was inhibited, while such bactericidal effect could be stably kept for at least 8 h. In vivo experiments also confirmed that the wound recovery could be accelerated by our therapy. Generation of radical oxygen species (ROS) including singlet oxygen (1O2) and superoxide anion (∙O2-) induced by PDT process, along with the metal ion release behavior, is considered as the mechanism for the excellent antibacterial performances of CuNPs@fullerenol. Our results will benefit the design and upgrade of nanocarbon-based antibacterial materials.

Binuclear VIV/V, MoVI and ZnII - hydroquinonate complexes: Synthesis, stability, oxidative activity and anticancer properties

Since the discovery of the anticancer properties of cis-platin the road for the development of less toxic and more specific metal ion based anticancer drugs has opened. Based on the low toxicity of VIV/V, MoVI and ZnII metal ions, their binuclear hydroquinonate complexes have been synthesized and their biological activity towards their anticancer properties on various cancerous and non-cancerous cell lines has been evaluated. The new complexes of ZnII with the ligands 2,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)benzene-1,4-diol (H2bpymah) and 2,2′-(((2,5-dihydroxy-1,4-phenylene)bis(methylene))bis((carboxymethyl)ammoniumdiyl))diacetate (H6bicah) have been synthesized and characterized by X-ray crystallography in solid state and 1H NMR in aqueous solution. The binuclear nature of the complexes increases their hydrolytic stability in aqueous solutions at pD 7.0, depending on the metal ion. The most hydrolytic stable VV and ZnII hydroquinonate complexes show to activate O2 towards oxidation of mercaptoethanol in aqueous solutions at physiological pHs. Only the strongest oxidant, the VV complex with bicah6−, significantly activates the intracellular radical oxygen species (ROS) generation. Apparently, the mercaptoethanol oxidation experiment vs time can be used as a preliminary experiment for the prediction of the in vitro ROS generation activity of the complexes in aqueous solutions.

Impact of General Anesthesia and Antioxidants on Cognitive, Static and Locomotor Functions During Laparoscopic Cholecystectomy

Aim of the study: to reduce cognitive impairment during laparoscopic cholecystectomy by perioperative administration of drugs with antihypoxic and antioxidant effects under the control of stabilography. Materials and methods. We studied the effect of general anesthesia and antioxidants on cognitive, static and locomotor functions during laparoscopic cholecystectomy. We studied 90 patients with acute calculous cholecystitis randomized into three experimental groups (n=30, each group). The control group was composed of 24 healthy individuals. Group 1 patients received no antioxidants, group 2 patients received the combination antioxidant drug (sodium fumarate+sodium chloride+potassium chloride+magnesium chloride) in the post-operative period, and group 3 patients received methylethylpyridinol. The patient groups were comparable in sex, age and type of inflammatory and destructive process in the gallbladder. Surgical intervention was performed under endotracheal anesthesia. Premedication with atropine and promedol was given, and anesthesia induction was carried out with propofol, fentanyl and suxamethonium. Sevoflurane, fentanyl andcisatracurium were employed to maintain anesthesia, analgesia and myorelaxation, respectively. The patients were examined before surgery, 24 and 48 hours after surgery. In the groups of patients who received antioxidant therapy, blood sampling for hematological and biochemical examinations was performed 30 min after the administration of antioxidants. Stabilographic studies and MoCA test (Montreal Scale) were performed before antioxidant administration prior to surgery and after surgery, on days 2 and 3. Results. Neuropsychological testing revealed postoperative cognitive dysfunction on standard therapy which included impaired attention and concentration, executive function, memory, speech, visual constructional skills, abstract thinking, counting, and orientation (21 points on the MoCA scale versus 28–30 points for normal). We found that the pathogenetic factors of cognitive dysfunction included insufficient antioxidant protection, decreased TNF-α and elevated interleukin-18 levels along with an increased level of C-reactive protein in plasma, which manifested as activation of free-radical oxidation processes and reduced antioxidant system and performance of nonspecific resistance. Perioperative use of the combination antioxidant drug and methylethylpyridinol antioxidants reduced the frequency and severity of postoperative cognitive impairment in patients after laparoscopic cholecystectomy. Conclusion. The most important pathogenetic factors of cognitive dysfunction after laparoscopic cholecystectomy include activation of free-radical oxidation, reduction of antioxidant defense system performance and lack of nonspecific resistance factors. Adding the combination antioxidant drug or methylethylpyridinol to the standard therapy reduces the intensity of radical oxygen species generation, maintains the antioxidant potential, activates production and secretion of nonspecific resistance factors, preventing the development and reducing the severity of cognitive disorders in the perioperative period. Neuropsychological testing and stabilographic examination allow identifying the risk of cognitive disorders in patients after laparoscopic cholecystectomy and provide a rationale for the use of antioxidant therapy for their prevention.

Boron Nitride Nanosheets Can Induce Water Channels Across Lipid Bilayers Leading to Lysosomal Permeabilization.

While the interaction between 2D materials and cells is of key importance to the development of nanomedicines and safe applications of nanotechnology, still little is known about the biological interactions of many emerging 2D materials. Here, an investigation of how hexagonal boron nitride (hBN) interacts with the cell membrane is carried out by combining molecular dynamics (MD), liquid-phase exfoliation, and in vitro imaging methods. MD simulations reveal that a sharp hBN wedge can penetrate a lipid bilayer and form a cross-membrane water channel along its exposed polar edges, while a round hBN sheet does not exhibit this behavior. It is hypothesized that such water channels can facilitate cross-membrane transport, with important consequences including lysosomal membrane permeabilization, an emerging mechanism of cellular toxicity that involves the release of cathepsin B and generation of radical oxygen species leading to cell apoptosis. To test this hypothesis, two types of hBN nanosheets, one with a rhomboidal, cornered morphology and one with a round morphology, are prepared, and human lung epithelial cells are exposed to both materials. The cornered hBN with lateral polar edges results in a dose-dependent cytotoxic effect, whereas round hBN does not cause significant toxicity, thus confirming ourpremise.

Animal-Free Human Whole Blood Sepsis Model to Study Changes in Innate Immunity.

Studying innate immunity in humans is crucial for understanding its role in the pathophysiology of systemic inflammation, particularly in the complex setting of sepsis. Therefore, we standardized a step-by-step process from the venipuncture to the transfer in a human model system, while closely monitoring the inflammatory response for up to three hours. We designed an animal-free, human whole blood sepsis model using a commercially available, simple to use, tubing system. First, we analyzed routine clinical parameters, including cell count and blood gas analysis. Second, we demonstrated that extracellular activation markers (e.g., CD11b and CD62l) as well as intracellular metabolic (intracellular pH) and functional (generation of radical oxygen species) features remained stable after incubation in the whole blood model. Third, we mimicked systemic inflammation during early sepsis by exposure of whole blood to pathogen-associated molecular patterns. Stimulation with lipopolysaccharide revealed the capability of the model system to evoke a sepsis-like inflammatory phenotype of innate immunity. In summary, the presented model serves as a convenient, economic, and reliable platform to study innate immunity in human whole blood, which may yield clinically important insights.

Effect of colloidal silver and copper nanoparticles on generation of radical oxygen species (ROS) in human neutrophils

Introduction: Silver and copper nanoparticles (AgNPs, CuNPs) applied as hydronanocolloids are known to produce strong antibacterial and antifungal activities. They are extensively used in a number of applications including pharmacy, medicine and cosmetology (especially for surface-applied treatment of skin lesions) as well as agriculture, industry (paint, construction, etc.) and home or office (mainly disinfection applications). Moreover, there is a promising perspective of an intra-systemic NP use, especially to optimize targeted drug delivery. For the above reasons NPs cause risk of penetrating human body and exerting toxic effects and/or stress reactions. This issue has inspired the authors to launch studies on the influence of colloidal AgNPs and CuNPs on physiological potential of neutrophils, blood-cells acting as the first line of immunological defense. Methods: Physiological activity of neutrophils was evaluated by measuring their ability to generate oxygen radicals (radical oxygen species, ROS) due to the respiratory (oxidative) burst mechanism. Human, peripheral blood-isolated neutrophils were stimulated with a standard activating agent (polystyrene latex particles) to develop high physiological potential revealed by enhanced ability to produce oxygen radicals. The cells were treated with silver and copper hydronanocolloids (each applied at concentrations ranging from 0.4 to 50 mg/kg) alternatively: in the absence and presence of the mentioned activator. The level of generated ROS upon oxidative burst was monitored chemiluminometrically. Results: The tested Ag and Cu-nanocolloids were not toxic against neutrophils although they hampered mitochondrial dehydrogenase activities when applied at higher levels. At lower concentrations they tended to stimulate ROS generation; however the treatment did not launch the oxidative burst. In the case of the latex-stimulated neutrophils, both types of nanoparticles in all experimental variants did not influence the levels of produced ROS. Conclusions: The obtained results indicate that the exposure of human neutrophils to colloidal AgNPs and CuNPs does not lead to an enhanced ROS generation, which may enable direct intra-blood application of the tested nanostructures, provided further necessary toxicological studies are carried out.

Assessment of Human Skin Gene Expression by Different Blends of Plant Extracts with Implications to Periorbital Skin Aging.

Since the skin is the major protective barrier of the body, it is affected by intrinsic and extrinsic factors. Environmental influences such as ultraviolet (UV) irradiation, pollution or dry/cold air are involved in the generation of radical oxygen species (ROS) and impact skin aging and dermal health. Assessment of human skin gene expression and other biomarkers including epigenetic factors are used to evaluate the biological/molecular activities of key compounds in cosmetic formulas. The objective of this study was to quantify human gene expression when epidermal full-thickness skin equivalents were exposed to: (a) a mixture of betaine, pentylene glycol, Saccharomyces cerevisiae and Rhodiola rosea root extract (BlendE) for antioxidant, skin barrier function and oxidative stress (with hydrogen peroxide challenge); and (b) a mixture of Narcissus tazetta bulb extract and Schisandra chinensis fruit extract (BlendIP) for various biomarkers and microRNA analysis. For BlendE, several antioxidants, protective oxidative stress biomarkers and many skin barrier function parameters were significantly increased. When BlendE was evaluated, the negative impact of the hydrogen peroxide was significantly reduced for the matrix metalloproteinases (MMP 3 and MMP 12), the skin aging and oxidative stress biomarkers, namely FBN2, ANXA1 and HGF. When BlendIP was tested for cell proliferation and dermal structural components to enhance the integrity of the skin around the eyes: 8 growth factors, 7 signaling, 7 structural/barrier function and 7 oxidative stress biomarkers were significantly increased. Finally, when BlendIP was tested via real-time RT-PCR for microRNA expression: miR-146a, miR-22, miR155, miR16 and miR21 were all significantly increased over control levels. Therefore, human skin gene expression studies are important tools to assess active ingredient compounds such as plant extract blends to advance dermal hypotheses toward validating cosmetic formulations with botanical molecules.

Time course of liver mitochondrial function and intrinsic changes in oxidative phosphorylation in a rat model of sepsis

BackgroundTissue ATP depletion and oxidative stress have been associated with the severe outcomes of septic shock. One of the compensatory mechanisms to alleviate the sepsis-induced mitochondrial dysfunction could be the increase in oxidative phosphorylation efficiency (ATP/O). We propose to study liver mitochondrial function and oxidative stress and the regulatory mechanism of mitochondrial oxidative phosphorylation efficiency in an animal model of sepsis.MethodsWe induced sepsis in rats by cecal ligation and perforation (CLP). Six, 24, or 36 h following CLP, we measured liver mitochondrial respiration, cytochrome c oxidase activity, and membrane permeability. We determine oxidative phosphorylation efficiency, by measuring ATP synthesis related to oxygen consumption at various exogenous ADP concentrations. Finally, we measured radical oxygen species (ROS) generation by liver mitochondria and mRNA concentrations of UCP2, biogenesis factors, and cytokines at the same end points.ResultsCLP rats presented hypotension, lactic acidosis, liver cytolysis, and upregulation of proinflammatory cytokines mRNA as compared to controls. Liver mitochondria showed a decrease in ATP synthesis and oxygen consumption at 24 h following CLP. A marked uncoupling of oxidative phosphorylation appeared 36 h following CLP and was associated with a decrease in cytochrome c oxidase activity and content and ATP synthase subunit β content (slip mechanism) and an increase in mitochondrial oligomycin-insensitive respiration, but no change in mitochondrial inner membrane permeability (no leak). Upregulation of UCP2 mRNA resulted in a decrease in mitochondrial ROS generation 24 h after the onset of CLP, whereas ROS over-generation associated with slip at cytochrome c oxidase observed at 36 h was concomitant with a decrease in UCP2 mRNA expression.ConclusionsDespite a compensatory increase in mitochondrial biogenesis factors, liver mitochondrial functions remain altered after CLP. This suggests that the functional compensatory mechanisms reported in the present study (slip at cytochrome c oxidase and biogenesis factors) were not strong enough to increase oxidative phosphorylation efficiency and failed to limit liver mitochondrial ROS over-generation. These data suggest that treatments based on cytochrome c infusion could have a role in mitochondrial dysfunction and/or ROS generation associated with sepsis.

Ischemic Preconditioning Preserves Liver Energy Charge and Function on Hepatic Ischemia/Reperfusion Injury in Rats

Cell energy during ischemia/reperfusion depends on mechanisms including adenosine diphosphate degradation, oxygen species and cytokine liberation, neutrophil infiltration, and endothelial dysfunction. Preconditioning-a brief ischemic episode that confers a state of protection against subsequent ischemia-reperfusion injury-involves NO and adenosine production, reduction in oxygen species liberation, and preservation of microcirculation. During hypoxia, constitutive NO production assures adequate oxygen delivery and reduced energy loss. The aim was to determine the role of ischemic preconditioning in the stimulation of constitutive endothelial nitric oxide (NO) production and its effect on energy charge, radical oxygen species generation, cytokine liberation, and neutrophil infiltration during reperfusion. Rats were assigned to one of four groups depending on the preconditioning protocol: hepatic ischemia/reperfusion, or hepatic ischemia/reperfusion and ischemic preconditioning, for 5, 10, or 20min. A portosystemic shunt was established between the portal and left jugular veins during ischemia. Preconditioning produced rises in plasma nitrites, but no rise in inducible nitric oxide synthase gene expression. A 5 or 10min preconditioning period allowed for higher energy charge, bile production, and glutathione levels, with less lipoperoxide, alanine aminotransferase, tumor necrosis factor-α, and interleukin-1 production and neutrophil infiltration, compared with 20min or control. Survival was 80% in the G10 group, 70 in G5, 10 in GC, and 0% in the G20 group. Ten-min liver preconditioning improves survival and prevents energy loss during hepatic ischemia/reperfusion by stimulating constitutive NO production, maintaining glutathione concentrations and reducing oxygen species and proinflammatory cytokine generation as well as neutrophil infiltration.

Cardiopreventive effect of ethanolic extract of Date Palm Pollen against isoproterenol induced myocardial infarction in rats through the inhibition of the angiotensin-converting enzyme

The present study aimed to examine the putative preventive effect of the ethanolic extract Date Palm Pollen (DPP, Phoenix dactylifera L., family Arecaceae) on isoproterenol-induced myocardial infarction (MI) in rats. Twenty four rats were randomly divided into four groups including control. They were treated with DPP extract (400mg/kg) and clopidogrel (0.2mg/kg) for 7days followed by myocardial injury induction using subcutaneous isoproterenol (100mg/kg) with an interval of 24h for two days (6th and 7th day). Administration of isoproterenol exhibited indicative changes in the ECG pattern evidenced by significant elevation of ST-segment and cardiac injury markers viz.; troponin-T, creatine phosphokinase (CPK), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) by 315%, 71%, 64% and 170%, respectively as compared to control. Additionally, the angiotensin-converting enzyme (ACE) activity in plasma was increased by 33% associated to histological myocardial necrosis. However, pre-co-treatment with DPP extract improved the cardiac biomarkers injury, normalized cardiac function indices and prevented the ventricular remodeling process through inhibition of ACE activity by 34% and the inhibition of the generation of radical oxygen species. Extensive characterization of this DPP extract using LC-HRMS revealed numerous flavonoids and phenols compounds which could be endowed with cardiopreventive actions. Overall, these results proved that DPP extract has preventive effects on cardiac remodeling process.

Malvidin's Effects on Rat Pial Microvascular Permeability Changes Due to Hypoperfusion and Reperfusion Injury.

The present study was aimed to evaluate the malvidin’s protective effects on damage induced by 30 min bilateral common carotid artery occlusion (BCCAO) and 60 min reperfusion (RE) in rat pial microcirculation. Rat pial microcirculation was observed using fluorescence microscopy through a closed cranial window. Western blotting analysis was performed to investigate the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) and matrix metalloproteinase 9 (MMP-9) expression. Moreover, MMP-9 activity was evaluated by zymography. Finally, neuronal damage and radical oxygen species (ROS) formation were assessed. In all animals, pial arterioles were classified in five orders of branching according to Strahler’s method. In hypoperfused rats, 30 min BCCAO and 60 min RE caused a decrease in arteriolar diameter, an increase in microvascular leakage and leukocyte adhesion, accompanied by decreased capillary perfusion and red blood cell velocity (VRBC). Moreover, marked neuronal damage and evident ROS generation were detected. Conversely, malvidin administration induced arteriolar dilation in dose-related manner, reducing microvascular leakage as well as leukocyte adhesion. Capillary perfusion and VRBC were protected. Nitric oxide (NO) synthase inhibition significantly attenuated malvidin’s effects on arteriolar diameter. Western blotting analysis revealed an increase in eNOS and p-eNOS expression, while zymography indicated a decrease in MMP-9 activity after malvidin’s administration. Furthermore, malvidin was able to prevent neuronal damage and to decrease ROS generation. In conclusion, malvidin protects rat pial microcirculation against BCCAO/RE injury, preventing blood-brain impairment and neuronal loss. Malvidin’s effects appear to be mediated by eNOS activation and scavenger activity.

Targeting the ataxia telangiectasia mutated pathway for effective therapy against hirsutine-resistant breast cancer cells.

The present authors have recently demonstrated that hirsutine, one of the major alkaloids in Uncaria species, promotes cell apoptosis by inducing DNA damage and suppresses metastasis of breast cancer cells. Despite its potent anti-cancer activity, certain types of human breast cancer cells exhibit resistance to hirsutine. To maximize the clinical utility of hirsutine therapy against breast cancer, it is critical to explore the underlying mechanism that protects hirsutine-resistant breast cancer cell lines. To identify potential targets for overcoming hirsutine-resistance, the present study investigated a library of kinase inhibitors in combination with hirsutine treatment in the hirsutine-resistant human breast carcinoma MCF-7 cell line. Amongst the 96 compounds tested, inhibitors of the ataxia telangiectasia mutated (ATM) pathway sensitized MCF-7 cells to hirsutine-induced cell death along with a sustained DNA damage response. This sensitization of MCF-7 cells to the hirsutine-induced DNA damage response by interfering with the ATM pathway did not require p53. Instead, radical oxygen species generation was significantly increased in hirsute and ATM inhibitor-treated MCF-7 cells. In conclusion, the present findings suggest the importance of the ATM pathway for optimizing the anti-cancer effect of hirsutine in breast cancer cells.

Upregulation of the N-formyl Peptide receptors in scleroderma fibroblasts fosters the switch to myofibroblasts.

Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis. N-Formyl peptide (fMLF) receptors (FPRs) are chemotactic receptors involved in inflammation. Three FPRs have been identified: FPR1, FPR2, and FPR3. We have examined, by RT-PCR, Western blot and immunohistochemistry, FPRs expression in skin fibroblasts from 10 normal subjects and 10 SSc patients, showing increased expression in SSc fibroblasts. Several functions of FPRs occur through the interaction with a region of the urokinase-type plasminogen activator receptor (uPAR88-92), able to interact with FPRs and to mediate urokinase (uPA) or fMLF-dependent cell migration. Soluble uPAR84-95 peptide can act as a direct ligand of FPRs. Furthermore, uPA or its aminoterminal fragment (ATF) can promote the exposure of the uPAR88-92 region. The WKYMVm peptide is a FPRs pan-agonist. We investigated the functional effects of these agonists on normal and SSc fibroblasts. ATF, uPAR84-95, and WKYMVm regulated adhesion, migration, and proliferation of normal fibroblasts. Despite FPR overexpression, the response of SSc fibroblasts to the same agonists was greatly reduced, except for the proliferative response to ATF. SSc fibroblasts showed increased α-smooth muscle actin expression and improved capability to induce wound closure. Indeed, they overexpressed a cleaved uPAR form, exposing the uPAR88-92 region, and vitronectin, both involved in fibrosis and in the fibroblast-to-myofibroblast transition. FPR stimulation promoted α-smooth muscle actin expression in normal fibroblasts as well as motility, matrix deposition, αvβ5 integrin expression, and radical oxygen species generation in normal and SSc fibroblasts. This study provides evidence that FPRs may play a role in fibrosis and in the fibroblast-to-myofibroblast transition.