Nitric Oxide Plays a Dual Role in Cardiorenal Syndrome in Vitro Model.

BACKGROUND/AIMS: Nitric oxide (NO) plays a dual role, acting as both an oxidant and a reducer, with various effects depending on its concentration and environment. Acute kidney injury's (AKI) pathogenesis observed in cardiorenal syndrome 3 (CRS 3) involves inflammatory responses and the production of reactive oxygen and nitrogen species. However, the role of NO on the development of CRS 3 is still not completely understood. The study aimed to mimic CRS 3 in vitro and investigate NO signaling and inflammatory molecules. METHODS: Thus, HEK293 cells were submitted to normoxia (NX) or hypoxia (HX) protocols for 16 h followed by 3 h of reoxygenation, treated or not with L-NAME. Conditionate medium by HEK293 was transferred to H9c2 for 24 h. Cellular viability was evaluated by MTT assay, real time PCR was used to analyze gene expression and NO content were evaluated in the intra and extracellular medium by amperimetry. RESULTS: Carbonic anhydrase 9 (CA9) expression increased 2.9-fold after hypoxia. Hypoxia reduced 18 % cell viability in HEK293 that was restored by L-NAME treatment. The sum of nitrite (NO2-) and S-nitrosothiol (S-NO) fractions in HEK293 cells showed a substantial decrease on NO intracellular content (38 %). Both IL-6 and IL-10 decreased in all groups compared to NX cells. Besides TNF-α and Bax/Bcl2 ratio increased in hypoxia (approximately 120-fold and 600-fold, respectively) and L-NAME restored this effect. Regarding H9c2 cells, the S-NO fractions showed a substantial decrease in extracellular content after HX (17%) that was not restored by L-NAME. IL-1ß decreases in cardiac cells treated with conditioned medium from HX/L-NAME. CONCLUSION: In conclusion this study highlights the complex interplay of NO and inflammatory factors in hypoxia-induced renal and cardiac cell responses, with potential implications for cardiorenal syndrome.

Implications of ZnO Nanoparticles and S-Nitrosoglutathione on Nitric Oxide, Reactive Oxidative Species, Photosynthetic Pigments, and Ionomic Profile in Rice.

Zinc is an important nutrient for several plants and humans. Nitric oxide (NO) is a free radical that is important to biological processes that mediate the growth and mitigation of biotic and abiotic stresses in plants. The present study investigated the enzymatic and photosynthetic profile and the accumulation of macro- and microelements in rice plants (Oryza sativa L.) that received foliar treatments of zinc oxide nanoparticles (ZnO NPs), nitric oxide donor (GSNO), and the association of both (GSNO-ZnO NPs). Zinc concentration in rice husks increased by 66% and 68% in plants treated with ZnO NPs and GSNO-ZnO NPs, respectively. The GSNO treatment caused an increase of 25% in the Fe concentration in the rice grains. Only a small disturbance of the antioxidant system was observed, with increases in H2O2, S-NO, and NO2-, mainly in the group treated with GSNO-ZnO NPs; however, the disturbance did not affect the yield, the growth, or vital processes, such as as photosynthetic pigments production. There was an increase in chlorophyll B of 290% and an increase in chlorophyll A of 187% when ZnO NPs was applied. GSNO-ZnO NPs increased chlorophyll B by 345% and chlorophyll A by 345%, indicating that the treatments GSNO, ZnO NPs, and GSNO-ZnO NPs reduced possible oxidative stress and helped as protective treatments.

Different vehicles containing CaneCPI-5 reduce erosive dentin wear in situ.

OBJECTIVE: This study evaluated the protective capacity of a sugarcane-derived cystatin (CaneCPI-5) in different vehicles (1-solution and 2-chitosan gel) against erosive dentin wear in situ. METHODS: In part-1, 15 volunteers participated in a crossover protocol (solutions): Water; Elmex™ and CaneCPI-5. The volunteers wore an appliance with 4 dentin samples for 5 days. These samples were treated with a drop of the solutions for 1 min (4X/d), then the acquired pellicle (AP) was formed and the samples were subjected to erosive challenges (EROSION: citric acid, for 90 s, 4X/day). 2X/day, half of the samples were also abraded for 15 s (ABRASION). In part-2, 16 volunteers participated in a crossover protocol (gel): No gel, Chitosan gel, Chitosan gel + NaF and Chitosan gel + CaneCPI-5. The volunteers also wore an appliance. The samples were treated once/day with the gel or not for 4 min, then the AP was formed and the samples were subjected to erosive and abrasive challenges, as reported in part-1. Dentin wear was measured by profilometry. Data were analyzed by two-way RM-ANOVA and Sidak's tests (p < 0.05). RESULTS: Part-1: Elmex™ and CaneCPI-5 significantly reduced dentin loss in comparison with Water for the EROSION/ABRASION conditions (p < 0.05). Part-2, all the treated groups significantly reduced the dentin loss in comparison to the No gel. The greatest reduction was found for the gel + CaneCPI-5 group for the EROSION/ABRASION (p < 0.05). CONCLUSION: The solution and chitosan gel containing CaneCPI-5 protected against erosive dentin wear in situ. CLINICAL RELEVANCE: These different vehicles are probably sufficient for protecting people with high risk of developing erosive dentin wear.

Iron oxide/silver hybrid nanoparticles impair the cholinergic system and cause reprotoxicity in Caenorhabditis elegans.

Iron oxide nanoparticles present superparamagnetic properties that enable their application in various areas, including drug delivery at specific locations in the organism. Silver nanoparticles have potent antimicrobial effects. Although the combination of Fe3O4-NPs and Ag-NPs in one hybrid nanostructure (Fe3O4@Ag-NPs) demonstrated promising targeted biomedical applications, their toxicological effects are unknown and need to be assessed. Caenorhabditis elegans is a promising model for nanotoxicological analysis, as it allows an initial screening of new substances. After exposure to Fe3O4-NPs, Ag-NPs and Fe3O4@Ag-NPs, we observed that hybrid NPs reduced the C. elegans survival and reproduction. Higher concentrations of Fe3O4@Ag-NPs caused an increase in cell apoptosis in the germline and a decrease in egg laying, which was associated with a decrease in worm swimming movements and abnormalities in the cholinergic neurons. Fe3O4@Ag-NPs caused an increase in reactive oxygen species, along with activation of DAF-16 transcription factor. A higher expression of the target genes GST-4::GFP and SOD-3::GFP were evidenced, which suggests the activation of the antioxidant system. Our results indicate the reprotoxicity caused by high levels of Fe3O4@Ag-NPs, as well as cholinergic neurotoxicity and activation of the antioxidant system in C. elegans, suggesting that high concentrations of these nanomaterials can be harmful to living organisms.

Chronic exposure to nitric oxide sensitizes prostate cancer cells and improved ZnO/CisPt NPs cytotoxicity and selectivity.

The co-therapy of common chemotherapeutics with nitric oxide (NO), an endogenous signaling molecule, is proposed as an alternative to sensitize cancer cells and enhance treatments' efficacy. Herein, we have synthesized cisplatin-releasing zinc oxide nanoparticles (ZnO/CisPt NPs), which promoted a sustained and pH targeted release, able to release a higher amount of CisPt at tumor microenvironment conditions. This material was combined with a chronic NO treatment, at low concentration, in prostate cancer cells (PC3). NO treatment enhanced the S-NO concentration in PC3 cells, suggesting the nitrosylation or transnitrosylation processes enhancement, which are directly related to S-NO binding to proteins, function alterations and cancer cells death. Indeed, these mechanisms directly impacted the cytotoxic effect of ZnO/CisPt NPs, inducing a 30 % higher viability reduction of PC3 cells after NO treatment, along with a higher selectivity index when compared to normal human fibroblasts (FN1).

Gels containing statherin-derived peptide protect against enamel and dentin erosive tooth wear in vitro.

The effect of gels containing a statherin-derived peptide (Stn) on the protection against enamel and dentin erosive tooth wear (ETW) in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 15 and 18/group for enamel and dentin, respectively) that were treated with Chitosan or Carboxymethylcellulose (CMC) gels containing Stn15pSpS at 1.88 × 10-5 M or 3.76 × 10-5 M. Chitosan or CMC gels without active ingredients served as negative controls, while chitosan gel containing 1.23% F (as NaF) and acidulated phosphate fluoride gel (1.23% F) served as positive controls. The gels were applied on the specimens for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.01 M HCl pH 2.0/45 s, artificial saliva/2 h, and artificial saliva overnight). The gels were applied again during pH cycling, 2 times/day for 4 min after the first and last erosive challenges. Enamel and dentin loss (µm) were assessed by contact profilometry. Scanning electron microscopy (SEM) was analyzed using a cold field emission. Data were analyzed by two-way ANOVA (for chitosan and CMC gels, separately) and Tukey's multiple comparison test. SEM images showed changes to enamel topography after application oft the gels containing Stn or F. Regarding CMC-based gels, for enamel, none of the treatments significantly reduced ETW in comparison with placebo; for dentin, however, gels containing Stn, regardless the concentration, significantly reduced the ETW. Moreover, Chitosan-based gels, regardless the Stn concentration, were able to protect enamel and dentin against ETW. Gels containing Stn might be a new approach to protect against ETW.

Cytotoxicity towards Breast Cancer Cells of Pluronic F-127/Hyaluronic Acid Hydrogel Containing Nitric Oxide Donor and Silica Nanoparticles Loaded with Cisplatin.

The incorporation of both nitric oxide (NO) donor (S-nitrosoglutathione, GSNO) and silica nanoparticles loaded with cisplatin (SiO2@CisPt NPs) into a polymeric matrix represents a suitable approach to creating a drug-delivery system with sustained and localized drug release against tumor cells. Herein, we report the synthesis, characterization, and cytotoxicity evaluation of Pluronic F-127/hyaluronic acid hydrogel containing GSNO and SiO2@CisPt NPs against breast cancer cells. SiO2@CisPt NPs were successfully synthesized, revealing a spherical morphology with an average size of 158 ± 20 nm. Both GSNO and SiO2@CisPt NPs were incorporated into the thermoresponsive Pluronic/hyaluronic hydrogel for sustained and localized release of both NO and cisplatin. The kinetics of NO release from a hydrogel matrix revealed spontaneous and sustained release of NO at the millimolar range for 24 h. The MTT assay showed concentration-dependent cytotoxicity of the hydrogel. The combination of GSNO and SiO2@CisPt incorporated into a polymeric matrix decreased the cell viability 20% more than the hydrogel containing only GSNO or SiO2@CisPt. At 200 µg/mL, this combination led to a critical cell viability of 30%, indicating a synergistic effect between GSNO and SiO2@CisPt NPs in the hydrogel matrix, and, therefore, highlighting the potential application of this drug-delivery system in the field of biomedicine.

Preventive effect of chitosan gel containing CaneCPI-5 against enamel erosive wear in situ.

OBJECTIVE: This study evaluated the preventive effect of a chitosan gel containing CaneCPI-5 against enamel erosion and erosion + abrasion in situ. METHODS: Sixteen volunteers participated in a crossover, double-blind protocol, comprising 4 phases: (1) no treatment (Nt); (2) chitosan gel (Cg); (3) chitosan gel + 12,300 ppm NaF (Cg + NaF); and (4) chitosan gel + 0.1 mg/mL CaneCPI-5 (Cg + Cane). Volunteers wore an appliance containing 4 specimens. Once/day, they applied the gel (except for Nt) (4 min/specimen). Erosive challenges were performed extra-orally (0.1% citric acid, 90 s, 4 × /day; ERO). Specimens were also abraded (toothbrush, 15 s/specimen, 2 × /day; ERO + ABR). Enamel wear was assessed by profilometry and relative surface reflection intensity (%SRI). Two-way RM-ANOVA/Sidak's tests and Spearman's correlation were used (p < 0.05). RESULTS: For profilometry, ERO + ABR promoted significantly greater wear when compared with ERO. There was a significant difference among all treatments. The lowest enamel loss occurred for Cg + Cane, followed by Cg + NaF, Cg, and Nt (p < 0.05). The %SRI was significantly lower for ERO + ABR when compared to ERO, only for the Nt group. The greatest %SRI was found for the Cg + NaF and Cg + Cane groups, which did not differ significantly, regardless of the conditions. The lowest %SRI was found for the Nt and Cg groups, which did not differ from each other, regardless of the conditions. The Nt group did not differ significantly from the Cg + NaF (ERO). There was a significant correlation between both analyses. CONCLUSION: The incorporation of CaneCPI-5 in the chitosan gel prevented erosive wear in situ. CLINICAL RELEVANCE: These results open a new perspective for the use of CaneCPI-5 in other application vehicles, such as chitosan gel.

Evaluation of collision/reaction gases in single-particle ICP-MS for sizing selenium nanoparticles and assessment of their antibacterial activity.

Selenium nanoparticles (SeNPs) have recently attracted attention because they combine the benefits of Se and lower toxicity compared to other chemical forms of this element. In this study, SeNPs were synthesized by a green method using ascorbic acid as the reducing agent and polyvinyl alcohol as stabilizer. The nanoparticles were widely characterized. To determine the total concentration of Se by ICP-MS, several isotopes and the use of He as collision gas were evaluated, which was effective in minimizing interferences. A method for sizing SeNPs by single particle ICP-MS (SP-ICP-MS) was developed. For this purpose, He and H2were evaluated as collision/reaction gases, and the second one showed promising results, providing an average diameter of 48 nm for the SeNPs. These results agree with those obtained by TEM (50.1 nm). Therefore, the SP-ICP-MS can be implemented for characterizing SeNPs in terms of size and size distribution, being an important analytical tool for Se and other widely studied nanoparticles (e.g. Ag, Au, Ce, Cu, Fe, Zn). Finally, the antibacterial activity of SeNPs was assessed. The SeNPs showed bacteriostatic activity against three strains of Gram-positive bacteria and were particularly efficient in inhibiting the growthE. faecaliseven at very low concentrations (MIC < 1.4 mg l-1). In addition, a bactericidal activity of SeNPs againstS. aureuswas observed. These nanoparticles may have potential application in pharmaceutical industry, biomedicine and agriculture.

Mitochondrial Dysfunction in Cardiorenal Syndrome 3: Renocardiac Effect of Vitamin C.

Cardiorenal syndrome (CRS) is a pathological link between the kidneys and heart, in which an insult in a kidney or heart leads the other organ to incur damage. CRS is classified into five subtypes, and type 3 (CRS3) is characterized by acute kidney injury as a precursor to subsequent cardiovascular changes. Mitochondrial dysfunction and oxidative and nitrosative stress have been reported in the pathophysiology of CRS3. It is known that vitamin C, an antioxidant, has proven protective capacity for cardiac, renal, and vascular endothelial tissues. Therefore, the present study aimed to assess whether vitamin C provides protection to heart and the kidneys in an in vivo CRS3 model. The unilateral renal ischemia and reperfusion (IR) protocol was performed for 60 min in the left kidney of adult mice, with and without vitamin C treatment, immediately after IR or 15 days after IR. Kidneys and hearts were subsequently collected, and the following analyses were conducted: renal morphometric evaluation, serum urea and creatinine levels, high-resolution respirometry, amperometry technique for NO measurement, gene expression of mitochondrial dynamic markers, and NOS. The analyses showed that the left kidney weight was reduced, urea and creatinine levels were increased, mitochondrial oxygen consumption was reduced, NO levels were elevated, and Mfn2 expression was reduced after 15 days of IR compared to the sham group. Oxygen consumption and NO levels in the heart were also reduced. The treatment with vitamin C preserved the left kidney weight, restored renal function, reduced NO levels, decreased iNOS expression, elevated constitutive NOS isoforms, and improved oxygen consumption. In the heart, oxygen consumption and NO levels were improved after vitamin C treatment, whereas the three NOS isoforms were overexpressed. These data indicate that vitamin C provides protection to the kidneys and some beneficial effects to the heart after IR, indicating it may be a preventive approach against cardiorenal insults.

Solutions and Gels Containing a Sugarcane-Derived Cystatin (CaneCPI-5) Reduce Enamel and Dentin Erosion in vitro.

The effect of solutions and gels containing a sugarcane-derived cystatin (CaneCPI-5) on the protection against enamel and dentin erosion in vitro was evaluated. Bovine enamel and dentin specimens were divided into 2 groups (n = 135 and 153/group for enamel and dentin, respectively) that were treated with solutions or chitosan gels containing 0.1 or 0.25 mg/mL CaneCPI-5. The positive controls for solutions and gels were Elmex Erosion Protection™ solution and NaF gel (12,300 ppm F), respectively. Deionized water and chitosan gel served as controls, respectively. The solutions were first applied on the specimens for 1 min and the gels for 4 min. Stimulated saliva was collected from 3 donors and used to form a 2-h acquired pellicle on the specimens. Then, the specimens were submitted to an erosive pH cycling protocol 4 times/day for 7 days (0.1% citric acid pH 2.5/90 s, artificial saliva/2 h, and artificial saliva overnight). The solutions and gels were applied again during pH cycling, 2 times/day for 1 min and 4 min, respectively, after the first and last erosive challenges. Enamel and dentin losses (µm) were assessed by contact profilometry. Data were analyzed by 2-way ANOVA and Tukey's test (p < 0.05). All the treatments significantly reduced enamel and dentin loss in comparison with controls. Both CaneCPI-5 concentrations had a similar protective effect against enamel erosion, but only the higher concentration was as effective against dentin erosion as the positive control. Regarding the vehicles, only the 0.1 mg/mL gel performed worse than the positive control for dentin. CaneCPI-5 reduced enamel and dentin erosion to a similar extent as the fluoride-containing vehicles. However, dentin requires higher CaneCPI-5 concentrations, in the case of gels. Solutions or gels containing CaneCPI-5 might be a new approach to protect against dental erosion.

Multifunctional hybrid nanoplatform based on Fe3O4@Ag NPs for nitric oxide delivery: development, characterization, therapeutic efficacy, and hemocompatibility.

The combination of Fe3O4@Ag superparamagnetic hybrid nanoparticles and nitric oxide (NO) represents an innovative strategy for a localized NO delivery with a simultaneous antibacterial and antitumoral actions. Here, we report the design of Fe3O4@Ag hybrid nanoparticles, coated with a modified and nitrosated chitosan polymer, able to release NO in a biological medium. After their synthesis, physicochemical characterization confirmed the obtention of small NO-functionalized superparamagnetic Fe3O4@Ag NPs. Antibacterial assays demonstrated enhanced effects compared to control. Bacteriostatic effect against Gram-positive strains and bactericidal effect against E. coli were demonstrated. Moreover, NO-functionalized Fe3O4@Ag NPs demonstrated improved ability to reduce cancer cells viability and less cytotoxicity against non-tumoral cells compared to Fe3O4@Ag NPs. These effects were associated to the ability of these NPs act simultaneous as cytotoxic (necrosis inductors) and cytostatic compounds inducing S-phase cell cycle arrest. NPs also demonstrated low hemolysis ratio (<10%) at ideal work range, evidencing their potential for biomedical applications. Targeted and hemocompatible nitric oxide-releasing multi-functional hybrid nanoparticles for antitumor and antimicrobial applications.

Characterization of the Oxidative Stress in Renal Ischemia/Reperfusion-Induced Cardiorenal Syndrome Type 3.

In kidney disease (KD), several factors released into the bloodstream can induce a series of changes in the heart, leading to a wide variety of clinical situations called cardiorenal syndrome (CRS). Reactive oxygen species (ROS) play an important role in the signaling and progression of systemic inflammatory conditions, as observed in KD. The aim of the present study was to characterize the redox balance in renal ischemia/reperfusion-induced cardiac remodeling. C57BL/6 male mice were subjected to occlusion of the left renal pedicle, unilateral, for 60 min, followed by reperfusion for 8 and 15 days, respectively. The following redox balance components were evaluated: catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (FRAP), NADPH oxidase (NOX), nitric oxide synthase (NOS), hydrogen peroxide (H2O2), and the tissue bioavailability of nitric oxide (NO) such as S-nitrosothiol (RSNO) and nitrite (NO2 -). The results indicated a process of renoprotection in both kidneys, indicated by the reduction of cellular damage and some oxidant agents. We also observed an increase in the activity of antioxidant enzymes, such as SOD, and an increase in NO bioavailability. In the heart, we noticed an increase in the activity of NOX and NOS, together with increased cell damage on day 8, followed by a reduction in protein damage on day 15. The present study concludes that the kidneys and heart undergo distinct processes of damage and repair at the analyzed times, since the heart is a secondary target of ischemic kidney injury. These results are important for a better understanding of the cellular mechanisms involved in CRS.

H2S- and NO-releasing gasotransmitter platform: A crosstalk signaling pathway in the treatment of acute kidney injury.

Acute kidney injury (AKI) is a syndrome affecting most patients hospitalized due to kidney disease; it accounts for 15 % of patients hospitalized in intensive care units worldwide. AKI is mainly caused by ischemia and reperfusion (IR) injury, which temporarily obstructs the blood flow, increases inflammation processes and induces oxidative stress. AKI treatments available nowadays present notable disadvantages, mostly for patients with other comorbidities. Thus, it is important to investigate different approaches to help minimizing side effects such as the ones observed in patients subjected to the aforementioned treatments. Therefore, the aim of the current review is to highlight the potential of two endogenous gasotransmitters - hydrogen sulfide (H2S) and nitric oxide (NO) - and their crosstalk in AKI treatment. Both H2S and NO are endogenous signalling molecules involved in several physiological and pathophysiological processes, such as the ones taking place in the renal system. Overall, these molecules act by decreasing inflammation, controlling reactive oxygen species (ROS) concentrations, activating/inactivating pro-inflammatory cytokines, as well as promoting vasodilation and decreasing apoptosis, hypertrophy and autophagy. Since these gasotransmitters are found in gaseous state at environmental conditions, they can be directly applied by inhalation, or in combination with H2S and NO donors, which are compounds capable of releasing these molecules at biological conditions, thus enabling higher stability and slow release of NO and H2S. Moreover, the combination between these donor compounds and nanomaterials has the potential to enable targeted treatments, reduce side effects and increase the potential of H2S and NO. Finally, it is essential highlighting challenges to, and perspectives in, pharmacological applications of H2S and NO to treat AKI, mainly in combination with nanoparticulated delivery platforms.