This study was aimed to investigate the effects of cobalt protoporphyrin (CoPP) on hyperexpression of heme oxygenase-1 (HO-1) and secretion of IL-10 in bone marrow-derived mesenchymal stem cells (BMMSC). The BMMSC were isolated from rats and cultured, then were randomly divided into 4 groups, and were induced in culture medium with CoPP of 0, 25, 50, and 75 µmol/L respectively. The expressions of CD34, CD45, CD44 and CD71 in BMMSC were detected by flow cytometry; the osteoblast and adipocyte induction of BMMSC was determined by alizarin red and oil red staining respectively; the proliferation status of BMMSC was assayed by MTT method; the HO-1 expression in test groups was detected by RT-PCR; the optimal concentration of CoPP for induction of HO-1 expression was screened. The ELISA kit was used to detect the HO-1 levels in each groups. The results showed that the expressions of CD34, CD45 in BMMSC were negative while the expressions of CD44 and CD71 in BMMSC were positive. After induced by osteoblast and adipocyte inductor, BMMSC were positive for alizarin red staining and oil red staining. Various concentrations of CoPP had no significant impact on cell proliferation. When the CoPP concentration of 50 µmol/L could induce strong expression of HO-1, expression level in test groups was significantly higher than that in control group, meanwhile the IL-10 secretion was significantly higher than that in control group. It is concluded that CoPP has no ability to promote cell proliferation, 50 µmol/L is the optimum concentration for up-regulation of CoPP-induced BMMSC HO-1 expression, the up-regulation of HO-1 expression can increased IL-10 secretion significantly.
Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Heme Oxygenase (Decyclizing) ; metabolism ; Interleukin-10 ; secretion ; Mesenchymal Stromal Cells ; metabolism ; Protoporphyrins ; pharmacology ; Rats ; Rats, Wistar ; Up-Regulation

OBJECTIVETo investigate the role of heme oxygenase-1 (HO-1) and its catalyst carbon monoxide (CO) in the development of myocardial damage and the effects of zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 on myocardium of mice with acute viral myocarditis.

METHODSA total of 112 inbred male Balb/C mice 4 - 6 weeks of age were divided randomly into 3 groups: the control group (C group, n = 32), the viral myocarditis group (V group, n = 40) and ZnPPIX group (Z group, n = 40). The Z and V groups were inoculated intraperitoneally (i.p.) with 0.1 ml of 10(-4.36) tissue culture infectious dose 50% (TCID(50))/ml Coxsackie virus B3 (CVB(3)) to produce viral myocarditis model on day 0, C group was injected i.p. with virus-free 1640 culture culture medium 0.1 ml at the same time, then operation was done as follows: the mice of group C and group V were injected i.p. with 0.1 ml NS each day. The mice of group Z were injected i.p. with 40 micromol per kilogram of body weight ZnPPIX (HO-1 inhibitor) qod. Eight mice of each group were sacrificed on days 4, 8, 15 and 21, respectively. The blood specimens were collected by taking out the eyeballs to test for the content of carboxyhemoglobin (COHb) using spectrophotometry and cardiac troponin I (cTnI) using chemiluminescent immunoassay. The hearts tissue slides were also stained by immunohistochemistry (IHC) for HO-1 and in situ hybridization (ISH) for HO-1 mRNA. The histological and ultrastructural changes were observed under light and electron microscopes.

RESULTS(1) The histopathological changes of myocardial cells: in the V and Z groups myocardial inflammatory cells infiltration reached the peak on day 8, the Z group histopathological scores were significantly lower than those in V group on day 8 (2.40 +/- 0.31 vs. 1.73 +/- 0.24, P < 0.01) and on day 15 (1.78 +/- 0.29 vs. 1.43 +/- 0.23, P < 0.05). No inflammation was present in group C. (2) The changes of serum cTnI level in both V and Z groups were significantly higher than those in C group on day 4, 8 and 15 (P < 0.01). The level in Z group was significantly lower than that in V group on day 4 [(6.074 +/- 1.475) ng/ml vs (7.911 +/- 1.225) ng/ml, P < 0.05] and day 8 [(0.821 +/- 0.294) ng/ml vs (1.480 +/- 0.454) ng/ml, P < 0.05]. (3) The changes of blood COHb level: compared with V group, in Z group the COHb level was lower on day 4 (P < 0.05) and day 15 (P < 0.01) after CVB(3) inoculation. Surprisingly, in Z group COHb level elevated suddenly on day 8 and showed conspicuously higher than that of V group (P < 0.01). (4) The result of HO-1 IHC staining: in both V and Z group myocardial cells had positive expression, while C group did not. (5) The results of HO-1 ISH were similar to those of HO-1 IHC, the A values of group Z was significantly lower than that of group V on day 4, 15 and 21(P < 0.01), but on day 8 it was higher than that of group C (P < 0.05).

CONCLUSIONHO-1 inhibitor, ZnPP not only could inhibit HO-1 overexpression but also could induce HO-1 expression temporarily and protect against myocardial injury at the early stage of acute viral myocarditis.

Animals ; Heme Oxygenase-1 ; antagonists & inhibitors ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; enzymology ; metabolism ; pathology ; virology ; Myocardium ; pathology ; ultrastructure ; Protoporphyrins ; pharmacology ; Virus Diseases ; metabolism ; pathology

OBJECTIVETo use heme oxygenase (HO) inducer hemin and a HO inhibitor zinc protoporphyrin (ZnPP) to investigate the effect of HO on apoptosis and apoptosis genes in hepatic ischemia reperfusion (IR) injury in rats.

METHODSNinety-six Sprague-Dawley rats were randomly divided into four groups (24 rats in each): a sham-operation group, an ischemia-reperfusion (IR) group, a hemin-IR group and a ZnPP-IR group. Liver functions, liver histology and hepatocellular apoptosis rates were observed at 0, 1.5, 4 and 8 hours after reperfusion. Hepatocellular apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; expressions of Bcl-2 and caspase-3 were determined by Western blot.

RESULTSCompared with the sham-operation group, the levels of ALT and AST were increased in the IR group. In the IR group the histological changes found in the livers were swelling of hepatocytes, narrowing of hepatic sinusoids, inflammatory cell infiltration and necrosis of hepatocytes in some areas of the livers. In the IR group rate of hepatocellular apoptosis was increased at 0, 1.5, 4 and 8 hours after reperfusion; expression of Bcl-2 was decreased and the expression of caspase-3 was increased. In the hemin-IR group, the levels of ALT and AST were lower, the pathological changes were milder and the rate of hepatocellular apoptosis was lower at 0, 1.5, 4 and 8 hours in comparison to those of the IR group. The expression of Bcl-2 was higher and the expression of caspase-3 was lower in the hemin-IR group in comparison to those of the IR group. The results in the ZnPP-IR group were just the opposite to those of the hemin-IR group.

CONCLUSIONHO might play a protective role in hepatic IR injury in rats, and this effect may be related to the inhibition of hepatocellular apoptosis.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Heme Oxygenase (Decyclizing) ; metabolism ; Hepatocytes ; metabolism ; pathology ; Liver Diseases ; metabolism ; pathology ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Protoporphyrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; pathology

OBJECTIVETo study the protective effect of islet xenograft and its possible mechanism of high expression of heme oxygenase-1 (HO-1) in donor pancreas islet induced by cobalt protoporphyrin (CoPP).

METHODSMale SD rats and C57BL/6 mouse were used as donors and recipients respectively. Donors were divided into 3 groups according to different pretreatment 24 hours before donation: control group (injected intraperitoneally with NaCl), induce group [injected intraperitoneally with cobalt-protoporphyrin (CoPP)], block group (injected intraperitoneally with CoPP and zinc protoporphyrin simultaneously). A modified approach was used for islet isolation.Recipients were rendered diabetic by intraperitoneal injection of streptozotocin. Islets were transplanted into mouse subrenal capsule. Postoperative mouse glycemia were monitored daily and normoglycemia time was compared among each group. The receptor mouse serum IL-10 was detected by ELISA approach, and real-time PCR was used to check the expression of IL-10 mRNA in islet graft tissues. The graft tissues were observed for the lymphocyte infiltration after HE staining.

RESULTSDiabetes mice accepted islets untreated, induced or blocked maintained the euglycemia for (9.3 +/- 1.4), (16.3 +/- 1.5) and (9.7 +/- 1.0) d respectively. The xeno-islets presented HO-1 over-expression survived much longer than that absent (P < 0.05), it was no significance between control group and block group (P > 0.05). The mouse islet serum IL-10 content after induction was (73.0 +/- 9.7) pg/ml, significantly higher than (30.6 +/- 3.9) pg/ml of the untreated group and (32.1 +/- 5.9) pg/ml of the blocked group (P < 0.05), there was no difference between control group and block group (P > 0.05). Moreover, the IL-10 mRNA expression up-regulated statistic significantly in HO-1 induced islet xeno-graft. Pathological examination showed that the graft lymphocyte infiltration of the induced group was obviously less serious than the other two groups.

CONCLUSIONSThe higher expression of HO-1 induced by CoPP in vivo would significantly prolong graft survival time and its mechanism could be related to immune modulation of IL-10.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; surgery ; Graft Survival ; Heme Oxygenase-1 ; drug effects ; metabolism ; Interleukin-10 ; metabolism ; Islets of Langerhans ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Pancreas Transplantation ; Protoporphyrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Subrenal Capsule Assay ; Transplantation, Heterologous

OBJECTIVETo investigate the protective effect of overexpressed heme oxygenase-1 (HO-1) in hypoxia and the correlation between HO-1 overexpressoin and hypoxia inducible factor-1alpha (HIF-1alpha) in human hepatoma HepG2 cells.

METHODSThe expressions of HO-1 and HIF-1alpha mRNA as well as the protein were detected by RT-PCR and Western blotting, respectively. MTT assay was used to examine the relative cell survival rate. The total superoxide dismutase (SOD) activity was examined with a SOD kit.

RESULTSHypoxia induced overexpression of HO-1 gene in HepG2 cells at transcriptional and translational levels. The relative survival rate of HepG2 cells under hypoxia was significantly decreased after the HO-1 protein overexpression was inhibited by ZnPPIX (P < 0.01). The total SOD activity of cells was significantly increased after cells were treated by hypoxia for 16 hours (P < 0.05), while decreased significantly by HO-1 inhibitor ZnPPIX treatment (P < 0.01). HIF-1alpha was upregulated under hypoxia. In addition, the HO-1 overexpression under hypoxia was decreased by HIF-1alpha inhibitor, while the HIF-1alpha expression level under hypoxia was not significantly changed after HO-1 expression was inhibited by ZnPPIX.

CONCLUSIONThe overexpression of HO-1 in hypoxic HepG2 cells is HIF-1alpha-dependent or at least partly HIF-1alpha-dependent. The relative survival rate of hypoxic hepatoma cells was significantly decreased by HO-1 inhibitor treatment. The results of this study may offer new thought and drug target for the therapy of human hepatoma in the future.

Cell Hypoxia ; Cell Survival ; Heme Oxygenase-1 ; antagonists & inhibitors ; metabolism ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Protoporphyrins ; pharmacology ; RNA, Messenger ; metabolism ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effects of CO release inhibitor zinc protoporphyria IX (ZnPP IX) and NO release inhibitor L-NAME on the content of cGMP in the penile tissue of rats.

METHODSThirty Wistar rats were randomly divided into a normal control, a ZnPP IX, and an L-NAME group, given saline (1 ml/kg/d), ZnPP IX (45 micromol/kg/d) and L-NAME (50 mg/kg/d), respectively, for 7 days. Then all the rats were killed, homogenate made from their penile tissues and detected for the contents of NOS, NO, CO and cGMP.

RESULTSThe contents of CO, NOS, NO and cGMP were all reduced in both the ZnPP IX and L-NAME groups as compared with the control group (P < 0.05).

CONCLUSIONZnPP IX and L-NAME can reduce the concentrations of CO and NO in the penile tissues of rats, and consequently the content of cGMP.

Animals ; Carbon Monoxide ; antagonists & inhibitors ; metabolism ; Cyclic GMP ; metabolism ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; antagonists & inhibitors ; metabolism ; Penis ; drug effects ; metabolism ; Protoporphyrins ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo study the change and role of heme oxygenase-1 (HO-1) in injured lungs following limb ischemia/reperfusion in rats.

METHODSA total of 96 healthy male Sprague-Dawley rats, weighing 250-300 g, were used in this study. Hind limb ischemia was made on 40 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, then limb reperfusion for 0, 4, 8, 16 and 24 hours (n=8 in each time point) was performed, respectively. Other 8 rats undergoing full surgical operation including isolation of the infrarenal aorta without occlusion were taken as the sham operation group. Lung tissues were obtained from the 48 animals and Northern blotting and Western blotting were employed to measure the changes of HO-1 mRNA and protein expression, respectively. Immunohistochemistry technique was used to determine the cell types responsible for HO-1 expression after limb ischemia/reperfusion. Then hind limb ischemia was made on other 12 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, among whom, 6 rats were given zinc protoporphyrin (ZnPP), an inhibitor of HO. Then limb reperfusion for 16 hours was performed on all the 12 rats. And other 12 rats underwent full surgical operation including isolation of the infrarenal aorta without occlusion, among whom, 6 rats were then given ZnPP. Then lung tissues were obtained from the 24 animals and lung injury markers, lung histology, polymorphonuclear leukocyte (PMN) count and malondialdehyde (MDA) content were detected, respectively. HO activity was determined through measuring the carboxyhemoglobin (COHb) level in artery blood with a CO-oximeter after limb ischemia/reperfusion. And the animal mortality was observed on the other 24 rats.

RESULTSNorthern blotting analysis showed that HO-1 mRNA increased significantly at 4 hours after reperfusion, peaked at 16 hours, and began to decrease at 24 hours. In contrast, no positive signal was observed in the sham and simple ischemia animals. Increased HO-1 mRNA levels were accompanied by similar increases in HO-1 protein. Lung PMNs and MDA content increased significantly at 4, 8, 16 and 24 hours after reperfusion, compared with the sham controls (P<0.001), while they decreased in rats with reperfusion for 16 hours when compared with rats with reperfusion for 4 hours (P<0.001). Immunohistochemical studies showed that HO-1 was expressed in a variety of cell types, including the airway epithelia, alveolar macrophages and vascular smooth muscular cells. The blood COHb level and animal mortality increased significantly after limb ischemia/reperfusion compared with the sham controls (P<0.001). ZnPP administrated to the ischemia/reperfusion animals led to a decrease in the COHb level and an increase in lung PMN number, MDA content and animal mortality (P<0.001 compared with ischemia/reperfusion group), and the lung injury was aggravated.

CONCLUSIONSLimb ischemia/reperfusion up-regulates pulmonary HO-1 expression, which serves as a compensatory protective response to the ischemia/reperfusion-induced lung injury in rats.

Animals ; Blotting, Northern ; Blotting, Western ; Heat-Shock Proteins ; metabolism ; Heme Oxygenase (Decyclizing) ; antagonists & inhibitors ; Immunohistochemistry ; Lung ; enzymology ; Male ; Oxygenases ; Protoporphyrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; Respiratory Insufficiency ; metabolism

BACKGROUNDThe hepatopulmonary syndrome (HPS) is a severe vascular complication in lungs resulting in systemic hypoxemia in patients with cirrhosis and portal hypertension. The underlying structural change in HPS is intrapulmonary vasodilation, which can lead to impaired oxygenation of pulmonary venous blood. It has been demonstrated that the heme oxygenase-1/carbon monoxide (HO-1/CO) system plays an important role in the control of vascular tone. The aim of this study was to further investigate the role of HO-1 in the pathogenesis of HPS in animal model.

METHODSTotally 35 rats were divided into liver cirrhosis, zinc protoporphyrin IX (ZnPP), cobalt protoporphyrin (CoPP) and sham groups. Biliary cirrhosis was established in the first three groups by bile duct ligation. Rats in the ZnPP and CoPP groups received once intraperitoneal injection of ZnPP and CoPP, respectively, 24 hours before sample collection. Expression of HO-1 mRNA in lung was detected by reverse-transcription polymerase chain reaction, while protein expression was determined by immunohistochemical analysis. Hematoxylin and eosin staining was performed to confirm the presence of liver cirrhosis and intrapulmonary vasodilation. Arterial blood gases, mean arterial pressure and portal vein pressure were also measured. Analysis of variance or Wilcoxon statistical methods were used to determine statistical significance.

RESULTSCompared with the sham group, the cirrhotic group demonstrated increased expression of pulmonary HO-1 mRNA and protein (P < 0.01). The level of arterial carboxyhemoglobin (COHb), alveolar-arterial oxygen gradient (A-aPO2), mean arterial pressure, portal vein pressure (P < 0.05, respectively), and intrapulmonary vasodilation were also significantly increased. Compared with the cirrhotic group, CoPP treatment increased pulmonary HO-1 mRNA and protein expression, the level of A-aPO2 (P < 0.05 respectively), COHb (P < 0.01), and intrapulmonary vasodilation, while ZnPP treatment decreased pulmonary HO-1 mRNA and protein expression, the level of COHb (P < 0.05 respectively), and intrapulmonary vasodilation, without obvious alteration of mean arterial pressure and portal vein pressure.

CONCLUSIONIncreased pulmonary HO-1 expression is an important contributor to the development of experimental HPS.

Animals ; Enzyme Inhibitors ; therapeutic use ; Heme Oxygenase-1 ; genetics ; metabolism ; Hemodynamics ; Immunohistochemistry ; Liver Cirrhosis ; enzymology ; genetics ; Lung ; drug effects ; metabolism ; Male ; Protoporphyrins ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

Erythropoietic protoporphyria (EPP) is a rare disorder of heme biosynthesis caused by mutations in the gene encoding the enzyme ferrochelatase. In EPP, deficient ferrochelatase activity leads to the excessive production and biliary excretion of protoporphyrin (PP). The major clinical features of EPP are photosensitivity and hepatobiliary disease that may progress to severe liver disease, that are caused by the toxicity of PP. EPP-related liver disease has been treated medically or surgically including liver transplantation. We described a 20-year-old male with severe liver disease who was diagnosed with EPP based on clinical and laboratory findings. He was treated with cholestyramine resin. Six months after the treatment, he was doing well without any abdominal pain or photosensitivity.
Bilirubin/blood ; Cholestyramine Resin/*therapeutic use ; Edema/complications ; Erythema/complications ; Ferrochelatase/genetics/metabolism ; Humans ; Liver Diseases/complications/*diagnosis/pathology ; Male ; Protoporphyria, Erythropoietic/complications/*diagnosis/drug therapy ; Protoporphyrins/metabolism ; Young Adult

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