Preventive effect of cyclosporin A on experimentally induced acute liver injury in rats

Abstract: We examined the effect of cyclosporin A (CsA) on the pathogenesis of acute experimental liver injury in rats induced by injection of heat-killed Propionibacterium acnes (P. acnes) and subsequent injection of lipopolysaccharide (LPS). Pretreatment with CsA significantly reduced serum alanine aminotransferase (ALT), serum tumor necrosis factor-α (TNF-α) production, without changing the TNF-α mRNA level in the liver, and plasma interferon-γ (IFN-γ), following LPS injection in this model. Twenty-four-hour mortality was also markedly improved, from 100% in the P. acnes plus LPS group to 0% in the CsA-pretreated group. Although direct addition of CsA to isolated hepatic macrophages from P. acnes-pretreated rats did not prevent the production of TNF-α and active oxygen species, isolated hepatic macrophages from P. acnes plus CsA-pretreated rats significantly reduced their production in response to the addition of LPS. These results suggest that CsA protects against P. acnes plus LPS-induced acute liver injury, not by direct inhibition of hepatic macrophage activation, but by indirect prevention of hepatic macrophage activation, presumably related to the reduction in plasma IFN-γ levels.     

Preventive Effect of FK 506 (Tacrolimus Hydrate) on Experimentally Induced Acute Liver Injury in Rats

The aim of the study was to investigate the effect of the immunosuppressant FK 506 (tacrolimus hydrate) on acute liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS). Acute liver injury was induced in male Wistar rats by injecting the animals with P. acnes (10 mg/rat), and administering LPS (10 g/rat) seven days later. One group was given FK 506 (1 mg/kg) 24 and 2 hr before administration of LPS, and the other group was given the same dose of saline. The 24-hr survival rate, serum alanine aminotransferase (ALT) concentration, and tumor necrosis factor (TNF) - mRNA and protein concentrations in the liver and spleen were then compared. Hepatic macrophages were also isolated from rats seven days after P. acnes injection, LPS, and FK 506 or saline were added to the culture supernatant, and TNF- production was studied. The 24-hr survival rate was 100% in the FK 506-treated group, in contrast with 16.6% in the saline group. Four hours after LPS injection, the serum ALT concentration was 755 ± 401 in the saline group versus 119 ± 42 units/ml (P < 0.01) in the FK 506-treated group. The serum TNF- concentration was lower in the FK 506-treated group (1419 ± 957 pg/ml) than in the saline group (9205 ± 2215) (P < 0.01). The mRNA and protein concentrations in the liver and spleen in the two groups did not differ significantly 1 hr after LPS injection but were significantly lower in the FK 506-treated group after 4 hr. FK 506 did not directly inhibit TNF- production by isolated cultured hepatic macrophages. FK 506 is unable to inhibit initial TNF- production by hepatic macrophages (or probably that by splenic macrophages either) stimulated by injection of LPS in P. acnes + LPS-induced acute liver injury. However, the immunosuppressant does limit hepatic damage by inhibiting subsequent aggravation of inflammation by the cytokine network.     

Role of adhesion molecules in the development of massive hepatic necrosis in rats

Massive hepatic necrosis develops after endotoxin administration in rats pretreated with heat-killed Propionibacterium acnes as a result of microcirculatory disturbance caused by endothelial cell destruction by activated macrophages in the hepatic sinusoids. Immunohistochemical hepatic expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen 1 alpha (LFA-1 alpha) and the effect of monoclonal antibodies against both adhesion molecules on liver necrosis provoked after endotoxin administration was studied in these rats. There were increased stains of ICAM-1 in endothelial cells and LFA-1 alpha in macrophages in the hepatic sinusoids in Propionibacterium acnes-pretreated rats compared with normal rats. Such stains were further increased soon after endotoxin administration, followed by development of hepatic necrosis. Monoclonal antibodies against both adhesion molecules significantly attenuated the extent of liver injury compared with controls, without affecting the infiltration and activation of hepatic macrophages. Polyclonal antibodies against polymorphonuclear leukocytes eradicated circulating neutrophils, but did not change such liver injury, although gum arabic, which suppressed macrophage activation, attenuated the extent of liver injury. Thus, adhesion between endothelial cells and activated macrophages in the hepatic sinusoids via ICAM-1 and LFA-1 alpha is essential for the initiation of massive hepatic necrosis of this type. Contribution of neutrophils seems less likely. (Hepatology 1996 Feb;23(2):320-8)     

Immunohistochemical study of tumor necrosis factor-alpha in acute liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

The effect of intravenous injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) on the distribution of tumor necrosis factor-α (TNF-α) in different organs have not previously been investigated. Immunohistochemistry and histological examination were employed in evaluating the distribution of TNF-α in the liver, spleen, lungs and bone marrow in rats injected intravenously with P. acnes followed by LPS 7 days later. Granulomas containing ED1-positive macrophages were observed in the liver 7 days after P. acnes injection. Subsequent LPS injection resulted in proliferation of ED1-positive macrophages in the sinusoids and coagulation necrosis of hepatocytes after 6 h. TNF-α was detected in ED2-positive macrophages (Kupffer cells) 1 day after P. acnes injection and in macrophages constituting the granulomas 7 days later, but prior to LPS injection. TNF-α was also detected in ED1-positive macrophages in the spleen, predominantly in the marginal zone. When granulomas were formed 7 days after P. acnes injection, TNF-α was observed in macrophages of the granulomas. TNF-α was also detected in macrophages of the granulomas found in the lung 1 day after P. acnes injection. No macrophages expressing TNF-α were found in the granulomas of bone marrow. The highest expression was in the liver at any time interval and in macrophages constituting granulomas. Our results suggest that the high expression of TNF-α in the liver results in selective hepatic necrosis. The expression of TNF-α in macrophages of the liver after P. acnes injection and the subsequent development of hepatic necrosis after LPS injection suggest that P. acnes acts as an inducer of TNF-α production in macrophages while LPS acts as a trigger for the release of TNF-α from macrophages.     

Studies on mechanisms of augmentation of liver regeneration by cyclosporine and FK 506.

Evidence could not be found of immune modulation of liver regeneration. The powerful immunosuppressive drug FK 506, which augments the response after partial hepatectomy in normal rats, had the same effect in T cell-deficient nude rats. The cytotoxicity of natural killer cells in treated nude rats was not significantly changed by FK 506 therapy. However, the serum of FK 506-treated nude rats increased hepatocyte proliferation when added to third-party hepatocyte cultures, suggesting that FK 506 had induced a serum growth factor in the nude rats or had suppressed an inhibitory factor. A hypothesis was advanced that FK 506 (and cyclosporine) affects hepatic growth by nonimmunological pathways.     

Sinusoidal endothelial cell injury by superoxide anion and iron in the Propionibacterium acnes‐pretreated and lipopolysaccharide‐stimulated rat liver

Abstract: Aims/Background: We attempted to measure the generation of superoxide anion, examine its site of release and determine its pathological role in Propionibacterium acnes‐lipopolysaccharide‐induced liver injury in the rat. Methods: The P. acnes‐pretreated (16 mg/kg i.v.) rat liver was perfused with buffer containing lipopolysaccharide (2.5 μg/ml). Chemiluminescence enhanced with Cypridina luciferin analog, MCLA, and reduction of nitro blue tetrazolium were used for detecting superoxide anion. Leakage of enzymes and release of cytokines into the perfusate, and histological specimens were also examined. Results: Superoxide dismutase‐inhibitable chemiluminescence peaked at 30 min of lipopolysaccharide infusion and blue formazan precipitate was histochemically deposited mainly on hepatic macrophages. Purine nucleoside phosphorylase (PNP) activity in the perfusate, as a marker of sinusoidal endothelial cell injury, reached its maximum at 50 min and aspartate aminotransferase (AST) activity, as a marker of hepatocyte injury, reached a plateau at 90 min. Simultaneous treatment with superoxide dismutase and deferoxamine mesylate significantly suppressed the leakage of PNP and AST. Release of tumor necrosis factor‐α and growth‐related oncogene/cytokine‐induced neutrophil chemoattractant‐1 lagged behind PNP leakage. Light microscopy showed destruction of the sinusoids followed by hepatocyte necrosis. Electron microscopy revealed adherence of hepatic macrophages to sinusoidal endothelial cells. Conclusion: These results indicate that superoxide anion released from hepatic macrophages may induce sinusoidal endothelial cell injury via interaction with iron in the P. acnes‐lipopolysaccharide‐treated liver.     

Adenovirus-mediated viral interleukin-10 gene transfer prevents concanavalin A-induced liver injury

Background and aimLiver injury is closely associated with immune inflammation. Lacking immunostimulatory functions, viral interleukin-10 (vIL-10), a cellular IL-10 homologue, has been an attractive molecule for immunomodulatory therapy. We aimed to reveal a protective effect of the gene transfer of an adenoviral vector encoding vIL-10 on liver injury induced by concanavalin A.MethodsC57BL/6J mice were intravenously injected with adenoviral vector encoding vIL-10 before concanavalin A challenge. Liver injury was assessed. Interferon-γ and interleukin-4 levels were measured by ELISA. The activation of splenic and hepatic immune cells was analysed using an MTT assay.ResultsAdenoviral vector encoding vIL-10 pretreatment significantly decreased concanavalin A-mediated elevations in serum alanine aminotransaminase and aspartate aminotransaminase activity, and necrotic area in liver tissues. The protective effect of adenoviral vector encoding vIL-10 was attributed to its inhibition of T cell activation, and production of interferon-γ and interleukin-4 by the immune cells. Recombinant mouse IL-10, a high homologous cytokine to vIL-10, effectively downregulated interferon-γ and interleukin-4 release by hepatic mononuclear cells.ConclusionAdenovirus vector-mediated vIL-10 gene transfer can prevent concanavalin A-induced hepatic injury, minimise pro-inflammatory cytokine release, and inhibit the activation of T lymphocytes.     

FK 506 ameliorates the hepatic injury associated with ischemia and reperfusion in rats.

The effect of FK 506 on regeneration of the liver was studied in rats after a two-thirds partial hepatectomy after 60 min of ischemia of the unresected liver. The animals were divided into three distinct groups of 10 rats each. Group 1 (controls) received 0.5 ml saline solution intravenously 30 min after the induction of ischemia. Groups 2 and 3 were injected with FK 506 (0.3 mg/kg) intravenously 30 min after and 24 min before the induction of hepatic ischemia, respectively. The hepatic content of ATP and serum levels of ALT and lactate dehydrogenase were determined on each animal. In addition, the histological appearance and mitotic activity of the remnant liver was determined at regular 24-hr intervals after hepatic ischemia. All 10 control animals died within 72 hr. Treatment with FK 506 resulted in improved survival in groups 2 and 3 (30% and 80%, respectively). The improved survival seen in the FK 506-treated animals was reflected by a restoration of hepatic ATP content, a reduction in the serum levels of ALT and lactate dehydrogenase, an amelioration of hepatic necrosis and neutrophilic infiltration and an increase in the mitotic activity of the liver. These results suggest that FK 506 ameliorates the hepatic injury associated with ischemia/reperfusion and has a potent stimulatory effect on liver cell regeneration that may make it valuable as a hepatoprotective agent when administered to organ donors before graft harvesting.     

All-trans retinoic acid suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

All-trans retinoic acid (ATRA) has been reported to exert major effects on the immune system, including monocytes/macrophages. The present study was designed to determine whether ATRA would modulate macrophage-associated liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS) in rats. All-trans retinoic acid administration alleviated the liver injury and reduced the incidence of death following hepatic failure. Serum alanine aminotransferase (ALT) levels 5 h after, and survival rates within 12 h after the administration of LPS were significantly lower in the ATRA-treated group (134 ± 119 IU/L and 72.7%) compared with the control group (713 ± 411 IU/L and 18.2%; P < 0.05). Histological findings supported these results. These effects may be due to suppression of tumour necrosis factor-α (TNF-α) and superoxide anions produced by activated macrophages. Serum levels of TNF-α 1 h after LPS administration were significantly lower in the ATRA-treated group (60.5 ± 7.0 ng/mL) as compared with the control group (105.2 ± 39.3 ng/mL; P < 0.05). Formazan deposition that was generated by the perfusion of the liver with nitroblue tetrazolium, also suggested suppression of the release of superoxide anions from hepatic macrophages. These results suggest that ATRA acts as an immunomodulator in liver injury by suppressing the activation of liver macrophages.     

Effect of endotoxin on release of reactive oxygen intermediates by rat hepatic macrophages

Reactive oxygen intermediates released by activated hepatic macrophages have been implicated in the pathogenesis of a rat model of liver injury induced by sequential administration of Corynebacterium parvum and endotoxin. In this model, C. parvum causes extensive infiltration of the liver with activated macrophages, but severe liver injury occurs only after subsequent exposure to endotoxin. We have therefore investigated the effects of endotoxin on the release of reactive oxygen intermediates by C. parvum-activated hepatic macrophages. After in vitro exposure to zymosan, opsonized zymosan, or phorbol myristate acetate, hepatic macrophages isolated from C. parvum- and endotoxin-treated rats demonstrated significantly (1.5-2-fold) increased release of superoxide and oxidation of [1-14C]glucose via the hexose monophosphate shunt compared with hepatic macrophages isolated from C. parvum- and saline-treated control rats. These results indicate that endotoxin enhances the state of activation of hepatic macrophages already partially activated by C. parvum. We suggest that the increased release of reactive oxygen intermediates by these cells promotes liver injury in this model.     

Interleukin-6 induces proliferation of rat hepatocytes in vivo

Background/Aims: The aim of this study was to assess the effect of interleukin-6 (IL-6) on the proliferation of hepatocytes and to study the interaction between IL-6 and hepatocyte growth factor (HGF) in vivo.Methods: IL-6 was injected at a dose of 200 μg/mg subcutaneously into rats every day for 14 days. Liver and blood samples were obtained at 1, 3, 7 and 14 days during IL-6 administration. Hepatocyte proliferative activity of sera was measured using ³H-thymidine incorporation into cultured rat hepatocytes. To evaluate the proliferative activity of the hepatocytes in tissue sections, hepatic DNA content and immunostaining of the liver tissue sections for proliferating cell nuclear antigen (PCNA) were performed. Plasma HGF levels were measured using specific EIA. In addition, total RNA was extracted from the liver and expression of HGF mRNA was detected by RT-PCR.Results: The DNA contents of liver taken from IL-6-treated rats were increased during IL-6 administration compared with untreated rats. Sera taken from IL-6-treated rats at various intervals during administration also significantly increased ³H-thymidine incorporation by cultured rat hepatocytes compared with sera from untreated rats, suppressing ³H-thymidine incorporation at day 1 and 3 by anti-HGF antibody. IL-6 itself did not increase ³H-thymidine incorporation. Increased expression of PCNA in these hepatocytes was noted from 1 day after IL-6 administration, and at 14 days, the number of PCNA-positive cells was sevenfold greater than in the livers of untreated rats. However, plasma HGF levels showed a peak at day 1, decreased gradually from day 3, and became undetectable by day 14. HGF mRNA expression in livers of IL-6-treated rats was suppressed from day 3 to day 14 of IL-6 administration.Conclusions: These data show that IL-6 induces an early phase of liver cell growth in vivo and suggest that an increase level of HGF mediates this effect.     

内毒素受体在肝星状细胞的表达及其作用

目的了解内毒素受体在肝星状细胞活化中的变化和作用。方法分离正常大鼠的肝星状细胞，以逆转录聚合酶链反应法检测其在体外培养过程中内毒素受体（CD14和TLR4）mRNA的表达变化。以细胞免疫染色法检测肝星状细胞内毒素受体CD14的表达。制作肝纤维化和肝硬化的大鼠模型，免疫组织化学法动态检测肝组织内CD14和α平滑肌肌动蛋白的表达变化和定位。结果初分离的肝星状细胞表达低水平的CD14 mRNA，不表达TLR4 mRNA，培养活化的肝星状细胞内毒素受体的表达增强，内毒素可上调这种表达。体外培养10d的肝星状细胞表达CD14蛋白，内毒素作用后CD14表达更明显。在肝纤维化的发展过程中，肝组织内CD14阳性细胞增多，阳性细胞多分布于肝窦周围，晚期CD14阳性细胞聚集在纤维隔内，与α平滑肌肌动蛋白阳性细胞的分布一致。结论肝星状细胞在体内外的活化过程中内毒素受体的表达增强，因此，内毒素受体可能参与肝星状细胞在肝脏炎症和纤维化中的作用。     

Cyclosporin a and fk-506 in inhibition of rat ito cell activation in vitro

Ito cells are the primary matrix-producing cells in the liver. In hepatic fibrosis in vivo or culture on plastic, these cells undergo activation, a process characterized by cell proliferation, fibrogenesis, and smooth muscle α-actin expression. The cytosolic-binding proteins of cyclosporin A (CsA) and FK506 accelerate folding of various proteins including collagen and become inactivated by binding to those agents. CsA is shown to inhibit collagen synthesis in cultured fibroblasts. These findings prompted us to examine the effect of cyclosporin A and FK506 on Ito cell activation. CsA and FK506 reduced DNA synthesis in a dose-related manner, to 26% and 45% of controls at 5 μmol/L, respectively, without affecting total protein synthesis. CsA reduced collagen synthesis in a dose-related manner, to 70% of controls at 5 μmol/ L without affecting noncollagenous protein synthesis, whereas FK506 changed neither collagen synthesis nor noncollagenous protein synthesis. Moreover, smooth muscle α-actin expression was reduced by 0.5 μmol/L CsA, but not by FK506. CsA merits consideration for the therapy of hepatic fibrosis. FK506 may also be a candidate for such therapy through inhibitory action on Ito cell proliferation.     

Alcohol-Induced Expression of the CD14 Endotoxin Receptor Protein in Rat Kupffer Cells

Gut-derived endotoxins (lipopolysaccharide, LPS) are believed to contribute to alcohol-induced liver disease (ALD) by stimulating Kupffer cells, the resident liver macrophages, to release proinflammatory cytokines. This activation is largely mediated by CD14, a high-affinity membrane-anchored receptor for LPS. We observed, by chemiluminescence-enhanced detection, an increase in immunoreactive CD14 protein in Kupffer cells isolated from rats treated with ethanol for 2 weeks. Immunocytofluorescence experiments confirmed that this increase was confined to the membranes of Kupffer cells from the alcohol-treated rats. The increase was regulated pretranslationally: a 3-fold elevation ( p < 0.01) in the hepatic level of CD14 mRNA was observed. The marked increase in CD14 expression suggests a new mechanism by which alcohol increases the LPS-mediated cytokine signaling by the liver macrophages, thus promoting the interaction between alcohol and endotoxins in the development of liver damage.     

Optimal immunosuppressor induces stable gut microbiota after liver transplantation

AIM To study the influence of different doses of tacrolimus(FK506)on gut microbiota after liver transplantation(LT)in rats.METHODS Specific pathogen-free Brown Norway(BN)rats and Lewis rats were separated into five groups:(1)Tolerance group(BN-BN LT,n=8);(2)rejection group(Lewis-BN LT,n=8);(3)high dosage FK506(FK506-H)group(Lewis-BN LT,n=8);(4)middle dosage FK506(FK506-M)group(Lewis-BN LT,n=8);and(5)low dosage FK506(FK506-L)group(LewisBN LT,n=8).FK506 was administered to recipients at a dose of 1.0 mg/kg,0.5 mg/kg,and 0.1 mg/kg body weight for 29 d after LT to the FK506-H,FK506-M,and FK506-L groups,respectively.On the 30~(th) day after LT,all rats were sampled and euthanized.Blood samples were harvested for liver function and plasma endotoxin testing.Hepatic graft and ileocecal tissues were collected for histopathology observation.Ileocecal contents were used for DNA extraction,Real-time quantitative polymerase chain reaction(RT-PCR)and digital processing of denaturing gradient gel electrophoresis(DGGE)profiles and analysis.RESULTS Compared to the FK506-H and FK506-L groups,FK506-M was optimal for maintaining immunosuppression and inducing normal graft function;the FK506-M maintained gut barrier integrity and low plasma endotoxin levels;furthermore,DGGE results showed that FK506-M induced stable gut microbiota.Diversity analysis indicated that FK506-M increased species richness and rare species abundance,and cluster analysis confirmed the stable gut microbiota induced by FK506-M.Phylogenetic tree analysis identified crucial bacteria associated with FK506-M;seven of the nine bacteria that were decreased corresponded to Bacteroidetes,while increased bacteria were of the Bifidobacterium species.FK506-M increased Faecalibacterium prausnitzii and Bifidobacterium spp.and decreased Bacteroides-Prevotella and Enterobacteriaceae,as assessed by RT-PCR,which confirmed the crucial bacterial alterations identified through DGGE.CONCLUSION Compared to the low or high dosage of FK506,an optimal dosage of FK506 induced immunosuppression,normal graft function and stable gut microbiota following LT in rats.The stable gut microbiota presented increased probiotics and decreased potential pathogenic endotoxin-producing bacteria.These findings provide a novel strategy based on gut microbiota for immunosuppressive dosage assessment for recipients following LT.     

Immunosuppressive effect of FK506 on experimental allergic neuritis in Lewis rats: change of T cell subsets.

The effect of a new immunosuppressant, FK506, on experimental allergic neuritis (EAN) was examined in Lewis rats. EAN was induced by inoculation with bovine peripheral myelin. The EAN rats were divided into two groups. FK506-treated EAN rats were prophylactically administered FK506 by injection into the peritoneal cavity at a dosage of 5.0 mg/kg/day for 13 days beginning the day after inoculation. The control EAN rats were injected with only saline solution. FK506 prevented the development of EAN, histologically and clinically. In FK506-treated EAN rats, flow cytometric analysis of T cell subsets of the lymph nodes showed a significant decrease in W3/13 positive T cells on the 14th and 21st day and a decrease in W3/25 positive T cells on the 21st day after inoculation when compared with the control EAN rats. The percentage of OX-8 positive T cells were not significantly different between the two groups. Our results suggest that FK506 prevented the development of EAN by decreasing W3/13 and W3/25 positive T cells.     

Peroxynitrite formation during rat hepatic allograft rejection

The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor-specific blood transfusion (DST). Serum nitrite/nitrate, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor-specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite.