BACKGROUND: Ischemic insult during operation could cause ischemic-reperfusion injuries in brain and memory impairments. Total intravenous anesthesia (TIVA) is preferred in brain surgery to promote the use of motor evoked potential monitoring and the use of opioids is common in TIVA. However there were few studies about ischemic protective effect of opioids to astrocytes. METHODS: We used astrocytes, which were derived from human brain. We divided groups by conditioning period; i) pre-culture, ii) post-culture, or iii) pre + post-culture. All groups were treated 100 nM hydromorphone. We measured reactive oxygen species (ROS) by flow cytometry with 2',7'-dichloroflurorescin diacetate. Then ROS in astrocytes which treated by opioid receptor antagonist were measured after treating 100 nM hydromorphone. RESULTS: ROS was reduced in hydromorphone treated group, as compared to the control group (only tert-butyl hydroperoxide [TBH] treated). There was no difference in pre-conditioned group and post-conditioned group. However, ROS was much more reduced in pre + post-conditioned group compared to pre-conditioned only or post-conditioned only group. Furthermore each selective micro-, delta- and kappa-opioid receptor antagonists partially negated the effect of hydromorphone. CONCLUSIONS: This study provides evidence that hydromorphone has both preconditioning and postconditioning effects on TBH-induced oxidative stress. Furthermore we proved each micro-, delta- and kappa-opioid receptor relates to protective mechanism of hydromorphone to astrocytes.
Analgesics, Opioid ; Anesthesia, Intravenous ; Astrocytes ; Brain ; Brain Ischemia* ; Evoked Potentials, Motor ; Flow Cytometry ; Humans ; Hydromorphone* ; Memory ; Oxidative Stress ; Reactive Oxygen Species ; Receptors, Opioid ; tert-Butylhydroperoxide

BACKGROUND/OBJECTIVES: The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. MATERIALS/METHODS: Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined. RESULTS: TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors. CONCLUSIONS: These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.
Apoptosis ; Biomarkers ; Caspase 3 ; Cell Death* ; Cell Survival ; Hep G2 Cells* ; Methanol ; Oxidative Stress ; Phosphotransferases ; Reactive Oxygen Species ; tert-Butylhydroperoxide

BACKGROUND/OBJECTIVES: This study investigated the antioxidant activities and hepatoprotective effects of Schisandra chinensis Baillon extract (SCE) against tert-butyl hydroperoxide (t-BHP)-induced oxidative hepatic damage in rats. MATERIALS/METHODS: Sprague-Dawley (SD) rats were pretreated with SCE (300, 600, and 1,200 mg/kg BW) or saline once daily for 14 consecutive days. On day 14, each animal, except those belonging to the normal control group, were injected with t-BHP (0.8 mmol/kg BW/i.p.), and all of the rats were sacrificed 16 h after t-BHP injection. RESULTS: Although no significant differences in AST and ALT levels were observed among the TC and SCE groups, the high-dose SCE group showed a decreasing tendency compared to the TC group. However, erythrocyte SOD activity showed a significant increase in the low-dose SCE group compared with the TC group. On the other hand, no significant differences in hepatic total glutathione (GSH) level, glutathione reductase (GR), and glutathione peroxidase (GSH-Px) activities were observed among the TC and SCE groups. Hepatic histopathological evaluation revealed that pretreatment with SCE resulted in reduced t-BHP-induced incidence of lesions, such as neutrophil infiltration, swelling of liver cells, and necrosis. In particular, treatment with a high dose of SCE resulted in induction of phase II antioxidant/detoxifying enzyme expression, such as glutathione S-transferase (GST) and glutamate-cysteine ligase catalytic subunit (GCLC). CONCLUSIONS: Based on these results, we conclude that SCE exerts protective effects against t-BHP induced oxidative hepatic damage through the reduction of neutrophil infiltration, swelling of liver cells, and necrosis. In addition, SCE regulates the gene expression of phase II antioxidant/detoxifying enzymes independent of hepatic antioxidant enzyme activity.
Animals ; Catalytic Domain ; Erythrocytes ; Gene Expression* ; Glutamate-Cysteine Ligase ; Glutathione ; Glutathione Peroxidase ; Glutathione Reductase ; Glutathione Transferase ; Hand ; Incidence ; Liver ; Necrosis ; Neutrophil Infiltration ; Rats* ; Rats, Sprague-Dawley ; Schisandra* ; tert-Butylhydroperoxide

PURPOSE: To investigate the effects of dipyridamole (DPD) on the production of reactive oxygen species (ROS) and oxidative stress in cultured human trabecular meshwork cells (HTMC). METHODS: Antioxidant activity of DPD was determined by DPPH assay. Primarily cultured HTMC were exposed to 0, 20, and 50 microm DPD using serum-deprived media. The effect of DPD on the production of ROS was assessed with the DCHFDA assay. The effect of DPD on the t-butyl hydroperoxide (tBHP)-induced oxidative stress was assessed with resazurin assay. RESULTS: DPD showed significant antioxidant activity. DPD significantly decreased the production of ROS (p < 0.05) and improved cellular activity significantly after treatment with t-BHP (p < 0.05). DPD did not affect the generation of nitric oxides. CONCLUSIONS: DPD suppressed the formation of ROS and possessed cytoprotective activity against the oxidative stress in HTMC.
Dipyridamole ; Humans ; Oxazines ; Oxidative Stress ; Reactive Oxygen Species ; tert-Butylhydroperoxide ; Trabecular Meshwork ; Xanthenes

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K+ current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 microM Ca2+ and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K+ current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K+ current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.
Antioxidants ; Benzimidazoles ; Constriction ; Endothelial Cells ; Endothelium ; Hand ; Human Umbilical Vein Endothelial Cells ; Humans ; Hydrogen ; Hydrogen Peroxide ; Ion Channels ; Muscle, Smooth, Vascular ; Oxidoreductases ; Pathologic Processes ; Phosphotransferases ; Reactive Oxygen Species ; Superoxides ; tert-Butylhydroperoxide ; Tissue Donors ; Transcription Factors

This paper was to explore the effect of blood oxygen saturation (SO2) on oxidative damages of erythrocytes under the condition of oxidative stress. Keeping SO2 of cultured erythrocytes in vitro at the states of 0.3, 0.5, 0.7, 0.9 and 0.98, respectively, we induced oxidative stress by tert-buthylhydroperoxide (BHP, 0.15 mmol/L of final concentration). After incubation, antioxidant capacity was assessed by measuring content of reduced glutathin hormone (GSH) in erythrocytes. Methemoglobin (MetHb) content, lipid peroxidation (thiobarbituric acid-reactive substances, TBARS) and denatured globin-chains on the plasma membrane were measured to assess the extent of oxidative damages. The results showed that in the presence of BHP, GSH contents increased from 0.3 to 0.98 groups; MetHb, TBARS and globin-chains levels all dropped with the rise of SO2. In conclusion, antioxidant capacity and oxidative damages of erythrocytes are closely related to SO2, declined SO2 could promote oxidative damages of erythrocytes.
Cells, Cultured ; Erythrocytes ; cytology ; metabolism ; physiology ; Glutathione ; blood ; Humans ; Methemoglobin ; metabolism ; Oxidative Stress ; drug effects ; Oximetry ; methods ; Oxygen ; blood ; Thiobarbituric Acid Reactive Substances ; metabolism ; tert-Butylhydroperoxide ; toxicity

OBJECTIVETo establish the oxidative damage model of cochlea hair cells using organic oxidant t-BHP in vitro.

METHODSHEI-OC1 cells were exposed to t-BHP at 8 doses (30~4000 µmol/L) for 12 h. Trypan blue test was used to detected the cellular viability and MTT assay was utilized to measured the cellular proliferation. The intracellular ROS levels were determined by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA).

RESULTSThe survival rates of HEI-OC1 cells started decrease significantly at the dose of 100 µmol/L t-BHP, the peak of decreased survival rates appeared at the doses of 200~800 µmol/L. The results of MTT assay demonstrated that 30 µmol/L t-BHP could promote cellular proliferation ability, when t-BHP concentrations were higher than 200 µmol/L, the cellular proliferation ability was inhibited. The results of DCFH-DA assay showed that there was no fluorescence in control group, the strong fluorescence was observed in positive control group, the weak fluorescence was observed in 30~50 µmol/L t-BHP groups, the bright fluorescence was observed in 100 µmol/L t-BHP group, still the stronger fluorescence was observed in 200~1000 µmol/L groups, but the cellular number decreased with the doses because of the lower cellular viability.

CONCLUSIONThe exposure to 100 µmol/L t-BHP for 12 h could simulate the oxidative damage induced by noise in cochlear hair cells.

Cell Survival ; Cells, Cultured ; Hair Cells, Auditory ; drug effects ; pathology ; Humans ; Noise ; adverse effects ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species ; analysis ; tert-Butylhydroperoxide ; toxicity

OBJECTIVETo investigate the regulatory effect of miR-181a with abnormal expression on the expression of c-fos in cochlear hair cells undergoing oxidative damage.

METHODSHouse Ear Institute-Organ of Corti1 (HEI-CO1) cells were assigned to 50, 100, and 200 µmol/L tert-butyl hydroperoxide (t-BHP) exposure groups and control group. The HEI-CO1 cells in the exposure groups were exposed to 50, 100, or 200 µmol/L t-BHP for 12 h. Then, total RNA and total protein were extracted from the HEI-CO1 cells, and the expression of miR-181a/-181d was measured by qPCR. The miR-181a with abnormal expression was selected as the subject of study. The putative miR-181a target sequence in the 3' untranslated region (3'-UTR) of c-fos was predicted by searching on a bioinformatics website. The HEI-CO1 cells were transfected with miR-181a mimics by lipofection, and the transfection efficiency was measured by qPCR. The mRNA and protein expression of c-fos was measured by qPCR and Western blot. The pGL3-c-fos-3'UTR-WT plasmid was constructed, and the luciferase activity of the plasmid in the case of high miR-181a expression was measured using the Dual-Luciferase Reporter Assay System.

RESULTSCompared with those in the control group, the expression of miR-181a in 100 and 200 µmol/L t-BHP exposure groups was significantly decreased, with expression ratios of 0.744 and 0.766 (P < 0.01), while the expression of miR-181d in 50 µmol/L t-BHP exposure group was significantly increased, with an expression ratio of 1.29 (P < 0.01). There was no significant difference in miR-181a expression between the 100 and 200 µmol/L t-BHP exposure groups (P > 0.05). The predication results revealed that c-fos was regulated by miR-181a in humans and mice, with complete complementarity to the seed region of miR-181a, and there was high degree of target sequence conservation across species. The expression of miR-181a in the HEI-OC1 cells transfected with miR-181a mimics was elevated 892.979 times at 24 hours after transfection. As compared with those of controls, the mRNA and protein expression levels of c-fos in the transfected HEI-OC1 cells were significantly increased (P < 0.05 and P < 0.01). The luciferase activity of pGL3-c-fos-3'UTR-WT plasmid was not suppressed but increased in the case of high miR-181a expression.

CONCLUSIONmiR-181a has no direct inhibitory effect on the mRNA and protein expression of c-fos, which may not be the target gene of miR-181a. Bioinformatic prediction might produce false-positive results.

Animals ; Apoptosis ; drug effects ; genetics ; Cell Line ; Hair Cells, Auditory ; cytology ; drug effects ; metabolism ; Mice ; MicroRNAs ; genetics ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; RNA, Messenger ; genetics ; Transfection ; tert-Butylhydroperoxide ; toxicity

This study was performed to investigate the effect of desalinated underground seawater (named as 'magma seawater', MSW) of Jeju Island in Korea on lipid metabolism and antioxidant activity. MSW was collected from underground of Han-Dong in Jeju Island, and freely given to high fat diet (HFD)-fed C57BL/6 mice for 10 weeks. Although there were no significant differences in the body weight changes and plasma lipid levels, hepatic triglyceride levels were significantly lower in the MSW group than in the normal tap water (TW)-drunken control group. Furthermore, the activity of fatty acid synthase (FAS) was significantly decreased and carnitine palmitoyltransferase (CPT) activity was increased in MSW group compared to TW group. Similarly, real-time PCR analysis revealed that mRNA expressions of lipogenic genes were lowered in MSW groups compared to the control group. In a morphometric observation on the liver tissue, accumulation of fats was remarkably reduced in MSW group. Meanwhile, in vitro assay, free radical scavenging activity measured by using diphenylpicrylhydrazyl (DPPH) was increased in MSW group. The 2'-7'-dichlorofluorescein diacetate (DCF-DA) staining followed with fluorescent microscopy showed a low intensity of fluorescence in MSW-treated HepG2 cells, compared to TW-treated HepG2 cells, which indicated that the production of reactive oxygen species by tert-butyl hydroperoxide (t-BHP) in HepG2 cells was decreased by MSW treatment. The antioxidant effect of MSW on t-BHP-induced oxidative stress in HepG2 cells was supported by the increased activities of intracellular antioxidant enzymes such as catalase and glutathione reductase. From these results, we speculate that MSW has an inhibitory effect on lipogenesis in liver and might play a protective role against cell damage by t-BHP-induced oxidative stress.
Animals ; Antioxidants ; Body Weight Changes ; Carnitine O-Palmitoyltransferase ; Catalase ; Diet ; Diet, High-Fat ; Fats ; Fatty Acid Synthetase Complex ; Fluorescence ; Glutathione Reductase ; Hep G2 Cells ; Korea ; Lipid Metabolism ; Lipogenesis ; Liver ; Mice ; Microscopy ; Oxidative Stress ; Plasma ; Reactive Oxygen Species ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Seawater ; tert-Butylhydroperoxide ; Water

AIMTo explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in murine MIN6 pancreatic beta-cells.

METHODSMIN6 cells were cultured in vitro. Cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin- V-PI staining. Nitric oxide levels were measured by Griess assay. Inducible nitric oxide synthase(iNOS) protein and NF-kappaBp65 fragment were detected by Western blot.

RESULTSExposure of 25 micromol/L t-BHP to MIN6 cells for 60 min, cell viability was reduced and the percentage of apoptosis was increased significantly. The levels of cytoplasmic iNOS protein and nitrite were elevated. Meanwhile, treatment with t-BHP resulted in nucleus NF-kappaBp65 fragment peaking at 20 min. Both L-NAME and N-Acetyl-l-cysteine (NAC) attenuated the elevated levels of nitrite and percentage of apoptosis due to t-BHP alone.

CONCLUSIONNF-kappa-iNOS-nitric oxide signalling pathway can mediated t-BHP induced apoptosis in MIN6 cells .

Animals ; Apoptosis ; Cell Line ; Insulin-Secreting Cells ; cytology ; Mice ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Oxidative Stress ; physiology ; Signal Transduction ; tert-Butylhydroperoxide ; pharmacology