Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.
Animals ; Antioxidants/*metabolism/*pharmacology ; Apoptosis/drug effects ; Caspase 3/metabolism ; Caspase 8/metabolism ; Cell Line ; Cisplatin/*toxicity ; Cysteine/*analogs & derivatives/pharmacology ; Gene Expression Regulation/*drug effects ; Gene Knockdown Techniques ; Glutathione/*metabolism ; Heme Oxygenase-1/genetics ; Intracellular Space/metabolism ; Male ; Metabolic Detoxication, Phase II/genetics ; Mice ; NF-E2-Related Factor 2/genetics ; Nitric Oxide/biosynthesis ; Organ of Corti/*drug effects/*metabolism ; RNA Interference ; Rats ; Reactive Oxygen Species/metabolism ; Superoxide Dismutase/genetics

Sulforaphane (SFN) is a naturally occurring compound which is known to induce the phase II antioxidant genes via Nrf2 activation, although the underlying mechanism has not been fully elucidated. In this study, we investigated Nrf2 induction in response to SFN in human bronchial epithelial BEAS-2B cells and determined the signaling pathways involved in this process. SFN treatment reduced cell viability. Prior to cell death, intracellular reactive oxygen species (ROS) were generated at a high rate within a minute of commencing SFN treatment. Pretreatment with antioxidant N-acetylcysteine (NAC) blocked SFN-induced decrease in cell growth. Erk1/2 was activated within 30 min of SFN addition, whereas Akt phosphorylation did not significantly change until the first 8 hr after SFN treatment but then became substantially low until 48 hr. Inhibition of Erk1/2 phosphorylation attenuated SFN-induced loss of cell viability. Nrf2 protein levels in both nuclear and whole cell lysates were increased by SFN treatment, which was dependent on ROS production. Knockdown of Nrf2 with siRNA attenuated SFN-induced heme oxygenase-1 (HO-1) up-regulation. Induction of the Nrf2/HO-1 after SFN treatment was potently suppressed by pretreatment with NAC. Overall, our results indicate that SFN mediates antioxidative and antiproliferative responses by generating ROS in BEAS-2B cells.
Acetylcysteine/pharmacology ; Anticarcinogenic Agents/pharmacology ; Antioxidants/*pharmacology ; Bronchi/cytology/*drug effects/metabolism ; Cell Line ; Cell Proliferation/*drug effects ; Epithelial Cells/drug effects/metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Free Radical Scavengers/pharmacology ; Heme Oxygenase-1/biosynthesis ; Humans ; NF-E2-Related Factor 2/biosynthesis/genetics ; Oxidative Stress/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; RNA, Small Interfering ; Reactive Oxygen Species/*metabolism ; Respiratory Mucosa/cytology/*drug effects/metabolism ; Signal Transduction/drug effects ; Thiocyanates/*pharmacology

Astrocytes are the most abundant cells in the central nervous system that play roles in maintaining the blood-brain-barrier and in neural injury, including cerebral malaria, a severe complication of Plasmodium falciparum infection. Prostaglandin (PG) D2 is abundantly produced in the brain and regulates the sleep response. Moreover, PGD2 is a potential factor derived from P. falciparum within erythrocytes. Heme oxygenase-1 (HO-1) is catalyzing enzyme in heme breakdown process to release iron, carbon monoxide, and biliverdin/bilirubin, and may influence iron supply to the P. falciparum parasites. Here, we showed that treatment of a human astrocyte cell line, CCF-STTG1, with PGD2 significantly increased the expression levels of HO-1 mRNA by RT-PCR. Western blot analysis showed that PGD2 treatment increased the level of HO-1 protein, in a dose- and time-dependent manner. Thus, PGD2 may be involved in the pathogenesis of cerebral malaria by inducing HO-1 expression in malaria patients.
Animals ; Astrocytes/*enzymology ; Blotting, Western ; Cell Line ; Gene Expression Profiling ; Heme Oxygenase-1/*biosynthesis ; Humans ; Malaria, Cerebral/*pathology ; Malaria, Falciparum/*complications/*pathology ; Plasmodium falciparum/*pathogenicity ; Prostaglandins/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDUnder an insulin resistance (IR) state, overproduction of reactive oxygen species (ROS) may be playing a major role in the pathogenesis of endothelial dysfunction, hypertension and atherosclerosis. Recently, increasing attention has been drawn to the beneficial effects of heme oxygenase-1 (HO-1) in the cardiovascular system. This study aimed to investigate the effects of HO-1 on vascular function of thoracic aorta in IR rats and demonstrate the probable mechanisms of HO-1 against endothelial dysfunction in IR states.

METHODSSprague-Dawley (SD) rats fed with high-fat diet for 6 weeks and the IR models were validated with hyperinsulinemic-euglycemic clamp test. Then the IR rat models (n = 44) were further randomized into 3 subgroups, namely, the IR control group (n = 26, in which 12 were sacrificed immediately and evaluated for all study measures), a hemin treated IR group (n = 10) and a zinc protoporphyrin-IX (ZnPP-IX) treated IR group (n = 8) that were fed with a high-fat diet. Rats with standardized chow diet were used as the normal control group (n = 12). The rats in IR control group, hemin treated IR group and ZnPP-IX treated IR group were subsequently treated every other day with an intraperitoneal injection of normal saline, hemin (inducer of HO-1, 30 micromol/kg) or ZnPP-IX (inhibitor of HO-1, 10 micromol/kg) for 4 weeks. Rats in the normal control group remained on a standardized chow diet and were treated with intraperitoneal injections of normal saline every other day for 4 weeks. Systolic arterial blood pressure (SABP) was measured by tail-cuffed microphotoelectric plethysmography. The blood carbon monoxide (CO) was measured by blood gas analysis. The levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), blood glucose (BG), insulin, total cholesterol (TC) and triglyceride (TG) in serum, and the levels of total antioxidant capacity (TAOC), malondialdehyde (MDA) and superoxide dismutase (SOD) in the aorta were measured. The expression of HO-1 mRNA and HO-1 protein in aortal tissue were detected by semi-quantitative RT-PCR and Western blot. The vasoreactive tensometry was performed with thoracic aortic rings (TARs).

RESULTSCompared with the normal control group, the levels of SABP, BG, insulin, TC, TG, NO, iNOS and MDA were higher, while the levels of CO, TAOC, SOD and eNOS were lower in IR control rats. After treatment of IR rats for 4 weeks a more intensive expression of HO-1 mRNA and HO-1 protein were observed in hemin treated IR group compared with the normal control group. And compared with 4-week IR control rats, the levels of CO, TAOC, SOD and eNOS were increased, while the levels of SABP and iNOS activity were lower in the hemin treated IR group. Administration of hemin in IR rats appeared to improve the disordered vasorelaxation of TARs to acetylcholine (ACh). Alternatively, the reverse results of SABP, CO, TAOC, SOD, iNOS and vasorelaxation responses to ACh were observed in IR rats with administration of ZnPP-IX.

CONCLUSIONSThe endothelial dysfunction in the aorta is present in the IR state. The protective effects of HO-1 against aortic endothelial dysfunction may be due to its antioxidation and regulative effect of vasoactive substances. It is proposed that hemin, inducer of HO-1, could be a potential therapeutic option for vascular dysfunction in IR states.

Animals ; Aorta ; drug effects ; physiology ; Carbon Monoxide ; blood ; Endothelium, Vascular ; drug effects ; physiology ; Enzyme Induction ; drug effects ; Heme Oxygenase-1 ; analysis ; biosynthesis ; genetics ; Hemin ; pharmacology ; Insulin Resistance ; Male ; Nitric Oxide ; blood ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Systole ; drug effects

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.
Active Transport, Cell Nucleus ; Animals ; Aorta/cytology ; Cell Nucleus/metabolism ; Cell Proliferation/*drug effects ; Cells, Cultured ; Curcumin/analogs & derivatives/*pharmacology ; Cyclin-Dependent Kinase Inhibitor p21/biosynthesis/metabolism ; Gene Expression Regulation ; Heme Oxygenase (Decyclizing)/biosynthesis/genetics/*physiology ; Heme Oxygenase-1/biosynthesis/genetics/*physiology ; Humans ; Metalloporphyrins/pharmacology ; Muscle, Smooth, Vascular/drug effects/*physiology ; Myocytes, Smooth Muscle/drug effects/*physiology ; NF-E2-Related Factor 2/metabolism ; Protoporphyrins/pharmacology ; Rats ; Regulatory Sequences, Nucleic Acid ; Response Elements ; Tumor Necrosis Factor-alpha/pharmacology

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is an animal model of posterior uveitis and heme oxygenase-1 (HO-1) is a well-known anti-oxidant factor. However, there is no report a protective role of HO-1 on EAU in vivo. To verify that HO-1 is induced in EAU by interphotoreceptor retinoid-binding protein (IRBP), that an HO-1 inducers ameliorates the associated inflammation, and that an HO-1 inhibitor exacerbates this inflammation. METHODS: Forty four Lewis rats were given either 40 mol/kg hemin or 40 mol/kg SnPP (tin protoporphyrin IX) by intraperitoneal injection and twenty two uveitis control rats were injected with 0.5 mL of saline once daily 5-20 days after IRBP immunization inducing EAU. Three normal control rats were used for Western blotting and ELISA assay of HO-1. The clinical uveitis signs of inflammation were scored in the three groups from 0 to 4 on alternate three days. To confirm the clinical results, histological and immunohistochemical stain of HO-1 were performed on the day of peak inflammation and Western blotting and ELISA assay of HO-1 were performed on 6th, 12th and 18th day after IRBP immunization. RESULTS: Hemin, an inducer of HO-1, ameliorated the clinical signs of EAU. In contrast, SnPP-treated rats show that the severity of the clinical sign were exacerbated at the peak period of the disease. These results are roughly compatible with histological, immunoblotting, and immunohistochemical evaluations and an ELISA assay of HO-1. CONCLUSIONS: We suggest that HO-1 plays an important protective role in EAU.
Animals ; Autoimmune Diseases/diagnosis/*drug therapy/metabolism ; Blotting, Western ; Disease Models, Animal ; Enzyme Inhibitors/*administration & dosage ; Enzyme-Linked Immunosorbent Assay ; Heme Oxygenase-1/*biosynthesis/drug effects ; Hemin/*administration & dosage ; Immunohistochemistry ; Injections, Intraperitoneal ; Male ; Metalloporphyrins/*administration & dosage ; Microscopy, Acoustic ; Protoporphyrins/*administration & dosage ; Rats ; Rats, Inbred Lew ; Retinitis/diagnosis/*drug therapy/metabolism ; Treatment Outcome ; Uveitis, Posterior/diagnosis/*drug therapy/metabolism

OBJECTIVETo investigate the effects of heme oxygenase 1 inducer hemin on protection of ischemia-reperfusion injury in rats and its mechanisms.

METHODSThe Langendorff model of isolated rat heart was used; the left anterior descending coronary artery was occluded for 30 min and subsequently reperfused for 2 h. Then the ventricular function and infarct size were measured.

RESULTHemin preconditioning prevented the increase in LVEDP, decrease in LVDP and +/- dp/dt(max) in the isolated ischemia-reperfusion rat hearts. The leakage of LDH and CK in the coronary effluent was significantly declined in hemin-treated rat hearts. And the infarct size was also reduced. Administration of a blocker of mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) 5-HD (5 mg/kg) before hemin preconditioning increased the LVEDP, and reduced the LVDP and +/- dp/dt(max). The leakage of LDH and CK in the coronary effluent and the infarct size were also increased compared with only hemin-treated rat hearts. Pretreatment of the rats with a blocker of sarcolemmal ATP-sensitive potassium channel (sarcK(ATP)) HMR-1098 (6 mg/kg) before hemin preconditioning also abolished the protective effect. Infusion of paxilline (1 micromol/L), a blocker of calcium activated potassium channel (K(Ca)) for 10 min before ischemia/reperfusion led to larger infarct size and poorer myocardial performance as compared with the hemin group. The leakage of LDH and CK in the coronary effluent was also increased.

CONCLUSIONBoth mitoK(ATP)and sarcK(ATP)channels activation are required for the delayed cardioprotection induced by hemin. The opening of K(Ca) channels-dependent mechanism may be involved in the protection.

Animals ; Cardiotonic Agents ; pharmacology ; Heme Oxygenase-1 ; biosynthesis ; Hemin ; pharmacology ; In Vitro Techniques ; Ischemic Preconditioning, Myocardial ; methods ; Male ; Myocardial Infarction ; metabolism ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; prevention & control ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate apoptosis and expression of heme oxygenase-1 (HO-1) induced by cadmium in human embryonic kidney cells (HEK239T).

METHODSThe MTT method was used for determining the cell proliferation activity. The apoptosis was determined by the flow cytometry. The HO-l mRNA expression and protein level were detected by RT-PCR method and Western blot respectively.

RESULTSThe ratios of apoptosis in HEK239T cells were 11.90% +/- 0.28%, 9.27% +/- 1.73%, 9.79% +/- 0.67% and 8 .97% +/- 1.60% at the concentration of 5.0, 10.0, 20.0 and 40.0 micromol/L CdCl(2) respectively, higher than those in the control group (6.69% +/- 0.46%) with the significant difference (P < 0.01). The CdCl(2) of between 10 and 40 micromol/L could highly induce the expression of HO-1 of the human embryonic kidney cells. The expression would increase slowly till the flat stage with the increase of the dosage and then would decrease slightly over time.

CONCLUSIONThe cadmium can induce the apoptosis of the human embryonic kidney cells and up-regulate the expression of HO-1.

Apoptosis ; drug effects ; Cadmium ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Heme Oxygenase-1 ; biosynthesis ; genetics ; Humans ; Kidney ; cytology ; embryology ; metabolism ; RNA, Messenger ; genetics

Animals ; Heme Oxygenase-1 ; biosynthesis ; Kidney ; drug effects ; metabolism ; Male ; Mercuric Chloride ; toxicity ; Oxidative Stress ; drug effects ; Random Allocation ; Rats ; Rats, Wistar

Carbon monoxide (CO), a metabolite of heme catalysis by heme oxygenase (HO), has been proposed to have anti-oxidative, anti-inflammatory and anti-apoptotic functions. Lipopolysaccharide (LPS)-induced lung injury (LI) is characterized by oxidative stress, inflammatory reaction and excessive pulmonary cell apoptosis. So we supposed that CO might have protection against LI. LI in rats was induced by intravenous injection of LPS (5 mg/kg). To observe the effect of CO inhalation, LI rats were exposed to 2.5 x 10(-4) (V/V) CO for 3 h. CO-induced changes of lung oxidative stress parameters, inflammatory cytokines, cell apoptosis, HO-1 expression and histology were examined. Results revealed that expressions of the tumor necrosis factor-alpha (TNF-alpha) and interlukin-6 (IL-6), activities of maleic dialdehyde (MDA) and myeloperoxidase (MPO), and cell apoptosis in LPS injection + CO inhalation group were (0.91+/-0.25) pg/mg protein, (0.64+/-0.05) pg/mg protein, (1.02+/-0.23) nmol/mg protein, (7.18+/-1.62) U/mg protein and (1.60+/-0.34)%, respectively, significantly lower than the corresponding values in LI group [(1.48+/-0.23) pg/mg protein, (1.16+/-0.26) pg/mg protein, (1.27+/-0.33) nmol/mg protein, (8.16+/-1.49) U/mg protein and (3.18+/-0.51) %, P<0.05]. Moreover, CO inhalation obviously increased the expressions of HO-1 and interlukin-10 (IL-10) and activity of superoxide dismutase (SOD) [(5.43+/-0.92), (0.26+/-0.07) pg/mg protein and (60.09+/-10.21) U/mg protein in LPS injection + CO inhalation group vs (3.08+/-0.82), (0.15+/-0.03) pg/mg protein and (50.98+/-6.88) U/mg protein in LI group, P<0.05]. LI was attenuated by CO inhalation. Our study demonstrates that inhalation of low concentration of CO protects lung against LPS-induced injury via anti-oxidant, anti-inflammation, anti-apoptosis and up-regulation of HO-1 expression.
Administration, Inhalation ; Animals ; Apoptosis ; drug effects ; Carbon Monoxide ; administration & dosage ; Carboxyhemoglobin ; analysis ; Cytokines ; biosynthesis ; Heme Oxygenase-1 ; genetics ; Lipopolysaccharides ; toxicity ; Lung ; drug effects ; metabolism ; pathology ; Male ; Oxidative Stress ; drug effects ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley