Fibroblast growth factor 15 deficiency increases susceptibility but does not improve repair to acetaminophen-induced liver injury in mice

The leading cause of acute liver failure (ALF) is hepatotoxicity from acetaminophen (APAP) overdose. However, limited options are available to treat this ALF so stimulating liver regeneration maybe a potential treatment. Our previous study has shown that fibroblast growth factor 15 (FGF15) plays a crucial role in liver regeneration, but the roles of FGF15 in liver injury and repair following APAP-overdose are unknown. In this study, treatment of FGF15 knockout (KO) male mice with APAP at 200, 250, or 300 mg/kg significantly increased the degree of liver injury compared to wild type (WT) mice. To determine the effects of FGF15 deficiency on liver repair following APAP overdose, a similar degree of liver injury was first obtained 24 h after treatment of WT and Fgf15 KO mice with APAP at different dosage. Fgf15 KO mice did not differ from WT mice in liver repair following similar degree of liver injury. In conclusion, we showed that FGF15 deficiency renders mice more susceptible to APAP-induced liver injury but did not seem to affect liver repair or regeneration. This study suggests that in contrast to the critical role that FGF15 plays in promoting liver regeneration following partial hepatectomy, this intestine factor is less involved in liver repair after APAP-induced liver injury.     

Activation of autophagy protects against acetaminophen-induced hepatotoxicity

Autophagy can selectively remove damaged organelles, including mitochondria, and, in turn, protect against mitochondria-damage-induced cell death. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing mitochondria damage and subsequent necrosis in hepatocytes. Although many detrimental mechanisms have been reported to be responsible for APAP-induced hepatotoxicity, it is not known whether APAP can modulate autophagy to regulate hepatotoxicity in hepatocytes. To test the hypothesis that autophagy may play a critical protective role against APAP-induced hepatotoxicity, primary cultured mouse hepatocytes and green fluorescent protein/light chain 3 transgenic mice were treated with APAP. By using a series of morphological and biochemical autophagic flux assays, we found that APAP induced autophagy both in the in vivo mouse liver and in primary cultured hepatocytes. We also found that APAP treatment might suppress mammalian target of rapamycin in hepatocytes and that APAP-induced autophagy was suppressed by N-acetylcysteine, suggesting APAP mitochondrial protein binding and the subsequent production of reactive oxygen species may play an important role in APAP-induced autophagy. Pharmacological inhibition of autophagy by 3-methyladenine or chloroquine further exacerbated APAP-induced hepatotoxicity. In contrast, induction of autophagy by rapamycin inhibited APAP-induced hepatotoxicity. CONCLUSION: APAP overdose induces autophagy, which attenuates APAP-induced liver cell death by removing damaged mitochondria.     

Curcumin attenuated paracetamol overdose induced hepatitis

AIM: To investigate whether curcumin could attenuate hepatitis in mice with paracetamol overdose. METHODS: Male mice were divided into four groups. Group 1 (control, n = 8); was fed with distilled water; Group 2 [N-acetyl-P-aminophenol (APAP), n = 8]; was fed with a single dose of 400 mg/kg APAP dissolved in distilled water; Group 3 [APAP + curcumin (CUR) 200, n = 8], was fed with a single dose of 400 mg/kg APAP and 200 mg/kg CUR; Group 4 (APAP + CUR 600, n = 8), was fed with a single dose of 400 mg/kg APAP and 600 mg/ kg CUR. Twenty-four hours later, the liver was removed to examine hepatic glutathione (GSH), hepatic malondialdehyde (MDA), and histopathologically. Then whole blood was withdrawn from heart to determine transaminase (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase) and inflammatory cytokines [interleukin (IL)-12 and IL-18] levels by enzyme linked immunosorbent assay.RESULTS: Serum transaminase, hepatic MDA, and inflammatory cytokines increased significantly in the APAP compared with the control group. Curcumin supplementation in APAP + CUR 200 and APAP + CUR 600 groups significantly decreased these parameters compared with the APAP group. The level of GSH decreased significantly in the APAP compared with the control group. Curcumin supplementation in APAP + CUR 200 and APAP + CUR 600 groups significantly increased these parameters compared with the APAP group. The histological appearance of the liver in the control group showed normal. In the APAP-treated group, the liver showed extensive hemorrhagic hepatic necrosis at all zones. Curcumin supplementation in APAP + CUR 200 and APAP + CUR 600 groups, caused the liver histopathology to improve. In the APAP + CUR 200 group, the liver showed focal necrosis and but the normal architecture was well preserved in APAP + CUR 600 group. CONCLUSION: APAP overdose can cause liver injury. Results indicate that curcumin prevents APAP-induced hepatitis through the improvement of liver histopathology by decreased oxidative stress,     

Mechanisms of protection by melatonin against acetaminophen-induced liver injury in mice

The present study was performed to determine whether melatonin protects mouse liver against severe damage induced by acetaminophen (APAP) administration and where melatonin primarily functions in the metabolic pathway of APAP to protect mouse liver against APAP-induced injury. Treatment of mice with melatonin (50 or 100 mg/kg, p.o.) 8 or 4 hr before APAP administration (750 mg/kg, p.o.) suppressed the increase in plasma alanine aminotransferase and aspartate aminotransferase activities in a dose- and a time-dependent manner. Melatonin treatment (100 mg/kg, p.o.) 4 hr before APAP administration remarkably inhibited centrilobular hepatic necrosis with inflammatory cell infiltration and increases in hepatic lipid peroxidation and myeloperoxidase activity, an index of tissue neutrophil infiltration, as well as release of nitric oxide and interleukin-6 into blood circulation at 9 hr after APAP administration. However, melatonin neither affected hepatic reduced glutathione (GSH) content nor spared hepatic GSH consumption by APAP treatment. Moreover, pretreatment with melatonin 4 hr before APAP administration did not influence the induction of hepatic heat shock protein 70 (HSP70) by APAP and melatonin alone did not induce HSP70 in mouse liver. These results indicate that exogenously administered melatonin exhibits a potent hepatoprotective effect against APAP-induced hepatic damage probably downstream of the activity of cytochrome P450 2E1, which works upstream of GSH conjugation in the pathway of APAP metabolism, via its anti-nitrosative and anti-inflammatory activities in addition to its antioxidant activity.     

Neutrophil depletion protects against murine acetaminophen hepatotoxicity

We previously reported that liver natural killer (NK) and NKT cells play a critical role in mouse model of acetaminophen (APAP)-induced liver injury by producing interferon gamma (IFN-gamma) and modulating chemokine production and subsequent recruitment of neutrophils into the liver. In this report, we examined the role of neutrophils in the progression of APAP hepatotoxicity. C57BL/6 mice were given an intraperitoneal toxic dose of APAP (500 mg/kg), which caused severe acute liver injury characterized by significant elevation of serum ALT, centrilobular hepatic necrosis, and increased hepatic inflammatory cell accumulation. Flow cytometric analysis of isolated hepatic leukocytes demonstrated that the major fraction of increased hepatic leukocytes at 6 and 24 hours after APAP was neutrophils (Mac-1+ Gr-1+). Depletion of neutrophils by in vivo treatment with anti-Gr-1 antibody (RB6-8C5) significantly protected mice against APAP-induced liver injury, as evidenced by markedly reduced serum ALT levels, centrilobular hepatic necrosis, and improved mouse survival. The protection was associated with decreased FasL-expressing cells, cytotoxicity against hepatocytes, and respiratory burst in hepatic leukocytes. In intracellular adhesion molecule (ICAM)-1-deficient mice, APAP caused markedly reduced liver injury when compared with wild-type mice. The marked protection in ICAM-1-deficient mice was associated with decreased accumulation of neutrophils in the liver. Hepatic GSH depletion and APAP-adducts showed no differences among the antibody-treated, ICAM-1-deficient, and normal mice. In conclusion, accumulated neutrophils in the liver contribute to the progression and severity of APAP-induced liver injury.     

Acetaminophen overdose-induced liver injury in mice is mediated by peroxynitrite independently of the cyclophilin D-regulated permeability transition

Acetaminophen (APAP) is safe at therapeutic dosage but can cause severe hepatotoxicity if used at overdose. The mechanisms of injury are not yet fully understood, but previous reports had suggested that the mitochondrial permeability transition (mPT) may be involved in triggering hepatocellular necrosis. We aimed at inhibiting mitochondrial cyclophilin D (CypD), a key regulator of the mPT, as a potential therapeutic target in APAP hepatotoxicity. Wildtype mice treated with a high dose of APAP (600 mg/kg, intraperitoneal) developed typical centrilobular necrosis, which could not, however, be prevented by cotreatment with the selective CypD inhibitor, Debio 025 (alisporivir, DEB025, a nonimmunosuppressive cyclosporin A analog). Similarly, genetic ablation of mitochondrial CypD in Ppif-null mice did not afford protection from APAP hepatotoxicity. To determine whether APAP-induced peroxynitrite stress might directly activate mitochondrial permeabilization, independently of the CypD-regulated mPT, we coadministered the peroxynitrite decomposition catalyst Fe-TMPyP (10 mg/kg, intraperitoneal, 90 minutes prior to APAP) to CypD-deficient mice. Liver injury was greatly attenuated by Fe-TMPyP pretreatment, and mitochondrial 3-nitrotyrosine adduct levels (peroxynitrite marker) were decreased. Acetaminophen treatment increased both the cytosolic and mitochondria-associated P-JNK levels, but the c-jun-N-terminal kinase (JNK) signaling inhibitor SP600125 was hepatoprotective in wildtype mice only, indicating that the JNK pathway may not be critically involved in the absence of CypD. Conclusion: These data support the concept that an overdose of APAP results in liver injury that is refractory to pharmacological inhibition or genetic depletion of CypD and that peroxynitrite-mediated cell injury predominates in the absence of CypD.     

Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase

Mechanistic study of idiosyncratic drug-induced hepatitis (DIH) continues to be a challenging problem because of the lack of animal models. The inability to produce this type of hepatotoxicity in animals, and its relative rarity in humans, may be linked to the production of anti-inflammatory factors that prevent drug-protein adducts from causing liver injury by immune and nonimmune mechanisms. We tested this hypothesis by using a model of acetaminophen (APAP)-induced liver injury in mice. After APAP treatment, a significant increase was observed in serum levels of interleukin (IL)-4, IL-10, and IL-13, cytokines that regulate inflammatory mediator production and cell-mediated autoimmunity. When IL-10 knockout (KO) mice were treated with APAP, most of these mice died within 24 to 48 hours from liver injury. This increased susceptibility to APAP-induced liver injury appeared to correlate with an elevated expression of liver proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, and IL-1, as well as inducible nitric oxide synthase (iNOS). In this regard, mice lacking both IL-10 and iNOS genes were protected from APAP-induced liver injury and lethality when compared with IL-10 KO mice. All strains, including wild-type animals, generated similar amounts of liver APAP-protein adducts, indicating that the increased susceptibility of IL-10 KO mice to APAP hepatotoxicity was not caused by an enhanced formation of APAP-protein adducts. In conclusion, these findings suggest that an important feature of the normal response to drug-induced liver injury may be the increased expression of anti-inflammatory factors such as IL-10. Certain polymorphisms of these factors may have a role in determining the susceptibility of individuals to idiosyncratic DIH.     

TLR介导的Nrf2抗氧化通路在对乙酰氨基酚药物性肝损伤中的保护作用

目的研究内毒素(LPS)及Toll样受体(TLR)在对乙酰氨基酚(APAP)药物性肝损伤中的保护作用及其相关机制。方法雄性C57BL/6小鼠40只,分为4组,每组10只。空白对照组腹腔注射0.9%氯化钠溶液,LPS组腹腔注射LPS 10μg/kg,APAP组腹腔注射APAP 300 mg/kg,LPS+APAP组在APAP造模前16 h给予LPS 10μg/kg预处理。通过比较各组血清ALT和AST水平,并通过HE染色评价肝组织损伤程度,观察LPS对小鼠肝损伤的保护作用。测定相应时间点的肝脏组织丙二醛(MDA)、还原型谷胱甘肽(GSH)的变化以及肝组织DHE染色,评价小鼠氧化应激水平。应用Western印迹及RT-PCR检测肝脏Nrf2,Gclc及HO-1的表达水平。结果 LPS预处理可明显减轻APAP所致的肝脏氧化应激反应及肝损伤程度。LPS预处理组的小鼠血清ALT(518.3±142.3对4542±498.4 U/L)、AST(643.3±105.6对5432.1±569.2 U/L)水平及肝组织MDA(78.0±14.5对141.7±26.4 mmoL/mg)水平与模型组相比明显降低,而GSH(6.2±1.7对3.5±1.0μmol/g)水平明显升高(P0.05),肝组织病理损伤明显减轻。同时,LPS预处理可明显促进Nrf2及其下游抗氧化基因的表达。结论 LPS在小鼠APAP肝损伤中起到保护作用,作用机制与Nrf2抗氧化通路的激活相关,可能成为药物性肝损伤的新的治疗策略。     

Green-tea Polyphenols Downregulate Cyclooxygenase and Bcl-2 Activity in Acetaminophen-induced Hepatotoxicity

Acetaminophen (APAP) elicits hepatotoxicity via multifactorial pathways, including increased apoptosis, cyclooxygenase (Cox-2) generation, reactive metabolite release, and glutathione (GSH) depletion. We previously showed that mice that consumed different antioxidants in their diets were protected against APAP-induced hepatotoxicity. We therefore further investigated the mechanisms by which green-tea polyphenols (GrTP) protect against APAP-induced hepatic damage. Mice were administered a diet supplemented with GrTP or vehicle for 5 consecutive days followed by intraperitoneal (IP) injection of a toxic dose of APAP or sham. APAP administration upregulated Cox-2 and B-cell lymphoma-2 (Bcl-2) production and had an effect, albeit minor, on Cox-1 and Fas expression in hepatic tissue. GrTP supplementation normalized APAP induced Cox-2 expression and Bcl-2 activation (P < 0.01), as evidenced by immunohistochemistry and Western blot analyses. Similarly, APAP administration elicited marked depletion (99%) in hepatic reduced GSH (rGSH) and endogenous S-adenosylmethionine (SAMe) concentrations (twofold) when compared with sham. APAP also caused severe centrilobular apoptosis and necrosis accompanied by leukocyte infiltration and marked elevations in the hepatic enzyme, alanine aminotransferase (ALT) released from damaged hepatocytes, and cytokine tumor necrosis factor alpha (TNF-α). GrTP improved hepatic histopathology (P < 0.01) and attenuated ALT activity (P < 0.05) and the depletion of rGSH (P < 0.05). In conclusion, GrTP supplementation attenuated hepatotoxicity by normalizing Cox-2 and Bcl-2 activation, suggesting a potential use for GrTP in treatng APAP toxicity.     

Role of the coagulation system in acetaminophen-induced hepatotoxicity in mice

Acetaminophen (N-acetyl-p-aminophenol [APAP]) is one of the leading causes of acute liver failure, and APAP hepatotoxicity is associated with coagulopathy in humans. We tested the hypothesis that activation of the coagulation system and downstream protease-activated receptor (PAR)-1 signaling contribute to APAP-induced liver injury. Fasted C57BL/J6 mice were treated with either saline or APAP (400 mg/kg intraperitoneally) and were euthanized 0.5-24 hours later. Hepatotoxicity and coagulation system activation occurred by 2 hours after administration of APAP. Treatment with APAP also caused a rapid and transient increase in liver procoagulant activity. In addition, significant deposition of fibrin was observed in the liver by 2 hours, and the concentration of plasminogen activator inhibitor-1 in plasma increased between 2 and 6 hours. Pretreatment with heparin attenuated the APAP-induced activation of the coagulation system and hepatocellular injury and diminished hepatic fibrin deposition at 6 hours. Loss of hepatocellular glutathione was similar in APAP-treated mice pretreated with saline or heparin, suggesting that heparin did not diminish bioactivation of APAP. In mice deficient in tissue factor, the principal cellular activator of coagulation, APAP-induced liver injury, activation of coagulation, and hepatic fibrin deposition were reduced at 6 hours. Formation of the tissue factor-factor VIIa complex leads to the generation of thrombin that can activate cells through cleavage of PAR-1. Mice lacking PAR-1 developed less injury and hepatic fibrin deposits at 6 hours in response to APAP than control mice. CONCLUSION: Activation of the coagulation system and PAR-1 signaling contribute significantly to APAP-induced liver injury.     

心肌去乙酰化酶SIRT3对缺血/再灌注小鼠心律失常的影响

目的:探讨心肌线粒体去乙酰化酶SIRT3对急性缺血再灌注(I/R)所致心律失常的影响。方法:以24只SIRT3基因敲除型小鼠为实验对象，用24只野生型小鼠为对照，两种小鼠均随机各分为对照组、假手术组、I/R模型组及I/R+烟酰胺腺嘌呤二核苷酸（NAD+）治疗组，每组6只小鼠(n=6)。采用冠脉左前降支结扎缺血30 min再灌注2 h建立在体大鼠急性心肌I/R模型，于术中监测心电指标。取心肌组织检测SIRT3、锰超氧化物歧化酶（MnSOD）和过氧化氢酶（Catalase）蛋白的表达和心肌内氧自由基(ROS)的水平。结果:与野生型对照小鼠相比，SIRT3基因敲除型小鼠心肌中SIRT3、MnSOD和Catalase蛋白表达的水平显著降低。心律失常评分的结果显示，SIRT3基因敲除型小鼠的假手术组即可观察到心律失常。SIRT3基因敲除可导致小鼠心肌I/R所致心律失常显著加重（与野生型模型组相比，P<0.05）。心肌I/R后，SIRT3基因敲除型小鼠心肌中ROS的增加程度明显高于野生型模型组小鼠（P<0.05）。预先采用NAD+治疗，可显著提高野生型I/R小鼠心肌SIRT3的活性（与野生型小鼠模型组相比，P<0.05），显著增加心肌MnSOD的活性，进而有效地抑制I/R小鼠心肌中ROS的水平，有效缓解I/R所致心律失常（与野生型小鼠模型组相比，均P<0.05）。但是，SIRT3基因敲除后，NAD+治疗引起的上述心肌保护作用基本消失。结论:心肌中SIRT3表达的降低可能是加重心肌I/R过程中氧化应激损伤并促发心律失常的重要机制。SIRT3正常活性的维持有助于对抗心肌I/R损伤(MIRI)的发生。     

大蒜素对自发性高血压大鼠肠系膜动脉平滑肌细胞钙电流的作用

目的:观察大蒜素(Gar)对自发性高血压大鼠(SHR)肠系膜动脉平滑肌细胞L-型钙电流(LCa,L)的影响。方法:利用双酶-两步法消化得到单个大鼠肠系膜动脉血管平滑肌细胞,用全细胞膜片钳记录钙电流。在细胞池中灌流含Gar的细胞外液,观察药物对LCa,L的作用和门控机制及门控动力学参数的改变。结果:1Gar对ICa,L的抑制效应呈浓度依赖性和电压依赖性特征。刺激电位0 mV时,200μmol/L Gar可使ICa,L峰值密度由(-8.4±0.4)pA/pF降低为(-6.1±0.3)pA/pF;2药物可使ICa,L半激活电压V1/2右移,半失活电压左移及失活后恢复动力学减慢等环节可减少通道的开放和重复开放,从而减少ICa,L峰值密度和窗口电流。结论:Gar可能通过减少细胞的钙电流发挥降压效应。     

Peroxisome proliferator-activated receptor alpha induction of uncoupling protein 2 protects against acetaminophen-induced liver toxicity

Acetaminophen (APAP) overdose causes acute liver failure in humans and rodents due in part to the destruction of mitochondria as a result of increased oxidative stress followed by hepatocellular necrosis. Activation of the peroxisome proliferator-activated receptor alpha (PPARα), a member of the nuclear receptor superfamily that controls the expression of genes encoding peroxisomal and mitochondrial fatty acid β-oxidation enzymes, with the experimental ligand Wy-14,643 or the clinically used fibrate drug fenofibrate, fully protects mice from APAP-induced hepatotoxicity. PPARα-humanized mice were also protected, whereas Ppara-null mice were not, thus indicating that the protection extends to human PPARα and is PPARα-dependent. This protection is due in part to induction of the PPARα target gene encoding mitochondrial uncoupling protein 2 (UCP2). Forced overexpression of UCP2 protected wildtype mice against APAP-induced hepatotoxicity in the absence of PPARα activation. Ucp2-null mice, however, were sensitive to APAP-induced hepatotoxicity despite activation of PPARα with Wy-14,643. Protection against hepatotoxicity by UCP2-induction through activation of PPARα is associated with decreased APAP-induced c-jun and c-fos expression, decreased phosphorylation of JNK and c-jun, lower mitochondrial H(2)O(2) levels, increased mitochondrial glutathione in liver, and decreased levels of circulating fatty acyl-carnitines. These studies indicate that the PPARα target gene UCP2 protects against elevated reactive oxygen species generated during drug-induced hepatotoxicity and suggest that induction of UCP2 may also be a general mechanism for protection of mitochondria during fatty acid β-oxidation.     

Dendritic cell depletion exacerbates acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure in the United States and is primarily mediated by toxic metabolites that accumulate in the liver upon depletion of glutathione stores. However, cells of the innate immune system, including natural killer (NK) cells, neutrophils, and Kupffer cells, have also been implicated in the centrilobular liver necrosis associated with APAP. We have recently shown that dendritic cells (DCs) regulate intrahepatic inflammation in chronic liver disease and, therefore, postulated that DC may also modulate the hepatotoxic effects of APAP. We found that DC immune-phenotype was markedly altered after APAP challenge. In particular, liver DC expressed higher MHC II, costimulatory molecules, and Toll-like receptors, and produced higher interleukin (IL)-6, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α). Conversely, spleen DC were unaltered. However, APAP-induced centrilobular necrosis, and its associated mortality, was markedly exacerbated upon DC depletion. Conversely, endogenous DC expansion using FMS-like tyrosine kinase 3 ligand (Flt3L) protected mice from APAP injury. Our mechanistic studies showed that APAP liver DC had the particular capacity to prevent NK cell activation and induced neutrophil apoptosis. Nevertheless, the exacerbated hepatic injury in DC-depleted mice challenged with APAP was independent of NK cells and neutrophils or numerous immune modulatory cytokines and chemokines. Conclusion: Taken together, these data indicate that liver DC protect against APAP toxicity, whereas their depletion is associated with exacerbated hepatotoxicity.     

Misoprostol protection against acetaminophen-induced hepatotoxicity in the rat

The hepatoprotective effects of misoprostol on acetaminophen (APAP)-induced toxicity were studied in the rat. Liver injury was evaluated at 36 hr after APAP administration by measuring serum ornithine carbamoyltransferase (OCT) and alanine aminotransferase (ALT) levels, by using tetranitroblue tetrazolium (TNBT) staining and by histological analysis. After APAP administration, peak serum levels of the drug were detected at 15 min. Liver GSH was depleted from control levels of 448±48 µg/g to 82±2 µg/g (P<0.01) within 3 hr. Serum ALT levels increased significantly after 16 hr and H&E staining revealed significant hepatic necrosis after 12 hr. Rats treated with misoprostol before and after APAP administration showed reduced OCT and ALT levels at 36 hr of overdose (454±446 IU/liter and 2571±2944 IU/liter, respectively) compared to those without misoprostol treatment (1348±480 IU/liter and 6077±3025 IU/liter, respectively,P<0.01). TNBT staining showed a reduced area of damage from 28.6±22.3% to 7.3±8.9% (P<0.01), and H&E staining also showed less extensive hepatic necrosis in rats treated with misoprostol before and after the overdose. In a time sequence study, misoprostol treatment starting within 10 hr of overdose showed the same protective effect as when it was given before and after APAP ingestion. No protection was detected when the treatment was started during the development of hepatic injury. However, misoprostol given when injury was established seemed to be protective. Our results show that misoprostol protects the liver against APAP-induced injury if given within 10 hr of overdose. Late administration of misoprostol may also be beneficial and thus may be considered in treating patients with APAP toxicity.This study is supported by the Australian National Health and Medical Research Council Grant 91/0662.     

Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity

BACKGROUND & AIMS: Inflammatory mediators released by nonparenchymal inflammatory cells in the liver have been implicated in the progression of acetaminophen (APAP) hepatotoxicity. Among hepatic nonparenchymal inflammatory cells, we examined the role of the abundant natural killer (NK) cells and NK cells with T-cell receptors (NKT cells) in APAP-induced liver injury. METHODS: C57BL/6 mice were administered a toxic dose of APAP intraperitoneally to cause liver injury with or without depletion of NK and NKT cells by anti-NK1.1 monoclonal antibody (MAb). Serum alanine transaminase (ALT) levels, liver histology, hepatic leukocyte accumulation, and cytokine/chemokine expression were assessed. RESULTS: Compared with APAP-treated control mice, depletion of both NK and NKT cells by anti-NK1.1 significantly protected mice from APAP-induced liver injury, as evidenced by decreased serum ALT level, improved survival of mice, decreased hepatic necrosis, inhibition of messenger RNA (mRNA) expression for interferon-gamma (IFN-gamma), Fas ligand (FasL), and chemokines including KC (Keratinocyte-derived chemokine); MIP-1 alpha (macrophage inflammatory protein-1 alpha); MCP-1 (monocyte chemoattractant protein-1); IP-10 (interferon-inducible protein); Mig (monokine induced by IFN-gamma) and decreased neutrophil accumulation in the liver. Hepatic NK and NKT cells were identified as the major source of IFN-gamma by intracellular cytokine staining. APAP induced much less liver injury in Fas-deficient (lpr) and FasL-deficient (gld) mice compared with that in wild-type mice. CONCLUSIONS: NK and NKT cells play a critical role in the progression of APAP-induced liver injury by secreting IFN-gamma, modulating chemokine production and accumulation of neutrophils, and up-regulating FasL expression in the liver, all of which may promote the inflammatory response of liver innate immune system, thus contributing to the severity and progression of liver injury downstream of the metabolism of APAP and depletion of reduced glutathione (GSH) in hepatocytes.     

Up-regulation of NAD（P）H quinone oxidoreductase 1 during human liver injury

AIM:To investigate the expression and activity ofNAD(P)H quinone oxidoreductase 1(NQO1)in humanliver specimens obtained from patients with liver damagedue to acetaminophen(APAP)overdose or primarybiliary cirrhosis(PBC).METHODS:NQO1 activity was determined in cytosolfrom normal,APAP and PBC liver specimens.Westernblot and immunohistochemical staining were used todetermine patterns of NQO1 expression using a specificantibody against NQO1.RESULTS:NQO1 protein was very low in normal humanlivers.In both APAP and PBC livers,there was stronginduction of NQO1 protein levels on Western blot.Correspondingly,significant up-regulation of enzymeactivity(16-and 22-fold,P<0.05)was also observed inAPAP and PBC livers,respectively.Immunohistochemicalanalysis highlighted injury-specific patterns of NQO1staining in both APAP and PBC livers.CONCLUSION:These data demonstrate that NQO1protein and activity are markedly induced in humanlivers during both APAP overdose and PBC.Up-regulationof this cytoprotective enzyme may represent an adaptivestress response tO limit further disease progression bydetoxifying reactive species.     

Early hepatic microvascular injury in response to acetaminophen toxicity

OBJECTIVE: The hepatic toxic response to acetaminophen (APAP) is characterized by centrilobular (CL) necrosis preceded by hepatic microvascular injury and congestion. The present study was conducted to examine changes in liver microcirculation after APAP dosing. METHODS: Male C57Bl/6 mice were treated with APAP (600 mg/kg body weight) by oral gavage. The livers of anesthetized mice were examined using established in vivo microscopic methods at 0, 0.5, 1, 2, 4, 6, 12 hours after APAP. RESULTS: The levels of hepatic transaminases (i.e., alanine aminotransferase [ALT] and aspartate transaminase) increased minimally for up to 2 hours. Thereafter, their levels were significantly and progressively increased. The numbers of swollen sinusoidal endothelial cells (SECs) in periportal regions were increased (3.5-fold) from 0.5 to 6 hours, and those in CL regions were increased (4.0-fold) at 0.5 and 1 hour. The intensity of in vivo staining for formaldehyde-treated serum albumin, which is a specific ligand for SECs, was reduced from 2 to 12 hours. Erythrocytes infiltrated into the space of Disse as early as 2 hours, and the area occupied by these cells was markedly increased at 6 hours. Sinusoidal perfusion was reduced from 1 through 12 hours, with a nadir (35% decrease) at 4 and 6 hours. Phagocytic Kupffer cell activity was significantly elevated from 0.5 through 12 hours. Although gadolinium chloride minimized the changes in sinusoidal blood flow and reduced ALT levels 6 hours after APAP, it failed to inhibit endothelial swelling, extravasation of erythrocytes, and CL parenchymal necrosis. CONCLUSIONS: These results confirm that APAP-induced SEC injury precedes hepatocellular injury, supporting the hypothesis that SECs are an early and direct target for APAP toxicity. These findings also suggest that reduced sinusoidal perfusion and increased Kupffer cell activity contribute to the development of APAP-induced liver injury.     

Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system

OBJECTIVE: Dnase1-deficient mice with the 129 x C57BL/6 genetic background develop symptoms of systemic lupus erythematosus, such as high titers of antinuclear autoantibodies directed against nucleosomes. In this study we analyzed a potential molecular pathomechanism leading to this autoimmunity, by exploring the influence of extracellular Dnase1 present in serum on the breakdown of chromatin in necrotic cells in vitro. METHODS: Human breast adenocarcinoma cells (MCF-7) and other cell lines were subjected to necrosis induced by hydrogen peroxide, streptolysin O, or freeze-thawing. Subsequently, the influence of sera from Dnase1-deficient and wild-type mice as well as the influence of purified enzymes present in the culture medium on the process of necrotic chromatin breakdown was investigated. RESULTS: Necrotic chromatin breakdown resembled apoptotic DNA laddering and was catalyzed by serum Dnase1 in conjunction with plasmin. During necrosis, Dnase1 and plasminogen penetrated the cell and accumulated in the cytoplasm and nucleus. Plasminogen bound to the cytoskeleton and nuclear structures, was activated to plasmin by either tissue-type or urokinase-type plasminogen activator, and degraded histone H1, thereby facilitating internucleosomal DNA cleavage by Dnase1. CONCLUSION: Our results suggest that serum Dnase1 in cooperation with the plasminogen system guarantees a fast and effective breakdown of chromatin during necrosis by the combined cleavage of DNA as well as of DNA binding proteins. The failure of such a clearance mechanism might lead to antinuclear autoimmunity similar to that observed in the Dnase1-deficient mouse.