ROS Reduction Research Articles

Melatonin synergistically enhances protective effect of atorvastatin against gentamicin-induced nephrotoxicity in rat kidney.

The risk of serious side-effects such as nephrotoxicity is the principal limitation of gentamicin (GEN) therapeutic efficacy. Oxidative stress is considered to be an important mediator of GEN-induced nephrotoxicity. The present study was designed to evaluate the efficacy of the combination of melatonin (MT) plus atorvastatin (ATO) against GEN-induced nephrotoxicity in rats. We utilized 30 male Wistar albino rats allocated in 5 groups, each containing 6 rats: control, GEN (100 mg/kg/day), ATO (10 mg/kg/day) + GEN, MT (20 mg/kg/day) + GEN, and ATO (10 mg/kg/day) plus MT (20 mg/kg/day) + GEN. Kidney weight, serum creatinine and urea concentration, renal ROS, MDA, GSH levels, SOD, and CAT activity were determined. GEN-induced nephrotoxicity was evidenced by marked elevations in serum urea and creatinine, kidney weight, renal ROS, and MDA levels and reduction in renal GSH level, SOD and CAT activity. MT pretreatment significantly lowered the elevated serum creatinine concentration, kidney weight, renal ROS and MDA levels. However ATO could not reduce these parameters, but similarly to MT, it was able to enhance the renal GSH level, CAT and SOD activity. In addition, a combination therapy of MT plus ATO enhanced the beneficial effects of ATO, while not changing the effects of MT effects or even improving them. The present study indicates that a combination therapy of MT plus ATO can attenuate renal injury in rats treated with GEN, possibly by reducing oxidative stress, and it seems that MT can enhance the beneficial effects of ATO.

Changes in Oxidative Stress and Vascular Reactivity of Thoracic and Abdominal Rat Aorta with Different Periods of Exposure to Hyperbaric Oxygenation

Oxidative stress and the antioxidant defense always seek balance in aerobic systems. Therefore, when O2 is added to an aerobic system during hyperbaric oxygenation, oxidative stress is induced and the antioxidant defense is stimulated. Dose-response curves were established for rat aorta exposed to different agonists. The aorta were from control rats and animals with 3, 10 or 20 HBO sessions. Parameters of vascular reactivity, oxidative stress and antioxidant defense were measured. With hyperbaric oxygenation, vascular reactivity was modified by the altered interaction between oxidative stress and antioxidant defense. The result was a low contractile response, possibly caused by endothelium dependent or independent pathways. Since, acetylcholine did not modify the vascular response, HBO seems to act by endothelium independent pathways related to nitric oxide. Hyperbaric oxygen therapy (HBO) led to an over 60% decrease in all parameters of oxidative stress and antioxidant defense (enzymatic and non-enzymatic) of thoracic aorta, an effect that lasted during the 20 sessions of HBO. This result indicates a probable drastic reduction in ROS and NO production, which could be explained by the capacity of an excess of oxygen to eliminate certain products that are normally involved in chain reactions leading to oxidative stress.

0194 : Angiotensin II type 2 receptor reduces metabolic and vascular effects of type 1 diabetes in the mouse

The renin-angiotensin system has a key role in cardiovascular homeostasis, mainly through activation of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors. Although AT2R opposes the effects of AT1R, with vasodilator and antitrophic properties, its effect in diabetes remains debated. Thus we determined the role of AT2R in endothelium-mediated dilation in a mouse model of type 1 diabetes. Diabetes was induced in three-month-old mice by streptozotocin (150mg/kg, ip). After 45 days, mesenteric resistance arteries were isolated and mounted on a wire myograph to measure vascular reactivity. The absence of AT2R reduced glucose tolerance and body weight and increased STZ-induced hyperglycemia, hyperinsulinemia, creatiniemia, blood thromboxaneA2, PGF2alpha and isoprostane. AT2R, COX-2 and NADPHoxidase subunits (gp91phox and p22phox) gene expression were higher in arteries of diabetic mice than in control animals. COX-2 level and oxidative stress were also higher in mesenteric and renal arteries in AT2R-/- mice. Endothelium-dependent relaxation, reduced in diabetic mice arteries, was normalized after cycloxygenase-2 (COX-2) (CAY10404) blockade or ROS reduction with tempol plus catalase in both AT2R-/- and AT2R+/+ mice. AT2R-dependent dilation in isolated perfused mesenteric arteries was greater in diabetic mice than in control animals. Thus, in a model of type 1 diabetes AT2R reduces the severity of diabetes possibly through a reduction of the production of ROS and COX-2-derived vasoconstrictor agents.

EGFR inhibition protects cardiac damage and remodeling through attenuating oxidative stress in STZ-induced diabetic mouse model

Diabetes mellitus is strongly associated with cardiomyopathy. The underlying mechanisms for the development of diabetic cardiomyopathy are complex and not completely understood. Recent studies showed that epidermal growth factor receptors (EGFRs) are involved in diabetes-induced cardiac injury. However, the role of EGFR in the diabetic heart has yet to be confirmed. The aim of the present study is to further determine the role of EGRF in the pathogenesis of diabetic heart injury. The type 1 diabetic mice induced by streptozotocin were treated with EGFR inhibitors (AG1478 and 451) for 8weeks, respectively. It was observed that diabetes induced phospohorylation of EGFR and AKT, increased cardiac ROS levels, and ultimately led to cardiac remodeling including cardiac hypertrophy, disorganization, apoptosis, and fibrosis, while all these molecular and pathological alterations were attenuated by the treatment with EGFR inhibitors. In vitro, either pharmacological inhibition of EGFR/AKT or sh-RNA silencing of EGFR significantly inhibited high concentration glucose (HG)-induced ROS generation and subsequently cell apoptosis in both cardiac H9C2 cells and primary rat cardiomyocytes, respectively. The ROS reduction by EGFR inhibitor was associated with the decreased NADPH oxidase activity and expression in H9c2 cells. HG-induced cardiomyocyte injuries were also reduced by NAC, an inhibitor of ROS. This study provides evidence that EGFR has a key role in the pathogenesis of STZ-induced diabetic cardiac damage and remodeling via ROS generation, and suggests that EGFR may be a potential target in treating diabetic cardiomyopathy.

Hyaluronic acid regulates a key redox control factor Nrf2 via phosphorylation of Akt in bovine articular chondrocytes

One important pharmacological function of hyaluronic acid (HA) in chondrocytes is reduction of cellular superoxide generation and accumulation. Here we demonstrated a relationship between HA supplementation and accumulation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), which is a master transcription factor in cellular redox reactions, in cultured chondrocytes derived from bovine joint cartilage. In HA-treated chondrocytes, expression of Nrf2 and its downstream genes was upregulated. In HA-treated chondrocytes, Akt was phosphorylated, and inhibition of Akt activity or suppression of HA receptors CD44 and/or RHAMM with siRNAs prevented HA-mediated Nrf2 accumulation. Furthermore, Nrf2 siRNA inhibited the HA effect on antioxidant enzymes. These results show that HA might contribute to ROS reduction through Nrf2 regulation by activating Akt. Our study suggests a new mechanism for extracellular matrix (ECM)-mediated redox systems in chondrocytes.

Exercise Increases Markers of Spermatogenesis in Rats Selectively Bred for Low Running Capacity.

The oxidative stress effect of exercise training on testis function is under debate. In the present study we used a unique rat model system developed by artificial selection for low and high intrinsic running capacity (LCR and HCR, respectively) to evaluate the effects of exercise training on apoptosis and spermatogenesis in testis. Twenty-four 13-month-old male rats were assigned to four groups: control LCR (LCR-C), trained LCR (LCR-T), control HCR (HCR-C), and trained HCR (HCR-T). Ten key proteins connecting aerobic exercise capacity and general testes function were assessed, including those that are vital for mitochondrial biogenesis. The VO2 max of LCR-C group was about 30% lower than that of HCR-C rats, and the SIRT1 levels were also significantly lower than HCR-C. Twelve weeks of training significantly increased maximal oxygen consumption in LCR by nearly 40% whereas HCR remained unchanged. LCR-T had significantly higher levels of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1α), decreased levels of reactive oxygen species and increased acetylated p53 compared to LCR-C, while training produced no significant changes for these measures in HCR rats. BAX and Blc-2 were not different among all four groups. The levels of outer dense fibers -1 (Odf-1), a marker of spermatogenesis, increased in LCR-T rats, but decreased in HCR-TR rats. Moreover, exercise training increased the levels of lactate dehydrogenase C (LDHC) only in LCR rats. These data suggest that rats with low inborn exercise capacity can increase whole body oxygen consumption and running exercise capacity with endurance training and, in turn, increase spermatogenesis function via reduction in ROS and heightened activity of p53 in testes.

Abstract 2433: Metabolic reprogramming by an epigenetic mechanism in endocrine therapy resistance of breast cancer

Abstract Metabolic reprogramming is a major mechanism of fueling cancer cell growth and proliferation. However, how tumor metabolism is altered to confer therapeutic resistance is poorly understood. Gene expression profiling of tamoxifen sensitive and resistant breast cancer cells revealed that transition to the resistance was associated with a specific expression reprogramming of genes involved in glucose/energy metabolism, which was accompanied by aberration of specific epigenetic regulators such as the histone methyltransferase NSD2/MMSET/WHSC1. In cell and xenograft tumor models, NSD2 overexpression alone was sufficient to confer tamoxifen resistance. Analysis of clinical specimens indicates that NSD2 was a strong predictive factor for early relapse of tamoxifen therapy. NSD2 wild type, but not its methylase-defective mutant form, coordinately stimulated the expression and enzymatic activity of HK2, TIGAR and G6PD and strongly augmented the pentose phosphate pathway (PPP) production of NADPH for ROS reduction and survival of tamoxifen treated cells and tumors. Further mechanistic studies demonstrated that elevated NSD2 stimulates the metabolic gene expression through H3K36 methylation at the target chromatin. Pharmacological targeting aberrant NSD2 by DZNeP, an S-adenosylhomocysteine (AdoHcy) hydrolase inhibitor, effectively blocked the growth of tamoxifen-resistant tumor. The study reveals a novel mechanism of cancer metabolic reprogramming and suggests targeting the key metabolic re-programmer as a viable option for effective treatment of endocrine resistant tumors. Note: This abstract was not presented at the meeting. Citation Format: June X. Zou, Junjian Wang, Zhijian Duan, Hongwu Chen, Hsing-Jien Kung, Xinbin Chen, Leigh C. Murphy, Alexander Borowsky. Metabolic reprogramming by an epigenetic mechanism in endocrine therapy resistance of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2433. doi:10.1158/1538-7445.AM2014-2433

Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion.

The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.

Chemopreventive effects of standardized papaya leaf fraction on oxidatively stressed human liver cells

Extract from papaya leaves, a waste material from fruit farms in Malaysia was previously reported to possess remarkable antioxidative activities. In this study, papaya leaf extract was separated into fractions of different polarities [petroleum ether (PE), ethyl acetate (EA), n-butanol (NB) and water (W) fractions]. The aim of this research was to determine the most active fraction in terms of its chemopreventive effects towards oxidative stress and the chemical constituents involved. The cytoprotective nature of the papaya fractions was observed against t-BOOH-induced oxidative stress on HepG2 liver cell line. ROS assay indicated that only PE and EA effectively reduced the increment of radical due to the pro-oxidant, t-BOOH. Nevertheless, PE was a stronger ROS scavenger by demonstrating ROS reducing activity in a dose-dependent manner to the basal level. This fraction was also found to inhibit cell death caused by t-BOOH toxicity, attenuating lactate dehydrogenase enzyme leakage by more than 90% (p<0.05). In addition, gene expression of phase II antioxidant enzymes (hmox-1 and nqo-1) and their transcription factor (nrf-2) were shown to be upregulated upon PE treatment during a time-course study. A GC-MS fingerprint of the active fraction was subsequently obtained with standardization using the marker compound; α-tocopherol, a well known antioxidant. However, this pure compound was not as effective as its corresponding PE concentrations in ROS reduction. Hence, PE of papaya leaf extract was a strong antioxidant and cytoprotectant with tremendous potential to be harnessed into the next therapeutic remedy against oxidative stress of the liver.

SIRT5 desuccinylates and activates SOD1 to eliminate ROS

Cu/Zn superoxide dismutase (SOD1) is a key antioxidant enzyme. Deficiency of SOD1 is associated with various human diseases, including cancer. Here, we report that SOD1 is succinylated and that succinylation decreases its activity. SIRT5 binds to, desuccinylates and activates SOD1. SOD1-mediated ROS reduction is increased when SIRT5 is co-expressed. Furthermore, mutation of the SOD1 succinylation site inhibits the growth of lung tumor cells. These results reveal a novel post-translational regulation of SOD1 by means of succinylation and SIRT5-dependent desuccinylation, which is important for the growth of lung tumor cells.

Ethyl gallate attenuates acute lung injury through Nrf2 signaling

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is the clinical syndrome of persistent lung inflammation caused by various direct and indirect stimuli. Despite advances in the understanding of disease pathogenesis, few therapeutic have emerged for ALI/ARDS. Thus, in the present study we evaluated the therapeutic potential of ethyl gallate (EG), a plant flavanoid in the context of ALI using in vivo (BALB/c) and in vitro models (human monocytes). Our in vivo data supports the view that EG alleviates inflammatory condition in ALI as significant reduction in BALF neutrophils, ROS, proinflammatory cytokines and albumin levels were observed with the single i.p of EG post LPS exposure. Also, histochemical analysis of mice lung tissue demonstrated that EG restored LPS stimulated cellular influx inside the lung airspaces. Unraveling the mechanism of action, our RT-PCR and western blot analysis suggest that enhanced expression of HO-1 underlies the protective effect of EG on ROS level in mice lung tissue. Induction of HO-1 in turn appears to be mediated by Nrf2 nuclear translocation and consequent activation and ablation of Nrf2 activity through siRNA notably abrogated the EG induced protective effect in LPS induced human monocytes. Furthermore, our results indicate that EG generated moderate amounts of H2O2 could induce Nrf2 translocation in the in vitro systems. However, given the insignificant amount of H2O2 recorded in the injected material in the in vivo system, additional mechanism for EG action could not be excluded. Nevertheless our results highlight the protective role of EG in ALI and provide the novel insight into its usefulness as a therapeutic tool for the treatment of ALI.

Exogenous trehalose differentially modulate antioxidant defense system in wheat callus during water deficit and subsequent recovery

To elucidate the resistance mechanism of exogenous trehalose on water deficit further, we investigated the effect of exogenous trehalose (50 mM) in wheat callus during water deficit and subsequent recovery. Enhanced levels of endogenous trehalose were detected in calli exposed to water deficit (W) and trehalose (T) medium, moreover, W plus T treatment showed an additive effect. Water deficit elevated the accumulation of ROS (hydrogen peroxide and formation rate of O 2 .− ) and the endogenous MDA (Malonaldehyde), and resulted in the decrease of cell viability and biomass. Exogenous trehalose (TW) could alleviate the damage induced by water deficit, which was involved in the decrease of MDA and the generation of ROS, and resulted in elevating cell viability and biomass. Additionally, water deficit induced activity of antioxidative enzymes (Peroxidase, POD; Catalase, CAT; Glutathione reductase, GR). Content of AsA (Reduced ascorbate) was also increased by water deficit, while the content of GSH (Glutathione) showed the opposite effect. The combined effect of T and W treatment led to a higher activity of enzymatic antioxidants including SOD (Superoxide dismutase) and GR, and elevated the content of nonenzymatic antioxidants including AsA and GSH, but had a negative effect on enzymatic antioxidants including POD and CAT in comparison to the water deficit treatment alone. During recovery, calli treated by TW showed a greater reduction in ROS resulted in enhancing a higher cell viability and biomass. The scavenging mechanism of ROS by exogenous trehalose is mainly dependent on nonenzymatic antioxidants, especially AsA-GSH cycle, rather than enzymatic mechanisms and trehalose itself.

Abstract 19478: The Protective Effect of Beta3-Adrenergic Signaling Occurs via Differential Phosphorylation of nNOS in Cardiac Myocytes

Rationale: Stimulation of β3-adrenoreceptor (β3-AR) signaling improves left ventricular function via a nNOS/NO-dependent manner in hypertrophied and failing animal hearts. nNOS, which is expressed in cardiac myocytes, is regulated by modification of stimulatory phosphorylation residue Ser1412 and inhibitory residue Ser847. However, how differential phosphorylation of these residues contribute to cardiomyocyte β3-AR-mediated NOS activation and signaling has yet to be explored. Objective: The aim of this study is to identify the molecular mechanisms involved in β3-AR-dependent regulation of nNOS activation and its downstream signaling in cardiomyocytes in the setting of hypertrophy. Methods and Results: In neonatal rat ventricular cardiomyocytes isolated from 4 day old pups, 48 hours of ET-1or 72 hours of norepinephrine lead to increased cell size and β3-AR mRNA. In hypertrophied cells, specific β3-AR agonist BRL-37433 induced NOS activity, nNOS phosphorylation at site Ser1412, dephosphorylation of site Ser847, and ROS suppression. BRL-dependent NOS activity and ROS suppression were attenuated by the nNOS inhibitor L-VNIO. NOS activity was also attenuated in phosphomimetic mutants Ser1412A (constitutively dephosphorylated) and Ser847D (constitutively phosphorylated). However, BRL failed to reduce ROS in the presence of Ser847D. In addition, Akt inhibition decreased BRL-induced Ser1412 phosphorylation and NOS activity, while Gi/o blockade altered BRL-regulation of both phosphorylation sites, NOS activity, and ROS reduction. In vivo, BRL administration completely dephosphorylated Ser847 and moderately increased Ser1412 phosphorylation in mice hearts under pressure overload. Conclusion: Our data suggest that β3-AR regulates myocardial NOS activity and ROS via alterations in nNOS phosphorylation in hypertrophied myocytes and failing hearts. This is the first study to demonstrate a role for nNOS phosphorylation as a key factor in cardiomyocyte and β3-AR signaling. These results contribute significantly to our understanding the protective properties of β3-AR signaling during cardiac sympathetic overdrive, and will ultimately aid in drug discoveries that target molecular mechanisms to treat heart failure.

Modulation of Cardiac Na+ Channels by PKC and Mitochondria

<h3>Background</h3> Elevated NADH downregulates cardiac Na⁺ channel (Na_v1.5) current (I_Na) acutely to a magnitude seen in Brugada syndrome by activating protein kinase C (PKC) and inducing mitochondrial ROS overproduction. Here, we studied the mechanism whereby this kinase alters I_Na. <h3>Methods</h3> HEK293 cells transfected with human cardiac Na_v1.5 wild type (WT), S1503A, and S1503D were utilized for whole-cell patch-clamp recording. Isolated mouse cardiomyocytes were used to monitor mitochondrial reactive oxygen species (mitoROS) with MitoSOX^TM Red. <h3>Results</h3> The peak I_Na of mutants was similar to that of the WT channel. Application of NADH reduced I_Na in PKC site mutant S1503D (42 ± 2% of untreated S1503D, <i>P</i> <.01) but not S1503A (81 ± 6% of untreated S1503A, <i>P</i> >.05). Similar results were seen with phorbol 12-myristate 13-acetate on S1503D (37 ± 6% of untreated S1503D, <i>P</i> <.01) and S1503A (87 ± 11% of untreated S1503A, <i>P</i> >.05). Inducing mitochondrial ROS with antimycin A decreased I_Na of WT SCN5A but not of S1503A. Antimycin A's effect on I_Na was not blocked chelerythrine, implying PKC activation alone is insufficient to reduce I_Na. Because δV1-1 (a specific inhibitor for PKCδ) but not PKCα or ε inhibitors blocked mitochondrial ROS overproduction and reduction of I_Na induced by elevated NADH, it would appear that PKCδ activation is upstream of mitochondrial ROS and that, in addition to PKC phosphorylation of the channel, mitochondrial ROS is required for current reduction with NADH. <h3>Conclusions</h3> PKC regulates Na_v1.5 in two ways: indirectly through modification of mitochondrial ROS production and directly through channel phosphorylation at S1503. PKCδ activation induces mitochondrial ROS overproduction. The I_Na of S1503D alone was equivalent to wild type, suggesting PKC phosphorylation of the Na_v1.5 channel is essential for channel downregulation and I_Na reduction but not sufficient for the NADH effect.

Carbohydrate derivatives from the roots of Brassica rapa ssp. campestris and their effects on ROS production and glutamate-induced cell death in HT-22 cells

Phytochemical investigation of the roots of Brassica rapa ssp. campestris led to the isolation of three new carbohydrate derivatives, namely sucrose 3,3′,4′-triisovalerate (2), sucrose 6,3′,4′-triisovalerate (3), and ethanone-1-C-β-d-glucopyranoside (3,7-anhydro-1-deoxy-d-glycero-d-gulo-2-octulose, 6), along with four known carbohydrate derivatives, 2,6,3′,4′-tetraisovalerate (1), ethyl β-d-glucopyranoside (4), n-butyl β-d-fructofuranoside (5), and n-pentyl β-d-fructofuranoside (7), which were initially isolated from plants of the Brassica genus. Structures of the isolated compounds were established by spectroscopic analyses, including UV, IR, MS, and NMR. All of the isolated carbohydrate derivatives were evaluated to determine their effect on ROS production and glutamate-induced cell death in HT-22 cells. Compound 6 showed the most significant ROS reduction and a protective effect with IC50 values of 69.4±3.8μM and 4.96±0.32μM, respectively, which were equivalent to those of the positive control, Trolox.

Exogenous Calcium Improves Viability of Biocontrol Yeasts Under Heat Stress by Reducing ROS Accumulation and Oxidative Damage of Cellular Protein

In this article, we investigated the effect of exogenous calcium on improving viability of Debaryomyces hansenii and Pichia membranaefaciens under heat stress, and evaluated the role of calcium in reducing oxidant damage of proteins in the yeast cells. The results indicated that high concentration of exogenous calcium in culture medium was beneficial for enhancing the tolerance of the biocontrol yeasts to heat stress. The possible mechanism of calcium improving the viability of yeasts was attributed to enhancement of antioxidant enzyme activities, decrease in ROS accumulation and reduction of oxidative damage of intracellular protein in yeast cells under heat stress. D. hansenii is more sensitive to calcium as compared to P. membranaefaciens. Our results suggest that application of exogenous calcium combined with biocontrol yeasts is a practical approach for the control of postharvest disease in fruit.

Interleukin-19 (IL-19) Induces Heme Oxygenase-1 (HO-1) Expression and Decreases Reactive Oxygen Species in Human Vascular Smooth Muscle Cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.

Growth Inhibition by Caffeic Acid, One of the Phenolic Constituents of Honey, in HCT 15 Colon Cancer Cells

Previous work from our laboratory showed that the mechanism of crude-honey induced apoptosis in colon cancer cells. Since phenolic constituents of honey were attributed to its apoptosis-inducing ability, we studied caffeic acid, one of the phenolic constituents of honey, induced effect on colon cancer cells. Antiproliferative effect of caffeic acid was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MTT assay signified the antiproliferative nature of caffeic acid against the HCT 15 colon cancer cells. A time-dependent inhibition of colony formation was evident with caffeic acid treatment. Cell-cycle analysis of caffeic acid- (CA-) treated cells indicated increasing accumulation of cells at sub-G1 phase. Photomicrograph images of treated cells showed membrane blebbing and cell shrinkage. Yo-pro-1 staining of caffeic-acid-treated cells confirmed apoptosis in dose- and time-dependent manner. Increasing ROS generation and reduction in the mitochondrial membrane potential were also accompanied in the caffeic acid-induced apoptosis. This work will promote caffeic acid as a likely candidate in the chemoprevention of colon cancer.

Preventive effect of Piracetam and Vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture

The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N 2, 5% CO 2) for 3 h and followed by 1 h of reoxygenation (21% O 2 and 5% CO 2) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia + Piracetam, Hypoxia + Vinpocetine, Normoxia + Piracetam and Normoxia + Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1 mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture.

Antioxidant effects of the chestnut ( Castanea crenata) inner shell extract in t-BHP-treated HepG2 cells, and CCl 4- and high-fat diet-treated mice

The antioxidant effects of chestnut inner shell extract (CISE) were investigated in a tert-butylhydroperoxide ( t-BHP)-treated HepG2 cells, and in mice that were administered carbon tetrachloride (CCl 4) and fed a high-fat diet (HFD). Pre-incubation with CISE significantly blocked the oxidative stress induced by t-BHP treatment in HepG2 cells ( P < 0.05) and preserved the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase compared to group treated with t-BHP only. Similarly, the CCl 4- and HFD-induced reduction of antioxidant enzymes activities in liver was prevented by CISE treatment compared to control groups. Furthermore, hepatic lipid peroxidation were remarkably lower ( P < 0.05) in the CISE-treated groups with t-BHP or HFD. To determine the active compound of CISE, the fractionation of CISE has been conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the t-BHP-induced ROS generation and reduction in antioxidant enzyme activity in an in vitro model system. From these results, it was demonstrated that CISE has the ability to protect against damage from oxidative stressors such as t-BHP, CCl 4 and HFD in in vitro and in vivo models. The CISE might be useful for the prevention of oxidative damage in liver cells and tissues.