Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

Effects of glutamine pretreatment on intestinal ischemia-reperfusion injury in rats

AIM：To determine the effects of glutamine (Gln) pretreatment on intestinal ischemia-reperfusion (I/R) injury in the rats. METHODS：Thirty male Wistar rats were randomly divided into 3 groups (n=10): sham group, I/R group and Gln pretreatment group. The rats in Gln pretreatment group were pretreated with 1 g·kg-1·d-1 Gln by orogastric route for 7 d, the rats in the other 2 groups were pretreated with normal saline. Intestinal I/R was induced by 30-min occlusion of the superior mesenteric artery followed by 24 h of reperfusion. After the operation, the plasma endotoxin, serum D-lactic acid, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured. The intestinal mucosal injury was observed with HE staining and evaluated using Chius scoring. RESULTS：Serum D-lactic acid, endotoxin level, MDA level and Chiu's score in I/R group were significantly higher than those in sham group and Gln group (all P<0.05). Serum SOD activity was significantly lower than that in sham group and Gln group (P<0.05). CONCLUSION：Glutamine has a protective effect on the intestines during ischemia-reperfusion injury. The mechanism may be related to oxidative stress response.     

Oxidative stress response and histopathological changes due to atrazine and chlorpyrifos exposure in common carp

Atrazine (ATR) and chlorpyrifos (CPF) are the most common pesticides found in freshwater ecosystems throughout the world. Herein, we investigated the oxidative stress responses and histopathological changes in the liver and gill of common carp after a 40-d exposure to CPF and ATR, alone or in combination, and a 20-d recovery treatment. We found that exposure to ATR, CPF or their mixture for 40 d could induce decrease in antioxidant enzyme (SOD, CAT and GSH-Px) activities and increase in MDA content in a dose-dependent manner in the liver and gill of common carp. Especially with regard to the pathological changes, the tissue damage increased in severity in a dose-dependent manner. The liver tissue of common carp revealed different degree of hydropic degeneration, vacuolisation, pyknotic nuclei, and fatty infiltration. The gills of common carp displayed varied degrees of epithelial hypertrophy, telangiectasis, oedema with epithelial separation from basement membranes, general necrosis, and epithelial desquamation. After a 20-d recovery treatment, the antioxidant enzyme activities and MDA content were significantly lower (p < 0.05) than in the corresponding exposure groups in all of the highest doses, but not in the lower doses. To our knowledge, this is the first report of subchronic oxidative stress and histopathological effects caused by ATR, CPF and their mixture in the common carp. Thus, the information presented in this study is helpful to understand the mechanism of ATR-, CPF- and ATR/CPF-mixture-induced oxidative stress in fish.     

Inhibitory effect of catalpol on inflammation and expression of RAGE in EA.hy926 cells induced by advanced glycation end products

AIM: To investigate the inhibitory effect of catalpol on inflammation in EA.hy926 cells induced by advanced glycation end products(AGEs) and to explore its antioxidant mechanisms.METHODS: Human endothelial cell line EA.hy926 was cultured and randomly divided into control group, catalpol(0.5 mmol/L) group, AGEs group, high-dose(0.5 mmol/L) catalpol+AGEs group, middle-dose(0.25 mmol/L) catalpol+AGEs group and low-dose(0.05 mmol/L) catalpol+AGEs group. Intracellular reative oxygen species(ROS) production was detected by laser scanning confocal microscopy. The levels of monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-α(TNF-α) and vascular cell adhesion molecule-1(VCAM-1) in culture supernatant were detected by commercial ELISA kits. The expression of MCP-1, TNF-α, VCAM-1 and receptor for advanced glycation end products(RAGE) in the EA.hy926 cells were detected by Western blot.RESULTS: In high-dose catalpol+AGEs and middle-dose catalpol+AGEs groups, the generation of ROS was decreased significantly. The levels of MCP-1, TNF-α and VCAM-1, and protein expression of MCP-1, TNF-α and VCAM-1 were significantly lower. The expression of RAGE protein in EA.hy926 cells were significantly inhibited(P<0.05).CONCLUSION: Catalpol effectively inhibits the AGEs-induced oxidative stress and inflammation in EA.hy926 cells, which may be associated with a decrease in the expression of RAGE.     

Effect of combined exposure of commonly used organophosphate pesticides on lipid peroxidation and antioxidant enzymes in rat tissues

Organophosphate compounds are among the most widely used synthetic chemicals for controlling a wide variety of pests. Organophosphate (OP) poisoning continues to be major cause of morbidity and mortality in the third world countries. Indiscriminate use of these pesticides tends to leave residues on the objects of the environment. Present study is aimed to compare the potential of three commonly used OP pesticides, chlorpyrifos (CPF), methyl parathion (MPT) and malathion (MLT), to generate oxidative stress in rat tissues and to evaluate whether the combined exposure of these pesticides exerts synergistic or antagonistic effects. Results of the present study showed that CPF, MPT and MLT exposure to rats caused accumulation of malondialdehyde (MDA) and 4-hydroxynonanal (4HNE), the two major end products of lipid peroxidation, in liver, kidney, brain and spleen of rats. Combined exposure of these pesticides also resulted in accumulation of MDA and 4HNE in rat tissues but the increase was almost of the same order as observed in rat tissues given these pesticides singly. Exposure with CPF, MPT and MLT singly or in mixture, caused dose-dependent decrease in the activities of antioxidant enzymes namely, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx), in rat tissues when compared with control, and the decrease observed was of the same order in all the groups. Acetylcholinesterase (AChE) activity, an indicator of OP poisoning, was also decreased in rat tissues in dose-dependent manner in CPF, MPT, MLT and mixture treated group. Differential increase in the levels of cytochrome P450 (cyt P450) in hepatic and extra-hepatic tissues of rats given CPF, MPT or MLT singly or in mixture, indicate different rates of metabolism of these pesticides. Results of the present study clearly show that CPF, MPT and MLT exposure singly or in mixture, induced oxidative stress in rat tissues which may be the major contributor of the overall toxicity of the OP pesticides. Combined exposure of these pesticides does not seem to potentiate the toxicity of each other and their toxic effects are not additive.     

Changes in antioxidative metabolism accompanying pitting development in stored blueberry fruit

Blueberries (Vaccinium spp. ‘LanFeng’) are harvested and consumed at maturation and have a short storage life at room temperature. Changes in blueberry quality and physiological parameters differ under room or low temperature storage conditions. The storage life of blueberries can be extended at low temperature, but pitting can develop associated with refrigeration, especially during subsequent shelf-life. The objective of this research was to understand the antioxidative metabolism accompanying pitting development of stored blueberry fruit, involving reactive oxygen species and antioxidant systems. Physiological and metabolic disorders, including low firmness, increased cell membrane electrolyte leakage and malondialdehyde (MDA) content, and changes in enzyme activity, were observed in pitting blueberries. Blueberries were stored at 20 °C and 0 °C for 10 days and 60 days, respectively. Hydrogen peroxide and superoxide radical production rate increased more rapidly during shelf-life after cold storage than at room temperature. On the other hand, blueberries during shelf-life after cold storage, when pitting occurred, had lower activity of antioxidant-related enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), than those stored at room temperature. The severity of pitting was paralleled by higher cell membrane electrolyte leakage and MDA content, and lower SOD, CAT, and APX activities.     

土壤产电信号与线性扫描伏安法联用模拟监测湿地铜污染

本研究采用产电信号与线性扫描伏安法(LSV)联用,探索快速响应和鉴定湿地铜污染的方法。采集湖岸土壤并淹水用以模拟湿地环境,构建土壤产电体系并实时连续记录土壤产电电压。土壤产电60 h后,分别向体积为145 ml的淹水层中加入5 ml Cu~(2+)浓度为300(T1)、600(T2)、1 200(T3)、2 400 mg/L(T4)的溶液。铜加入后,T1和T2处理的电压迅速降低,T3和T4处理的电压迅速上升,之后所有电压缓慢回到加铜前的水平。加铜后2 h,在淹水土壤中设置三电极体系并采用线性扫描伏安法(LSV)对污染物进行定性检测,结果显示,LSV曲线在0.162~0.182 V范围内出现单个氧化峰,峰电流值随溶液中Cu~(2+)浓度增加而升高。为解释土壤产电信号变化特征,对加铜前后电流、阴极电势和内阻进行了检测,结果显示,加铜后T1处理电流降低,阴极电势无明显变化;T2处理电流无明显变化,阴极电势上升;而T3、T4处理电流和阴极电势都出现了明显的升高,且T4处理的内阻最低。本研究为实现土壤产电信号在监测污染方面的实际应用提供了参考。     

基于 AFM 的人参水提物对 VERO 细胞氧化损伤作用研究

[目的]研究人参水提物对VERO细胞在体外培养中的氧化损伤。[方法]采用细胞体外培养技术、MTF比色法、SOD和MDA试剂盒法、原子显微镜（AFM）观察法,研究人参水提物对VERO细胞氧化损伤的机制。[结果]人参水提物对VERO细胞能够起到明显的增殖抑制作用。人参水提物对VERO细胞具有一定的的氧化损伤作用且有一定的剂量及时间依赖性。原子力显微扫描成像结果表明正常生长的VERO细胞表面光滑、细胞体积饱满,而人参水提物处理组细胞膜损坏现象明显,细胞表面的粗糙度较大。[结论]人参水提物对VERO细胞具有一定的氧化损伤作用,其作用机制可能与细胞膜的损伤有关。