Inducer of heme oxygenase‐1 cobalt protoporphyrin accelerates autophagy and suppresses oxidative damages during lipopolysaccharide treatment in rat liver

Aim: Mitochondrial damage and subsequent oxidative stresses play important roles in the pathogenesis of sepsis‐induced organ failure. Recently, autophagy, the major degradation pathway involved in mitochondrial quality control, was reported as a cellular adaptive response to oxidative stresses. The aim of the present study was to elucidate the molecular mechanism that underlies hepatic damage during lipopolysaccharide (LPS) treatment. We also try to determine if the damage can be attenuated by administration of cobalt protoporphyrin (CoPP), a potent heme oxygenase‐1 (HO‐1) inducer. Methods: Five‐week‐old male Sprague–Dawley rats were injected i.p. with 15 mg/kg LPS. To determine if hepatic damage following LPS administration can be attenuated by HO‐1, CoPP was injected s.c. for 4 days consecutively at 24‐h intervals. After treatment with LPS, the liver was obtained and analyzed. Results: A large reduction in liver mitochondrial protein and induction of autophagy were observed in LPS‐treated rats. Electron microscopic and immunohistochemical analyses demonstrated autophagic vacuoles in LPS‐treated rat liver. Oxidative stress markers (4‐hydroxy‐2‐nonenal and 8‐hydroxy‐2′‐deoxyguanosine) were increased in LPS‐treated animals; CoPP treatment ablated these alterations. An inhibitor for the opening of mitochondrial permeability transition pore, cyclosporine A, suppressed oxidative stress as well as liver damage during LPS administration. CoPP promoted autophagy and prevented rats from liver damage during LPS administration. Conclusion: HO‐1 promotes autophagy and elimination of damaged mitochondria thereby repressing oxidative stress in LPS‐treated rat liver, revealing a novel mechanism for protection by HO‐1 against septic liver damage.     

Tumor growth-promoting cellular host response during liver atrophy after portal occlusion.

BACKGROUND/AIMS: Clinical observations suggest cancer progression after preoperative segmental portal vein occlusion, a procedure to prevent liver failure after major hepatic resections. The aim of this study was to determine whether portal occlusion induces host reactions which promote cancer invasion and angiogenesis. METHODS: The rat model of portal branch ligation (PBL) was compared with partial hepatectomy (PH) and sham operation (SO) and evaluated for the expression of heat shock protein-70 (hsp70), heme oxygenase-1 (hmox1), early growth response gene-1 (Egr-1) and urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1) and receptor (uPAR). RESULTS: Portal deprivation after PBL was associated with a regression of liver tissue to 25% of its original mass within 8 days with only modest fibrotic changes. During the progression of atrophy, there were significant inductions of hsp70-, hmox1- and Egr-1-mRNA in comparison with regenerating liver tissue. PAI-1-specific mRNA was transiently elevated at 3 - 48 h after PBL in the atrophying lobes, whereas uPA and uPAR were unaffected in comparison with PH or SO. CONCLUSION: Hepatic atrophy caused by PBL is associated with increased expression of genes known to promote tumor growth. These host events represent a possible explanation for the tumor progression after portal occlusion and require further evaluation.     

Preconditioning donor liver with Nodosin perfusion lessens rat ischemia reperfusion injury via heme oxygenase-1 upregulation

Background and Aim: Ischemia reperfusion injury (IRI) remains a major cause of graft injury, dysfunction and even failure post‐transplantation. Heme oxygenase 1 (HO‐1) has been found to be an attractive target for anti‐inflammatory therapies and a potential candidate responsible for cell injury. The objective of this study was to investigate whether preconditioning the donor liver with Nodosin perfusion upregulates HO‐1 and then lessens IRI in rat models. Methods: Wistar rats were divided into four groups: experimental group, control group, positive control group and negative control group in which the donor liver was preconditioned with Nodosin, lactated ringer's solution, cobalt protoporphyrin and zinc protoporphyrin perfusion, respectively. We measured HO‐1 expression and enzyme activity in rat livers of each group ex vivo at 0, 1 and 2 h after perfusion. At 1 h after perfusion, donor livers of Wistar rats were transplanted into Sprague–Dawley rats orthotopically. Serum transaminase levels, degree of cell apoptosis and Suzuki's score were used to assess ischemia/reperfusion injury in recipients at 24 h after transplantation. Results: Ex vivo, donor liver preconditioning with Nodosin perfusion induced HO‐1 expression and enzyme activity significantly, compared with the control group (P < 0.05). In vivo, serum transaminase levels, cell apoptosis degree and Suzuki's score of representative recipients in the Nodosin group were lower than that in the control group (P < 0.05). Preconditioning with Nodosin perfusion induced HO‐1 protein mainly in Kupffer cells. Conclusions: This study suggests that preconditioning with Nodosin perfusion provides a potential protective effect through inducing HO‐1 expression to attenuate ischemia/reperfusion injury in liver transplantation.     

Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.     

Ferritin/alanine aminotransferase ratio as a possible marker for predicting the prognosis of acute liver injury

Background and Aims: Serum levels of ferritin and heme oxygenase (HO)‐1 are both markers of macrophage activation. We evaluated simple markers for predicting the prognosis of severe acute liver injury in which macrophage activation plays an important role. Methods: Subjects comprised 114 patients with acute liver injury, admitted to the liver unit of Nagasaki Medical Center between January 2001 and September 2010. Subjects included 11 patients with fulminant hepatic failure (FHF), 82 patients with ordinary acute hepatitis (AH), and 21 patients with severe‐form AH (AHS). We determined serum levels of ferritin, HO‐1 and other biochemical makers, and analyzed relationships between clinical outcomes of patients and each of these parameters alone and in combination. Results: Median serum ferritin levels were significantly higher in FHF (25 900 ng/mL) and AHS (3060 ng/mL) than in AH (700 ng/mL; P < 0.01 each). Median HO‐1 levels were also significantly higher in FHF (123 ng/mL) and AHS (51 ng/mL) than in AH (19 ng/mL; P < 0.01 each). Similarly, median ferritin/alanine aminotransferase (F/A) ratio was significantly higher in FHF (6.7) than in AHS (1.6, P < 0.05) or AH (0.5, P < 0.01). Among the 11 FHF patients, three recovered, seven died and one underwent liver transplantation. The ability of F/A ratio to distinguish non‐survivors from survivors was analyzed using receiver operating characteristics curves. A cut‐off level of 3.12 provided high sensitivity (87.5%) and specificity (81.2%). Conclusion: These results suggest that F/A ratio offer a quick and simple marker for predicting the prognosis of acute liver injury.     

Pulmonary Expression of iNOS and HO-1 Protein Is Upregulated in a Rat Model of Prehepatic Portal Hypertension

Portal hypertension is associated with a wide range of pulmonary pathophysiologies, ranging from portopulmonary hypertension to hepatopulmonary syndrome. Although the clinical and pathological features of pulmonary dysfunction in this setting have been extensively characterized, the underlying biology is not well understood. Specifically, the role of mediators that regulate mesenteric vascular hemodynamics in portal hypertension, such as nitric oxide and endothelin, have not been studied in the lung. Using a rat model of prehepatic portal hypertension with preserved hepatic function, we examined pulmonary elaboration of endothelial nitric oxide synthase (NOS), inducible NOS, heme oxygenase- 1 (HO-1), heme oxygenase-2 (HO-2), endothelin-1 mRNA, and protein. In comparison to sham controls, portal hypertensive animals exhibited significantly increased pulmonary iNOS and HO-1 mRNA and protein. Cyclic GMP was significantly increased in portal hypertensive lung tissue, suggesting activation of guanylyl cyclase by the endproducts of iNOS and/or HO-1 activity. Using immunohistochemical analysis, iNOS expression was localized to the vascular endothelium, while HO-1 localized to bronchiolar epithelium and macrophages. These results suggest that production of nitric oxide and carbon monoxide may contribute to the pulmonary pathology associated with portal hypertension.     

Study on changes of heme oxygenase-1 expression in patients with coronary heart disease

BACKGROUND: Heme oxygenase (HO) is a rate-limiting enzyme of endogenetic carbon monoxide (CO) that degrades heme into carbon monoxide, bilirubin, and iron. These products have important physiologic effects: bilirubin is a potent antioxidant that can act against ischemia/reperfusion injury; there is a negative correlation between the content of HO-1 and the incidence of coronary heart disease (CHD). HYPOTHESIS: This study was undertaken to investigate the changes of HO-1 in patients with CHD. METHODS: Thirty-five patients with acute myocardial infarction (AMI), 40 patients with unstable angina pectoris (UAP, diagnosed by coronary angiography), and 30 patients with stable angina pectoris (AP, diagnosed by coronary angiography) were selected for the study; another 30 patients with normal coronary artery (diagnosed by coronary angiography) were selected as controls. The levels of HO-1 protein expression in monocyte and lymphocyte in the subjects were tested by immunohistochemistry and western blot. Computer picture analyzing systems were also used to measure the levels of HO-1 protein expression. RESULTS: Heme oxygenase-1 protein is located in cell plasma. The levels of HO-1 protein expression in patients with CHD were significantly higher than in those without CHD (p < 0.01). There were significant differences of HO-1 expression among the three groups of patients with CHD. The group with AMI was the highest, followed by the group with UAP and finally by the group with AP. CONCLUSIONS: There is a higher expression of HO-1 in patients with CHD. The levels of HO-1 protein are associated with the severity of CHD.     

Administration of Bacterial Lipopolysaccharide to Rats Induces Heme Oxygenase-1 and Formation of Antioxidant Bilirubin in the Intestinal Mucosa

Heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation, is induced by oxidative stress and its major end product, bilirubin, is a potent physiological antioxidant. We studied the induction of HO-1 and bilirubin production in intestinal mucosa using a rat model of sepsis. E. coli lipopolysaccharide was administered intraperitonealy to male Wistar rats and intestinal mucosa was harvested. Intestinal lipid peroxides increased significantly at 1 hr and peaked at 170% of the control value at 5 hr. GSH significantly decreased at 3 hr, reaching the nadir of 50% of the control value at 5 hr. HO-1 mRNA was maximally induced fivefold at 3 hr and HO-1 protein maximally increased to 10 times the control value at 7.5 hr. Both bilirubin and bilirubin oxidative metabolites were maximally increased at 10 hr, to 4.3 and 3.7 times the control value, respectively. These data suggest that oxidative stress in sepsis quickly induces HO-1 in intestinal mucosa and that subsequent production of bilirubin works as an antioxidant. The small intestinal mucosa is an active participant in the general response to sepsis.     

Subcellular location of heme oxygenase 1 and 2 and divalent metal transporter 1 in relation to endocytotic markers during heme iron absorption

Background and Aim: Heme is an important dietary micronutrient, although its absorptive mechanisms are poorly understood. One hypothesis suggests enterocytes take up heme by receptor‐mediated endocytosis (RME) which then undergoes catabolism by heme oxygenase (HO) inside internalized vesicles. This would require the translocation of HO‐1 or HO‐2 to endosomes and/or lysosomes and the presence of a transporter, possibly divalent metal transporter 1 (DMT1), to transfer released iron to the cytoplasm. Currently, the location of HO‐1 and HO‐2 in enterocytes is unknown. Methods: We studied the subcellular location of HO‐1, HO‐2, and DMT1 in the proximal small intestine of rats by confocal immunofluorescence microscopy up to 4 h after a dose of heme or ferrous iron. Double‐labeling was performed with endocytotic (EEA1, Lamp1) and structural markers (F‐actin). Results: HO‐1 was distributed evenly throughout the cytoplasm of enterocytes and did not colocalize with endocytotic markers in any condition. HO‐2 staining remained constant with dosing, presenting as a dense band in the apical cytoplasm that colocalized extensively with endosomes. DMT1 staining was markedly reduced by ferrous iron, but not heme and did not exhibit colocalization with endocytotic markers. Conclusion: The subcellular location of HO‐2 is consistent with the RME hypothesis for heme uptake and may suggest a possible role for this enzyme in heme degradation. The lack of translocation of DMT1 with heme dosing suggests another protein may be present to transport iron released from heme.     

Melatonin synergistically increases resveratrol-induced heme oxygenase-1 expression through the inhibition of ubiquitin-dependent proteasome pathway: a possible role in neuroprotection

Abstract: Melatonin is an indoleamine secreted by the pineal gland as well as a plant‐derived product, and resveratrol (RSV) is a naturally occurring polyphenol synthesized by a variety of plant species; both molecules act as a neuroprotector and antioxidant. Recent studies have demonstrated that RSV reduced the incidence of Alzheimer’s disease and stroke, while melatonin supplementation was found to reduce the progression of the cognitive impairment in AD. The heme oxygenase‐1 (HO‐1) is an inducible and redox‐regulated enzyme that provides tissue‐specific antioxidant effects. We assessed whether the co‐administration of melatonin and RSV shows synergistic effects in terms of their neuroprotective properties through HO‐1. RSV significantly increased the expression levels of HO‐1 protein in a concentration‐dependent manner both in primary cortical neurons and in astrocytes, while melatonin per se did not. Melatonin + RSV showed a synergistic increase in the expression levels of HO‐1 protein but not in the HO‐1 mRNA level compared to either melatonin or RSV alone, which is mediated by the activation of PI3K‐Akt pathway. Treatment of melatonin + RSV significantly attenuated the neurotoxicity induced by H₂O₂ in primary cortical neurons and also in organotypic hippocampal slice culture. The blockade of HO‐1 induction by shRNA attenuated HO‐1 induction by melatonin + RSV and hindered the neuroprotective effects against oxidative stress induced by H₂O₂. The treatment of MG132 + RSV mimicked the effects of melatonin + RSV, and melatonin + RSV inhibited ubiquitination of HO‐1. These data suggest that melatonin potentiates the neuroprotective effect of RSV against oxidative injury, by enhancing HO‐1 induction through inhibiting ubiquitination‐dependent proteasome pathway, which may provide an effective means to treat neurodegenerative disorders.     

Role of naofen in apoptosis of hepatocytes induced by lipopolysaccharide through mitochondrial signaling in rats

Aim: Lipopolysaccharide (LPS) causes apoptosis of hepatocytes, which is probably mediated by inflammatory substances released from Kupffer cells (KCs). Recently, we have reported that naofen, a newly found intracellular WD40-repeat protein, has a role in inducing the apoptosis in HEK293 cells. Hence, the present study was undertaken to investigate a role of naofen in the LPS-induced apoptosis of rat hepatocytes. Methods: Rats were treated with i.v. injections of LPS, and livers were extirpated to evaluate expression of naofen and apoptosis. In in vitro experiments, hepatocytes and KCs were separately isolated from rat livers. The incubation medium for KCs treated with LPS (KC-CM) was used for hepatocyte culture. Results: Intravenous injections of LPS enhanced the expression of naofen in the livers. Livers showed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive staining, and elevated caspase-3 activity. In isolated KCs or hepatocytes, LPS hardly affected naofen expression and caspase-3 activity, whereas incubation of hepatocytes with KC-CM enhanced both naofen expression and caspase-3 activation. Transfection of hepatocyte with naofen siRNA prevented such effects of KC-CM, and clearly eliminated KC-CM-induced reduction of Bcl-2 and Bcl-xL. In contrast, overexpression of naofen in hepatocytes downregulated Bcl-2 and Bcl-xL, released cytochrome c from mitochondria, and activated caspase-3. Conclusion: These results indicate that LPS may induce the hepatic apoptosis in association with enhanced naofen expression, and that naofen may mediate the activation of caspase-3 through downregulating the Bcl-2 and Bcl-xL expression, and releasing cytochrome c from mitochondria to cytoplasm.     

Genetic variants of GSNOR and ADRB2 influence response to albuterol in African‐American children with severe asthma

African Americans are disproportionately affected by asthma. Social and economic factors play a role in this disparity, but there is evidence that genetic factors may also influence the development of asthma and response to therapy in African American children. Our hypothesis is that variations in asthma related genes contribute to the observed asthma disparities by influencing the response to asthma‐specific therapy. In order to test this hypothesis, we characterized the clinical response to asthma‐specific therapy in 107 African American children who presented to the emergency room in status asthmaticus, with a primary outcome indicator of length of time on continuous albuterol. Single locus analysis indicated that genotype variation in glutathione‐dependent S‐nitrosoglutathione reductase (GSNOR) is associated with a decreased response to asthma treatment in African American children. A post hoc multi‐locus analysis revealed that a combination of four single nucleotide polymorphisms (SNPs) within GSNOR, adrenergic receptor beta 2, and carbamoyl phosphate synthetase‐1 give a 70% predictive value for lack of response to therapy. This predictive model needs replication in other cohorts of patients with asthma, but suggests gene–gene interactions may have greater significance than that identified with single variants. Our findings also suggest that genetic variants may contribute to the observed population disparities in asthma. Pediatr Pulmonol. 2009; 44:649–654. © 2009 Wiley‐Liss, Inc.     

Estradiol-17β modifies the induction of spermidine/spermine N1-acetyltransferase activity in the liver of lipopolysaccharide-treated mice

ABSTRACT— In an attempt to elucidate the effects of estrogen on polyamine metabolism in lipopolysaccharide (LPS)-treated mice, we assayed polyamine content and the activity of spermidine/spermine N¹-acetyltransferase (SAT) and ornithine decarboxylase (ODC) in some organs. LPS elevated N'-acetylspermidine levels in the liver and lung and putrescine levels in the liver, lung and spleen. LPS increased the activity of ODC at 6 h and that of SAT at 12 h in the liver. When estradiol-17β was simultaneously administered with LPS, the maximum increase in hepatic N¹-acetylspermidine levels was found 6 h earlier than in the LPS control. Likewise, the peak of the hepatic SAT activity after LPS-treatment was observed 6 h earlier in the estradiol-17β-treated mice than in the LPS control. No such effect of estradiol-17β was found in the lung and spleen. The LPS-induced ODC activity was not affected by estradiol-17β in the liver, lung or spleen. Estrone and 16β-ethylestradiol (an anti-estrogen) were also effective in enhancing the LPS-induced elevation of N¹-acetyl-spermidine and putrescine in the liver, while both diethylstilbestrol, which has a potent estrogenic activity without steroid structure and estradiol-17α (a non-estrogenic isomer of estradiol-17β) were without effect. Tamoxifen (an estrogen receptor antagonist) did not suppress the estrogen-induced increase in hepatic N¹-acetylspermidine levels.     

Methylprednisolone injection via the portal vein suppresses inflammation in acute liver failure induced in rats by lipopolysaccharide and d-galactosamine.

BACKGROUND: We have reported that hepatic arterial steroid injection is an effective therapy to rescue patients from fulminant or severe acute hepatic failure. We speculate that a high concentration of steroid suppresses inflammatory processes in the liver directly by restraining activated inflammatory cells, including macrophages. To analyse the detailed mechanism, steroid injection via the portal vein was performed in an experimental model of liver damage. METHODS: Rats subjected to lipopolysaccharide and d-galactosamine injection were treated with a methylprednisolone injection via the tail vein or the portal vein. The survival rate, serum levels of inflammatory cytokines and apoptotic cell counts in the liver were analysed. RESULTS: The survival rate was significantly improved by steroid injection, especially via the portal vein. Serum values of alanine aminotransferase, tumor necrosis factor-alpha and interferon-gamma were reduced in the treated groups, especially the group given portal venous injections. Apoptotic cell counts in the liver were significantly lower in the group injected with steroid via the portal vein. CONCLUSION: In the model rats, high concentrations of steroid in the liver acted on inflammatory cells and suppressed inflammatory cytokines and liver cell death. The mechanism is suggested to be the same for arterial steroid injection therapy in patients with acute hepatic failure.     

Remodeling in the microcirculation of rat skeletal muscle during chronic ischemia

OBJECTIVE: To establish the time course and extent of remodeling of terminal microcirculation in ischemic rat skeletal muscle during prolonged low flow that does not lead to inflammation. METHODS: One common iliac artery was ligated via laparotomy in adult Sprague-Dawley rats and extensor digitorum longus (EDL) muscles removed at intervals (1, 2, and 5 weeks) postsurgery. Serial frozen EDL sections were stained to show capillaries (alkaline phosphatase), cell proliferation (antibody to proliferating cell nuclear antigen [PCNA]), terminal microvessels (antibodies to alpha-smooth muscle actin (alpha-SMA) or endothelial nitric oxide synthase [eNOS]), and macrophages (antibodies to infiltrating and resident macrophages). Total muscle eNOS protein was quantified by standard Western blotting techniques. RESULTS: Capillary proliferation was very limited in ischemic EDLs, with a modest 12% increase in the capillary/fiber ratio after 5 weeks, preceded at 2 weeks by increased numbers of PCNA-positive nuclei at capillary sites. There was no muscle necrosis or evidence of inflammation, based on macrophage staining. The number of terminal microvessels that were positive for alpha-SMA and <10 microm in diameter was fewer in ischemic EDLs at all time points, whereas the number of larger positive vessels was unchanged. eNOS-positive vessels <10 microm in diameter were stained similarly throughout ischemic muscles as the controls, and showed a similar increase in vessel/fiber ratio as the capillaries. The total eNOS protein level was similar to that in controls in ischemic EDLs after 1 and 2 weeks, but was 28% lower after 5 weeks. CONCLUSIONS: Prolonged, moderate flow reduction to skeletal muscles does not necessarily lead to inflammation or extensive capillary growth. Based on eNOS staining, the terminal microcirculation remains intact, but the loss of alpha-SMA immunoreactivity may indicate remodeling involving the "deinvestment" of microvessels by smooth muscle.