Heat shock response in a rat model of septic multiple organ dysfunction syndrome

Severe sepsis is known to result in multiple organ dysfunction syndrome (MODS), which is thought to be mediated by oxidative stress, as a result of excessive systemic inflammation. Heme Oxygenase-1 (HO-1), the rate limiting enzyme in heme catabolism, is also known as HSP32. HO-1 is induced not only by its substrate heme but also by oxidative stress. We investigated gene expression of HO-1 and physiological significance of HO-1 induction in a rat model of septic MODS induced by intraperitoneal injection of bacterial lipopolysaccharide (LPS). Following administration of LPS, HO-1 mRNA was significantly induced in the liver, lung and kidney in an organ-specific manner. Hepatic HO-1 induction appears to be mediated by an increase in hepatic free heme concentration. Inhibition of HO activity by tin mesoporphyrin significantly exacerbated lung injury. These results suggest that HO-1 induction may play an important role in conferring protection on cells from oxidative damage not only by catalyzing heme, a pro-oxidant, but also by producing bilirubin, an anti-oxidant. Furthermore, HO-1 mRNA is induced markedly in the buffy coat of the blood at 3 h after LPS administration, coinciding with the increase in serum IL-6 level, suggesting that HO-1 may be one of the key markers of septic MODS.     

Coordinate up-regulation of CYP1A1 and heme oxygenase-1 (HO-1) expression and modulation of delta-aminolevulinic acid synthase and tryptophan pyrrolase activities in pyridine-treated rats.

To determine the changes in heme metabolism associated with induction of cytochrome P450 expression by pyridine, we compared the time course of CYP1A expression with the time course of (i) expression of heme oxygenase-1 (HO-1) (EC 1.14.99.3), (ii) activity of delta-aminolevulinic acid synthetase (ALAS) (EC 2.3.1.37), and (iii) heme saturation of tryptophan pyrrolase (TPO) (EC 1.13.11.11) in tissues of rats administered a single 100 or 150 mg/kg i.p. dose of pyridine. Both mRNA and protein of HO-1 and CYP1A1 were induced in the liver, kidney, and lung, with the induction of HO-1 mRNA preceding and paralleling that of CYP1A1 mRNA in the liver and lung but not kidney. Induction of CYP1A1 mRNA expression peaked within 9-12 hr and returned to control levels by 24 hr in all tissues examined, whereas induction of HO-1 mRNA expression was sustained for 48 hr in the lung and liver. In contrast to the transient up-regulation of CYP1A1 mRNA, increased microsomal CYP1A1 protein was sustained in all three tissues. Similar to the induction of HO-1 expression, lipid peroxidation was stimulated by pyridine treatment in the kidney, lung, and liver, but with the stimulation being more persistent in the liver and lung than in the kidney. Increased hepatic CYP1A1 or CYP1A2 activity was preceded by increased activities of HO-1 and ALAS. Pyridine treatment negatively modulated heme saturation of hepatic TPO. The findings indicate that pyridine stimulates the synthesis, utilization, and degradation of heme in a coordinate manner, and suggest that these alterations in heme metabolism may contribute to CYP1A1 induction by pyridine.     

Prevention of hemorrhagic shock-induced lung injury by heme arginate treatment in rats

Hemorrhagic shock followed by resuscitation (HSR) induces oxidative stress, which leads to acute lung injury. Heme oxygenase (HO)-1 (EC 1.14.99.3), the rate-limiting enzyme in heme catabolism, is inducible by oxidative stress and is thought to play an important role in the protection from oxidative tissue injuries. In this study, we examined expression of HO-1 as well as tissue injuries in the lung, liver, and kidney after HSR in rats. We also pretreated animals with heme arginate (HA), a strong inducer of HO-1, and examined its effect on the HSR-induced lung injury. HO-1 expression significantly increased in the liver and kidney following HSR, while its expression in the lung was very low and unchanged after HSR. In contrast to HO-1 expression, tissue injury and tumor necrosis factor-alpha (TNF-alpha) gene expression was more prominent in the lung compared with those in the liver and kidney. HA pretreatment markedly induced HO-1 in pulmonary epithelial cells, and ameliorated the lung injury induced by HSR as judged by the improvement of histological changes, while it decreased TNF-alpha and inducible nitric oxide synthase gene expression, lung wet weight to dry weight ratio, and myeloperoxidase activity. In contrast, inhibition of HO-1 by tin-mesoporphyrin administration abolished the beneficial effect of HA pretreatment. These findings suggest that tissues with higher HO-1 may be better protected than those with lower HO-1 from oxidative tissue injury induced by HSR. Our findings also indicate that HA pretreatment can significantly suppress the HSR-induced lung injury by virtue of its ability to induce HO-1.     

Tissue-dependent induction of heme oxygenase-1 and metallothionein-1/2 by methyl methanesulfonate

Methyl methanesulfonate (MMS), a methylating agent, is known to be a genotoxicant in testis. The purpose of this study was to investigate roles of oxidative stress-responsive proteins, heme oxygenase-1 (HO-1) and metallothionein-1/2 (MT-1/2), in genotoxicity of MMS. Cadmium, a potent genotoxicity inducer, induced HO-1 and MT-1/2 in rat livers and kidneys. Then we comparatively investigated MMS-induced HO-1 and MT-1/2 in rat livers, kidneys and testes. We found that a single administration of MMS (40 mg/kg) resulted in the induction of MT-1/2 mRNA in the liver, but not HO-1 mRNA, reaching maximum level at 6 hr and returning to the control levels by 24 hr. Interestingly, MMS induced both HO-1 and MT-1/2 mRNAs in the kidney. In contrast, MMS induced HO-1 mRNA, but not MT-1/2 mRNA in the testis. Since HO-1 and MT-1/2 have been recognized to respond to various oxidative stimuli, we further examined the inducing effect of MMS on these two proteins. MMS at dosages of 20 to 40 mg/kg for 2 consecutive weeks induced HO-1 mRNA (123 to 187% of the control) and protein (274 to 404% of the control) in rat testes. However, MT-1/2 mRNA was not induced by MMS administration, although a high level of expression was observed in comparison with the liver and kidney. These findings suggest that MMS induces HO-1 and/or MT-1/2 mRNA and its protein tissue-dependently, and the heme catabolites by HO-1 in the testis may contribute in some manner to its genotoxicity.     

HO-1在原发性肝癌中的表达及意义

目的探讨血红素氧合酶-1(HO-1)在肝癌组织中的表达及意义。方法分别用RT-PCR、免疫组化检测35例肝癌标本、癌旁组织、正常肝组织的HO-1mRNA及HO-1蛋白表达情况,探讨HO-1在肝癌组织中表达及意义。结果肝癌组织中的HO-1mRNA表达明显高于癌旁组织、正常肝组织(P<0.05),肝癌组织中的HO-1表达高于癌旁组织及正常肝组织(P<0.05)。结论HO-1mRNA、蛋白表达在原发性肝癌组织中表达上调与肝癌演变、发展密切相关,并提示HO-1对肝癌细胞存在保护作用。     

Upregulation of heme oxygenase-1 with hemin prevents D-galactosamine and lipopolysaccharide-induced acute hepatic injury in rats

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been shown to be induced during oxidative injury, and its induction acts as an important cellular defense mechanism against such injuries. In this study, we examined the functional roles of HO-1 induction in a rat model of d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced liver injury. We found that GalN/LPS treatment of rats produced severe hepatic injury, whereas upregulation of HO-1 by hemin pretreatment prevented rats from liver damage, as evidenced by decreased serum ALT, AST levels and ameliorated histological signs in the liver. Induction of HO-1 resulted in a significant decrease in hepatic malondialdehyde (MDA) contents, tumor necrosis factor-alpha (TNF-alpha) levels, iNOS/NO production, as well as the levels of caspase-3. In contrast, inhibition of HO activity by zinc protoporphyrin-9 (ZnPP, a specific inhibitor of HO) completely reversed HO-1-induced hepatoprotective effect. These data therefore suggested that HO-1 induction provided critical protection against GalN/LPS-induced liver injury, and the protection seemed to be mediated through the anti-oxidant, anti-inflammatory and anti-apoptotic functions.     

Prevention of halothane-induced hepatotoxicity by hemin pretreatment: protective role of heme oxygenase-1 induction

Reductive metabolism of halothane in phenobarbital-pretreated rats is known to increase free radical formation that results in hepatotoxicity. It also is associated with a marked induction of microsomal heme oxygenase-1 (HO-1), suggesting that there is an alteration in heme metabolism. In this study, we examined heme metabolism in rats pretreated with phenobarbital, followed by exposure to halothane-hypoxia. In this model, there was a significant decrease in microsomal cytochrome P450 content in the liver, followed by a rapid increase in free heme concentration and a decrease in the level of mRNA for the nonspecific delta-aminolevulinate synthase. A transient but dramatic induction of HO-1 mRNA and a prolonged induction of heat shock protein 70 mRNA also occurred. The HO-1 protein was detected principally in the hepatocytes around the central vein. Serum alanine transaminase (ALT) activity, an indicator of hepatic dysfunction, increased continuously throughout the experiment. Hemin pretreatment induced hepatic HO-1 with abrogation of the halothane-induced hepatotoxicity in this model, as judged by ALT activity and normal histology. Our findings in this study thus indicate that halothane-induced hepatotoxicity is due not only to its reductive metabolite formation, but also to an increase in hepatic free heme concentration, which is a potent prooxidant; HO-1 induction is an important protective response against such changes. This is also the first study to demonstrate that hemin pretreatment, which induces HO-1 prior to exposure to halothane, effectively prevents halothane-induced hepatotoxicity.     

Tissue- and dose-dependent alteration of stress-inducible proteins by beta2-adrenoceptor agonist, salbutamol, in rats

The effects of selective beta(2)-adrenoceptor agonists on proinflammatory cytokines and the expression of stress-inducible proteins have not yet been clarified. We investigated the effect of a higher dose (60 mg/kg intravenously) of salbutamol, a selective beta(2)-adrenoceptor agonist, on the induction of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in plasma and the expression of protein and mRNA of metallothioein-1 (MT-1), heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) in heart, lung, liver and spleen in rats. The plasma IL-6 concentration was significantly increased after administration with a maximum increase at 3 hr in a dose-dependent manner, but IL-1beta and TNF-alpha concentrations were not changed. MT-1 mRNA increased in heart, lung and liver, but not in spleen, and MT-1 protein increased in endocardium, fibroblasts of lung and periportal regions in liver. HO-1 mRNA was not changed in lung, decreased at 3 hr in liver and spleen, and increased at 6 hr in liver. Contrary to liver, HO-1 mRNA in the heart increased at 3 hr and decreased at 6 hr. HO-1 protein increased in cardiomyocytes and centrilobular regions in the liver. iNOS mRNA increased in lung, liver and spleen, but decreased in the heart, and iNOS protein increased in alveolar type II cells and hepatocytes, and decreased in necrotic cardiomyocytes. In contrast, a lower dose (6 mg/kg intravenously) of salbutamol suppressed lipopolysaccharide-induced HO-1 and iNOS mRNA. We conclude that salbutamol tissue- and dose-dependently alters the expression of stress-inducible proteins.     

血红素加氧酶-1的表达对实验性肝硬化内毒素血症大鼠的影响

目的:探讨血红素加氧酶-1(HO-1)的表达对实验性肝硬化内毒素血症大鼠的影响。方法:32只健康雄性wistar大鼠,随机分为4组,正常+脂多糖(LPS)(对照组)、肝硬化+生理盐水对照组(TAA组)、肝硬化+LPS组(TAA+LPS组)、肝硬化+LPS+hemin(氯化高铁血红素)(HM组)。用硫代乙酰胺(TAA)诱导肝硬化模型时间共计10周,对照组自由饮清水。于模型造成后,向对照组、TAA+LPS组、HM组大鼠腹腔内注入LPS3 mg/kg;TAA组大鼠腹腔内注入等量的生理盐水;HM组于注射LPS 12 h前,腹腔注射HM(40 mg/kg),并在注入LPS 6 h后,各组动物经腹主动脉穿刺采全血观察血浆中丙氨酸氨基转移酶(ALT)、天门冬氨基酸转移酶(AST)、一氧化氮(NO)及丙二醛(MDA)的表达。留肝组织用免疫组织化学法观察HO-1的表达。结果:对照组、TAA组、TAA+LPS组、HM组血浆中的ALT/AST、MDA、NO依次升高差异有显著性(P<0.05),对照组、TAA、TAA+LPS、HM组各组大鼠的灰阶值显著依次降低,且有明显差别(P<0.05)。结论:在实验性的肝硬化内毒素中尽管HO-1的表达增加,但它未起到保护作用,它与内毒素对机体的损伤有关。     

血晶素诱导HO-1表达增加及对急性肺损伤小鼠MMP-2、MMP-9 mRNA的影响

目的 通过复制急性肺损伤模型,探讨血晶素诱导血红素加氧酶-1（heme oxygenase-1, HO-1）的表达对于小鼠急性肺损伤的影响.方法 利用逆转录-聚合酶链反应和蛋白质印迹方法检测急性肺损伤小鼠肺组织基质金属蛋白酶-2 （matrix metalloproteinas-2, MMP-2）、MMP-9 mRNA和HO-1蛋白的表达.结果 血晶素加脂多糖组和对照组比较,HO-1蛋白表达明显增高 （P〈0.05）;脂多糖组与对照组比较,MMP-2 mRNA和MMP-9 mRNA表达差异有统计学意义（P〈0.05）,血晶素加脂多糖组与对照组比较,差异无统计学意义（P〉0.05）.结论 HO-1降低了急性肺损伤小鼠肺组织MMP-2、MMP-9 mRNA的表达,对于小鼠的急性肺损伤有保护作用.     

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO₂), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200 mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.     

Maternally administered lipopolysaccharide (LPS) upregulates the expression of heme oxygenase-1 in fetal liver: the role of reactive oxygen species

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the rate-limiting step in the degradation of heme to biliverdin, carbon monoxide and iron. Previous studies have demonstrated that lipopolysaccharide (LPS) upregulates the expression of HO-1 in adult mouse liver. The present study aimed to investigate the effects of maternal LPS exposure on the expression of HO-1 in fetal liver. The pregnant mice were intraperitoneally injected with different doses of LPS (1, 10, 75 microg/kg) on gestational day 17. Results showed that the expression of HO-1 in fetal liver was increased, beginning 2h after LPS, being at the highest level 24h after LPS, and remaining elevated up to 48h after LPS, whereas HO-2, the constitutive form, did not change at the various time points observed. LPS-induced upregulation of HO-1 was blocked by alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin trapping agent. Correspondingly, PBN pretreatment significantly attenuated LPS-induced lipid peroxidation and glutathione (GSH) depletion in fetal liver. However, aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and pentoxifylline (PTX), an inhibitor of tumor necrosis factor alpha (TNF-alpha) synthesis, had no effect on LPS-induced upregulation of HO-1 in fetal liver. In conclusion, reactive oxygen species (ROS), rather than TNF-alpha or nitric oxide (NO), are involved in LPS-induced upregulation of HO-1 in fetal liver. These results provide new evidence that maternal LPS exposure results in oxidative stress in fetuses, which may contribute to LPS-induced developmental toxicity.     

Protective effect of baicalin against lipopolysaccharide/D-galactosamine-induced liver injury in mice by up-regulation of heme oxygenase-1

Baicalin, a traditional anti-inflammatory drug, has been found to protect against liver injury in several experimental animal hepatitis models; however, the mechanisms underlying the hepatoprotective properties of baicalin are poorly understood. In the present study,we investigated the effects of baicalin on the acute liver injury in mice induced by Lipopolysaccharide/D-galactosamine (LPS/D-GalN). Baicalin (50, 150, and 300 mg/kg) was pretreated intraperitoneally (i.p.) at 2, 24, and 48 h respectively before LPS/D-GalN injected in mice.The mortality, hepatic tissue histology, hepatic tissue Tumor necrosis factor-alpha (TNF-alpha) and myeloperoxidase (MPO), plasma levels of TNF-alpha and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. Besides, western blotting analyses of nuclear factor kappa B (NF-kappaB) translocation and Heme oxygenase-1(HO-1) protein expression, as well as HO-1 activity were determined. The results showed that baicalin protected against LPS/D-GalN-induced liver injury, including dose-dependent alleviation of mortality and hepatic pathological damage, decrease of ALT/AST release and the rise of MPO. Baicalin reduced nuclear translocation of NF-kappa B, TNF-alpha mRNA and protein levels in hepatic tissues and plasma levels of TNF-alpha induced by LPS/D-GalN. Moreover, baicalin dose-dependently increased HO-1 protein expression and activity. Further, inhibition of HO-1 activity significantly reversed the protective effect of baicalin against LPS/D-GalN-induced liver injury. These results suggest that baicalin can effectively prevent LPS/D-GalN-induced liver injury by inhibition of NF-kappa B activity to reduce TNF-alpha production and the underlying mechanism may be related to up-regulation of HO-1 protein and activity.     

Serum iron increases with acute induction of hepatic heme oxygenase-1 in mice

Heme oxygenase (HO)-1 is induced by oxidative stress and protects against oxidant injury. We examined the effect of rapid induction of hepatic HO-1 on serum iron level. Serum iron was approximately doubled within 6 h when HO-1 was induced by phenobarbital treatment of selenium-deficient mice. Blocking heme synthesis with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) prevented the induction of HO-1 and the rise in serum iron. DDC did not block HO-1 induction by hemin. Inhibition of HO activity by tin protoporphyrin prevented a rise in serum iron that occurred following phorone treatment. These results indicate that heme synthesis or an exogenous source of heme is needed to allow induction of HO-1. Further, they link HO-1 induction with a rise in serum iron, suggesting that the iron resulting from catabolism of heme by HO-1 is released by the liver.     

Serum Iron Increases with Acute Inductionof Hepatic Heme Oxygenase-1 in Mice

Heme oxygenase (HO)-1 is induced by oxidative stress and protects against oxidant injury. We examined the effect of rapid induction of hepatic HO-1 on serum iron level. Serum iron was approximately doubled within 6 h when HO-1 was induced by phenobarbital treatment of selenium-deficient mice. Blocking heme synthesis with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) prevented the induction of HO-1 and the rise in serum iron. DDC did not block HO-1 induction by hemin. Inhibition of HO activity by tin protoporphyrin prevented a rise in serum iron that occurred following phorone treatment. These results indicate that heme synthesis or an exogenous source of heme is needed to allow induction of HO-1. Further, they link HO-1 induction with a rise in serum iron, suggesting that the iron resulting from catabolism of heme by HO-1 is released by the liver.     

Down-regulation of mouse organic anion-transporting polypeptide 4 (Oatp4; Oatp1b2; Slc21a10) mRNA by lipopolysaccharide through the toll-like receptor 4 (TLR4).

Lipopolysaccharide (LPS) causes a systemic reaction known as sepsis, which is frequently associated with cholestasis. Many biological effects produced by LPS are thought to be mediated by Toll-like receptor 4 (TLR4). Organic anion-transporting polypeptide 4 (Oatp4; Slc21a10) mediates hepatic uptake of bile acids and other organic anions. The purpose of this study was to determine 1) whether LPS decreases Oatp4 mRNA levels; 2) the role of TLR4 in the LPS-induced down-regulation of Oatp4; and 3) the time course of serum concentrations of tumor necrosis factor alpha, interleukin (IL) 1beta, and IL-6 after LPS administration. For the dose-response study, LPS (1 mg/kg i.p.) produced a significant decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice, and higher doses produced slightly greater decreases. However, none of the doses of LPS examined significantly decreased Oatp4 mRNA levels in TLR4-mutant C3H/HeJ mice. For the time-response study, LPS (5 mg/kg i.p.) produced a rapid decrease in Oatp4 mRNA levels in TLR4-normal C3H/OuJ mice. The maximal decrease in Oatp4 mRNA levels (80%) was observed 12 h after LPS administration and returned to control levels thereafter. In contrast, LPS did not produce a significant decrease in Oatp4 mRNA levels at any time in TLR4-mutant C3H/HeJ mice. These findings demonstrate that LPS decreases Oatp4 mRNA levels in mice, and the decrease is mediated through TLR4.     

Anti-inflammatory activity of 20(S)-protopanaxadiol: enhanced heme oxygenase 1 expression in RAW 264.7 cells

20( S)-Protopanaxadiol (PPD) is one of the metabolites of ginsenosides from Panax ginseng. In this study, we demonstrate that PPD inhibits the increase in lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression through inactivation of nuclear factor-kappaB by preventing degradation of inhibitory factor-kappaBalpha. PPD also induces heme oxygenase 1 (HO-1) expression in RAW 264.7 cells, at the mRNA and protein levels, in the presence and absence of LPS. This effect is associated with suppression of LPS-induced nitric oxide (NO) production and iNOS expression. The HO-1 inducer hemin is associated with the suppression of LPS-induced NO production in a dose-dependent manner, and the HO-1 inhibitor tin protoporphyrin attenuates the inhibitory activity of PPD on LPS-induced NO production. These results provide evidence for the role of HO-1 in the inhibition of LPS-induced NO production by PPD.