Zinc Protoporphyrin Group Research Articles

Influence of Heme Oxygenase-1 on Rats with Diabetic Retinopathy Through ERK1/2 Signaling Pathway.

The study aimed to investigate the influence of heme oxygenase-1 (HO-1) on rats with diabetic retinopathy (DR) through the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway. 40 rats were selected and divided into Control group (n=10), diabetes mellitus (DM) group (n=10), cobalt protoporphyrin (CoPP) group (n=10) and zinc protoporphyrin (ZnPP) group (n=10) according to weight. Streptozotocin (STZ) was intraperitoneally injected to establish the DM model in DM, CoPP and ZnPP groups, and CoPP and ZnPP solution was intraperitoneally injected in CoPP and ZnPP groups, respectively. Blood was drawn to determine fasting blood glucose. The changes in the protein and messenger ribonucleic acid (mRNA) levels were evaluated via Western blotting and polymerase chain reaction (qRT-PCR), respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to measure antioxidant capacity and the levels of total reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GPx). The weight of rats was notably higher in the CoPP group and lower inZnPP group than in the DM group (p<0.05). After induction of DM, compared with those in the DM group, the protein expression levels of Nrf2 and pERK were considerably elevated in the CoPP group (p<0.05) but declined remarkably in the ZnPP group (p<0.05). The levels of total ROS and MDA were notably elevated (p<0.05) in DM and ZnPP groups, with a lowered level of GPx and distinctly elevated levels of MDA and total ROS (p<0.05). Moreover, the mRNA expression level of HO-1 in the retinas of rats was remarkably raised in the DM group and CoPP group (p<0.05), but it declined markedly in the ZnPP group (p<0.05). The red fluorescent aggregation of Nrf2 and pERK proteins was overtly less in the ZnPP group than that in the DM group (p<0.05). HO-1 can affect the level of oxidative stress and intervene in retinopathy in DM rats through the Nrf2/ERK pathway.

Protection Against Post-resuscitation Acute Kidney Injury by N-Acetylcysteine via Activation of the Nrf2/HO-1 Pathway.

Background and ObjectiveAcute kidney injury (AKI), the common complication after cardiopulmonary resuscitation (CPR), seriously affects the prognosis of cardiac arrest (CA) patients. However, there are limited studies on post-resuscitation AKI. In addition, it has been demonstrated that N-acetylcysteine (N-AC) as an ROS scavenger, has multiorgan-protective effects on systemic and regional ischaemia-reperfusion injuries. However, no studies have reported its protective effects against post-resuscitation AKI and potential mechanisms. This study aimed to clarify the protective effects of N-AC on post-resuscitation AKI and investigate whether its potential mechanism was mediated by activating Nrf-2/HO-1 pathway in the kidney.MethodsWe established cardiac arrest models in rats. All animals were divided into four groups: the sham, control, N-AC, and ZnPP groups. Animals in each group except for the ZnPP group were assigned into two subgroups based on the survival time: 6 and 48 h. The rats in the control, N-AC, and ZnPP groups underwent induction of ventricular fibrillation (VF), 8 min untreated VF and cardiopulmonary resuscitation. Renal function indicators, were detected using commercial kits. Renal pathologic changes were assessed by haematoxylin–eosin (HE) staining. Oxidative stress and inflammatory responses were measured using the corresponding indicators. Apoptosis was evaluated using terminal uridine nick-end labeling (TUNEL) staining, and expression of proteins associated with apoptosis and the Nrf-2/HO-1 pathway was measured by western blotting.ResultsN-AC inhibited post-resuscitation AKI. We observed that N-AC reduced the levels of biomarkers of renal function derangement; improved renal pathological changes; and suppressed apoptosis, oxidative stress, and inflammatory response. Additionally, the production of ROS in the kidneys markedly decreased by N-AC. More importantly, compared with the control group, N-AC further upregulated the expression of nuclear Nrf2 and endogenous HO-1 in N-AC group. However, N-AC-determined protective effects on post-resuscitation AKI were markedly reversed after pretreatment of the HO-1 inhibitor zinc protoporphyrin (ZnPP).ConclusionsN-AC alleviated renal dysfunction and prolonged survival in animal models of CA. N-AC partially exerts beneficial renal protection via activation of the Nrf-2/HO-1 pathway. Altogether, all these findings indicated that N-AC as a common clinical agent, may have the potentially clinical utility to improve patients the outcomes in cardiac arrest.

Shenmai Injection Upregulates Heme Oxygenase-1 to Confer Protection Against Severe Acute Pancreatitis

BackgroundTo explore the mechanism of Shenmai injection (SMI) on severe acute pancreatitis (SAP) through heme oxygenase-1 (HO-1) signaling. MethodsA total of 40 male Sprague–Dawley (SD) rats (220-260 g) were grouped into the following four categories (n = 10): SAP + SMI + Zinc protoporphyrin (ZnPP), SAP + SMI, SAP, and sham surgery groups. ZnPP is a specific inhibitor of HO-1. Four percent of sodium taurocholate (1 mL/kg) was retrogradely injected via the pancreatic duct to induce the SAP model. The SAP group rats received 1.6 mL/kg saline by intravenous injection 30 min after the induction of SAP. The SAP + SMI group rats received 1.6 mL/kg SMI by intravenous injection 30 min after the induction of SAP. The SAP + SMI + ZnPP group rats received an intravenous injection of 1.6 mL/kg SMI and intraperitoneal administration of 30 mg/kg ZnPP 30 min after the SAP induction. Twenty-four hours after the SAP induction, blood samples were collected for the measurement of amylase, lipase, creatinine, myeloperoxidase, interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and HO-1 level, while tissue specimens were harvested for the determination of HO-1, TNF-α, and IL-10 mRNA level. Meanwhile, histopathological changes in organs (pancreas, lung, and kidney) were stored. ResultsThe serum concentration of amylase, lipase, creatinine, and myeloperoxidase was higher in the SAP group than in the SAP + SMI group. Treatment with SMI increased HO-1 and IL-10 level and reduced TNF-α level in serum and tissues compared to the SAP group (P < 0.05). Treatment with SMI abolished the organ-damaging effects of SAP (P < 0.05). Furthermore, suppression of HO-1 expression by ZnPP canceled the aforementioned effects. ConclusionsSMI confers protection against the SAP-induced systemic inflammatory response and multiple organs damage via HO-1 upregulation.

Dexmedetomidine attenuates oxidative/nitrative stress in lung tissues of septic mice partly via activating heme oxygenase-1.

Excessive reactive oxygen/nitrogen species are considered to be one of the primary events that cause lung injury during sepsis. The present study aimed to determine whether dexmedetomidine (Dex) exhibits antioxidative and antinitrative effects on sepsis-induced lung injury and its effect on heme oxygenase-1 (HO-1) activation. The cecal ligation and puncture (CLP) mouse model was used, where male C57BL/6J mice were randomized into groups: Sham, CLP, Dex and Dex + zinc protoporphyrin (ZnPP). Following CLP or sham operation, intraperitoneal injections of 40 µg/kg Dex or saline were administered in the Dex + ZnPP group, intraperitoneal injections of ZnPP (40 mg/kg) were administered 1 h prior to the CLP operation. Subsequently, histopathological examination of the lungs and measurement of HO-1 activity in the lung, as well as oxidative and nitrative stress were determined 24 h following CLP. Dex significantly decreased the levels of oxidative and nitrative stress, as demonstrated by the decreased levels of malondialdehyde and nitrotyrosine, and the protein expression of inducible nitric oxide synthase, as well as increased superoxide dismutase in lung tissues. Also Dex inhibited the elevation of serum interleukin-6 and tumor necrosis factor-α and increased lung HO-1 activity. Furthermore, the effects of Dex were partially reverted by the HO-1 inhibitor ZnPP. In conclusion, Dex inhibited oxidative/nitrative stress in sepsis and attenuated sepsis-induced acute lung injury partially by increasing HO-1 activity.

Cobalt-protoporphyrin enhances heme oxygenase 1 expression and attenuates liver ischemia/reperfusion injury by inhibiting apoptosis.

The aim of the present study was to investigate the preconditioning effect and underlying mechanisms of cobalt-protoporphyrin (CoPP) in a mouse model of liver ischemia‑reperfusion (I/R) injury. Mice were divided into five groups: Sham‑operated (control), I/R, I/R + CoPP, I/R + CoPP and zinc‑protoporphyrin (ZnPP) and I/R + ZnPP. Serum levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) were detected using commercial kits. The expression of the pro‑apoptotic protein caspase‑3 was detected by immunohistochemistry and the expression levels of the anti‑apoptotic protein B‑cell lymphoma 2 (Bcl‑2) and heme oxygenase 1 (HO‑1) were analyzed by western blotting. Sections of liver tissue were stained with hematoxylin and eosin to observe pathologic alterations. Furthermore, hepatocyte apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. AST and ALT levels of the CoPP preconditioned group were significantly reduced compared with the IR injury group (P<0.05) and liver damage was attenuated. The expression levels of the pro‑apoptotic protein caspase3 was inhibited and those of HO‑1 and Bcl‑2 were increased in the CoPP group compared with the I/R group; the opposite results were observed in the ZnPP group. Furthermore, the percentage of apoptotic cells as detected by TUNEL was significantly decreased in the CoPP group compared with the I/R group (P<0.05); these protective effects were abrogated by ZnPP. In conclusion, the results of the present study suggested that CoPP may induce HO‑1 overexpression and produce anti‑apoptotic effects in liver I/R injury.

Role of heme oxygenase-1 in sevoflurane-induced reduction of airway injury in asthmatic mice

Objective To evaluate the role of heme oxygenase-1(HO-1)in sevoflurane-induced reduction of airway injury in asthmatic mice. Methods Forty SPF female C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups(n=10 each)using a random number table: control group(group C), asthma group(group A), asthma plus sevoflurane group(group A+ S)and asthma plus sevoflurane plus zinc protoporphyrin group(group A+ S+ Z). The mice were sensitized and repeatedly challenged with ovalbumin to establish the asthma model.In group A+ S, 3% sevoflurane was inhaled for 1 h after every challenge.In group A+ S+ Z, zinc protoporphyrin 10 mg/kg was intraperitoneally injected after every challenge and before sevoflurane inhalation.The airway responsiveness were assessed at 24 h after the last challenge.The bronchoalvelor lavage fluid(BALF)was collected for determination of cell type and count and concentrations of interleukin-13(IL-13)and IL-33(by enzyme-linked immunosorbent assay). The right lung tissues were obtained for measurement of the superoxide dismutase(SOD)activity, contents of malondialdehyde(MDA)and glutathione(GSH)and expression of HO-1(by Western blot). Lung tissues were stained with haematoxylin and eosin and inflammatory cell infiltration was assessed and scored. Results Compared with group C, the airway responsiveness, inflammatory cell count and concentrations of IL-13 and IL-33 in BALF, and MDA content and infiltration score in lung tissues were significantly increased, the SOD activity and GSH content were decreased, and the expression of HO-1 was up-regulated in group A(P 0.05). Compared with group A+ S, the airway responsiveness, inflammatory cell count and concentrations of IL-13 and IL-33 in BALF, and MDA content and infiltration score in lung tissues were significantly increased, the SOD activity and GSH content were decreased, and the expression of HO-1 was down-regulated in group A+ S+ Z(P<0.05). Conclusion HO-1 is involved in the pathophysiological mechanism by which sevoflurane attenuates airway injury in asthmatic mice. Key words: Asthma; Anesthetics, inhalation; Heme oxygenase-1

Study on the role of autophagy in heme oxygenase 1 preventing hepatic ischemia/reperfusion injury in rats

To identify the role of autopahgy in the protective mechanism of heme oxygenase 1 (HO-1) against hepatic ischemia/reperfusion (I/R) injury. Forty healthy male Sprague-Dawley (SD) rats were randomly (random number table) divided into five groups (n = 8 in each group), namely sham group, model group, cobalt protoporphyrin (CoPP) group, zinc protoporphyrin (ZnPP) group and 6-amino-3-methylpurine (3-MA) group. Partial hepatic I/R model was established by clamping the pedicles of left and median lobes for 1 hour and reopening for 6 hours in rats, and the rats in sham group were only received celiotomp without hepatic I/R. In the CoPP group, CoPP (a HO-1 inducer, 5 mg/kg) was administered i.p 24 hours before I/R. In the ZnPP or 3-MA group, besides pretreatment with CoPP, the rats were given ZnPP (a HO-1 inhibitor, 25 mg/kg) or 3-MA (an autophagy inhibitor, 30 mg/kg) i.p 1 hour before I/R. Serum alanine aminotransferase (ALT) was determined with automatic biochemistry analyzer. The hepatic pathological scores (PS) were determined under light microscope using hematoxylin-eosin (HE) staining. The hepatocyte apoptosis index (AI) was assessed with terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Autophagosomes in liver tissue were counted under electron microscope. The mRNA expressions of HO-1, caspase-3, Beclin-1 and Atg-5 in the liver were determined by reverse transcription-polymerase chain reaction (RT-PCR). The HO-1 activity was also measured by the generation of bilirubin with the method of double-wave spectrophotometry. Compared with the sham group, the level of serum ALT significantly increased in the I/R group (U/L: 560.3±73.6 vs. 49.1±13.8, P < 0.01), HE staining showed a severe hepatic injury (PS: 12.0±2.0 vs. 1.3±0.9, P < 0.01), TUNEL staining showed a higher hepatocytes apoptosis and the expression of caspase-3 significantly increased [AI: (19.38±3.07)% vs. (3.25±1.28)%, caspase-3 mRNA (2-ΔΔCt): 4.62±0.40 vs. 1.05±0.15, both P < 0.01]. However, there was no significant difference in the expression of HO-1 and the genes associated with autophagy between the two groups. In the CoPP group, the hepatic injury was blunted compared with that in the I/R group [ALT (U/L): 223.3±34.4 vs. 560.3±73.6, PS: 5.6±2.3 vs. 12.0±2.0, AI: (11.38±2.39)% vs. (19.38±3.07)%, caspase-3 mRNA (2-ΔΔCt): 2.42±0.33 vs. 4.62±0.40, all P < 0.01]. HO-1 was induced in the CoPP group and autophagy was also increased significantly after I/R when compared with those in the I/R group [HO-1 mRNA (2-ΔΔCt): 3.01±0.71 vs. 1.14±0.20, HO-1 activity (pmol×mg-1×h-1): 259±37 vs. 113±26, the number of autophagosomes: 8.75±0.87 vs. 1.25±0.71, Beclin-1 mRNA (2-ΔΔCt): 2.85±0.28 vs. 1.15±0.11, Atg-5 mRNA (2-ΔΔCt): 2.44±0.25 vs. 1.14±0.12, all P < 0.01]. In the ZnPP group, the activity of HO-1 was much lower than that in the CoPP group, and as a result autophagy was decreased and liver injury was increased. In the 3-MA group, although there was no difference in the activity of HO-1 compared with that in the CoPP group, autophagy was inhibited, and the protective effect of CoPP was eliminated. HO-1 could regulate the level of autophagy during liver I/R, and in turn autophagy might mediate the protective effects of HO-1 against liver I/R injury.

Luteolin protects mice from severe acute pancreatitis by exerting HO-1-mediated anti-inflammatory and antioxidant effects.

Reseda odorata L. has long been used in traditional Asian medicine for the treatment of diseases associated with oxidative injury and acute inflammation, such as endotoxemia, acute lung injury, acute myocardial infarction and hepatitis. Luteolin, the main component of Reseda odorata L., which is also widely found in many natural herbs and vege tables, has been shown to induce heme oxygenase-1 (HO-1) expression to exert anti-inflammatory and antioxidant effects. In this study, we aimed to examine the effects of luteolin on mice with severe acute pancreatitis (SAP), and to explore the underlying mechanisms. Cerulein and lipopolysaccharide were used to induce SAP in male Institute of Cancer Research (ICR) mice in the SAP group. The SAP group was divided into 4 subgroups, as follows: the vehicle, luteolin, zinc protoporphyrin (ZnPP) only, and luteolin (Lut) + ZnPP (luteolin plus zinc protoporphyrin treatment) groups. The wet/dry weight ratios, hematoxylin and eosin staining and pathological scores of pancreatic tissues were assessed and compared to those of the control mice. Amylase, lipase, nuclear factor-κB (NF-κB) and myeloperoxidase activities, and malondialdehyde, tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-10 and HO-1 levels, as well as the expression of HO-1 were determined in serum and/or pancreatic tissue samples. SAP was successfully induced in male mice compared to normal control mice. The wet/dry weight ratios, pathological scores, and amylase and lipase activity, as well as the levels of TNFα and IL-6 were significantly reduced in the pancreatic tissues of the mice in the Lut group compared with those of the mice in the vehicle group. The Lut group exhibited a significant increase in HO-1 expression in the pancreas and enhanced serum HO-1 and IL-10 levels compared with the vehicle group. The suppression of HO-1 activity in the ZnPP group significantly abolished the protective effects of luteolin. NF-κB expression in the pancreatic tissues from the mice in the Lut + ZnPP group was significantly increased following the suppression of HO-1 activity. On the whole, our findings demonstrate that luteolin protects mice from SAP by inducing HO-1-mediated anti-inflammatory and antioxidant activities, in association with the suppression of the activation of the NF-κB pathway.

The therapeutic effect and mechanism of interleukin-10 on cerebral vasospasm caused by subarachnoid hemorrhage

Objective To study the effect of interleukin-10(IL-10)on cerebral vasospasm following subarachnoid hemorrhage(SAH) in rabbit and explore the mechanism. Methods Thirty Japanese rabbits were randomly divided into Sham operation group(A), SAH group(B), SAH+ IL-10 group(C), SAH+ Zinc protoporphyrin(ZnPP)+ IL-10 group(D) and SAH+ ZnPP group (E).The animal model of cerebral vasospasmfollowingsubarachnoid hemorrhage was made by infusing autologous artery blood twice into the cisterna magna.The C, D, E group were respectively received IL-10, ZnPP through intraperitoneal injection after the SAH model was established. The tumor necrosis factor(TNF)-αand IL-6 levels in serum were detected by enzyme linked immunosorbent assay(ELISA) method 5 days postoperatively.The diameter and expression of heme oxygenase-1(HO-1) protein in basilar artery were observed. Results The animal model of cerebral vasospasm after subarachnoid hemorrhage was success.The basilar artery diameter of SAH groups was significantly shorter than the sham operation group(P< 0. 05)and the TNF-αand IL-6 levels were higher in SAH group(P< 0. 05), The diameter of group C and D[(733. 94±17. 28), (646. 11±9. 79)μm]was increased than group B and E[(595. 64±10. 15),(532. 81±17. 09) μm], the TNF-αand IL-6 levels in group C[(26. 27±1. 64),(58. 15±1. 38) ng/L] and D[ (43. 45±1. 77),(77. 17±1. 09) ng/L]were lower than group B[(53. 56±1. 27),(115. 93±1. 47) ng/L]and E[(60. 56±1. 79),(136. 45±1. 73) ng/L].There wasn't the expression of protein HO-1 in sham operation group and the content of HO-1 in C group(0. 446±0. 019) was higher than group B, D, E(0. 314±0. 014, 0. 251±0. 018, 0. 160±0. 011). Conclusion IL-10 can alleviate the cerebral vasospasm caused by subarachnoid hemorrhage and may be mediated by the HO-1 protein. Key words: Subarachnoid hemorrhage; Cerebral vasospasm; Interleukin-10; Heme oxygenase-1

Second Pathways in the Pathophysiology of Ischemic Priapism and Treatment Alternatives

To evaluate the early therapeutic alternatives such as bosentan, an endothelin receptor blocker, theophylline, an adenosin receptor blocker, and a nonselective phosphodiesterase enzyme inhibitor, zinc protoporphyrin (ZnPP), a heme oxygenase 1 inhibitor, for the therapy of ischemic priapism in the rat models. Twenty-four Sprague-Dawley rats were randomly divided into 4 equal groups: control group, ZnPP group, bosentan group, and theophylline group. Erection was provided by vacuum constriction method and maintained for 4 hours for achieving the priapism in all groups. The rats in the control group were administered 1 mL/kg saline intraperitoneally (ip). The rats in group 2 were administered 25 mg/kg ZnPP ip. The rats in group 3 were administered 0.25 mg/kg bosentan ip. The rats in group 4 were administered 100 mg/kg theophylline ip. Six rats from each group were decapitated after 6 hours of drug administration. Then endothelin 1, adenosine deaminase, heme oxygenase 1 enzymatic activity, and apoptosis index in the cavernous tissues were estimated. Cavernous tissue endothelin 1, adenosine deaminase, heme oxygenase 1 enzymatic activity levels, and apoptosis index were significantly decreased in bosentan, theophylline, and ZnPP-treated rats compared with the controls. Inhibition of priapism induced apoptosis with bosentan, theophylline, and ZnPP seems promising on preserving erectile function.

Neuro-protective function of cytoglobin on neonatal rats with hypoxic-ischemic brain damage

Objective To investigate the protective function of cytoglobin (CYGB) on neurodevelopment of the neonatal rats with hypoxic-ischemic brain damage (HIBD).Methods Healthy seven day old Sprague-Dawlay rats were randomly divided into sham operated group,HIBD group,HIBD+Hemin group and HIBD+zinc protoporphyrin (ZnPP) group.The rats of HIBD,HIBD+Heminand HIBD+ZnPPgroup were given normal saline (0.5 ml),Hemin (50 mg/kg) and ZnPP (50 mg/kg) intraperitoneally respectively,and 12 hours later the left carotid arteries of these rats were ligated and cut off,then hypoxic treated for 2 hours to establish the HIBD models.At 0,24,48 h after HIBD models were established,the expressions of CYGB in the cerebral cortex and hippocampus were observed by immunohistochemistry analysis.At 48 h after HIBD,histopathological changes of brain were observed after HE staining.At 72 h after HIBD,the water content of the brain was observed.At 28 days after HIBD,long term study memory outcome was assessed by Morris water maze.Analysis of variance and Bonferroni test were applied as statistical methods.Results (1)The expression of CYGB in brain(expressed by average gray value which negatively correlated with protein levels):At 0 h,the average gray value of CYGB in the cerebral cortex in HIBD+ Hemin and HIBD group were 166.7±5.1 and 207.1±5.1,which were lower than that in sham operated group (232.3±3.4); but in HIBD+ZnPP group,it was higher (234.9±4.5)(P＜0.05).The average gray value of CYGB was decreased with the extension of hypoxic-ischemic time.At 48 h,the average gray value of CYGB was the lowest in HIBD+Hemin group (126.0± 2.6),followed by HIBD group (150.9±4.5) and HIBD+ZnPP group (163.7±6.3),and the highest was in sham operated group (232.1±5.8)(all P＜0.01).(2) Histopathologic changes of the brain:At 48 h,typical cerebral infarction and hemorrhage were seen in HIBD,HIBD+Hemin and HIBD+ZnPP group,but those were less severe in HIBD + Hemin group than in HIBD + ZnPP group.(3) The water content of the brain:At 72 h,the water content of the left brain in HIBD and HIBD+ZnPP group was (86.5±0.4)％ and (87.3±0.3)％,which was higher than that in right brain [(85.6±0.2)％ and (85.9±0.2)％] (t 12.57 and 11.32,P＜0.01,respectively).(4)Function of the hippocampus:Morris water-maze showed that the longest average escape latency in HIBD+ZnPP group [(76.7±29.8) s],followed by HIBD group [(71.0±30.5) s],HIBD+ Hemin group [(46.7±34.0) s],and sham operated group [(38.3±30.3) s] (all P＜0.01).(5) Long-term histopathologic changes of the brain:At 34 d,brain atrophy rate was the highest in HIBD+ZnPP group [(34.07± 6.75) ％],and then in HIBD group [(29.73± 6.53) ％] and HIBD+ Hemin group [(18.33±4.52)％],which were all higher than that in the sham operated group [(1.55±1.32)％](all P＜0.01).HE staining showed that the hippocampal stratum pgramidale was getting thinner and a large number of neurons was lost in HIBD and HIBD+ ZnPP group,but only a small amout of neurons was lost in HIBD+ Hemin group.Conclusions Increased expression of CYGB in HIBD brain could mitigate the short term and long term pathological injury,and protect the long-term study memory function of the hippocampus. Key words: Hypoxia ischemia, brain; Globins; Rats

Inhibiting heme oxygenase-1 attenuates rat liver fibrosis by removing iron accumulation

To investigate the effects of the heme oxygenase (HO)-1/carbon monoxide system on iron deposition and portal pressure in rats with hepatic fibrosis induced by bile duct ligation (BDL). Male Sprague-Dawley rats were divided randomly into a Sham group, BDL group, Fe group, deferoxamine (DFX) group, zinc protoporphyrin (ZnPP) group and cobalt protoporphyrin (CoPP) group. The levels of HO-1 were detected using different methods. The serum carboxyhemoglobin (COHb), iron, and portal vein pressure (PVP) were also quantified. The plasma and mRNA levels of hepcidin were measured. Hepatic fibrosis and its main pathway were assessed using Van Gieson's stain, hydroxyproline, transforming growth factor-β1 (TGF-β1), nuclear factor-E2-related factor 2 (Nrf2), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Serum COHb and protein and mRNA expression levels of HO-1 and Nrf2 were increased in the BDL group compared with the Sham group and were much higher in the CoPP group. The ZnPP group showed lower expression of HO-1 and Nrf2 and lower COHb. The levels of iron and PVP were enhanced in the BDL group but were lower in the ZnPP and DFX groups and were higher in the CoPP and Fe groups. Hepcidin levels were higher, whereas superoxide dismutase levels were increased and malonaldehyde levels were decreased in the ZnPP and DFX groups. The ZnPP group also showed inhibited TGF-β1 expression and regulated TIMP-1/MMP-2 expression, as well as obviously attenuated liver fibrosis. Reducing hepatic iron deposition and CO levels by inhibiting HO-1 activity though the Nrf2/Keap pathway could be helpful in improving hepatic fibrosis and regulating PVP.

The protective effect of glutamine on endotoxemic intestinal injury and expression of heme oxygenase-1 in rats

To investigate the protective effect of glutamine (Glu) pretreatment on intestinal injury induced by endotoxin and expression of heme oxygenase-1 (HO-1) in rats. Thirty-two male Sprague-Dawley (SD) rats were randomly divided into four groups (n=8 in each group): normal control group, model group, Glu group and Glu+zinc protoporphyrin (ZnPP) group. In model group, endotoxemia was produced by intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). In Glu group, the rats received intragastrically 1 g/kg of Glu 12 hours before LPS intraperitoneal injection. In Glu+ZnPP group, the rats received 1 g/kg of Glu by gavage 12 hours before LPS intraperitoneal injection and ZnPP 10 mmol/kg intravenously via tail vein 1 hour before LPS injection. The distal ileum was harvested in full thickness 12 hours after LPS injection. The myeloperoxidase (MPO) activity, tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the intestine were determined, the pathologic changes were observed and expressed in Chiu grade. The expression of HO-1 was evaluated by immunohistochemistry method. Compared with normal control group, the Chiu grade, MPO activity, the content of TNF-α and IL-10 were significantly increased in model group [Chiu grade: 3.3±0.4 vs. 1.1±0.6, MPO activity (U/g): 0.40±0.08 vs. 0.26±0.07, TNF-α (ng/g): 25.2±6.9 vs. 6.5±2.8, IL-10 (ng/g): 27.6±10.2 vs. 5.7±2.9, all P<0.01], and the expression of HO-1 was decreased. Compared with model group, the Chiu grade, MPO activity, the content of TNF-α in Glu group were significantly decreased [Chiu grade: 1.6±0.5 vs. 3.3±0.4, MPO activity (U/g): 0.25±0.05 vs. 0.40±0.08, the content of TNF-α (ng/g): 13.4±3.2 vs. 25.2±6.9, all P<0.01], while the level of IL-10 (ng/g) elevated (47.3±5.5 vs. 27.6±10.2, P<0.01), and the expression of HO-1 was increased. There was no difference in above mentioned indexes between model group and Glu+ZnPP group. Glu pretreatment significantly ameliorates the expression of HO-1 of intestinal tissue induced by LPS in rats, and intestinal mucosa is protected with alleviation of inflammatory reaction in intestinal tract.

Heme oxygenase-1 protects donor livers from ischemia/reperfusion injury: the role of Kupffer cells.

To examine whether heme oxygenase (HO)-1 overexpression would exert direct or indirect effects on Kupffer cells activation, which lead to aggravation of reperfusion injury. Donors were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4 degrees C for 24 h before transplantation. Kupffer cells were isolated and cultured for 6 h after liver reperfusion. Postoperatively, serum transaminases were significantly lower and associated with less liver injury when donors were pretreated with CoPP, as compared with the ZnPP group. Production of the cytokines tumor necrosis factor-alpha and interleukin-6 generated by Kupffer cells decreased in the CoPP group. The CD14 expression levels (RT-PCR/Western blots) of Kupffer cells from CoPP-pretreated liver grafts reduced. The study suggests that the potential utility of HO-1 overexpression in preventing ischemia/reperfusion injury results from inhibition of Kupffer cells activation.

Heme Oxygenase-1 Prevents Hyperthyroidism Induced Hepatic Damage via an Antioxidant and Antiapoptotic Pathway

The exact pathogenesis of hepatic dysfunction in hyperthyroidism is still unknown. We aimed to investigate the pathogenesis of liver dysfunction caused by hyperthyroidism through inducing heme oxygenase-1 (HO-1) expression, which has antioxidant and anti-apoptotic properties. Rats were divided into six groups: untreated (group 1), treated with zinc protoporphyrin (ZnPP) (group 2), treated with hemin (group 3), treated with tri-iodothyronine (T3) (group 4), treated with T3 and ZnPP (group 5), and treated with T3 and hemin (group 6). After 22 d, oxidative stress and antioxidant enzymes and the expression of HO-1, mitochondrial permeability transition, cytochrome c, Bax, Bcl-2, caspase-3, caspase-8, and caspase-3 activity, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were examined. Hyperthyroidism induced oxidative stress of liver tissue was ameliorated by HO-1 induction. Administration of hemin (HO-1 inducer) increased Bcl-2 expression. Decreased expression of cytochrome c was accompanied by a decrease in caspase-3, caspase-8, Bax expression, and caspase-3 activity. The apoptotic activity and oxidative damage were found to be increased by the administration of ZnPP (HO-1 inhibitor). Immunohistochemistry findings supported these results. HO-1 induction plays a protective role in the pathogenesis of the liver dysfunction in hyperthyroidism. This effect is dependent on modulation of the antiapoptotic and antioxidative pathways by HO-1 expression.

Effects of zinc protoporphyrin and NO synthase inhibitor on cyclic guanosine monophosphate content in penile tissue of rats

The present study was undertaken to investigate the influence of carbon monoxide (CO) synthase inhibitor zinc protoporphyrin (ZnPP) and nitrous oxide (NO) synthase inhibitor L‐NAME on cGMP content in penile tissue of rats and the relation of them to erectile dysfunction. Thirty male Sprage‐Dawley rats were divided into 3 groups (normal control group, ZnPP group and L‐NAME group) and given the physiological saline, ZnPP (45µmol/kg) and L‐NAME (10mg/kg) each group, then the rats sacrificed 3 days later. The penis were made into homogenate to determine the content of nitric oxide synthase(NOS), NO, CO and cGMP. Compared with control group, the content of CO, NOS, NO and cGMP decreased in the ZnPP group and L‐NAME group, and the statistical difference was significant. The results suggest that the CO synthase inhibitor ZnPP and NO synthase inhibitor L‐NAME can reduce the content of CO, NO and cGMP that contain in the rat penis, however, the concrete mechanism still needs further research.

Pharmacological Preconditioning With Simvastatin Protects Liver From Ischemia-Reperfusion Injury by Heme Oxygenase-1 Induction

The protective role of heme oxygenase-1 (HO-1) against liver ischemia-reperfusion (I/R) injury in models of hypoxic and remote preconditioning has been proved. The feasible candidates who induce HO-1 and thorough which exert the protective effects are under investigation. The aim was to study the role of HO-1 in pharmacological preconditioning by simvastatin in a rat model. Pharmacological preconditioning by intraperitoneal injection of simvastatin (5 mg/kg) was tested on a partial liver I/R model on rats. The expression of HO-1 protein and enzyme activities in livers, serum alanine transaminase (ALT) levels, and TUNEL staining of liver after I/R injury were measured in rats with and without simvastatin preconditioning. HO-1 was induced and persistently overexpressed in the hepatocytes 24 hr after simvastatin treatment. Simvastatin preconditioning diminished the elevation of serum ALT levels 4 hr after I/R injury (69.6+/-26.3 U/L) (P<0.05 vs. other groups) when compared with control (403.8+/-261.9 U/L) and zinc protoporphyrin (ZnPP)-pretreated (717.5+/-205.6 U/L) groups. Simvastatin preconditioning diminished the apoptosis after I/R injury as well (apoptosis index: 26.4+/-8 for Simvastatin, 78+/-7 for control, and 85.3+/-2 for ZnPP group; P<0.05). The addition of ZnPP negated the protective effects of simvastatin as evidenced in the ALT level (406.2+/-243.0 U/L) and apoptosis index (75.6+/-6). The heme oxygenase activity in treated rats correlated with these results. The induction of HO-1 by simvastatin preconditioning played a protective role against hepatic I/R injury.

Protective effects of the induction of heme oxygenase-1 on ischemia reperfusion lung injury: in vivo experiment with rats

To investigate the protective effects of the induction of heme oxygenase-1 (HO-1) on ischemia/reperfusion lung injury. Forty Sprague-Dawley rats were randomly divided into four equal groups: sham group, lung ischemia/reperfusion injury (I/R) group, undergoing ligaturing of the left lung hilum for 30 minutes followed by reperfusion for 120 minutes; hemin group, undergoing intraperitoneal injection of hemin, an inducer of HO-1, 48 hours before the ligation and reperfusion; zinc protoporphyrin (ZnPP) group, undergoing intravenous injection of ZnPP, an inhibitor of heme oxygenase, 15 min after the ischemia-reperfusion; and sham operation group, undergoing sham operation. Two hours after the I/R arterial blood samples were collected and then the left lungs of the rats were taken out. Plasma tumor necrosis factor-alpha (TNF-alpha) and lung superoxide dismutase (SOD) activity were examined. Lung wet-to-dry weight (W/D) ratio was measured. The ultrastructure of the pulmonary alveoli and its capillaries were studied by using transmissional electronmicroscopy. The lung W/D ratio of the hemin group was 5.92 +/- 0.66, significantly lower than that of the I/R group (7.55 +/- 0.66, P < 0.01), and that of the ZnPP group (7.34 +/- 0.39, P < 0.01). The SOD activity of the hemin group was 6.5 +/- 0.6 U/mg protein, significantly higher than those of the I/R group and ZnPP group (2.8 +/- 0.4 U/mg protein and 3.0 +/- 0.4 U/mg protein respectively, both P < 0.01). The plasma TNF-alpha was 180.36 +/- 12.46, significantly lower than those of the I/R and ZnPP groups (452.26 +/- 22.59 and 438.59 +/- 30.26 respectively, both P < 0.01). Transmissional electronmicroscopy showed that the microscopic structure of the sham group was normal and that the pathological changes of hemin group were milder then those of the T/R and ZnPP groups. The induction of heme oxygenase-1 can protect effectively the lesion of lung pathology in ischemia reperfusion in vivo.

Pyrrolidine dithiocarbamate protects the small bowel from warm ischaemia/reperfusion injury of the intestine: the role of haem oxygenase

IR (ischaemia/reperfusion) injury of the intestine occurs commonly during abdominal surgery. We have previously shown that PDTC (pyrrolidine dithiocarbamate), an HO-1 (haem oxygenase-1) donor, improves intestinal microvascular perfusion. In the present study, we have investigated the effects of PDTC on the intestinal microcirculation following IR (ischaemia/reperfusion) injury of the intestine. Male Sprague-Dawley rats (n=72) were randomly assigned to four groups (n=18/group): (i) sham-operated group, who underwent laparotomy without induction of IR of the intestine; (ii) IR group, who were subjected to 30 min of superior mesenteric artery occlusion and 2 h of reperfusion; (iii) PDTC+IR group, who received PDTC prior to IR; and (iv) ZnPP group, who received the HO-1 inhibitor ZnPP (zinc protoporphyrin) followed by procedures as in group (iii). The ileum was evaluated for changes in tissue cytochrome c oxidase redox status, RBC (red blood cell) dynamics and leucocyte-endothelial interactions. The expression of HO-1 in the ileal tissue was examined at the end of the reperfusion. PDTC significantly improved the intestinal tissue oxygenation, mucosal perfusion index and RBC velocity compared with the IR and ZnPP groups. PDTC also decreased the leucocyte-endothelial interactions (P<0.05 compared with the IR and ZnPP groups). PDTC induced the expression of HO-1, whereas ZnPP abolished this effect.

Heme Oxygenase-1 Overexpression Protects Rat Livers from Ischemia/Reperfusion Injury with Extended Cold Preservation

This study analyzes the effects and mechanisms of heme oxygenase-1 (HO-1)-mediated cytoprotection in rat livers exposed to cold preservation. In the first series, rats were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4°C for 24 h, and then perfused ex vivo for 2 h. Livers pretreated with CoPP had significantly higher portal venous blood flow and increased total bile production, as compared with the ZnPP group. This correlated with histologic (Banff) criteria of hepatocyte injury/liver function. In the second series, rat livers were stored at 4 °C for 24 h or 40 h, and then transplanted into syngeneic recipients. After 24 h of preservation, 80% of rats bearing CoPP-pretreated liver grafts survived 21 days (vs. 50% in controls). After 40 h of cold preservation, liver transplant survival at day 1, 7 and 21 for the CoPP group was: 100%, 71% and 57%, respectively (vs. 50%, 50% and 33% in controls). This correlated with improved hepatic function/histologic (Suzuki) criteria of hepatocyte injury after HO-1 overexpression (immunohistology/Western blots) by infiltrating macrophages. This study documents the potential utility of HO-1-inducing agents in preventing ischemia/reperfusion injury resulting from prolonged storage of liver transplants.