To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Animals ; Carbon Monoxide ; metabolism ; Cell Hypoxia ; Cyclic GMP ; metabolism ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Up-Regulation

To explore the expression of heme oxygenase-1 (HO-1) in the peripheral blood mononuclear cells (PBMCs) and its relationship with pulmonary ventilation function in asthmatic patients, 18 asthmatic patients and 18 healthy subjects were selected. HO-1 protein and mRNA levels in PBMCs were measured by immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Blood carbon monoxide Hb (COHb), serum total IgE and pulmonary ventilatory function were observed. Our results showed that the percentage of cells positive for immunohistochemical staining of HO-1 were significantly higher in asthmatic patients (41.72 +/- 7.44) % than that in with healthy subjects (10.45 +/- 4.36) % (P < 0.001) and the optical density of PBMC HO-1 mRNA was higher in asthmatic patients (26.05 +/- 4.14) than that in healthy subjects (10. 82 +/- 4.26) (P < 0.001). The relation analysis showed that PBMC HO-1 protein and mRNA levels had significantly negative relation with FEV1%, PEFR, MEFR50%, respectively (r = -0.51-0.89, P < 0.05-0.001, respectively) and a positive relation with COHb and serum total IgE (r = 0.48-0. 85, 0.05-0.001, respectively). It is concluded that the expression of PBMC HO-1 protein and mRNA increased significantly in asthmatic patients, and HO-1 may play a significant role in the pathogenesis of asthma. The expression of HO-1 may bear a relation with severity of asthma.
Asthma/blood ; Asthma/*enzymology ; Carbon Monoxide/blood ; Heme Oxygenase-1/*biosynthesis ; Heme Oxygenase-1/blood ; Immunoglobulin E/*blood ; Leukocytes, Mononuclear/*enzymology ; RNA, Messenger/blood

To investigate the expression of the HO-1 gene in PC12 cells in hypoxic environment and gain further insight to the role of HO-1 in cerebral ischemia, PC12 cells were exposed to hypoxia environment (95% N2, 5% CO2) for 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h respectively. The level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR); the volume of COHb in the media were measured spectrophotometrically and the cGMP concentration of PC12 cell extracts was determined by radioimmunoassay. We found that after exposure to hypoxia for 1 h, 4 h, 8 h, 12 h, 24 h, HO-1 mRNA increased by 3%, 4%, 17%, 31% 36% as compared with that in control group respectively (P < 0.01 or P < 0.05); the COHb increased by 12%, 29%, 59%, 88%, 94% as compared with that in control group respectively (P < 0.01 or P < 0.05), and the cGMP concentration were 2.2, 3.4, 5.2, 8.1, 10.9-fold as that of the control group (P < 0.01). We are led to conclude that hypoxia induced the expression of HO-1 gene, the production of endogenous CO, and the concentration of cGMP was elevated as well.
Carbon Monoxide/metabolism ; Cell Hypoxia ; Cyclic GMP/metabolism ; Heme Oxygenase (Decyclizing)/biosynthesis ; Heme Oxygenase (Decyclizing)/*genetics ; Heme Oxygenase-1 ; PC12 Cells ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Up-Regulation

OBJECTIVESTo observe the function of heme oxygenase (HO) in the lung damage in hepatic cirrhosis rats.

METHODSLiver cirrhosis model rats were made by CCl4. Lung samples taken from normal and cirrhotic rats were examined for HO-1 and HO-2 protein and expression distribution with immunohistochemical staining and western blot.

RESULTSLiver cirrhosis model rats were successfully constructed. There was a notable increase of HO-1 staining (0.062+/-0.021 vs 0.185+/-0.044, t=11.24, P<0.01) and protein expression (0 vs 5294.92+/-46.02, t=11.45, P<0.01) in both vascular and bronchial smooth muscle cells and endothelium in cirrhotic rats, however, no statistical difference of HO-2 between cirrhotic and normal rats was observed.

CONCLUSIONThe HO-CO pathway is probably involved in the pathogenesis of lung damage in hepatic cirrhosis rats.

Animals ; Carbon Tetrachloride Poisoning ; Heme Oxygenase (Decyclizing) ; analysis ; biosynthesis ; Heme Oxygenase-1 ; Liver Cirrhosis, Experimental ; enzymology ; Lung ; enzymology ; Male ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley

Heme oxygenase (HO) is a rate-limiting enzyme for endogenous carbon monoxide (CO) production. Recently it has been suggested that endogenous CO plays an important role in regulating smooth muscle tone. The development of bladder outlet obstruction in men with benign prostates hyperplasia was shown to be related to the prostates smooth muscle tone, but it was not clear whether endogenous HO/CO system mediates prostates smooth muscle activity. To investigate the influence of sex hormone on the expression of heme oxygenase (HO) gene in rat ventral prostate, we created the model of castrated male rats to test the mRNA levels of HO-1 and HO-2 by RT-PCR, and used immunohistochemical staining procedures with image analytic technical system to confirm the effects of exogenous androgen and estrogen on the expression of HO-1 and HO-2 protein in rat ventral prostate. The results showed that two isoforms of HO were present in rat ventral prostate. The epithelial cells of acini and fibromuscular stroma of the rat prostate displayed HO-1 immunoreactivity, whereas HO-2 immunostaining was only examined to be in the acinar cells. Both at protein and transcript levels, HO-1 in castrated group was markedly decreased compared with the normal control group (p<0.01). In groups of exogenous administration of androgen and estrogen HO-1 was much higher than that in the control groups (p<0.01). However, estrogen increased HO-1 protein level in prostate stroma, while the levels of HO-2 did not give any evidence of change among all groups (p>0.05). These findings suggest that expression of HO-1 gene is induced by sex hormones, in contrast, there is no change in HO-2 expression. We speculate that CO-HO system is possibly involved in the pathologic processes of prostates abnormal proliferation induced by sex hormones and that CO derived from HO-1 may play an important role in the regulation of smooth muscle activity in rat prostate.
Animals ; Carbon Monoxide ; metabolism ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Male ; Orchiectomy ; Prostate ; enzymology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone Propionate ; pharmacology

We investigated the expression of heme oxygenase-1 (HO-1) gene and production of endogenous carbon monoxide (CO) in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin on the expression of HO-1 gene and pulmonary hypertension. A rat model of hypoxic pulmonary hypertension was recreated by exposure to intermittent normobaric hypoxic environment (10% O2). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the level of HO-1 mRNA in the rat lung tissue and double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood. Cardiac catheterization was employed to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathological changes of the intra-acinar pulmonary arteries (IAPA). It was found that (1) There was a low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P<0.01). The quantity of COHb was 2-3 times those of control group (P<0.01 or P<0.05). These were accompanied by the increased of RVSP and the thickened IAPA; (2) Hemin could keep the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, thereby ameliorating the pathological changes of IAPA. In conclusion, the upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.
Animals ; Carbon Monoxide ; metabolism ; physiology ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Hypertension, Pulmonary ; etiology ; metabolism ; Hypoxia ; complications ; Lung ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; pathology ; Pulmonary Artery ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats

In order to investigate the role of heme oxygenase-1 (HO-1) in the molecular mechanism of experimental allergic encephalomyelitis (EAE), which was induced by guinea pig spinal cord homogenate + complete freund adjuvant on Wistar rats, we observed the gene of HO-1 and its protein expression with reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistry 1, 7, 14, and 21 d after EAE induction in rats. The relationship between HO-1 and the symptoms of EAE was also observed. The results showed that the levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On day 7, the level of HO-1 mRNA reached the peak, but the expression level of HO-1 protein in the brains reached the peak on day 14. The immunoreactive cells of HO-1 were mainly located at the choroid plexuses and subfornical organ (SFO), as well as in regions around the "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The levels of HO-1 mRNA and its protein expression were lowered gradually on day 21, which were in parallel with the severities of symptoms and signs of EAE. After a specific inhibitor of HO-1, Snpp-9, was applied, both of the symptoms and pathological lesions of EAE in the rat brains were mitigated markedly. Therefore, these results may suggest that the dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brain. In conclusion, the levels of HO-1 mRNA and its protein expression in brains may play an important role in the pathogenesis of EAE, and application of inhibitors of HO-1 may be one of the potential therapeutic ways for the prevention and treatment of EAE.
Animals ; Brain ; enzymology ; metabolism ; Encephalomyelitis, Autoimmune, Experimental ; enzymology ; genetics ; physiopathology ; Female ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Subfornical Organ ; metabolism ; pathology

The heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme metabolism, has been recently defined as a novel stress-stimulated protein, since the intracellular expression of HO-1 in response to various stimuli as oxidation, ischemia and endotoxin injury has been proved to be able to protect the cells from damage. In this study, a retroviral vector containing human HO-1 gene was constructed and transfected to rat vascular smooth muscle cells (VSMCs). Using Southern and Northern blot analyses, the integration and mRNA expression of HO-1 gene in the transfected cells were confirmed. The profound protein expression of HO-1 as well as HO enzyme activity in the transfected cells increased by 1.8-fold and 2.0-fold respectively as compared with the non-transfected cells. It was found that the HO-1 transfected-VSMCs presented dominant resistance to toxicity produced by exposure to H2O2, as a significant protective effect of HO-1 marked by cell survival and LDH leakage was observed when 200, 400 and 600 micromol/L of H2O2 were used. The protection of HO-1 rapidly declined after the transfected-VSMCs were pretreated 24 h with an HO-1 specific inhibitor (ZnPP-IX). The results of this investigation suggest that the functional expression of HO-1 gene within VSMCs raises an alternative ability to protect the vascular cells against active oxygen injury.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Vectors ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; Hydrogen Peroxide ; toxicity ; Muscle, Smooth, Vascular ; enzymology ; pathology ; physiology ; Oxidants ; toxicity ; Rats ; Rats, Inbred WKY ; Retroviridae ; genetics ; Transfection

We investigated the expression of heme oxygenase-1 (HO-1) gene and production of endogenous carbon monoxide (CO) in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin on the expression of HO-1 gene and pulmonary hypertension. A rat model of hypoxic pulmonary hypertension was recreated by exposure to intermittent normobaric hypoxic environment (10% O2). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the level of HO-1 mRNA in the rat lung tissue and double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood. Cardiac catheterization was employed to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathological changes of the intra-acinar pulmonary arteries (IAPA). It was found that (1) There was a low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P<0.01). The quantity of COHb was 2-3 times those of control group (P<0.01 or P<0.05). These were accompanied by the increased of RVSP and the thickened IAPA; (2) Hemin could keep the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, thereby ameliorating the pathological changes of IAPA. In conclusion, the upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.
Anoxia/complications ; Carbon Monoxide/*metabolism ; Carbon Monoxide/physiology ; Heme Oxygenase (Decyclizing)/*biosynthesis ; Heme Oxygenase (Decyclizing)/genetics ; Heme Oxygenase-1 ; Hypertension, Pulmonary/etiology ; Hypertension, Pulmonary/*metabolism ; Lung/metabolism ; Myocytes, Smooth Muscle/drug effects ; Myocytes, Smooth Muscle/pathology ; Pulmonary Artery/metabolism ; Pulmonary Artery/*pathology ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics

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