This study investigated the effects of N-acetylcysteine (NAC) and ascorbic acid (AA) on hemin-induced K562 cell erythroid differentiation and the role of reactive oxygen species (ROS) in this process. Hemin increased ROS levels in a concentration-dependent manner, whereas NAC and AA had opposite effects. Both NAC and AA eliminated transient increased ROS levels after hemin treatment, inhibited hemin-induced hemoglobin synthesis, and decreased mRNA expression levels of β-globin, γ-globin, and GATA-1 genes significantly. Pretreatment with 5,000 μmol/L AA for 2 h resulted in a considerably lower inhibition ratio of hemoglobin synthesis than that when pretreated for 24 h, whereas the ROS levels were the lowest when treated with 5,000 μmol/L AA for 2 h. These results show that NAC and AA might inhibit hemin-induced K562 cell erythroid differentiation by downregulating ROS levels.
Acetylcysteine ; pharmacology ; Antioxidants ; pharmacology ; Ascorbic Acid ; pharmacology ; Cell Differentiation ; drug effects ; Down-Regulation ; Erythroid Cells ; drug effects ; Hemin ; pharmacology ; Humans ; K562 Cells ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the effect of simulated microgravity on erythroid differentiation of K562 cells and explore the possible mechanism.

METHODSThe fourth generation rotating cell culture system was used to generate the simulated microgravity environment. Benzidine staining was used to evaluate the cell inhibition rate, and real-time quantitative PCR (qRT-PCR) was used to detect GATA-1, GATA-2, Ets-1, F-actin, β-Tubulin and vimentin mRNA expressions. The changes of cytoskeleton were observed by fluorescence microscopy, and Western blotting was employed to assay F-actin, β-tubulin and vimentin protein expression levels.

RESULTSBenzidine staining showed that simulated microgravity inhibited erythroid differentiation of K562 cells. K562 cells treated with Hemin presented with increased mRNA expression of GATA-1 and reduced GATA-2 and Ets-1 mRNA expressions. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1, F-actin, β-tubulin and vimentin mRNA expressions and up-regulated mRNA expressions of GATA-2 and Ets-1, and reduced F-actin, β-tubulin and vimentin protein expressions. Exposure to simulated microgravity caused decreased fluorescence intensities of cytoskeletal filament F-actin, β-tubulin and vimentin in the cells.

CONCLUSIONSimulated microgravity inhibits erythroid differentiation of K562 cells possibly by causing cytoskeleton damages to result in down-regulation of GATA-1 and up-regulation of GATA-2 and Ets-1 expressions.

Actins ; metabolism ; Cell Differentiation ; Down-Regulation ; GATA1 Transcription Factor ; metabolism ; GATA2 Transcription Factor ; metabolism ; Hemin ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Protein c-ets-1 ; metabolism ; Tubulin ; metabolism ; Up-Regulation ; Vimentin ; metabolism ; Weightlessness Simulation

OBJECTIVETo investigate the therapeutic effects of hemin, an inducer of heme oxygenase, in a rat model of gestational hypertension and explore the possible mechanism.

METHODSEighteen pregnant SD rats at day 12 of gestation were randomized equally into gestational hypertension model group, hemin treatment group, and normal pregnancy (control) group. In the former two groups, the rats were subjected to daily nitro-L-arginine methyl ester (L-NAME, 80 mg/kg) gavage since gestational day 14 for 7 consecutive days to induce gestational hypertension; saline was administered in the same manner in the control rats. The rats in hemin group received daily intraperitoneal injection of hemin (30 mg/kg) starting from gestational day 16. HO activity and carboxyhemoglobin (COHb) level in rat placental tissue were detected with spectrophotometric method, and soluble vascular endothelial growth factor receptor-1 (sFlt-1) and vascular endothelial growth factor (VEGF) level in the placental tissue homogenate supernatant were detected using ELSIA.

RESULTSAt gestational day 20, the blood pressure and 24-h urinary protein were significantly higher in the model group than in the other two groups (P<0.05), and were higher in hemin group than in the control group (P<0.05); HO activity and COHb content in the placenta tissue were the lowest in the model group (P<0.05), and was lower in hemin group than in the control group (P<0.05). The level of sFlt-1 was significantly higher and VEGF level significantly lower in the model group than in the other two groups (P<0.05); sFlt-1 level remained higher and VEGF lower in hemin group than in the control group (P<0.05).

CONCLUSIONHemin can reduce blood pressure and urinary protein in rats with gestational hypertension possibly by up-regulating HO activity, enhancing carbon monoxide production, reducing sFlt-1 and increasing VEGF in the placental tissue.

Animals ; Blood Pressure ; Carbon Monoxide ; metabolism ; Disease Models, Animal ; Female ; Heme Oxygenase (Decyclizing) ; Hemin ; pharmacology ; Hypertension, Pregnancy-Induced ; drug therapy ; Placenta ; drug effects ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism

OBJECTIVEInflammation plays a critical role in secondary brain damage after intracerebral hemorrhage (ICH). However, the mechanisms of inflammatory injury following ICH are still unclear, particularly the involvement of NLRP3 inflammasome, which are crucial to sterile inflammatory responses. In this study, we aim to test the hypothesis that NLRP3 signaling pathway takes a vital position in ICH-induced secondary inflammatory damage and detect the role of N-methyl-D-aspartic acid receptor 1 (NMDAR1) in this progress.

METHODSICH was induced in mice by microinjection of hemin into the striatum. The protein levels of NMDAR1, NMDAR1 phosphorylation, NLRP3 and IL-1b were measured by Western blot. The binding of NMDAR1 to NLRP3 was detected by immunoprecipitation.

RESULTSThe expression of NMDAR1, NMDAR1 phosphorylation, NLRP3 and IL-1b were rapidly increased after ICH. Hemin treatment enhanced NMDAR1 expression and NMDAR1 phosphorylation, as well in cultured microglial cells treated by hemin. Hemin up regulated NLRP3 and IL-1b level, which was reversed by MK801 (NMDAR antagonist) in vitro. Hemin also promoted the binding of NMDAR1 to NLRP3.

CONCLUSIONOur findings suggest that NMDAR1 plays a pivotal role in hemin-induced NLRP3-mediated inflammatory damage through synergistic activation.

Animals ; Cells, Cultured ; Cerebral Hemorrhage ; complications ; Hemin ; pharmacology ; Inflammation ; etiology ; Mice ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein ; physiology ; Phosphorylation ; Receptors, N-Methyl-D-Aspartate ; physiology ; Signal Transduction

The role of ROS in hydroquinone-induced inhibition of K562 cell erythroid differentiation was investigated. After K562 cells were treated with hydroquinone for 24 h, and hemin was later added to induce erythroid differentiation for 48 h, hydroquinone inhibited hemin-induced hemoglobin synthesis and mRNA expression of γ-globin in K562 cells in a concentration-dependent manner. The 24-h exposure to hydroquinone also caused a concentration-dependent increase at an intracellular ROS level, while the presence of N- acetyl-L-cysteine prevented hydroquinone- induced ROS production in K562 cells. The presence of N-acetyl-L-cysteine also prevented hydroquinone inhibiting hemin-induced hemoglobin synthesis and mRNA expression of γ-globin in K562 cells. These evidences indicated that ROS production played a role in hydroquinone-induced inhibition of erythroid differentiation.
Acetylcysteine ; pharmacology ; Cell Differentiation ; drug effects ; Dose-Response Relationship, Drug ; Hemin ; pharmacology ; Humans ; Hydroquinones ; pharmacology ; K562 Cells ; drug effects ; Reactive Oxygen Species ; metabolism ; gamma-Globins ; genetics

OBJECTIVETo investigate whether the cardioprotective effect of hemin against ischemia/reperfusion (I/R) injury is through the inhibition of calpain activity, and to explore its underlying mechanism.

METHODSSixty-four SD rats were randomly divided into eight groups (n = 8): sham, I/R, MDL+ I/R, MDL, hemin + I/R, hemin, and ZnPP + hemin+ I/R, ZnPP. Iangendorff isolated rat heart perfusion model was used. The rat hearts were suffered from 40 min of ischemia followed by 30 min of reperfusion. After that, left ventricular developed pressure (LVDP) was recorded. Infarct size and release of lactate dehydrogenase (LDH) were measured. Calpain, heme oxygenase (HO), and caspase 3 activities were evaluated. Expression of calpastatin protein was detected by Western blot.

RESULTS(1) After suffered from ischemia/reperfusion, the calpain activity and caspase 3 activity increased. MDL28170, an inhibitor of calpain, prevented ischemia/reperfusion induced increases in LDH and infarct size, improved the LVDP recovery. (2) Compared with ischema/reperfusion rat hearts, pretreatment of hemin enhanced the HO-1 activity, decreased the calpain and caspase 3 activities, declined LDH release and infarct size, and improved LVDP recovery. (3) Ischemia/reperfusion reduced the expression of calpastatin protein in rat hearts, which was inhibited by hemin pretreatment. And HO-1 inhibitor could abolish the cardioprotection of hemin.

CONCLUSIONCardioprotective effect of hemin against ischemia/reperfusion injury is through the inhibition of calpain activity, the mechanism might be involved in the increase in calpastatin protein expression.

Animals ; Calpain ; metabolism ; Cardiotonic Agents ; pharmacology ; Caspase 3 ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hemin ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Myocardial Reperfusion Injury ; drug therapy ; Rats ; Rats, Sprague-Dawley

OBJECTIVEIn order to get the method to improve the salt resistance of seeds and seedlings for Cassia obtusbifolia under NaCl stress, seed germination and physiological characteristics of C. obtusifolia seedlings were studied.

METHODSeveral physiological indexes of C. obtusifolia seeds treated with exogenous carbon monoxide donor hematin under NaCl stress like the germination vigor, germination rate, germination index and vigor index were measured. And other indexes like the relative water content, the contents of photosynthetic pigment, chlorophyll fluorescence parameters, the contents of soluble sugar, protein and proline, malondialdehyde (MDA), the activities of superoxide (SOD), peroxidase (POD) and catalase (CAT) were also measured.

RESULTThe germination indexes of C. obtusifolia seeds under NaCl stress had been inhibited obviously. But after the treatment of hematin, every germination indexes were all increased. The result showed that the treatment of exogenous CO donor hematin obviously improved the germination vigor, germination rate, germination index and vigor index, increased the content of chlorophyll a, chlorophyll b, total chlorophyll, improved the photochemical efficiency of photosystem II (Fv/Fm), photochemical efficiency (Fv'/Fm'), PS II actual photochemical efficiency (phiPS II), photochemical quench coefficient (qP), decreased non-photochemical quenching coefficient (NPQ) and the content of malondialdehyde (MDA) , increased the relative water content of leaves and the content of soluble surge, protein and proline. Meanwhile, the results also indicated that CO improved the activities of superoxide (SOD), peroxidase (POD) and catalase (CAT). The effects of CO could be reversed when CO scavenger Hb is added.

CONCLUSIONExogenous CO donor hematin with appropriate concentration could significantly alleviate the damages to the seeds and seedlings of C. obtusifolia under NaCl stress and promote the salt resistance of the seeds and seedlings through improving the germination indexes, the photochemical efficiency and the antioxidase activities of the seedlings.

Carbohydrates ; analysis ; Carbon Monoxide ; metabolism ; Cassia ; drug effects ; growth & development ; metabolism ; Catalase ; metabolism ; Chlorophyll ; metabolism ; Germination ; drug effects ; physiology ; Hemin ; metabolism ; pharmacology ; Malondialdehyde ; metabolism ; Peroxidase ; metabolism ; Photosystem II Protein Complex ; metabolism ; Plant Proteins ; metabolism ; Proline ; metabolism ; Seedlings ; drug effects ; growth & development ; metabolism ; Seeds ; growth & development ; Sodium Chloride ; pharmacology ; Superoxide Dismutase ; metabolism ; Time Factors ; Water ; metabolism

OBJECTIVETo investigate the effect of heme oxygenase/carbon monoxide (HO-1/CO) system on lipid deposition at aortic intima and the mechanism involved in hyperlipidemic rabbits.

METHODSTotally 32 rabbits, were divided into four groups. One group as control. Three groups for the following treatments: 1.5% cholesterol ration (Ch group, n = 8); 1.5% cholesterol ration plus HO-1 inducer hemin (Hm group, n = 8); and instead of hemin, the HO-1 inhibitor, zinc protoporphyrin IX (Zn group, n = 8) was given by injection into the abdominal cavity. Experiments were lasted for 12 weeks. Rabbit aortas were then isolated as the samples for histopathologic and ultrastructural examination. The protein expressions of HO-1 and endothelin-1 (ET-1) were investigated by immunohistochemical staining and Western blot analysis.

RESULTSComparing with the Ch group, rabbits of the Hm group showed a remarkably less extent of lipid deposition at the aortic intima [(17.9 ± 3.0)% vs (54.0 ± 4.2)%], and rabbits of the Zn group had a marked extent of lesion development [(61.1 ± 3.5)%]. Lipid deposition, endothelial damage and neo-intimal formation were less severe in rabbits of the Hm group than those in the Zn or Ch group, respectively. Comparing with the control group, rabbits of the Ch group showed a significant decrease of aortic NO production and cNOS activity. However, there were an enhancement of CO production and HO-1 activity (P < 0.01). Compared with Ch group, rabbits of the Hm group showed a remarkable elevation of aortic HO activity and CO production, whereas rabbits of the Zn group showed a marked decrease of both parameters. Compared with the Ch group, rabbits of the Hm group demonstrated a marked reduction of aorta ET-1 expression, whereas Zn group had a significantly higher ET-1 expression.

CONCLUSIONSModulation of HO-1/CO system may improve vascular endothelial function and inhibit smooth muscle cell proliferation in hypercholesterolemic rabbits, likely through a compensatory mechanism and a reduction of ET-1 expression, eventually leading to an inhibition of atherosclerotic plaque development.

Animals ; Aorta ; metabolism ; pathology ; Carbon Monoxide ; metabolism ; Cholesterol ; pharmacology ; Endothelin-1 ; metabolism ; Enzyme Inhibitors ; pharmacology ; Heme Oxygenase-1 ; antagonists & inhibitors ; metabolism ; Hemin ; pharmacology ; Hyperlipidemias ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Plaque, Atherosclerotic ; metabolism ; pathology ; prevention & control ; Protoporphyrins ; pharmacology ; Rabbits ; Tunica Intima ; metabolism ; pathology

OBJECTIVETo identify the gene expression profiles associated with the apoptosis of pulmonary arterial smooth muscle cells stimulated by carbon monoxide (CO).

METHODSPrimary cultured Sprague-Dawley rat pulmonary arterial smooth muscle cells (PASMC) were stimulated by platelet-derived growth factor (PDGF, 20 ng/mL) and hemin (20 μmol/L). Cells were harvested after 2 hrs and Affymetrix microarrays were used to detect the gene expression profile.

RESULTSSome genes associated with Map2k3 (P38) signal pathway, such as CyclinD1, CyclinH, CyclinL1, MAP2K3, Kras and Nras, were upregulated, but P27 expression was downregulated after PDGF treatment. After endogenous CO treatment, some genes associated with P53 pathway, such as Gadd45α, P21 and Trp53inp1, were upregulated.

CONCLUSIONSP53 pathway probably plays an important role in apoptosis of pulmonary arterial smooth muscle cells treated with endogenous CO.

Animals ; Apoptosis ; Carbon Monoxide ; physiology ; Gene Expression Profiling ; Hemin ; pharmacology ; Male ; Muscle, Smooth, Vascular ; pathology ; Myocytes, Smooth Muscle ; pathology ; Pulmonary Artery ; pathology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Suppressor Protein p53 ; physiology ; p38 Mitogen-Activated Protein Kinases ; physiology

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