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

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

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

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

OBJECTIVE: To investigate the effect of hemin on lung injury following severe acute pancreatitis (SAP) in rats and to explore its rudimentary mechanism. METHODS: Thirty-six rats were randomly divided into 3 groups: a control group, a SAP model group, and a hemin-pretreated group. Rats were sacrificed 12 hours after inducing SAP model. The pathological changes of the pancreas and lungs were observed under light microscope. Expression of heme oxygenase (HO-1) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), NF-kappaB activity in the lung tissues was detected by enzyme linked immunosorbent assay (ELISA), and the serum levels of TNF-alpha and IL-6 were measured by ELISA. RESULTS: HO-1 was induced during experimental SAP, NF-kappaB activity in the lung tissues was elevated after the induction of SAP and the serum levels of TNF-alpha and IL-6 were significantly elevated. Hemin further upregulated the expression of HO-1 mRNA, decreased NF-kappaB activity drastically, and inhibited the serum levels of TNF-alpha and IL-6 significantly (P < 0.05). Hemin could treat SAP by alleviating the pancreatic and lung injury. CONCLUSION Hemin moderates the inflammatory reaction and decreases the lung injury following SAP, the mechanism of which may be closely related to the upregulation of expression of HO-1 mRNA, the inhibitory effect on NF-kappaB, and adjustment of cytokines.
Acute Lung Injury ; drug therapy ; etiology ; Animals ; Cytokines ; genetics ; metabolism ; Female ; Heme Oxygenase (Decyclizing) ; genetics ; metabolism ; Hemin ; therapeutic use ; Male ; NF-kappa B ; genetics ; metabolism ; Pancreatitis, Acute Necrotizing ; complications ; drug therapy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the protection of Heme oxygenase-1 (HO-1) from lipopolysaccharide (LPS)-induced cardiocyte injury and its mechanism.

METHODSCardiocyte was isolated from SD neonate rat and cultured in vitro, and was divided into control group (normal culture), LPS group (with stimulation of 30 micromoL/L LPS for 1 hour), LPS + Hemin group (with same treatment to LPS group after stimulation of 5 micromoL/L Hemin for 1 hour), and LPS + ZnPP group (with same treatment to LPS group after stimulation of 3 micromoL/L ZnPP for 1 hour). The level of lactic-dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) were measured by thio-barbituric acid and xanthine oxidase techniques. The cell heart rhythm, survival rate and apoptosis rate were examined. The expressions of nuclear factor kappaB (NF-kappaB), HO-1 and tumor necrosis factor-alpha (TNF-alpha) were measured with Western blotting. The HO-1 mRNA was examined by RT-PCR.

RESULTSThe level of LDH and MDA in LPS, LPS + Hemin, and LPS + ZnPP groups were (113 +/- 15), (79 +/- 13), (154 +/- 22) U/L, and (1.88 +/- 0.36), (1.16 +/- 0.32), (2.84 +/- 0.44) mmoL/L respectively, which were all obviously higher than those in control group [(69 +/- 10) U/L, (0.87 +/- 0.25) mmol/L, P < 0.05]. The level of SOD in LPS, PS + Hemin, and LPS + ZnPP groups (17.8 +/- 1.8, 22.5 +/- 2.4, 13.4 +/- 1.5 U/mL, respectively) was all obviously lower than that in control group (24.3 +/- 3.6 U/mL, P < 0.05). The apoptosis rate and heart rhythm were obviously higher and survival rate significantly lower in LPS, LPS + Hemin, and LPS + ZnPP groups than those in control group (P < 0.05). The level of HO-1mRNA in LPS, LPS + Hemin, and LPS + ZnPP groups was higher than that in control group (P < 0.01), among which LPS + Hemin group was the highest. The level of HO-1, TNF-alpha and NF-kappaB in LPS, LPS + Hemin, and LPS + ZnPP groups was higher than those in control group (P < 0.05), among which the level of HO-1 protein in LPS + Hemin group was the highest, the level of TNF-alpha and NF-kappaB in LPS + ZnPP group was highest.

CONCLUSIONLPS can induce cardiocyte injury, which can be inhibited through the anti-inflammatory, anti-oxidant, and anti-apoptosis functions by HO-1.

Animals ; Caspase 3 ; metabolism ; Cells, Cultured ; Heme Oxygenase (Decyclizing) ; metabolism ; Hemin ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; Malondialdehyde ; metabolism ; Myocytes, Cardiac ; metabolism ; NF-kappa B ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDUrinary trypsin inhibitor inhibits the enhanced production of pro-inflammatory molecules. Hemeoxygenase-1 induction protects against ischemia/reperfusion injury, oxidative stress, inflammation, transplant rejection, apoptosis, and other conditions. However, it is unknown if a combined hemin and ulinastatin pretreatment could result in protective effects for septic shock. In this study, we investigated the role of hemin pretreatment combined with ulinastatin on septic shock in rats.

METHODSEighty healthy, male Sprague-Dawley rats were randomly divided into four groups: group S, group H, group U and group HU. Groups S and U received 1 ml normal saline intraperitoneally, while groups H and HU both received 1 ml (100 mg /kg) hemin. Twenty-four hours later, 0.5 ml (10 mg/kg) E. coli lipopolysaccharide was injected intravenously to replicate the experimental model of septic shock. After an initial 25% decrease in the mean arterial pressure, corresponding to time point 0, groups HU and U received 0.5 ml 10 000 U/kg ulinastatin intravenously, and the others received 0.5 ml normal saline.

RESULTSThe number of deaths in groups H and U was lower than that in the group S (P < 0.05), and was higher than that in group HU (all P < 0.05) respectively. The mean arterial pressure (MAP) in the group S was significantly greater than that in group H (P < 0.05), and was lower than that in group HU and group U (P < 0.05). The plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr) and blood urea nitrogen (BUN), the malondial-dehyde (MDA) of liver, kidney and lung, and the lung Evans blue (EB) contents in groups H and U, were greater than that in group HU (all P < 0.05), and were lower than that in group S (all P < 0.05). In contrast, the plasma levels of CO in groups H and HU were higher than that in groups S and U (all P < 0.05), and SOD of liver, kidney and lung in groups H and U were higher than that in group S, and were lower than that in group HU (all P < 0.05). The levels of TNF-alpha, IL-6, IL-8 and beta-glucuronidase (GCD) activity of plasma in groups U and HU were lower than those in groups H and S, all having a P < 0.05, while there were no significant differences between group H and group S, or between group HU and group U (all P > 0.05). The HO-1 mRNA and HO-1 protein levels from hepatic, renal, and pulmonary tissue in groups S and U were lower than those in groups H and HU (all P < 0.05), but there were no significant differences between groups S and U, or between groups H and HU (all P > 0.05). The HO-2 mRNA and HO-2 protein were not significantly different among the four groups (all P > 0.05).

CONCLUSIONSCombined pretreatment with hemin and ulinastatin in septic shock rats results in an improved response by the upregulation of HO-1 protein followed by increasing CO with resistance to increased oxidative stress, restraining the release of inflammatory mediators, and inhibiting beta-GCD activity.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Blood Pressure ; drug effects ; Blood Urea Nitrogen ; Creatinine ; blood ; Cytokines ; blood ; Glycoproteins ; therapeutic use ; Heme Oxygenase (Decyclizing) ; analysis ; genetics ; Hemin ; therapeutic use ; Male ; Malondialdehyde ; blood ; Rats ; Rats, Sprague-Dawley ; Shock, Septic ; drug therapy ; physiopathology ; Superoxide Dismutase ; metabolism