Elevated branched-chain amino acid promotes atherosclerosis progression by enhancing mitochondrial-to-nuclear H2O2-disulfide HMGB1 in macrophages.

As the essential amino acids, branched-chain amino acid (BCAA) from diets is indispensable for health. BCAA supplementation is often recommended for patients with consumptive diseases or healthy people who exercise regularly. Latest studies and ours reported that elevated BCAA level was positively correlated with metabolic syndrome, diabetes, thrombosis and heart failure. However, the adverse effect of BCAA in atherosclerosis (AS) and its underlying mechanism remain unknown. Here, we found elevated plasma BCAA level was an independent risk factor for CHD patients by a human cohort study. By employing the HCD-fed ApoE-/- mice of AS model, ingestion of BCAA significantly increased plaque volume, instability and inflammation in AS. Elevated BCAA due to high dietary BCAA intake or BCAA catabolic defects promoted AS progression. Furthermore, BCAA catabolic defects were found in the monocytes of patients with CHD and abdominal macrophages in AS mice. Improvement of BCAA catabolism in macrophages alleviated AS burden in mice. The protein screening assay revealed HMGB1 as a potential molecular target of BCAA in activating proinflammatory macrophages. Excessive BCAA induced the formation and secretion of disulfide HMGB1 as well as subsequent inflammatory cascade of macrophages in a mitochondrial-nuclear H2O2 dependent manner. Scavenging nuclear H2O2 by overexpression of nucleus-targeting catalase (nCAT) effectively inhibited BCAA-induced inflammation in macrophages. All of the results above illustrate that elevated BCAA promotes AS progression by inducing redox-regulated HMGB1 translocation and further proinflammatory macrophage activation. Our findings provide novel insights into the role of animo acids as the daily dietary nutrients in AS development, and also suggest that restricting excessive dietary BCAA consuming and promoting BCAA catabolism may serve as promising strategies to alleviate and prevent AS and its subsequent CHD.

Saponins from Aralia taibaiensis attenuate D-galactose-induced aging in rats by activating FOXO3a and Nrf2 pathways.

Reactive oxygen species (ROS) are closely related to the aging process. In our previous studies, we found that the saponins from Aralia taibaiensis have potent antioxidant activity, suggesting the potential protective activity on the aging. However, the protective effect of the saponins and the possible underlying molecular mechanism remain unknown. In the present study, we employed a D-galactose-induced aging rat model to investigate the protective effect of the saponins. We found that D-galactose treatment induced obvious aging-related changes such as the decreased thymus and spleen coefficients, the increased advanced glycation end products (AGEs) level, senescence-associated ß-galactosidase (SAß-gal) activity, and malondialdehyde (MDA) level. Further results showed that Forkhead box O3a (FOXO3a), nuclear factor-erythroid 2-related factor 2 (Nrf2), and their targeted antioxidants such as superoxide dismutase 2 (SOD2), catalase (CAT), glutathione reductase (GR), glutathione (GSH), glutamate-cysteine ligase (GCL), and heme oxygenase 1 (HO-1) were all inhibited in the aging rats induced by D-galactose treatment. Saponins supplementation showed effective protection on these changes. These results demonstrate that saponins from Aralia taibaiensis attenuate the D-galactose-induced rat aging. By activating FOXO3a and Nrf2 pathways, saponins increase their downstream multiple antioxidants expression and function, at least in part contributing to the protection on the D-galactose-induced aging in rats.

Ischemic neurons activate astrocytes to disrupt endothelial barrier via increasing VEGF expression.

Blood-brain barrier (BBB) disruption occurring within the first few hours of ischemic stroke onset is closely associated with hemorrhagic transformation following thrombolytic therapy. However, the mechanism of this acute BBB disruption remains unclear. In the neurovascular unit, neurons do not have direct contact with the endothelial barrier; however, they are highly sensitive and vulnerable to ischemic injury, and may act as the initiator for disrupting BBB when cerebral ischemia occurs. Herein, we employed oxygen-glucose deprivation (OGD) and an in vitro BBB system consisting of brain microvascular cells and astrocytes to test this hypothesis. Neurons (CATH.a cells) were exposed to OGD for 3-h before co-culturing with endothelial monolayer (bEnd 3 cells), or endothelial cells plus astrocytes (C8-D1A cells). Incubation of OGD-treated neurons with endothelial monolayer alone did not increase endothelial permeability. However, when astrocytes were present, the endothelial permeability was significantly increased, which was accompanied by loss of occludin and claudin-5 proteins as well as increased vascular endothelial growth factor (VEGF) secretion into the conditioned medium. Importantly, all these changes were abolished when VEGF was knocked down in astrocytes by siRNA. Our findings suggest that ischemic neurons activate astrocytes to increase VEGF production, which in turn induces endothelial barrier disruption.

Mechanism of acute lung injury due to phosgene exposition and its protection by cafeic acid phenethyl ester in the rat.

The mechanism of phosgene-induced acute lung injury (ALI) remains unclear and it is still lack of effective treatments. Previous study indicated that oxidative stress was involved in phosgene-induced ALI. Caffeic acid phenethyl ester (CAPE) has been proved to be an anti-inflammatory agent and a potent free radical scavenger. The purpose of this study was to investigate the protective effects of CAPE on phosgene-induced ALI and identify the mechanism, in which oxidative stress and inflammation were involved. The phosgene was used to induce ALI in rats. The results showed that after phosgene exposure, total protein content in BALF was not significantly changed. The increase of MDA level and SOD activity induced by phosgene was significantly reduced by CAPE administration, and the decrease of GSH level in BALF and lung were significantly reversed by CAPE. CAPE also partially blocked the translocation of NF-κB p65 to the nucleus, but it had little effect on the phosphorylation of p38 MAPK. In conclusion, CAPE showed protective effects on lung against phosgene-induced ALI, which may be related with a combination of the antioxidant and anti-inflammatory functions of CAPE.

Ethyl pyruvate protects rats from phosgene-induced pulmonary edema by inhibiting cyclooxygenase2 and inducible nitric oxide synthase expression.

Phosgene is a poorly water-soluble gas penetrating the lower respiratory tract which can induce acute lung injury characterized by a latent phase of fatal pulmonary edema. Pulmonary edema caused by phosgene is believed to be a consequence of oxidative stress and inflammatory responses. Ethyl pyruvate (EP) has been demonstrated to have anti-inflammatory and anti-oxidative properties in vivo and in vitro. The potential therapeutic role of EP in phosgene-induced pulmonary edema has not been addressed so far. In the present study, we aim to investigate the protective effects of EP on phosgene-induced pulmonary edema and the underlying mechanisms. Rats were administered with EP (40 mg kg(-1)) and RAW264.7 cells were also incubated with it (0, 2, 5 or 10 µm) immediately after phosgene (400 ppm, 1 min) or air exposure. Wet-to-dry lung weight ratio (W:D ratio), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, cyclooxygenase2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, and mitogen-activated protein kinases activities (MAPKs) were measured. Our results showed that EP treatment attenuated phosgene-induced pulmonary edema and decreased the level of NO and PGE(2) dose-dependently. Furthermore, EP significantly reduced COX-2 expression, iNOS expression and MAPK activation induced by phosgene. Moreover, specific inhibitors of MAPKs reduced COX-2 and iNOS expression induced by phosgene. These findings suggested that EP has a protective role against phosgene-induced pulmonary edema, which is mediated in part by inhibiting MAPK activation and subsequently down-regulating COX-2 and iNOS expression as well as decreasing the production of NO and PGE(2).

Poly(ADP-ribose) polymerase-1 inhibition by arsenite promotes the survival of cells with unrepaired DNA lesions induced by UV exposure.

Human arsenic exposure is associated with increased risk of skin cancer, and arsenite greatly enhances ultraviolet (UV)-induced skin tumors in a mouse model of carcinogenesis. Inhibition of DNA repair is one proposed mechanism for the observed cocarcinogenicity. We have previously demonstrated that low concentrations of arsenite inhibit poly(ADP-ribose) polymerase (PARP)-1, thus interfering with DNA repair process triggered by UV radiation. Because overactivation of PARP-1 often leads to apoptotic cell death, and unrepaired DNA lesions promote genomic instability and carcinogenesis, we hypothesized that inhibition of PARP-1 by arsenic may promote the survival of potentially "initiated carcinogenic cells," i.e., cells with unrepaired DNA lesions. In the present study, we tested this hypothesis on UV-challenged HaCat cells. Cells were pretreated with 2µM arsenite for 24 h before UV exposure. Outcome parameters included apoptotic death rate, PARP-1 activation, apoptotic molecules, and retention of DNA lesions. UV exposure induced PARP-1 activation and associated poly(ADP-ribose) production, apoptosis-inducing factor release, cytochrome C release, and caspases activation, which led to apoptotic death in HaCat cells. Pretreatment with 2µM arsenite significantly inhibited UV-induced cell death as well as the associated molecular events. Notably, knockdown of PARP-1 with small interfering RNA completely abolished the antagonism of arsenite. Furthermore, arsenite pretreatment led to long-term retention of UV-induced cyclobutane pyrimidine dimers. Together, these results suggest that low concentration of arsenite reduces UV-induced apoptosis via inhibiting PARP-1, thus promoting the survival of cells with unrepaired DNA lesions, which may be an important mechanism underlying arsenic cocarcinogenic action.

Pentoxifylline inhibits intercellular adhesion molecule-1 (ICAM-1) and lung injury in experimental phosgene-exposure rats.

Phosgene inhalation results in acute lung injury (ALI) mostly, pulmonary edema and even acute respiratory distress syndrome, but there is no specific antidote. Inflammatory cells play an important role in the ALI caused by phosgene. Intercellular adhesion molecule-1 (ICAM-1) is a critical factor for inflammatory organ injury. We hypothesized that pentoxifylline (PTX), an inhibitor of leukocyte activation, would have a protective effect on experimental phosgene-induced lung injury rats by inhibiting ICAM-1. To prove this hypothesis, we used rat models of phosgene (400 ppm x 1 min)-induced injury to investigate: (1) the time course of lung injury (control 1, 3, 6, 12, 24, and 48 h group), including pathological changes in hematoxylin and eosin staining and transmission electron microscope, myeloperoxidase (MPO) activity by colorimetric method and ICAM-1 protein level detected by western blot, (2) At 3 h after phosgene exposure, protective effects of different dosages of PTX (50 mg/kg and 100 mg/kg) administration were evaluated by MPO activity, ICAM-1 differential expression and WBC count in bronchoalveolar lavage fluid. The results showed that inflammatory cells emerged out of lung blood vessels at 3 h after phosgene exposure. The MPO activity of lung tissue increased significantly from 3 to 48 h after phosgene exposure (P < 0.05) and ICAM-1 expression presented a similar change, especially at 3 h and 24 h (P < 0.05). After pretreatment and treatment with PTX (100 mg/kg), significant protective effects were shown (P < 0.05). These data supported our hypothesis that PTX reduced phosgene-induced lung injury, possibly by inhibiting ICAM-1 differential expression.

Correlation between sPLA2-IIA and phosgene-induced rat acute lung injury.

Secreted phospholipase A(2) of group IIA (sPLA(2)-IIA) has been involved in a variety of inflammatory diseases, including acute lung injury. However, the specific role of sPLA(2)-IIA in phosgene-induced acute lung injury remains unidentified. The aim of the present study was to investigate the correlation between sPLA(2)-IIA activity and the severity of phosgene-induced acute lung injury. Adult male rats were randomly exposed to either normal room air (control group) or a concentration of 400 ppm phosgene (phosgene-exposed group) for there are 5 phosgene-exposed groups altogether. For the time points of 1, 3, 6, 12 and 24 h post-exposure, one phosgene-exposed group was sacrificed at each time point. The severity of acute lung injury was assessed by Pa(O2)/F(IO2) ratio, wet-to-dry lung-weight ratio, and bronchoalveolar lavage (BAL) fluid protein concentration. sPLA(2)-IIA activity in BAL fluid markedly increased between 1 h and 12 h after phosgene exposure, and reached its highest level at 6 h. Moreover, the trend of this elevation correlated well with the severity of lung injury. These results indicate that sPLA(2)-IIA probably participates in phosgene-induced acute lung injury.

Inhibition of poly(ADP-ribose) polymerase-1 by arsenite interferes with repair of oxidative DNA damage.

Arsenic enhances skin tumor formation when combined with other carcinogens, including UV radiation (UVR). In this study we report that low micromolar concentrations of arsenite synergistically increases UVR-induced oxidative DNA damage in human keratinocytes as detected by 8-hydroxyl-2'-deoxyguanine (8-OHdG) formation. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in base excision repair, a process that repairs 8-OHdG lesions. Arsenite suppresses UVR-induced PARP-1 activation in a concentration-dependent manner. Inhibition of PARP-1 activity by 3-aminobenzamide or small interfering RNA silencing of PARP-1 expression significantly increases UVR-induced 8-OHdG formation, suggesting that inhibition of PARP-1 activity by arsenite contributes to oxidative DNA damage. PARP-1 is a zinc finger protein, and mass spectrometry analysis reveals that arsenite can occupy a synthetic apopeptide representing the first zinc finger of PARP-1 (PARPzf). When the PARPzf peptide is preincubated with Zn(II) followed by incubation with increasing concentrations of arsenite, the ZnPARPzf signal is decreased while the AsPARPzf signal intensity is increased as a function of arsenite dose, suggesting a competition between zinc and arsenite for the same binding site. Addition of Zn(II) abolished arsenite enhancement of UVR-stimulated 8-OHdG generation and restored PARP-1 activity. Our findings demonstrate that arsenite inhibits oxidative DNA damage repair and suggest that interaction of arsenite with the PARP-1 zinc finger domain contributes to the inhibition of PARP-1 activity by arsenite. Arsenite inhibition of poly(ADP-ribosyl)ation is one likely mechanism for the reported co-carcinogenic activities of arsenic in UVR-induced skin carcinogenesis.

Dual actions involved in arsenite-induced oxidative DNA damage.

Arsenic is a recognized human carcinogen, but the mechanism of carcinogenesis is not well understood. Oxidative stress and inhibition of DNA damage repair have been postulated as potential carcinogenic actions of arsenic. The present study tests the hypothesis that arsenite not only induces oxidative stress but also inhibits the activity of the DNA base excision repair protein, poly(ADP-ribose) polymerase-1 (PARP-1), leading to exacerbation of the oxidative DNA damage induced by arsenic. HaCat cells were treated with arsenite for 24 h before measuring 8-hydroxyl-2'-deoxyguanosine (8-OHdG), PARP-1 activity, and reactive oxygen species (ROS). Zinc supplementation and PARP-1 siRNA were used to increase or decrease, respectively, the PARP-1 protein's physiological function. At high concentrations (10 microM or higher), arsenite greatly induced oxidative DNA damage, as indicated by 8-OHdG formation. At lower concentrations (1 microM), arsenite did not produce detectable 8-OHdG, but was still able to effectively inhibit PARP-1 activity. Zinc supplementation reduced the formation of 8-OHdG, restored the PARP-1 activity inhibited by arsenite, but did not decrease ROS production. SiRNA knockdown of PARP-1 did not affect the 8-OHdG level induced by arsenic, while it greatly increased the 8-OHdG level produced by hydrogen peroxide indicating that PARP-1 is a molecular target of arsenite. Our findings demonstrate that in addition to inducing oxidative stress at higher concentrations, arsenite can also inhibit the function of a key DNA repair protein, PARP-1, even at very low concentrations, thus exacerbating the overall oxidative DNA damage produced by arsenite, and potentially, by other oxidants as well.

Low concentration of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity.

Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA-repair processes. Poly(ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA-repair protein, which can promptly sense DNA strand breaks and initiate DNA-repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/or UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite (

Time course for expression of VEGF and its receptor and regulator levels of contraction and relaxation in increased vascular permeability of lung induced by phosgene.

Acute lung injury (ALI) induced by phosgene increases risk of serious edema and mortality. Increased permeability of the microvascular endothelium is implicated in the progression of ALI, but the processing interaction and time course activity of the vascular regulators in exudation are still not understood. The main aim of this study was to investigate the time course and potential role for vascular endothelial growth factor (VEGF), its receptors, and some vascular function regulators related to increased vascular permeability of lung induced by phosgene. Sprague Dawley rats were randomly divided into seven groups according to time post phosgene exposure (control, and 1, 3, 6, 12, 24, and 48 h groups). Lung tissue was removed to evaluate VEGF isoforms, fms-like tyrosine kinase receptor 1 (Flt-1), and kinase insert domain containing region (KDR/Flk-1) by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Blood samples were collected for measurement of plasma endothelin-1 (ET-1) and nitric oxide (NO) level. The results showed that the mRNA and protein expression profile of the VEGF system after phosgene exposure was time dependent. The VEGF system expression in lung tissue was related closely to the level of ET-1 and NO. In conclusion, increased permeability of the lung microvascular endothelium induced by phosgene was primarily a result of differential expression of VEGF and its receptors, and was related to the level of ET-1 and NO. The results suggest that the cooperation of VEGF system, ET-1, and NO plays a critical role, and all those parameters emerge as time dependent in the early phase of the permeability process induced by phosgene exposure.

The dysfunction of ATPases due to impaired mitochondrial respiration in phosgene-induced pulmonary edema.

Phosgene is a toxic gas that is widely used in modern industry, and its inhalation can cause severe pulmonary edema. There is no effective clinical treatment because the mechanism of phosgene-induced pulmonary edema still remains unclear. Many studies have demonstrated that the Na(+)/K(+)-ATPase plays a critical role in clearing pulmonary edema and the inhibition of Na(+)/K(+)-ATPase protein expression has been found in many other pulmonary edema models. In the present study, after the mice were exposed to phosgene, there was serious pulmonary edema, indicating the dysfunction of the ATPases in mice. However, in vitro enzyme study showed that there were increases in the activities of the Na(+)/K(+)-ATPase and Ca(2+)-ATPase. Further investigation showed that the ATP content and mitochondrial respiratory control ratio (RCR) in the lungs decreased significantly. The oxidative stress product, malondialdehyde (MDA), increased while the antioxidants (GSH, SOD, and TAC) decreased significantly. These results indicate that mitochondrial respiration is the target of phosgene. The dysfunction of ATPases due to impaired mitochondrial respiration may be a new mechanism of phosgene-induced pulmonary edema.

Protection of multiple antioxidants Chinese herbal medicine on the oxidative stress induced by adriamycin chemotherapy.

Adriamycin is an effective anthracycline anti-tumor antibiotic. However, the clinical use of adriamycin has been restricted by its serious side effects. Some reports indicated that the side effects of adriamycin could cause systemic injury, in which reactive oxygen species (ROS) play an important role. ROS are a large family of oxygen free radical and non-free radical active oxygen-containing molecules, including superoxide radical, hydrogen peroxide and hydroxyl radical, which contribute to oxidative stress. Although antioxidant treatment is a promising method to prevent the side effects, protection by a single antioxidant is limited. The Chinese herbal medicine ANTIOXIN is a multiple antioxidant that can effectively block oxidative stress. It was hypothesized that ANTIOXIN could effectively reduce the side effects of adriamycin. A rat tumor model with a transplanted tumor in the liver was treated with adriamycin and ANTIOXIN was used as a protection. Oxidative stress and antioxidant enzymes were evaluated. The results showed that adriamycin chemotherapy increased the level of malondialdehyde (MDA), nitrogen oxide (NO) and decreased the activities of total superoxide dismutase (T-SOD), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione (GSH) and total antioxidant capacity (TAC). Adriamycin chemotherapy also decreased the expression of Bcl-2, increased the expression of iNOS and cell apoptosis in the liver and kidney. Multiple antioxidants ANTIOXIN had an antagonistic effect on these changes and significantly decreased the mortality of the experimental rats. These data demonstrated that adriamycin chemotherapy could cause oxidative stress to the whole body, on which multiple antioxidants based on the theory of 'multiple antioxidant chain' had effective protection.

[Effect of phosgene on apoptosis of alveolar type II cells and vascular endothelial growth factor in exposed mice].

OBJECTIVE: To study the apoptosis of alveolar type II cells, alterations of vascular endothelial growth factor (VEGF), VEGF receptor (Flt1) in serum and lung and expression of VEGF mRNA in lung in pulmonary edema mice induced by phosgene. METHODS: Twenty-six BALB/C mice were randomly divided into 2 groups: control group, exposed group (13 mice in each group). Mice of exposed group were intoxicated by inhalation of phosgene 11.9 mg/L for 5 minutes. Mice of control group were treated as the same way by inhalation of air. Isolation of mice alveolus type II cells 4 h after intoxication was carried out to observe their apoptosis under electron microscope. Contents of VEGF and Flt1 in lung and serum by ELISA, and expression of VEGF mRNA were determined. RESULTS: Alveolar type II cells were identified by tannic acid staining and electron microscopy. After exposed to 11.9 mg/L of phosgene for 5 minutes, the apoptotic body in alveolus type II cells was found in exposed group. The contents of VEGF in serum and lung and Flt1 in lung of exposed mice [(134.07 +/- 120.26), (477.76 +/- 98.06), (1,2818.48 +/- 2,304.15) pg/ml] were significantly lower than those of control group [(445.57 +/- 173.30), (1,026.87 +/- 474.56), (21,976.51 +/- 7,421.01) pg/ml, P < 0.05] but the content of Flt1 in serum [(2,369.56 +/- 381.70) pg/ml] was higher than that in control group [(1,898.00 +/- 453.69) pg/ml, P < 0.05]. The expression of VEGF mRNA in pulmonary edema mice was decreased. CONCLUSION: Phosgene can induce apoptosis of alveolar type II cells, and decrease in the content of VEGF and Flt1, and expression of VEGF mRNA in lung.