Hepatic Heme Oxygenase-1 mRNA Research Articles

Cobalt protoporphyrin protects the liver against apoptosis in rats of brain death

Brain death (BD) leads to a marked increase in apoptosis, which influences the viability of donor organs. Induction of heme oxygenase 1 (HO-1) has been shown to exert beneficial effects in different liver injury models. Therefore, we examined the effect of pretreating rats with cobalt protoporphyrin (CoPP), an HO-1 inducer, on apoptosis in liver during BD and elucidated the mechanisms involved. First, rats were killed at 0, 1, 2, 4 and 6 h after BD induction to examine the expression of hepatic HO-1. Second, rats were randomly divided into four groups (n=6): (S group) rats undergoing sham operation, (CS group) rats pretreated with CoPP for 24 h before the sham operation, (B group) rats undergoing BD for 6 h, (CB group) rats pretreated with CoPP for 24 h before BD induction. The expression levels of hepatic HO-1 mRNA and protein in rats increased at 0, 1, 2, 4 and 6h after BD induction, compared with sham operated rats. In the CB group compared with the B group, the increased hepatic expression of HO-1 correlated with a significant decrease in serum ALT/AST levels, fewer apoptotic cells in liver, increased hepatic expression of Mcl-1 and Bcl-2, and decreased hepatic expression of Bax, cytosolic cytochrome c and cleaved caspase-3. CoPP inhibits apoptosis in liver of BD rats in part via modulating the mitochondrial apoptosis pathway. HO-1 may serve as a potential target for improving the quality of organs from BD donors.

Octreotide attenuates liver fibrosis by inhibiting hepatic heme oxygenase-1 expression.

The aim of the present study was to investigate the effects of octreotide treatment on hepatic heme oxygenase-1 (HO-1) expression, together with the influence of altered hepatic HO-1 expression levels on hepatic function and fibrosis in bile duct-ligated rats. The rats were divided randomly into sham, cirrhotic, cobalt protoporphyrin and octreotide treatment groups. The expression levels of hepatic HO-1 mRNA were measured by reverse-transcription polymerase chain reaction, while the protein expression was determined by western blotting and immunohistochemical analysis. Hematoxylin and eosin, and Van Gieson’s staining, along with determination of the hydroxyproline content in the liver, were performed to determine the degree of liver fibrosis. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and carboxyhemoglobin (COHb) in arterial blood, and the mean arterial pressure and portal vein pressure were also measured. As compared with the sham group, hepatic HO-1 mRNA and protein expression levels, serum levels of ALT, AST and TBIL, COHb in arterial blood, hydroxyproline and collagen type I content were all significantly increased in the cirrhotic group. As compared with the cirrhotic group, the octreotide-treated group exhibited significantly reduced hepatic HO-1 expression levels, serum levels of ALT, AST and TBIL, COHb in arterial blood and the extent of hepatic fibrosis, whereas the cobalt protoporphyrin group exhibited significantly increased hepatic HO-1 expression levels, as well as aggravated hepatic function and fibrosis (P<0.05). In conclusion, octreotide inhibited hepatic HO-1 overexpression in cirrhotic rats, reduced hepatic HO-1 expression levels to relieve liver injury and attenuated liver fibrosis.

Endothelial Deficiency Augments and Inhaled Nitric Oxide Prevents the Adverse Hemodynamic Effects of Transfusing Syngeneic Stored Blood in Mice

Abstract 38 Introduction:During storage, red blood cells (RBC) undergo progressive deleterious functional, biochemical and structural changes, producing a “storage lesion”. The storage lesion includes reversible and irreversible changes that begin within hours of storage, progress during storage, release free hemoglobin (Hb) and Hb-containing microvesicles, and impair RBC function and lifespan after transfusion. Our recent studies in mice with endothelial dysfunction revealed an enhanced systemic vasoconstrictor response to infusion of tetrameric Hb or Hb-based oxygen carriers. Therefore, we sought to test the hypothesis that endothelial dysfunction would predispose mice to the vasoconstrictor effects of infusion of fresh and stored murine blood. Methods:Murine leukoreduced RBC from C57BL/6 mice were prepared with 14% CPDA-1 anticoagulant and stored at 4°C for either ≤24 h (fresh red blood cells, FRBC) or 2 weeks (stored red blood cells, SRBC). RBC morphology, as well as ATP levels, 2,3-diphosphoglycerate (2,3-DPG) levels, and P50 were measured in FRBC and SRBC before transfusion. We also prepared murine RBC storage components, i.e. supernatant from FRBC or SRBC, oxidized supernatant from SRBC, and washed SRBC. We studied three groups of mice, awake wild-type mice (WT, C57BL/6) fed a standard diet, WT mice fed a high-fat diet (HFD) for 4–6 weeks (to induce endothelial dysfunction), and diabetic (db/db, C57BL/6 background) mice. Each group was transfused with FRBC, SRBC, and db/db mice also received RBC components (10% of total blood volume). Systolic blood pressure (SBP) was measured every 10 min in awake mice before and 2 h after transfusion with FRBC, SRBC or RBC components. A subgroup of mice transfused with SRBC also breathed air (FiO2=0.21) supplement with nitric oxide (NO, 80 parts per million (ppm)). Invasive hemodynamic measurements were performed in anesthetized mice in order to obtain mean arterial blood pressure, heart rate, and cardiac output. Blood and tissue samples were collected 2 h after FRBC or SRBC transfusion for determination of plasma Hb and iron levels, and measurement of lung and liver levels of mRNA encoding inflammatory cytokines. In addition, heme oxygenase-1 (HO-1) in lung and liver was measured 2 h after FRBC or SRBC transfusion. Results:SRBC were characterized by altered RBC morphology, decreased ATP and 2,3-DPG levels, and a reduced P50. Transfusion of SRBC into awake WT mice fed a standard diet or HFD produced no systemic hemodynamic changes. In contrast, transfusion of SRBC or supernatant from SRBC into db/db mice induced systemic hypertension that was prevented by concurrent inhalation of NO. Infusion of washed SRBC or oxidized SRBC supernatant into db/db mice did not induce systemic vasoconstriction or hypertension. Invasive hemodynamic studies confirmed that transfusion of SRBC and SRBC supernatant induced systemic vasoconstriction and hypertension, but transfusion of washed SRBC did not. Plasma Hb levels were greater in all mouse groups at 2 h after transfusion of SRBC but not after FRBC transfusion. Two hours after transfusion of SRBC, plasma interleukin-6 and iron levels, as well as hepatic HO-1 mRNA levels, were increased in all mouse groups. Conclusions:Syngeneic transfusion of SRBC or only the supernatant from SRBC but not washed SRBC produces systemic hypertension and vasoconstriction in db/db mice, which is prevented by oxidizing the supernatant of SRBC or breathing NO during SRBC transfusion. Infusion of SRBC induced a mild systemic inflammatory response in WT fed a standard diet or HFD, and db/db mice. Transfused cell-free oxyHb in the supernatant released from RBC during storage appears to be responsible for the vasoconstriction produced in db/db mice, since it is prevented by oxidizing the supernatant. Our current data support examining the link between the RBC storage lesion and cardiovascular and immunological perturbations in highly susceptible recipients with endothelial dysfunction. Disclosures:Yu:Massachusetts General Hospital: patents on inhaled nitric oxide and blood transfusion. Bloch:MGH has received sponsored research grant funding from Ikaria LLC, the maker of nitric oxide gas for inhalation in the US, in support of Dr. Bloch’s research program.: Research Funding. Zapol:Dr. Warren Zapol receives royalties from patents on inhaled nitric oxide licensed by Massachusetts General Hospital to Linde Corp, Munich, Germany, and Ikaria Corp, Clinton, New Jersey. Dr. Zapol has applied for patents on inhaled nitric oxide and blood t: Patents & Royalties.

Hepatic steatosis prevents heme oxygenase-1 induction by isoflurane in the rat liver

To characterize the inductive effects of isoflurane (ISO) on hepatic heme oxygenase-1 (HO-1) in an animal model of hepatic steatosis. Lean (LEAN) and obese (FAT) Zucker rats were randomized into 4 groups: 1: LEAN + pentobarbital sodium (PEN); 2: LEAN + ISO; 3: FAT + PEN; 4: FAT + ISO. The animals were mechanically ventilated for 6 h. In vitro analyses of liver tissue included determination of HO-1 mRNA and protein expression as well as measurement of HO enzyme activity and immunohistochemical analyses. Compared to PEN treatment, ISO administration profoundly induced hepatic HO-1 mRNA and protein expression and significantly increased HO enzyme activity in lean Zucker rats. In contrast, no difference in HO-1 gene expression was observed after ISO or PEN anesthesia in obese Zucker rats. The present study demonstrates that ISO is an inducer of hepatic HO-1 gene expression in non-steatotic organs but failed to upregulate HO-1 in steatotic livers.

Effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on heme oxygenase-1, biliverdin IXα reductase and δ-aminolevulinic acid synthetase 1 in rats with wild-type or variant AH receptor

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its monoglucuronide in moderately TCDD-resistant line B rats, but not in highly TCDD-resistant line A rats. In the mammalian heme degradation process, heme is cleaved to biliverdin by the rate-limiting enzyme heme oxygenase-1 (HO-1). Subsequently, biliverdin IXα reductase (BVRA) catalyzes the reduction of biliverdin to bilirubin. In heme biosynthesis, the rate-limiting enzyme is δ-aminolevulinic acid synthetase 1 (ALAS1). The effect of TCDD on HO-1, BVRA and ALAS1 was studied at the levels of mRNA (all three enzymes), protein expression (HO-1), and enzymatic activity (BVRA, liver only) in order to determine whether the accumulation of biliverdin could be due to their altered expression. In both lines A and B, 300 μg/kg TCDD transiently repressed hepatic HO-1 mRNA on day 2 but induced HO-1 protein expression at later time-points; however, the impact emerged earlier (day 14 vs. day 35) in line B rats. In spleen, TCDD repressed HO-1 mRNA and protein expression in lines A and B through days 2–35, but did not affect its mRNA levels in TCDD-sensitive L-E rats (10 days after 100 μg/kg). In all rat strains/lines, there was a strong repression of ALAS1 and a moderate induction of BVRA mRNA in liver, but mostly not in spleen. Hepatic BVRA activity was increased in lines A and B on day 14. At 5 weeks, it was still elevated in line A but reduced to 51% of control in line B. The results suggest that hepatic heme degradation is induced by TCDD in rats; however, this does not alone explain the accumulation of biliverdin in line B rats. Other factors such as the late repression of BVRA found here and possibly oxidative stress may be important contributors to biliverdin accumulation in these rats.

S1899 Genetic Ablation of Bach1 Followed By Upregulation of Heme Oxygenase-1 Improved Nonalcoholic Steatohepatitis in Rodent Model

Backgrounds/Aims: Bach1 is a transcriptional repressor of heme oxygenase-1 (HO-1). HO1 has cytoprotective activities through its antioxidant and anti-inflammatory properties. In this study, we investigated the effect of bach1 knockout on an animal model of NASH. Methods: Ten-week old male C57BL/6J mice (WT) and bach1 deficient mice (KO) were pair-fed either a chow or methionine/choline deficient (MCD) diet for 8 weeks (n=15 each). At fixed periods, mice were sacrificed, and livers and blood were collected. Histological findings were assessed by HE and Azan-Mallory staining. Serum transaminases were analyzed enzymatically. Hepatic mRNA expression of HO-1, TGF-β1, αSMA, peroxisome proliferator activated receptor α (PPARα) and microsomal triglyceride transfer protein (MTP) were analyzed by real-time PCR. Hepatic content of triglycerides (TG) was analyzed enzymatically. As the determination of hepatic oxidative stress generation, hepatic malondialdehyde (MDA) content was analyzed enzymatically. Results: Whereas MCD diet promoted significantly greater weight loss (chow -15%; MCD -40%, P < 0.05), significant change was not observed between WT and KO mice on each diet. At 8 weeks on MCD diet, liver inflammation and steatosis were pathohistologically significantly attenuated in KO mice when compared with WT mice, but changes found in the serum level of ALT were not significant. The expression of hepatic HO-1 mRNA was increased in KO mice when compared with WT mice (6.1fold). Upregulation of hepatic MDA content by MCD diet (4.2-fold) was inhibited in KO mice thoroughly. MCD diet feeding increased hepatic content of TG in WT mice (6.1-fold). But, in KO mice, such change was repressed significantly. In WT mice, MCD diet reduced hepatic PPARα and MTP mRNA expression significantly, whereas such a change was not observed in KO mice. Hepatic expression of TGF-β1 and αSMA mRNA was expressed at higher levels in WT mice after 1 and 4 weeks on the MCD diet when compared to KO mice. Such changes were not observed in WT and KO mice on chow diet. Conclusions: Up-regulation of HO-1 induced by Gene ablation of bach1 improved steatosis, at least in part, through themechanism of hepatic secretion of TG associated with PPARα-MTP pathway. HO-1 also inhibited ROS generation in the liver and suppressed the activation of hepatic stellate cells, suggesting its causal relation to liver fibrosis. We speculate that HO-1 plays a protective role in the development of NASH through its cytoprotective and anti-oxidant activities.

Heme Oxygenase-1 Genotype of the Donor Is Associated With Graft Survival After Liver Transplantation

Heme oxygenase-1 (HO-1) has been suggested as a cytoprotective gene during liver transplantation. Inducibility of HO-1 is modulated by a (GT)(n) polymorphism and a single nucleotide polymorphism (SNP) A(-413)T in the promoter. Both a short (GT)(n) allele and the A-allele have been associated with increased HO-1 promoter activity. In 308 liver transplantations, we assessed donor HO-1 genotype and correlated this with outcome variables. For (GT)(n) genotype, livers were divided into two classes: short alleles (<25 repeats; class S) and long alleles (> or =25 repeats; class L). In a subset, hepatic messenger ribonucleic acid (mRNA) expression was correlated with genotypes. Graft survival at 1 year was significantly better for A-allele genotype compared to TT-genotype (84% vs. 63%, p = 0.004). Graft loss due to primary dysfunction (PDF) occurred more frequently in TT-genotype compared to A-receivers (p = 0.03). Recipients of a liver with TT-genotype had significantly higher serum transaminases after transplantation and hepatic HO-1 mRNA levels were significantly lower compared to the A-allele livers (p = 0.03). No differences were found for any outcome variable between class S and LL-variant of the (GT)(n) polymorphism. Haplotype analysis confirmed dominance of the A(-413)T SNP over the (GT)(n) polymorphism. In conclusion, HO-1 genotype is associated with outcome after liver transplantation. These findings suggest that HO-1 mediates graft survival after liver transplantation.

Heme Oxygenase-1 Induction by the Clinically Used Anesthetic Isoflurane Protects Rat Livers From Ischemia/Reperfusion Injury

It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and alpha-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.

Tissue-specific gene expression of heme oxygenase-1 (HO-1) and non-specific δ-aminolevulinate synthase (ALAS-N) in a rat model of septic multiple organ dysfunction syndrome

Reactive oxygen species are thought to be involved in the pathogenesis of septic multiple organ dysfunction syndrome (MODS). It has been reported that heme oxygenase-1 (HO-1) (EC 1.14.99.3) is induced in septic animal models and is thought to confer protection against oxidative tissue injury. In this study, we examined changes in gene expression of HO-1 and non-specific δ-aminolevulinate synthase (ALAS-N) (EC 2.3.1.37), the rate-limiting enzymes in heme catabolism and heme synthesis, respectively, after intraperitoneal administration of bacterial lipopolysaccharide (LPS) to rats. LPS treatment caused the elevation of body temperature, increases in white blood cell counts, and marked elevation of serum interleukin-6 levels associated with liver, lung, and kidney injuries, characteristic of septic MODS. LPS administration significantly induced HO-1 mRNA, protein, and enzyme activity in the liver, lung, and kidney. In contrast, ALAS-N mRNA was decreased rapidly in the liver, followed by an oscillating recovery pattern. Induction of hepatic HO-1 mRNA and rapid suppression of ALAS-N mRNA were likely the result of a rapid increase in hepatic free heme concentration as judged by the increase in heme saturation of tryptophan pyrrolase. In contrast to that in the liver, the ALAS-N mRNA level in the lung and kidney was increased significantly after LPS administration, suggesting a novel mechanism of ALAS-N regulation in these tissues. These findings suggest that HO-1 and ALAS-N mRNA are regulated in a tissue-specific manner in a rat model of septic MODS.

Kupffer cell depletion partially prevents hepatic heme oxygenase 1 messenger RNA accumulation in systemic inflammation in mice: role of interleukin 1beta.

The heme oxygenase 1 (HO-1) gene is rapidly activated in the liver after lipopolysaccharide (LPS) treatment. Ninety minutes after LPS treatment (0.1 mg/kg, intraperitoneally) hepatic HO-1 messenger RNA (mRNA) of mice was 40 times the control value. To investigate the hepatic cellular source of the increased HO-1 transcript, we treated mice with LPS and galactosamine (700 mg/kg, intraperitoneally), a selective transcriptional inhibitor of hepatocytes. Galactosamine prevented the LPS-mediated increase of HO-1 mRNA in the liver, indicating that hepatocytes are the main cell type in which HO-1 mRNA accumulates after LPS treatment. We then tested in vitro and in vivo the hypothesis that LPS-mediated hepatic accumulation of HO-1 mRNA is caused by intercellular communication between Kupffer cells and hepatocytes. Isolated rat hepatocytes showed an increase in HO-1 mRNA compared with controls after 90 minutes of exposure to a LPS stimulated Kupffer cell-conditioned medium. This suggests that soluble mediators from Kupffer cells were responsible for this effect. To study the role of Kupffer cells in vivo, we treated mice with Kupffer cell-inactivating or -depleting agents and LPS. Gadolinium chloride and liposome-encapsulated dichloromethylene diphosphonate lowered LPS-mediated HO-1 mRNA accumulation (by about 50%); in these groups hepatic levels of interleukin (IL)-1beta were decreased, by more than 75%. Methylpalmitate hardly affected hepatic HO-1 mRNA accumulation or IL-1beta content after LPS treatment. There was no relationship between HO-1 mRNA and serum TNF or IL-6 levels. These results suggest that LPS-mediated hepatic HO-1 mRNA accumulation is a hepatocyte response partly caused by soluble mediators, particularly IL-1beta, released from Kupffer cells.