Intracellular Reactive Oxygen Species Release Research Articles

Sodium Selenite Ameliorates Silver Nanoparticles Induced Vascular Endothelial Cytotoxic Injury by Antioxidative Properties and Suppressing Inflammation Through Activating the Nrf2 Signaling Pathway.

Silver nanoparticles (AgNP) are the dominant nanomaterials in commercial products and the medical field, but the widespread occurrence of AgNP has become a global threat to human health. Growing studies indicate that AgNP exposure can induce vascular endothelial toxicity by excessive oxidative stress and inflammation, which is closely related to cardiovascular disease (CVD), but the potential intrinsic mechanism remains poorly elucidated. Thus, it has been crucial to control the toxicological effects of AgNP in order to improve their safety and increase the outcome of their applications.Multiple researches have demonstrated that sodium selenite (Se) possesses the capability to counteract the toxicity of AgNP, but the functional role of Se in AgNP-induced CVD is largely unexplored. The aim of this study was to explore the potential protective effect of Se on AgNP-induced vascular endothelial lesion and elucidate the underlying mechanisms. An in vivo model of toxicity in animals was established by the instillation of 200 µL of AgNP into the trachea of rats both with (0.2 mg/kg/day) and without Se treated. In vitro experiments, human umbilical vein endothelial cells (HUVECs) were incubated with AgNP (0.3 µg/mL ) and Se for a duration of 24 h. Utilizing transmission electron microscopy, we observed that the internalization of AgNP-induced endothelial cells was desquamated from the internal elastic lamina, the endoplasmic reticulum was dilated, and the medullary vesicle formed. Se treatment reduced the levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), inhibited the release of pro-inflammatory cytokines (specifically tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6), improved endothelial cell permeability, integrity, and dysfunction, and prevented damage to the aortic endothelium caused by AgNP. Importantly, we found that Se showed the capacity against AgNP with biological functions in guiding the intracellular reactive oxygen species (ROS) scavenging and meanwhile exhibiting anti-inflammation effects. Se supplementation decreased the intracellular ROS release and suppressed NOD-like receptor protein 3 (NLRP3) and nuclear factor kappa-B (NF-κB) mediated inflammation within AgNP-intoxicated rats and HUVECs. The anti-oxidant stress and anti-inflammatory effects of Se were at least partly dependent on nuclear factor erythroid 2-related factor 2 (Nrf2). Overall, our results indicated that the protectiveness of Se against AgNP-induced vascular endothelial toxicity injury was at least attributed to the inhibition of oxidative ROS and pro-inflammatory NF-κB/NLRP3 inflammasome by activating the Nrf2 and antioxidant enzyme (HO-1) signal pathway.

LLDPE/TiO<sub>2</sub>-ZnO Nanocomposite Films induces Transitory Oxidative Stress Response on Human Fibroblast and Blood Cell Lines Models

Healthcare-associated infections (HAIs) are a major safety concern globally that contribute to mortality rates amongst patients especially associated with indwelling or implanted medical devices. The advanced metal-oxide nanocomposites (MNPs) embedded in polymer matrix present an outstanding antibacterial profile, especially for MDR strains owing to reactive oxygen species (ROS) and free radicals’ mode of action. To date, there is still a lack of knowledge on the implication of external reactive species from MNPs-based polymers to humans. This study investigates the bio-interaction of TiO2-ZnO nanocomposite films embedded in linear low-density polyethylene (LLDPE/ TiO2-ZnO) on human fibroblast and blood cell lines model at molecular genes and protein level. The initial analysis of the in vitro bio-interaction responses on fibroblast and blood cell line models showed signs of cell membrane integrity disturbance, which might be due to free radicals’ activities, such as the release of intracellular ROS and Zn ions (Zn2+) during the initial cellular adaptation process on the TiO2–ZnO polymer nanocomposite film. Further findings found that cell–polymer nanocomposite film interaction could possibly trigger transitory oxidative stress response and cellular redox regulation via NF-kβ interactions. However, further comprehensive studies are needed to support this study, especially involving animal models.

Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Potassium iodide enhances the anti-hepatocellular carcinoma effect of copper-cysteamine nanoparticle mediated photodynamic therapy on cancer treatment

As a novel photosensitizer, copper-cysteamine (Cu-Cy) nanoparticles (NPs) can enable photodynamic therapy (PDT) for cancer treatment with improved efficiency. Previous studies have demonstrated that potassium iodide (KI) can enhance anti-infection ability by increasing the release of reactive oxygen species (ROS). Herein, for the first time, we investigate whether KI can enhance the anti-hepatocellular carcinoma effect by Cu-Cy NPs based PDT. The results show that Cu-Cy NPs based PDT inhibited the growth, migration, and clone formation ability of HepG2, Hep3B, and Huh7 cells, and the inhibitory effect is enhanced largely after adding KI. The observations from calcein-AM and EthD-1 double fluorescence staining method, DAPI staining, and Annexin V-FITC/PI technology show that KI enhances the mortality rate of HepG2 cells through Cu-Cy NPs-mediated PDT and confirm the cell apoptosis with a significant rate. Further studies through flow cytometry have been found that KI can affect mitochondrial function by leading the mitochondrial membrane potential (MMP) decrease and enhance the intracellular ROS release through Cu-Cy NPs-mediated PDT. It also up-regulated p53 gene and protein expression, down-regulated surviving, decreased Bcl-2/Bax ratio, and activated caspase-3 and caspase-9 through PCR, and WB technology in cells and tissues. We theorize that the apoptosis mechanism may be caused by the up-regulation of p53 to activate the mitochondrial endogenous apoptosis pathway. Finally, using HepG2 bearing tumor model, we further confirmed that the combination of KI with Cu-Cy NPs-mediated PDT can inhibit tumor cell growth, which is a safe and effective modality without damaging healthy tissues. This study proposes Cu-Cy NPs combined with KI can enhance the efficacy of PDT anti-tumor therapy. The most likely products of the combination of Cu-Cy NPs and KI that are responsible for the enhanced PDT effects are singlet oxygen (1O2), biocidal triiodide (I3−), and H2O2. Our research offers a new strategy for combined applications to improve the anti-tumor effect.

Protective effect of dihydromyricetin on vascular smooth muscle cell apoptosis induced by hydrogen peroxide in rats.

Dihydromyricetin (DMY), also called Ampelopsin, which was extracted from Ampelopsis grossedentata, has been demonstrated to have a protective effect against cell oxidative injury and cell apoptosis in vitro. In the present study, we tried to study the role of DMY on apoptosis of vascular smooth muscle cells (VSMCs) induced by hydrogen peroxide (H2O2) and explore the underlying mechanisms. Apoptotic cells were detected by Hematoxylin and Eosin (H.E.) staining, Hoechst 33342 staining, and Annexin V-fluorescein isothiocyanate binding assay. The intracellular reactive oxygen species (ROS) level was estimated through fluorescence assay. The mRNA and protein expression of Caspase-3, Caspase-9, Bcl-2, and Bax were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The results showed that the pretreatment of VSMCs with DMY not only significantly increased cell viability, reduced intracellular ROS release, alleviated the morphological changes of apoptosis, and decreased the apoptosis rate, but also upregulated Bcl-2 expression and downregulated Caspase-3, Caspase-9, Bax expression, and ultimately attenuated the H2O2-stimulated apoptosis. The inhibition of DMY on VSMC apoptosis may be mediated by ROS scavenging and the activation of the mitochondrial apoptotic signaling pathway.

Singlet oxygen from endoperoxide initiates an intracellular reactive oxygen species release in HaCaT keratinocytes.

Singlet oxygen (‍1O2) is a selective intermediate reactive oxygen species generated naturally in biological systems by light- and non-light mediated processes. Although ‍1O2 plays an important role in cell signaling and in maintaining homeostasis, it can be toxic due to its ability to diffuse across considerable distances. Several in vitro studies have investigated the pathways by which ‍1O2 mediates oxidation of biological molecules and potential pathogenesis. However, understanding how singlet oxygen exerts cell injury through the production of subsequent reactive oxygen species remains unexplored. To study this, we used a hydrophobic endoperoxide as a source of ‍1O2. Endoperoxides are reagents that quantitatively generate singlet oxygen in solution at 35°C by thermal decomposition. Our chemiluminescence and cell viability assay data revealed that ‍1O2 stimulated a secondary intracellular reactive oxygen species production in a very short time. To determine the source of these reactive oxygen species with endo-peroxide exposure, cells were treated with inhibitors targeting NADPH oxidases and platelet activating factor receptors. Our results showed that addition of the platelet activating factor receptor antagonist, Apafant (WEB2086), alleviated cell injury and hydrogen peroxide levels following endoperoxide stimulation. Furthermore, intracellular calcium assay data demonstrated a potential calcium sensitive production of intracellular reactive oxygen species.

Enzymatic Hydrolysis of Broken Rice Protein: Antioxidant Activities by Chemical and Cellular Antioxidant Methods.

Excessive reactive oxygen species (ROS) is an important cause of aging, and supplementing antioxidants through diet is one of the important ways to delay aging. Some studies have confirmed that rice protease hydrolysate has antioxidant activity, but was rarely been investigated on cells. Thus, commercial enzymes, alkaline enzyme, neutral enzyme, pepsin, chymotrypsin, and trypsin were selected to hydrolyze broken rice protein (BRP) to obtain the corresponding hydrolysates, which were A-broken rice protein hydrolysate (BRPH), N-BRPH, P-BRPH, C-BRPH, and T-BRPH, respectively. Then the antioxidant properties of BRPHs were evaluated by different chemical and cellular antioxidation. Molecular weight, peptide length distribution, and amino acid sequence were detected to insight into the antioxidant properties. Among BRPHs, the A-BRPH displayed the strongest hydroxyl radical scavenging activity (IC50 = 1.159 mg/ml) and metal ion-chelating activities (IC50 = 0.391 mg/ml). Furthermore, cellular antioxidation confirmed that A-BRPH significantly increased cell viability and inhibited the intracellular ROS release in both aging cells and cell-aging processes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that peptides with molecular weight <14.5 KDa were produced by enzymatic hydrolysis. Additionally, A-BRPH rich in low molecular weight (<3 kDa) and short-length peptides with some specific amino acids, such as aromatic and hydrophobic amino acids, contributes to the antioxidant properties. This study provided theoretical to the utilization of broken rice and confirmed that A-BRPH could be used in new anti-aging food and health products for human consumption.

Silver Nanoparticles Genotoxicity on Normal Human Melanocyte Cell Line (HBF-4 cells).

Nanotechnology has been rapidly used in diverse consumer products in both medical and industrial fields; which has raised various concerns. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials due to their excellent broad-spectrum antibacterial properties. However the studies on their cyto- and geno-toxic effects are scarce. The present study aim to evaluate the DNA damaging effects of AgNPs and estimate the release of intracellular reactive oxygen species (ROS) on HBF-4 normal human melanocyte cells. The study was conducted on HBF-4 cells, they will be treated with AgNPs at different levels (0.01 ,0.1,1, 10 and 100 μg/mL), LC50 was determined using MTT viability assay. Cells were then divided into two groups: the first was treated with the LC50 dose and the second was treated with 0.1 LC50 dose. DNA fragmentation assay using diphenylamine (DPA) technique and Intracellular ROS assay were measured and compared to control group. Significant increases in DNA damage and release of intracellular ROS were observed on treated HBF-4 cells with LC50 and its 1/10 LC50 value in comparison to the control group. Conclusion: silver nanoparticles can induce oxidative stress and DNA damage in normal human melanocyte cells with a potential carcinogenic effect to be considered. Further investigations form their genetic alterations mechanisms are required.

Glaesserella parasuis induces inflammatory response in 3D4/21 cells through activation of NLRP3 inflammasome signaling pathway via ROS

Glaesserella parasuis (G. parasuis) is an important pathogenic bacterium that can cause Glässer’s disease, and it has resulted in tremendous economic losses to the global swine industry. The intensive pulmonary inflammatory response caused by G. parasuis infection is the main cause of lung injury and death in pigs. However, the exact mechanism by which it causes severe pulmonary inflammation is not fully understood yet. In this study, severe pneumonia was observed in piglets infected with G. parasuis; and an infection cell model was established using porcine alveolar macrophages cell line 3D4/21, which was determined to be susceptible to G. parasuis infection in vitro. G. parasuis infection of 3D4/21 cells induced upregulation of proinflammatory cytokines TNF-α, IL-1β, IL-18 and production of intracellular reactive oxygen species (ROS). The expression of IL-1β related to activation of the NLRP3 inflammasome signaling pathway, which had not been shown before in G. parasuis infection. Furthermore, it was first found that release of intracellular ROS, which was mediated by NADPH oxidase in 3D4/21 cells, was found crucial for the activation of the NLRP3 signaling pathway and promoted the expression of proinflammatory cytokines, such as TNF-α and IL-1. In general, this study explored the specific mechanism of severe pulmonary inflammation caused by G. parasuis infection, and provides a foundation for further elucidating the pathogenic mechanism of G. parasuis.

Biological Assessment of Laser-Synthesized Silicon Nanoparticles Effect in Two-Photon Photodynamic Therapy on Breast Cancer MCF-7 Cells.

Driven by their distinctive physiological activities, biological properties and unique theranostic modalities, silicon nanoparticles (SiNPs) are one of the promising materials for the development of novel multifunctional nanoplatforms for biomedical applications. In this work, we assessed the possibility to use laser-synthesized Si NPs as photosensitizers in two-photon excited photodynamic therapy (TPE-PDT) modality. Herein, we used an easy strategy to synthesize ultraclean and monodispersed SiNPs using laser ablation and fragmentation sequences of silicon wafer in aqueous solution, which prevent any specific purification step. Structural analysis revealed the spherical shape of the nanoparticles with a narrow size distribution centered at the mean size diameter of 62 nm ± 0.42 nm, while the negative surface charge of −40 ± 0.3 mV ensured a great stability without sedimentation over a long period of time. In vitro studies on human cancer cell lines (breast and liver) and healthy cells revealed their low cytotoxicity without any light stimulus and their therapeutic potential under TPE-PDT mode at 900 nm with a promising cell death of 45% in case of MCF-7 breast cancer cells, as a consequence of intracellular reactive oxygen species release. Their luminescence emission inside the cells was clearly observed at UV-Vis region. Compared to Si nanoparticles synthesized via chemical routes, which are often linked to additional modules with photochemical and photobiological properties to boost photodynamic effect, laser-synthesized SiNPs exhibit promising intrinsic therapeutic and imaging properties to develop advanced strategy in nanomedicine field.

The curcuminoid, EF-24, reduces cisplatin-mediated reactive oxygen species in zebrafish inner ear auditory and vestibular tissues

Cisplatin is a widely used chemotherapy drug that can damage auditory and vestibular tissue and cause hearing and balance loss through the intracellular release of reactive oxygen species (ROS). Curcumin has anticancer efficacy and can also counteract cisplatin’s damaging effect against sensory tissue by scavenging intracellular ROS, but curcumin’s applicability is limited due to its low bioavailability. EF-24 is a synthetic curcumin analog that is more bioavailable than curcumin and can target cancer, but its effects against cisplatin-mediated ROS in auditory and vestibular tissue is currently unknown. In this study, we employed a novel zebrafish inner ear tissue culture system to determine if EF-24 counteracted cisplatin-mediated ROS release in two sensory endorgans, the saccule and the utricle. The zebrafish saccule is associated with auditory function and the utricle with vestibular function. Trimmed endorgans were placed in tissue culture media with a fluorescent reactive oxygen species indicator dye, and intracellular ROS release was measured using a spectrophotometer. We found that cisplatin treatment significantly increased ROS compared to controls, but that EF-24 treatment did not alter or even decreased ROS. Importantly, when equimolar cisplatin and EF-24 treatments are combined, ROS did not increase compared to controls. This suggests that EF-24 may be able to prevent intracellular ROS caused by cisplatin treatment in inner ear tissue.

Blood clotting and traumatic injury with shock mediates complement-dependent neutrophil priming for extracellular ROS, ROS-dependent organ injury and coagulopathy.

Polymorphonuclear (PMN) leucocytes participate in acute inflammatory pathologies such as acute respiratory distress syndrome (ARDS) following traumatic injury and shock, which also activates the coagulation system systemically. Trauma can prime the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex for an enhanced respiratory burst, but the relative role of various priming agents in this process remains incompletely understood. We therefore set out to identify mediators of PMN priming during coagulation and trauma-shock and determine whether PMN reactive oxygen species (ROS) generated in this manner could influence organ injury and coagulation. Initial experiments demonstrated that PMN are primed for predominantly extracellular ROS production by products of coagulation, which was abrogated by CD88/C5a receptor(C5aR) inhibition. The importance of this was highlighted further by demonstrating that known PMN priming agents result in fractionally different amounts of extracellular versus intracellular ROS release depending on the agent used. Plasma from trauma patients in haemodynamic shock (n=10) also primed PMN for extracellular ROS in a C5a-dependent manner, which correlated with both complement alternative pathway activation and thrombin generation. Furthermore, PMN primed by preincubation with products of blood coagulation directly caused loss of endothelial barrier function in vitro that was abrogated by C5aR blockade or NADPH oxidase inhibition. Finally, we show in a murine model of trauma-shock that p47phox knock-out (KO) mice with PMN incapable of generating ROS were protected from inflammatory end-organ injury and activated protein C-mediated coagulopathy. In summary, we demonstrate that trauma-shock and coagulation primes PMN for predominantly extracellular ROS production in a C5a-dependent manner that contributes to endothelial barrier loss and organ injury, and potentially enhances traumatic coagulopathy.

Targeting biofilm inhibition using Quercetin – Interaction with bacterial cell membrane and ROS mediated biofilm control

Background: Quercetin is an active nutraceutical ingredient widely distributed in foods, vegetables, fruits, and more. Quercetin is a versatile functional food with extensive protective effects against many infectious and degenerative diseases due to their antioxidant activities. Apsergillus niger is a filamentous fungus and the most abundant mold found in the environment. This fungus has been the source of several bioactive compounds and industrial enzymes through biotransformation.Aim: In this report we emphasized the potential of Aspergillus species for the selective conversion of rutin to quercetin, which involved stereoselective and regiospecific reactions with enhanced production and minimization of the formation of toxic wastes. This fungal microbe was able to transform the complex structure of rutin to quercetin with remarkable catalytic activity for the reaction with high product yield. The quercetin produced demonstrated the ability to inhibit biofilm formation and eradicate established biofilm involving the production of reactive oxygen species (ROS) indicative of membrane activity. These results suggest quercetin may have implications in biofilm control targeting reactive oxygen species as a novel therapeutic strategy.Methods: Quercetin was synthesized by microbial biotransformation recruiting Aspergillus niger. The synthesis of quercetin was compared with the chemical process. Furthermore, the quercetin produced by the biotransformation process was characterized by high performance thin layer liquid chromatography. The quercetin produced was assessed for biological activities. The antimicrobial activity, hemolytic activity, inhibition of biofilm by crystal violet staining, and cell viability by confocal laser scanning microscope was assessed. The membrane interaction effect and oxidant scavenging effect by DPPH, Intracellular ROS release, and lipid peroxidation was measured.Results: Quercetin produced by microbial transformation demonstrated antimicrobial activity against S. aureus by effectively inhibiting the growth and dispersion of preformed biofilms. Quercetin demonstrated a significant free radical scavenging activity and significant inhibition of lipid peroxidation. Significant release of reactive oxygen species was observed in bacterial cells.Conclusion: In conclusion, the bio transformed quercetin exhibited disruptive potential of biofilm formation by preventing cell surface attachment and biofilm growth. Therefore, it can be suggested that the major public health benefits could be achieved by substantially increasing the consumption of quercetin rich foods.

Effect of ethnomedicinal extracts used for wound healing on cellular migration and intracellular reactive oxygen species release in SC-1 fibroblasts

The inhibition of reactive oxygen species (ROS) and the migration of fibroblasts are key processes involved in wound healing. In this study, the ability of aqueous ethnomedicinal plant extracts prepared from Aspilia africana CD Adams, Boerhavia diffusa L. and Erythrina senegalensis DC. to mediate fibroblast migration and ROS release was determined. Phytochemical composition was assessed using thin-layer chromatography (TLC), whereas phytochemical markers were detected using ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC-TOF-MS). Sulforhodamine B staining and morphological examination via microscopy was conducted to determine cytotoxic effects on SC-1 fibroblasts. The effect on AAPH-induced oxidative stress was assessed by measuring ROS release using dichlorofluorescein diacetate activation. The scratch wound assay was used to estimate the rate of cellular migration. Alkaloids, flavonoids and phenols were detected in all three extracts using TLC, whilst UPLC-TOF-MS revealed the presence of neobavaisoflavone in E. senegalensis. None of the extracts was cytotoxic to the SC-1 cells at the highest in-well concentration tested (100 μg/mL). E. senegalensis extract reduced intracellular ROS and cellular migration by 35% and 32.5%, respectively. Although these plant extracts have the potential to minimise oxidation, they do not facilitate fibroblast migration. Further investigation into their mechanism of wound healing is required.

Contradictory effects of chemical filters in UV/ROS-stressed human keratinocyte and fibroblast cells.

Chemical UV-filters are frequently applied as active ingredients in sunscreen to protect from detrimental effects of UV radiation. Regardless, many of these compounds are not well characterized concerning their capacity to counteract UV induced reactive oxygen species (ROS). Intracellular ROS release is an early event upon UV exposure and a crucial trigger of reaction cascades that may provoke adverse effects both in short- and long-term. We report a strategy to assess the capacity of UV-filters (ecamsule, oxybenzone and menthyl anthranilate) to counteract UVA/UVB stress in the human keratinocyte HaCaT and the wildtype Fibs E6/E7 fibroblast cell lines. The reduction of ROS levels was taken as primary endpoint. The effect of treatment on the cells' metabolic activity was analyzed as an indicator of viability post-treatment, to investigate potential immediate and late (photo)toxicity. Additionally, the compounds' antioxidative capacity was investigated using an azo-based radical generator. Established antioxidants, quercetin and N-acetylcysteine, were used as controls. Data showed remarkable differences in the mode of action of the chemical UV-filters, ranging from protective to pro-oxidative properties, indicating the need for more detailed mode of action-based investigations. Certainly, additional consideration and evaluation will be necessary to further extrapolate these in vitro data for the assessment of in vivo exposure situations. However, the presented approach enables parallel investigations of photoprotective and phototoxic effects of UV-filters, and thus can complement and extent existing in vitro testing strategies.

Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated via mTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.

Modulation of Host Cellular Pathways by HBV-Induced ROS in Modulating Autophagy and Inflammation in Hepatic Cells

Background and Aim: Hepatitis B virus, a ds-DNA Virus, is one of the major risk for the development of chronic liver disease. HBV modulates several cellular pathways for its own survival and replication in the host cells. Reactive oxygen species (ROS) that are produced in the host cells during HBV infection, was considered to inhibit the cellular antioxidants and SIRT-1 expression. However, the role for HBV-induced ROS in modulating autophagy, proliferation and viral replication is not known. Hence, we hypothesized that HBV might modulate host factors through ROS in hepatic cells. Methods: HepG2.2.15 cells (Hep G2 cells expressing HBV genome stably) were treated with the N-acetyl cysteine (NAC, 20 μm) and Sirt-1 inhibitor Butyrate (5 mM) overnight. Western blotting was performed for the proteins SIRT-1, HBx, p-akt, akt, p-mTOR, mTOR, Beclin-1, LC-3, ATG5, COX-2, iNOS and β-actin using specific primary and secondary antibodies. H2-DCFDA was employed to analyse the intracellular release of ROS. Autophagy assay was done using the Cyto- ID autophagy detection kit. ELISA was used to determine HBsAg and real-time RT PCR was done to quantitate HBV-DNA. Results: HBV infection increased the ROS, COX-2, iNOS expression and formation of autophagic bodies in Hep G2.2.1.5 cells. Addition of NAC inhabited the inflammatory markers, autophagy and ROS production. Further, Western blot results showed that SIRT-1 beclin-1, LC3-II and ATG-5 expression were induced by ROS, which was inhibited by incubating cells with NAC, butyrate or SIRT-1 siRNA. Inhibition of the SIRT-1 expression by butyrate or SIRT-1 si-RNA inhibited the viral replication and HBV-induced autophagy. Conclusion: In this study, the data showed that HBV infection induces autophagy and inflammatory proteins via ROS production, which can be inhibited by butyrate or NAC treatment. It may be possible that butyrate might be considered for possible therapeutic applications. The authors have none to declare.

The effect of phosphatidylinositol-3 kinase inhibition on matrix metalloproteinase-9 and reactive oxygen species release from chronic obstructive pulmonary disease neutrophils

BackgroundChronic Obstructive Pulmonary Disease (COPD) is characterised by increased neutrophilic inflammation. A potential novel anti-inflammatory target in COPD is phosphatidylinositol-3 kinase (PI3 kinase), which targets neutrophil function. This study evaluated the effects of selective PI3Kδ inhibition on COPD blood and sputum neutrophils both in the stable state and during exacerbations. MethodsBlood and sputum neutrophils from stable and exacerbating COPD patients were cultured with the corticosteroid dexamethasone, a pan PI3 kinase inhibitor (ZSTK474), a δ selective PI3 kinase inhibitor (GSK045) and a p38 mitogen activated protein (MAP) kinase inhibitor (BIRB 796); matrix metalloproteinase (MMP)-9 and reactive oxygen species (ROS) release were analysed. ResultsPI3Kδ inhibition significantly reduced MMP-9, intracellular ROS and extracellular ROS release from blood neutrophils (45.6%, 30.1% and 47.4% respectively; p<0.05) and intracellular ROS release from sputum neutrophils (16.6%; p<0.05) in stable patients. PI3Kδ selective inhibition significantly reduced stimulated MMP-9 (36.4%; p<0.05) and unstimulated and stimulated ROS release (12.6 and 26.7%; p<0.05) from blood neutrophils from exacerbating patients. The effects of the p38 MAP kinase inhibitor and dexamethasone in these experiments were generally lower than PI3Kδ inhibition. ConclusionPI3Kδ selective inhibition is a potential strategy for targeting glucocorticoid insensitive MMP-9 and ROS secretion from COPD neutrophils, both in the stable state and during exacerbations.

TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.

Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax

Catalpol, an iridoid glucoside found in the root of Rehmannia glutinosa Libosch, has been demonstrated to reduce apoptosis in neuronal cell lines. Recent data suggests that catalpol also exerts anti-apoptotic effects on other cell types. The aim of the present study was to investigate whether catalpol protects against hydrogen peroxide (H 2O 2) induced apoptosis in human umbilical vein endothelial cells (HUVECs). Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and by assessment of caspase-3 activity. The level of intracellular reactive oxygen species was quantified by 2′, 7′-dichlorofluorescein diacetate assay. Expression of Akt, Bad, Bcl-2 and Bax mRNA and protein was determined by real-time semiquantitative reverse transcription-polymerase chain reaction and Western blotting. Apoptosis in HUVECs was associated with increased Bax, decreased Bcl-2 activity and inactivated phosphorylation of Akt and Bad after 24 h of H 2O 2 exposure. Pre-treatment of HUVECs with catalpol significantly reduced H 2O 2-induced intracellular reactive oxygen species release. Catalpol not only increased the expression of Bcl-2, while decreasing Bax expression, but also induced Akt activation and Bad phosphorylation, and ultimately reduced H 2O 2-induced apoptosis. The protective effects of catalpol were partially inhibited by the phosphatidylinositol 3-kinase (PI3K) antagonist wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Taken together, these results suggest that pre-treatment of HUVECs with catalpol can block H 2O 2-induced apoptosis, and that the underlying mechanism involves reactive oxygen species scavenging, activation of the PI3K/Akt–Bad signaling pathway and increased Bcl-2 and decreased Bax expression.