Protective effects of peoniflorin against hydrogen peroxide-induced oxidative stress in human umbilical vein endothelial cells

Peoniflorin (PF), extracted from the root of Paeonia lactiflora Pall., has been reported to have anti-inflammation and antioxidant effects in several animal models. Herein, we investigated the protective effects of PF against hydrogen peroxide (H(2)O(2))-induced oxidative damage in human umbilical vein endothelial cells (HUVECs). HUVECs were treated by H(2)O(2) (240?μmol/L) with or without PF. PF significantly increased the percent cell viability of HUVECs injured by H(2)O(2) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. By flow cytometric analysis, PF markedly attenuated H(2)O(2)-induced apoptosis and intracellular reactive oxygen species production. In addition, PF also displayed a dose-dependent reduction of lactate dehydrogenase leakage, malondialdehyde formation, and caspase-3 proteolytic activities in H(2)O(2)-treated cells, which was accompanied with a restoration of the activities of endogenous antioxidants, including total superoxide dismutase and glutathione peroxidase. Finally, Western blot data revealed that H(2)O(2) upregulated phosphorylation of extracellular signal-regulated kinase 1/2 in HUVECs, which was almost completely reversed by PF. Taken together, our data provide the first evidence that PF has a protective ability against oxidative damage in HUVECs. PF may be a candidate medicine for the treatment of vascular diseases associated with oxidative stress.     

Phosphocreatine protects against LPS-induced human umbilical vein endothelial cell apoptosis by regulating mitochondrial oxidative phosphorylation

Phosphocreatine (PCr) is an exogenous energy substance, which provides phosphate groups for adenosine triphosphate (ATP) cycle and promotes energy metabolism in cells. However, it is still unclear whether PCr has influenced on mitochondrial energy metabolism as well as oxidative phosphorylation (OXPHO) in previous studies. Therefore, the aim of the present study was to investigate the regulation of PCr on lipopolsaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs) and mitochondrial OXPHO pathway. PCr protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c (Cyt C), Ca²⁺, reactive oxygen species and subsequent activation of caspases, and increasing Bcl₂ expression, while suppressing Bax expression. More importantly, PCr significantly improved mitochondrial swelling and membrane potential, enhanced the activities of ATP synthase and mitochondrial creatine kinase (CK_mt) in creatine shuttle, influenced on respiratory chain enzymes, respiratory control ratio, phosphorus/oxygen ratio and ATP production of OXPHO. Above PCr-mediated mitochondrial events were effectively more favorable to reduced form of flavin adenine dinucleotide (FADH₂) pathway than reduced form of nicotinamide-adenine dinucleotid pathway in the mitochondrial respiratory chain. Our results revealed that PCr protects against LPS-induced HUVECs apoptosis, which probably related to stabilization of intracellular energy metabolism, especially for FADH₂ pathway in mitochondrial respiratory chain, ATP synthase and CK_mt. Our findings suggest that PCr may play a certain role in the treatment of atherosclerosis via protecting endothelial cell function.     

Salidroside protects retinal endothelial cells against hydrogen peroxide-induced injury via modulating oxidative status and apoptosis

Oxidative stress can cause injury in retinal endothelial cells. Salidroside is a strong antioxidative and cytoprotective supplement in Chinese traditional medicine. In this study, we investigated the effects of salidroside on H₂O₂-induced primary retinal endothelial cells injury. Salidroside decreased H₂O₂-induced cell death, and efficiently suppressed cellular ROS production, malondialdehyde generation, and cell apoptosis induced by H₂O₂ treatment. Salidroside induced the intracellular mRNA expression, protein expression, and enzymatic activities of catalase and Mn-SOD and increased the ratio of Bcl2/Bax. Our results demonstrated that salidroside protected retinal endothelial cells against oxidative injury through increasing the Bcl2/Bax signaling pathway and activation of endogenous antioxidant enzymes. This finding presents salidroside as an attractive agent with potential to attenuate retinopathic diseases.     

Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress

In this paper, we evaluate the extent to which flavonoids in red wine (catechin, epicatechin, quercetin and procyanidins) protect against hydrogen peroxide-induced oxidative stress in Fao cells. When cells were exposed to H(2)O(2), malondialdehyde (MDA) levels, oxidized glutathione (GSSG) levels and lactate dehydrogenase (LDH) release increased, indicating membrane damage and oxidative stress. All the flavonoids studied, and in particular epicatechin and quercetin, protected the plasma membrane. Only procyanidins lowered MDA levels and LDH leakage, maintained a higher reduced/oxidized glutathione ratio, and increased catalase/superoxide dismutase and glutathione peroxidase/superoxide dismutase ratios, and glutathione reductase and glutathione transferase activities. These results show that the procyanidin mixture has a greater antioxidant effect than the individual flavonoids studied, probably due to its oligomer content and/or the additive/synergistic effect of its compounds. This suggests that the mixture of flavonoids found in wine has a greater effect than individual phenols, which may explain many of the healthy effects attributed to wine.     

Exogenous hydrogen sulfide delays the senescence of human umbilical vein endothelial cells by lessening oxidative stress

The present study was aimed to investigate the effect of pretreatment with hydrogen sulfide (H2S) on human umbilical vein endothelial cells (HUVECs) senescence and the underlying mechanism. Cultured HUVECs at twelfth and fourth passages were taken as old and young groups, respectively. Sodium hydrosulfide (NaHS, donor of H2S) group was treated with NaHS from fourth to twelfth passage. The cell senescence was determined by senescence-associated β-galactosidase (SA β-gal) staining. DAPI fluorescent dye was used to detect cellular apoptosis. Western blot was used to analyze the expression levels of xanthine oxidase (XOD), manganese-superoxide dismutase (Mn-SOD) and the subunits p67(phox) of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the HUVECs. Colorimetric method was used to detect SOD activity and cellular hydrogen peroxide (H2O2) level. The results showed that, compared with young group, the old group exhibited higher SA β-gal positive rate and cellular apoptosis, while NaHS pretreatment decreased SA β-gal positive rate and cellular apoptosis. Compared with the young group, the old group showed increased expression levels of XOD and p67(phox), as well as lower Mn-SOD expression level. With the pretreatment of NaHS, the up-regulations of XOD and p67(phox) levels and down-regulation of Mn-SOD level were inhibited. Compared with the young group, the old group showed lower SOD activity and higher H2O2 level, whereas NaHS pretreatment reversed the changes of SOD activity and H2O2 level. These results suggest that H2S delays senescence of HUVECs through lessening oxidative stress.     

Hsa-miRNA-29a protects against high glucose-induced damage in human umbilical vein endothelial cells

Sustained exposure to high glucose (HG) results in dysfunction of vascular endothelial cells. Hence, diabetic patients often suffer from secondary vascular damages, such as vascular sclerosis and thrombogenesis, which may eventually cause cardiovascular problems. Thus, elucidating how HG results in vascular endothelial cell damage and finding an approach for prevention are important to prevent and treat vascular damages in diabetic patients. In the current study, we first showed that 72-hour exposure to HG-decreased hsa-miRNA-29a and increased the expression of Bcl-2 associated X protein (Bax), which subsequently inhibited Bcl-2 and promoted the expression of apoptotic protease activating factor-1 and activation of caspase-3, thus directly triggering the mitochondrial apoptotic pathway in human umbilical vein endothelial cells (HUVECs). Study of the underlying mechanism showed that hsa-miRNA-29a/Bax plays an essential role in the decreased proliferation and increased apoptosis of HUVECs induced by HG, and overexpression of hsa-miRNA-29a effectively inhibits HG-induced apoptosis and restores the proliferation and tube formation of HUVECs exposed to HG by inhibiting its target gene Bax. In short, our study demonstrates that hsa-miRNA-29a is a promising target for the prevention and treatment of vascular injury in diabetic patients.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

An aqueous extract of Zingiber officinale Roscoe protects mouse primary hepatic cells against hydrogen peroxide-induced oxidative stress

Hepatocytes exposed to an oxidative stressor such as hydrogen peroxide (H₂O₂) are potentially sensitized to cell death; thus, reactive oxygen species (ROS) are considered to be critical mediators of liver damage. Zingiber officinale Roscoe (ZO), also known as ginger, is cultivated commercially in China, India, Korea, and other parts of the world. In addition, it is used as a spice and flavoring agent and is also purported to possess a number of medicinal properties. In the present study, we examined the protective effect of ZO against cell damage caused by H₂O₂-induced oxidative stress. ZO reduced H₂O₂-induced apoptotic signals and the levels of intracellular ROS. ZO pretreatment also increased the phosphorylation of c-Jun, and JNK kinase. The expression of heme oxygenase-1 (HO-1) and heat shock protein 72 (HSP72) were increased by ZO pretreatment more than H₂O₂ treatment. In addition, siRNA-mediated knockdown of HO-1 and HSP72 decreased protective effect of ZO pretreatment. Our data suggest that ZO decreases ROS levels and the expressions of HO-1 and HSP72 are involved in the hepatocyte protective function of ZO against H₂O₂.     

Thymoquinone protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis

Oxidative stress in retinal pigment epithelium (RPE) cells may contribute to the progression of age-related macular degeneration. Thymoquinone (TQ), an active component derived from Nigella sativa, possesses antioxidative effect. However, the role of TQ in RPE cells under oxidative stress condition remains unclear. The present study aimed to examine the protective effect of TQ against hydrogen peroxide (H₂O₂)-induced oxidative stress in human RPE cells. Our results showed that TQ improved the cell viability and apoptosis in H₂O₂-induced ARPE cells. We also found that the levels of reactive oxygen species and malondialdehyde induced by H₂O₂ were reduced after the pretreatment of TQ. In addition, the inhibitory effect of H₂O₂ on the glutathione (GSH) level and superoxide dismutase activity was markedly attenuated by TQ pretreatment. Moreover, TQ enhanced the activation of Nrf2/heme oxygenase 1 (HO-1) signaling pathway in H₂O₂-induced ARPE cells. Knockdown of Nrf2 abolished the protective effect of TQ on H₂O₂-induced oxidative damage. These results suggested that TQ protected ARPE cells from H₂O₂-induced oxidative stress and apoptosis via the Nrf2/HO-1 signaling pathway.     

Water-soluble fractions from defatted sesame seeds protect human neuroblast cells against peroxyl radicals and hydrogen peroxide-induced oxidative stress

Oxidative stress is involved in the development of aging-related diseases, such as neurodegenerative diseases. Dietary antioxidants that can protect neuronal cells from oxidative damage play an important role in preventing such diseases. Previously, we reported that water-soluble fractions purified from defatted sesame seed flour exhibit good antioxidant activity in vitro. In the present study, we investigated the protective effects of white and gold sesame seed water-soluble fractions (WS-wsf and GS-wsf, respectively) against 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H₂O₂) induced oxidative stress in human neuroblast SH-SY5Y cells. Pretreatment with WS-wsf and GS-wsf did not protect cells against AAPH-induced cytotoxicity, while simultaneous co-treatment with AAPH significantly improved cell viability and inhibited membrane lipid peroxidation. These results suggest that WS-wsf and GS-wsf protect cells from AAPH-induced extracellular oxidative damage via direct scavenging of peroxyl radicals. When oxidative stress was induced by H₂O₂, pretreatment WS-wsf and GS-wsf significantly enhanced cell viability. These results suggest that in addition to radical scavenging, WS-wsf and GS-wsf enhance cellular resistance to intracellular oxidative stress by activation of the Nrf-2/ARE pathway as confirmed by the increased Nrf2 protein level in the nucleus and increased heme oxygenase 1 (HO-1) mRNA expression. The roles of ferulic and vanillic acids as bioactive antioxidants in these fractions were also confirmed. In conclusion, our results indicated that WS-wsf and GS-wsf, which showed antioxidant activity in vitro, are also efficient antioxidants in a cell system protecting SH-SY5Y cells against both extracellular and intracellular oxidative stress.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H₂O₂) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H₂O₂-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H₂O₂-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H₂O₂, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca^{2 +}) chelator, overexpression of Homer 1a had no significant effects on H₂O₂-induced oxidative stress. These results suggest that Homer 1a has protective effects against H₂O₂-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca^{2 +} homeostasis.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.     

Overexpression of human metallothionein-III prevents hydrogen peroxide-induced oxidative stress in human fibroblasts

Incorporation of inter- or intramolecular covalent cross-links into food proteins with microbial transglutaminase (MTG) improves the physical and textural properties of many food proteins, such as tofu, boiled fish paste, and sausage. By using nuclear magnetic resonance, we have shown that the residues exhibiting relatively high flexibility in MTG are localized in the N-terminal region; however, the N-terminal region influences the microenvironment of the active site. These results suggest that the N-terminal region is not of primary importance for the global fold, but influences the substrate binding. Therefore, in order to increase the transglutaminase activity, the N-terminal residues were chosen as candidates for site-directed replacement and deletion. We obtained several mutants with higher activity, del1-2, del1-3, and S2R. We propose a strategy for enzyme engineering targeted toward flexible regions involved in the enzymatic activity. In addition, we also briefly describe how the number of glutamine residues in a substrate protein can be increased by mixing more than two kinds of TGases with different substrate specificities.     

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

Acetaminophen protects human erythrocytes against oxidative stress

Acetaminophen protects human erythrocytes against various modes of oxidative stress. Protection against ozone-induced damage can be explained by a direct scavenging reaction between the drug and ozone. With t-butylhydroperoxide acetaminophen appeared to be an effective scavenger of radicals, generated in secondary reactions. The protection by acetaminophen against t-butylhydroperoxide- and hydrogen peroxide-induced lipid peroxidation and K+-leakage can be explained along these lines. In all cases the protective effect of acetaminophen was attended with covalent binding of acetaminophen to membrane proteins.     

The stingless bee honey protects against hydrogen peroxide-induced oxidative damage and lipopolysaccharide-induced inflammation in vitro

Oxidative stress, DNA damage, and unresolved inflammation are the predisposing factors of many chronic and degenerative diseases, including cancer. Stingless bee honey (SBH) is recognized to have high medicinal value by traditional medicine practitioners and has been used to treat various illnesses traditionally. This study aimed to determine the antioxidant, anti-inflammatory, and genoprotective effects of SBH by using in vitro cell culture models. The sugar content, total phenolic content, radical scavenging activity, and ferric reducing antioxidant power (FRAP) of SBH were determined in this study. Then, the protective effect of SBH against hydrogen peroxide (H₂O₂)-induced cell death and DNA damage was studied by using WIL2-NS human lymphoblastoid cell line, while the lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages cell line was used to study the anti-inflammatory effects of SBH. Results from this present study showed that the major sugar contents of SBH were fructose (19.39 + 0.01%) and glucose (14.03 ± 0.03%). Besides, the total phenolic content, the radical scavenging activity, and the FRAP value of SBH were 15.38 ± 0.02 mg GAE/100 g of honey, 34.04 ± 0.21%, and 206.77 + 1.76 μM AAE/100 g honey respectively. Pretreatment with SBH protected WIL2-NS cells from H₂O₂-induced cell death and DNA damage (p < 0.001). Moreover, SBH was also able to attenuate the production of nitric oxide by inhibiting the expression of inducible nitric oxide synthase in LPS-induced RAW 264.7 cells (p < 0.001). In conclusion, SBH is rich in total phenolic content and possesses strong antioxidant, anti-inflammatory, and genoprotective properties. Our current findings suggest that SBH might be useful in the prevention and treatment of many diseases caused by oxidative stress and inflammation assuming the observed effects are also achievable in vivo.     

Autotaxin protects microglial cells against oxidative stress

Oxidative stress occurs when antioxidant defenses are overwhelmed by oxygen-reactive species and can lead to cellular damage, as seen in several neurodegenerative disorders. Microglia are specialized cells in the central nervous system that act as the first and main form of active immune defense in the response to pathological events. Autotaxin (ATX) plays an important role in the modulation of critical cellular functions, through its enzymatic production of lysophosphatidic acid (LPA). In this study, we investigated the potential role of ATX in the response of microglial cells to oxidative stress. We show that treatment of a microglial BV2 cell line with hydrogen peroxide (H(2)O(2)) stimulates ATX expression and LPA production. Stable overexpression of ATX inhibits microglial activation (CD11b expression) and protects against H(2)O(2)-treatment-induced cellular damage. This protective effect of ATX was partially reduced in the presence of the LPA-receptor antagonist Ki16425. ATX overexpression was also associated with a reduction in intracellular ROS formation, carbonylated protein accumulation, proteasomal activity, and catalase expression. Our results suggest that up-regulation of ATX expression in microglia could be a mechanism for protection against oxidative stress, thereby reducing inflammation in the nervous system.     

Protective effect of whey protein hydrolysates against hydrogen peroxide-induced oxidative stress on PC12 cells

The protective effect of whey protein hydrolysates (WPHs) against H₂O₂-induced oxidative damage on rat pheochromocytoma line 12 (PC12) cells was studied. Whey protein was hydrolyzed by pepsin and trypsin and purified by macrospore absorption resins. PC12 cells were pretreated with WPHs (from 369 to 1,980?Da) at different concentrations for 2?h, then washed and incubated with 100?μM H₂O₂ in the presence of WPHs for another 24?h. With 100–400?μg WPH/ml the viable cells increased by 20–30?% when incubated with H₂O₂ suggesting that they may play a role as antioxidant in foods.