Role of heme oxygenase 1 in TNF/TNF receptor-mediated apoptosis after hepatic ischemia/reperfusion in rats.

Hepatocellular apoptosis commonly occurs in ischemia/reperfusion (I/R) injury. The binding of tumor necrosis factor (TNF) to TNF receptor 1 (TNFR1) leads to the formation of a death-inducing signaling complex (DISC), which subsequently initiates a caspase cascade resulting in apoptosis. Heme oxygenase 1 (HO-1) confers cytoprotection against cell death in I/R injury and inhibits stress-induced apoptotic pathways in vitro. This study investigated the role of HO-1 in modulating TNF/TNFR1-mediated cell death pathways in hepatic I/R injury. Rats were pretreated with hemin, an HO-1 inducer, and zinc protoporphyrin (ZnPP), an HO-1 inhibitor, before undergoing hepatic I/R. Heme oxygenase 1 activity increased after reperfusion. Ischemia/reperfusion-induced hepatocellular apoptosis was attenuated by hemin, as determined by the caspase-3 and -8 activity assays and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). Zinc protoporphyrin eliminated the cytoprotective effect of hemin. Hepatic TNFR1 protein expression was unchanged among the experimental groups, whereas mitochondrial TNFR1 protein increased after I/R. Ischemia/reperfusion increased the quantity of DISC components, including TRADD (TNFR1-associated death domain), FADD (Fas-associated death domain), and caspase-8, as well as the assembly of DISCs within the liver. In the mitochondrial fraction, TNFR1-associated caspase-8 was increased after I/R. These increases were attenuated by hemin; zinc protoporphyrin eliminated this effect. Our findings suggest that the cytoprotective effects of HO-1 are mediated by suppression of TNF/TNFR1-mediated apoptotic signaling, specifically by modulating apoptotic DISC formation and mitochondrial TNFR1 translocation during hepatic I/R.

Characterization of DISC formation and TNFR1 translocation to mitochondria in TNF-α-treated hepatocytes.

Tumor necrosis factor receptor 1 (TNFR1) activation in hepatocytes can trigger apoptotic or inflammatory signaling. The factors that determine which signaling pathway dominates are not clear and are thought to relate to the efficiency of death-inducing signaling complex (DISC) formation. However, the steps involved in DISC formation in hepatocytes are poorly understood. In characterizing DISC formation within cultured hepatocytes, we demonstrated that TNF-α exposure leads to the rapid formation of a DISC involving TNF-α, the TNFR-associated death domain adaptor molecule (TRADD), the Fas-associated death domain adaptor molecule (FADD), caspase-8, TNFR-associated factor 2 (TRAF2), and receptor-interacting protein (RIP). The inclusion of the sensitizing agent actinomycin D both accelerated and amplified the appearance of the DISC. Notably, TNFR1 along with some DISC components also appeared within mitochondria within 30 minutes. Whereas TNFR1 consistently co-localized with the TRADD, FADD, the caspase-8, and TRAF2 in the cytosolic fraction, TNFR1 in the mitochondria was associated only with caspase-8 after TNF-α exposure. Similar observations were made in vivo using TNF-α with D-galactosamine. Actinomycin D alone also enhanced the appearance of DISC components in both cytosol and the mitochondria. Thus the DISC that includes TNFR1 forms in the cytosol of hepatocytes under both survival and pro-apoptotic conditions. The observations also suggest that TNF-α-mediated signaling includes the translocation of TNFR1 to mitochondria.

Protective effect of Ssanghwa-tang fermented by Lactobacillus fermentum against carbon tetrachloride-induced acute hepatotoxicity in rats.

Ssanghwa-tang (SHT) is a traditional herbal medicine formula that has been used for the development of physical strength, relief of pain, and the reduction of fatigue. In this study, we fermented SHT with Lactobacillus fermentum (L. fermentum), Lactobacillus gasseri (L.gasseri), or Lactobacillus casei (L.casei) to investigate the hepatoprotective effects of SHT and fermented SHT with Lactobacillus on carbon tetrachloride (CCl(4))-induced liver injury in rats. Rats were given CCl(4) (1 ml/kg, 50% CCl(4) in olive oil) intraperitoneally and either SHT or fermented SHTs (15 ml/kg) was administered 30 min before CCl(4). At 24 hr after CCl(4) injection, the levels of transaminases in the serum were markedly increased. These increases were significantly attenuated by either SHT + L. fermentum or SHT+ L.gasseri. However, SHT and SHT + L.casei showed slight suppression of the increase of transaminases. The liver histological changes were diminished by treatment with SHT + L. fermentum. Additionally, the potential hepatoprotective effect of fermented-SHTs correlated with the amount of unknown metabolite which is produced during fermentation process with L. fermentum, L.gasseri, or L.casei. Therefore, these results suggest that the hepatoprotective effect of SHT may be improved by fermentation with L. fermentum and the intestinal bacterial enzyme activities may likely play an important role in the pharmacological action of herbal medicines.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome.

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

Protective role of heme oxygenase-1 against liver damage caused by hepatic ischemia and reperfusion in rats.

This study investigated the time course of heme oxygenase (HO)-1 expression and the role of endogenous HO-1 in hepatic ischemia and reperfusion (I/R). Rats were pretreated with hemin, an HO-1 inducer, and zinc protoporphyrin (ZnPP), an HO-1 inhibitor. Hepatic HO activity increased at 1 h after reperfusion, reaching a maximum at 6 h after reperfusion and then declined. HO-1 mRNA and protein expression in I/R liver were upregulated prior to reperfusion and highly induced again by reperfusion. The ALT level was upregulated at all time points, with a peak at 4-6 h. This increase was augmented by ZnPP but attenuated by hemin. Lipid peroxidation and serum HMGB1 release significantly increased at 1 h after reperfusion and remained elevated throughout the 24 h of reperfusion period, whereas the glutathione content decreased markedly at 4-6 h after reperfusion. These changes were attenuated by hemin but augmented by ZnPP. The levels of serum TNF-α, iNOS, and COX-2 protein and mRNA expressions were upregulated after reperfusion, further enhanced by ZnPP, and suppressed by hemin. HO-1 overexpression protects the liver against I/R injury by modulating oxidative stress and proinflammatory mediators.

Role of nitric oxide in the expression of hepatic vascular stress genes in response to sepsis.

This study examined the role of nitric oxide (NO) on the expression of the hepatic vasoregulatory gene during polymicrobial sepsis. Aminoguanidine (AG, 100 mg/kg) or Nomega-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg) was injected intraperitoneally at 0, 3, 6, 10, and 20 h after a cecal ligation and puncture (CLP). The heart rate increased 24 h after the CLP, and this increase was attenuated by L-NAME and further attenuated by AG. The mean arterial pressure in the CLP animals did not change significantly 24 h after the onset of sepsis but was increased after the L-NAME injection. Sepsis increased the serum aminotransferase levels, which were attenuated by AG but augmented by L-NAME. CLP increased the mRNA level of the ET-1 and ETB receptors in the liver. This increase was prevented by AG but augmented by L-NAME. The level of iNOS and HO-1 mRNA expression were increased by CLP, which was prevented by both AG and L-NAME. The level of TNF-alpha and COX-2 mRNA expression increased after CLP, and was attenuated by AG. These results show that iNOS and eNOS are regulated differently in sepsis. While eNOS appears to have a protective role in liver microcirculation, the strong upregulation of iNOS might contribute to a microvascular dysfunction and hepatic injury.

Necrosis and apoptosis: sequence of liver damage following reperfusion after 60 min ischemia in rats.

This study evaluated the time-dependent modes of cell death that occur during the course of reperfusion after 60 min ischemia. The serum ALT level increased immediately after reperfusion, peaked at 6 h and then declined gradually thereafter. This was supported by the H&E staining of the liver tissues taken at 2 h reperfusion, which revealed massive peri-portal necrosis. The succinate driven mitochondrial-swelling rate, release of cytochrome c into the cytoplasm, increase in caspase-3 activity and TUNEL stained tissue were measured to determine the changes in the biochemical markers of apoptosis. The biochemical markers of apoptosis increased by 2 h of reperfusion, peaked at 6 h and remained elevated throughout the 24 h reperfusion period. Cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), inhibited MPT opening, the release of cytochrome c and caspase-3 activation. This indicates that necrotic death occurs particularly in the peri-portal region in the initial period of reperfusion, and delayed apoptotic death occurs primarily in the peri-central region in the liver tissues undergoing I/R.

Role of nitric oxide in the inhibition of liver cytochrome P450 during sepsis.

Nitric oxide (NO) plays an important role in the pathophysiology of sepsis and septic shock but the mechanism is not well understood. The aim of this study was to investigate the role of NO in the cytochrome P450 (CYP) isozyme activity and the expression of its gene during polymicrobial sepsis. The rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Aminoguanidine (AG, 100 mg/kg body weight) or N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg body weight) was injected intraperitoneally at 0, 3, 6, 10, and 20 h after CLP. The plasma nitrite/nitrate concentration increased 24 h after CLP, and this increase was almost completely abolished by AG and L-NAME. Sepsis increased the serum aminotransferase and lipid peroxidation levels, which were attenuated by AG but augmented by L-NAME. The hepatic concentration of the reduced gluthathione decreased in the CLP rats, which was inhibited by AG but augmented by L-NAME. The total CYP content decreased after CLP, which was restored by AG and L-NAME. The CYP1A1, 1A2, and 2E1 activities, along with their protein levels, decreased 24 h after CLP but these decreases were reversed by AG and L-NAME. The CYP1A1, 1A2, 2B1, and 2E1 mRNA expression levels decreased 24 h after CLP, and L-NAME inhibited this decrease. NO plays a key role in the sepsis-mediated decrease in CYP via the interplay of two different mechanisms: NO-dependent suppression of protein via the enhanced inducible NO synthase, and NO-dependent transcriptional suppression via endothelial NO synthase.

Anti-inflammatory activity of CML-1: an herbal formulation.

CML-1 is a purified extract from a mixture of 13 Oriental herbs (Achyranthis Radix, Angelicae Gigantis Radix, Cinnamomi Cortex Spissus, Eucommiae Cortex, Glycyrrhizae Radix, Hoelen, Lycii Fructus, Paeoniae Radix, Rehmanniae Radix Preparata and Atractylodis Rhizoma, Zingiberis Rhizoma, Zizyphi Semen, Acori Graminei Rhizoma) that have been widely used for the treatment of inflammatory diseases in Asia. The aim of this study was to investigate the anti-inflammatory and analgesic potential of CML-1. The animals used in this study were administered either vehicle or CML-1 (30, 100, 300 and 600 mg/kg) orally. The vascular permeability induced by acetic acid was significantly reduced by CML-1 in all doses. The swelling of the rat's hind paw induced by carrageenan was significantly inhibited by CML-1 in doses of 100, 300 and 600 mg/kg. In the case of rheumatoid arthritis induced by complete Freund's adjuvant in rats, the treatment with CML-1 at a dose level of 300 mg/kg inhibited edema. CML-1 at a dose level of 600 mg/kg inhibited acetic acid-induced writhing syndrome, however it did not have any anti-nociceptive action in the Randall-Selitto assay or the hot plate test. Our findings suggest that CML-1 has a potent anti-inflammatory activity.

Effects of Trolox on the activity and gene expression of cytochrome P450 in hepatic ischemia/reperfusion.

1. The aim of this study was to investigate the effect of Trolox on hepatic microsomal cytochrome P450 (CYP) activity and gene expression during ischemia and reperfusion (I/R). 2. Rats were subjected to 60 min of hepatic ischemia, and 5 h (acute phase) and 24 h (subacute phase) of reperfusion. Rats were treated intravenously with Trolox (2.5 mg kg(-1)) or vehicle, 5 min before reperfusion. 3. The serum alanine aminotransferase level and lipid peroxidation were increased as a result of I/R. These increases were attenuated by Trolox. Reduced glutathione concentration decreased in I/R group, and this decrease was inhibited by Trolox. 4. Both total hepatic CYP content and NADPH-cytochrome P450 reductase activity decreased after I/R, which were restored by Trolox. 5. CYP1A1 activity and its protein level decreased 24 h after reperfusion; decreases which were prevented by Trolox. Both the activity and mRNA expression of CYP1A2 decreased 24 h after reperfusion. The decrease in CYP1A2 mRNA was prevented by Trolox. CYP2B1 activity and mRNA expression decreased 5 h after reperfusion. The decrease in CYP2B1 activity was prevented by Trolox. In contrast, the CYP2E1 activity and its protein level increased 5 h after reperfusion and this increase was prevented by Trolox. 6. The expression of TNF-alpha and iNOS mRNAs increased after I/R. Trolox inhibited increase in iNOS mRNA expression. 7. Trolox ameliorates hepatic drug-metabolizing dysfunction, as indicated by abnormalities in CYP isoforms during I/R, and this protection is likely due to the scavenging of reactive oxygen species.

Effect of Trolox on altered vasoregulatory gene expression in hepatic ischemia/reperfusion.

This study was designed to investigate the effect of Trolox, a hydrophilic analogue of vitamin E, on the alteration of vasoregulatory gene expression during hepatic ischemia and reperfusion (I/R). Rats were subjected to 60 min of hepatic ischemia in vivo. The rats were treated intravenously with Trolox (2.5 mg/kg) or the vehicle as a control 5 min before reperfusion. Liver samples were obtained 5 h after reperfusion for a RT-PCR analysis on the mRNA for the genes of interest. These mRNA peptides are endothelin-1 (ET-1), potent vasoconstrictor peptide, its receptor ET(A) and ET(B), vasodilator endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2). It was seen that serum alanine aminotransferase and lipid peroxidation levels were markedly increased after I/R and Trolox significantly suppressed this increase. In contrast, the glutathione concentration decreased in the I/R group, and this decrease was inhibited by Trolox. ET-1 mRNA expression was increased by I/R, an increase which was prevented by Trolox. The mRNA levels for ET(A) receptor was significantly decreased, whereas ET(B) receptor transcript increased in the I/R group. The increase in ET(A) was prevented by Trolox. The mRNA levels for iNOS and HO-1 significantly increased in the I/R group and Trolox attenuated this increase. There were no significant differences in eNOS mRNA expression among any of the experimental groups. The mRNA levels for COX-2 and TNF-alpha significantly increased in I/R group and Trolox also attenuated this increase. Our findings suggest that I/R induces an imbalanced hepatic vasoregulatory gene expression and Trolox ameliorates this change through its free radical scavenging activity.

Trolox C ameliorates hepatic drug metabolizing dysfunction after ischemia/reperfusion.

The present study was done to determine the effect of trolox C, a hydrophilic analogue of vitamin E, on hepatic injury, especially the alteration in cytochrome P-450 (CYP)-dependent drug metabolism during ischemia and reperfusion (I/R). Rats were subjected to 60 min of hepatic ischemia and 5 h of reperfusion. Rats were treated intravenously with trolox C (2.5 mg/kg) or vehide (PBS, pH 7.4), 5 min before reperfusion. Serum alanine aminotransferase and lipid peroxidation levels were markedly increased after I/R. This increase was significantly suppressed by trolox C. Cytochrome P-450 content was decreased after I/R but was restored by trolox C. There were no significant differences in ethoxyresorufin O-dealkylase (CYP 1A1) and methoxyresorufin O-dealkylase (CYP 1A2) activities among any of the experimental groups. Pentoxyresorufin O-dealkylase (CYP 2B1) activity was decreased and aniline p-hydroxylase (CYP 2E1) activity was increased after I/R. Both these changes were prevented by trolox C. Our findings suggest that trolox C reduces hepatocellular damage as indicated by abnormalities in microsomal drug-metabolizing function during I/R, and that this protection is, in part, caused by decreased lipid peroxidation.