Cobalt Protoporphyrin Group Research Articles

Cobalt protoporphyrin promotes heme oxygenase 1 expression and ameliorates cardiac dysfunction in long-term fasting mice

BackgroundThe association between malnutrition and cardiac dysfunction has been reported. Heme oxygenase (HO)-1 played protective roles in the animals functioning as a myocardial infarction, heart failure, or cardiomyopathy model. We hypothesized that the administration of HO-1 inducer, cobalt protoporphyrin (CoPP) reduces oxidative stress and ameliorates cardiac systolic dysfunction in long-term fasting mice. MethodsC57BL/6 J mice were classified into three groups: fed mice (fed group), 48-h fasting mice with a single intraperitoneal injection of the corresponding vehicle (fasting group), and 48-h fasting mice with a single intraperitoneal injection of 5 mg/kg CoPP (CoPP group). ResultsThe fasting group showed a significant increase in heme and 4-hydroxy-2-nonenal (4HNE) protein in the heart tissue, and reduced left ventricular ejection fraction (LVEF) when compared with the fed group. The CoPP group showed significantly increased protein levels of nuclear factor-erythroid 2-related factor 2 and HO-1, and increased mRNA expression levels of HO-1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, forkhead box protein O1, sirtuin-1, cyclooxygenase 2, and superoxide dismutase 2, and reduced levels of heme and 4HNE protein when compared with the fasting group. LVEF were significantly higher in the CoPP group than in the fasting group. ConclusionsAdministration of CoPP reduced heme accumulation and oxidative stress, and ameliorated cardiac systolic dysfunction in long-term fasting mice. This study suggests that heme accumulation may be associated with impaired cardiac function induced by long-term fasting and that HO-1 may be a key factor or therapeutic target.

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.

Effects of heme oxygenase-1 upregulation on isoproterenol-induced myocardial infarction

The present study was designed to examine the effect of heme oxygenase-1 (HO-1) induction by cobalt protoporphyrin (CoPP) on the cardiac functions and morphology, electrocardiogram (ECG) changes, myocardial antioxidants (superoxide dismutase [SOD] and glutathione [GSH]), and expression of heat shock protein (Hsp) 70 and connexin 43 (Cx-43) in myocardial muscles in isoproterenol (ISO) induced myocardial infarction (MI). Thirty two adult male Sprague Dawely rats were divided into 4 groups (each 8 rats): normal control (NC) group, ISO group: received ISO at dose of 150 mg/kg body weight intraperitoneally (i.p.) for 2 successive days; ISO + Trizma group: received (ISO) and Trizma (solvent of CoPP) at dose of 5 mg/kg i.p. injection 2 days before injection of ISO, with ISO at day 0 and at day 2 after ISO injections; and ISO + CoPP group: received ISO and CoPP at a dose of 5 mg/kg dissolved in Trizma i.p. injection as Trizma. We found that, administration of ISO caused significant increase in heart rate, corrected QT interval, ST segment, cardiac enzymes (lactate dehydrogenase, creatine kinase-muscle/brain), cardiac HO-1, Hsp70 with significant attenuation in myocardial GSH, SOD, and Cx-43. On the other hand, administration of CoPP caused significant improvement in ECG parameters, cardiac enzymes, cardiac morphology; antioxidants induced by ISO with significant increase in HO-1, Cx-43, and Hsp70 expression in myocardium. In conclusions, we concluded that induction of HO-1 by CoPP ameliorates ISO-induced myocardial injury, which might be due to up-regulation of Hsp70 and gap junction protein (Cx-43).

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.

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.

Endogenous carbon monoxide downregulates hepatic cystathionine-γ-lyase in rats with liver cirrhosis.

The aim of the present study was to investigate the effect of endogenous carbon monoxide (CO) on the hydrogen sulfide/cystathionine-γ-lyase (H2S/CSE) pathway in cirrhotic rat livers. The rats were allocated at random into four groups: Sham, cirrhosis, cobalt protoporphyrin (CoPP) and zinc protoporphyrin IX (ZnPP). The expression of hepatic CSE mRNA was evaluated using a quantitative polymerase chain reaction, while CSE protein expression was determined using immunohistochemical analysis. Hematoxylin and eosin staining was performed for the histological evaluation of liver fibrosis. The levels of H2S, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and carboxyhemoglobin (COHb) in the arterial blood were determined, in addition to the portal vein pressure. The mRNA and protein expression levels of hepatic CSE and the serum levels of H2S were significantly decreased in the cirrhosis group compared with those in the sham group (P<0.05). Compared with the cirrhosis group, rats in the ZnPP group had significantly lower levels of serum ALT, AST and TBIL, arterial COHb and hepatic fibrosis, while hepatic CSE expression and the production of H2S were significantly increased (P<0.05). The CoPP group exhibited decreased hepatic CSE expression and H2S production, but aggravated hepatic function and fibrosis (P<0.05). In conclusion, the H2S/CSE pathway is involved in the formation of liver cirrhosis and serves a crucial function in protecting liver cells against the progression of liver fibrosis. Endogenous CO downregulates hepatic CSE mRNA and protein expression and the production of H2S in rats with liver cirrhosis.

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.

Role of the heme oxygenase/carbon monoxide pathway in the pathogenesis and prevention of hepatic encephalopathy

Hepatic encephalopathy (HE) is a severe complication of liver cirrhosis and its pathogenesis has yet to be fully elucidated. Previous studies have demonstrated that heme oxygenase-1 (HO-1) is important in the induction of liver cirrhosis. The present study aimed to investigate the role of HO-1 in the pathogenesis of HE. Rats were divided into 5treatment groups; sham, bile duct ligation (BDL), HE, zinc protoporphyrin (ZnPP) and cobalt protoporphyrin (CoPP). The levels of HO-1 were examined by western blotting and quantitative real-time PCR (qRT-PCR). Serum levels of carboxyhemoglobin (COHb), ammonia levels in the plasma and brain, brain water content and portal vein pressure (PVP) were also quantified. Aquaporin-4 expression levels were measured by immunohistochemistry and qRT-PCR. The results demonstrated that the levels of HO-1 in the brain and the serum levels of COHb were significantly increased in the HE group compared with the BDL group. Brain water content, PVP and ammonia levels in the plasma and brain were increased in the HE and CoPP groups; however, these were reduced following the treatment with ZnPP. The levels of AQP-4 expression and oxidative stress in the brain were reduced following treatment with ZnPP and increased following treatment with CoPP. In conclusion, following the inhibition of HO-1 expression, treatment with ZnPP improved HE due to reducing the expression levels of AQP-4 and oxidative stress. Therefore, ZnPP treatment may represent a novel therapeutic approach for HE.

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.

Influence of heme oxygenase-1 expression on immune liver fibrosis induced by cobalt protoporphyrin in rats

To investigate the effect of heme oxygenase-1 (HO-1) expression on immune liver fibrosis induced by cobalt protoporphyrin (CoPP) in rats. An immune liver fibrosis model of rat was established by administering human serum albumin (HSA). The rats were divided into CoPP, liver fibrosis and normal control groups. Rats in the CoPP group received intraperitoneal CoPP concurrently with HSA. Expression of HO-1 protein was observed by Western blotting and immunohistochemistry. Hematoxylin and eosin (HE) staining was performed to assess fibrosis proliferation and distribution, proliferation extent of fibroblasts, and alterations in hepatocytes and inflammatory cells. Type I and III collagens were detected with Van Gieson's (VG) staining and Foot's reticular fiber staining, respectively. In addition, spindle-shaped cells existing at perisinusoidal locations beyond portal and septa areas were investigated with HE staining. Western blotting and immunohistochemistry showed that the expression of HO-1 protein was higher in the CoPP group than in the liver fibrosis group (P < 0.05). Compared with the liver fibrosis group, the serological index of hepatic fibrosis in the CoPP group decreased significantly (P < 0.05). HE, VG and Foot's staining revealed that administration of CoPP reduced the extent of hepatic fibrosis. The levels of serological indicators and the number of spindle-shaped cells at perisinuous locations beyond the portal and septa areas were reduced in the CoPP group. Only a few inflammatory cells were seen around the portal areas and central veins in the CoPP group. Increased endogenous HO-1 may suppress liver fibrosis by protecting liver cells, inhibiting inflammatory cell infiltration and hepatic stellate cell transformation.

Cobalt-protoporphyrin treatment enhances murine isoislets engraftment

To study the effect of treatment with cobalt-protoporphyrin (CoPP) for the induction of the heme oxygenase-1 (HO-1) enzyme on islet engraftment donor mice received either a single intraperitoneal injection of CoPP (20 mg/kg body weight) 1 day prior to islet isolation or this injection plus a 9 day posttransplantation course of Copp. After a single injection of CoPP, the CoPP-induced islets contained higher HO-1 proteins than did the normal islets both at 12 (5.3 ± 1.5 vs 0.1 ± 0.1 ng/mg protein, P < .01) and at 30 hours (6.8 ± 2.1 vs 0.4 ± 0.3 ng/mg protein, P < .05), but not at 56 hours (1.9 ± 0.8 vs 1.6 ± 0.8 ng/mg protein, P > .05). In contrast, diabetic mice that received 75 CoPP-induced islets and a 9-day CoPP injection course posttransplantation showed better improvement in blood glucose levels and body weights than did the mice that only received CoPP-induced islets. Mice of both CoPP-treated groups displayed better improvement in glycemic control than mice that received control islets. At 8 weeks after transplantation, the insulin content of grafts from both CoPP groups was significantly higher than that in the control group. In conclusion, CoPP treatment for the induction of HO-1 enhances engraftment of islets in a syngeneic murine transplantation model.

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.