Edaravone protects endotoxin-induced liver injury by inhibiting apoptosis and reducing proinflammatory cytokines

Studies have shown that edaravone may prevent liver injury. This study aimed to investigate the effects of edaravone on the liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in female BALB/c mice. Edaravone was injected into mice 30 min before and 4 h after GalN/LPS injection. The survival rate was determined within the first 24 h. Animals were killed 8 h after GalN/LPS injection, and liver injury was biochemically and histologically assessed. Hepatocyte apoptosis was measured by TUNEL staining; proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the liver were assayed by ELISA; expression of caspase-8 and caspase-3 proteins was detected by Western blot assay; and caspase-3 activity was also determined. Results showed that GalN/LPS induced marked elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Edaravone significantly inhibited elevation of serum AST and ALT, accompanied by an improvement in histological findings. Edaravone lowered the levels of TNF-α and IL-6 and reduced the number of TUNEL-positive cells. In addition, 24 h after edaravone treatment, caspase-3 activity and mortality were reduced. Edaravone may effectively ameliorate GalN/LPS-induced liver injury in mice by reducing proinflammatory cytokines and inhibiting apoptosis.     

Intrinsic resistance to TNF-alpha-induced hepatocyte apoptosis in ICR mice correlates with expression of a short form of c-FLIP

The administration of tumor necrosis factor-alpha (TNF-alpha) or the anti-Fas antibody (Jo-2) to mice causes acute liver failure, which is lethal within hours as a result of the induction of apoptosis in hepatocytes. It was recently reported that nonobese diabetic (NOD) mice are less sensitive to TNF-alpha/D-galactosamine (GalN)-induced liver failure than C57BL/6J (B6) mice, whereas both NOD and B6 mice were sensitive to the lethal effect of Jo-2. In the present study, we investigated the differences between the apoptotic liver cell death induced by TNF-alpha/GalN and that induced by Jo-2. B6, NOD, and Jcl-Imperial Cancer Research (ICR) mice were injected intravenously with TNF-alpha/GalN or Jo-2. ICR mice were less sensitive to TNF-alpha/GalN-induced liver failure than NOD and B6 mice (P<0.0001). In contrast, ICR mice were more sensitive to Jo-2-induced liver failure than B6 mice (P=0.0003). The liver caspase-3, -8 activity, serum transaminase levels, and the number of apoptotic liver nuclei all decreased in ICR in comparison to B6 mice treated with TNF-alpha/GalN. The mRNA expression of TNFR-associated death domain, Fas associated protein with death domain, and Bcl family and nuclear factor-kappaB activation induced by TNF-alpha/GalN were similar in both mice. Interestingly, the short form of cellular FLICE/caspase-8-inhibitory protein (c-FLIP(S)) was constitutively upregulated in ICR mice. In conclusion, these results suggest that ICR mice have an intrinsic resistance to TNF-alpha-induced hepatocyte apoptosis, and that c-FLIP(S) may play a role in TNF-alpha/GalN-induced liver failure, but not in Fas-induced liver failure.     

Dual effects of heat stress on tumor necrosis factor-alpha-induced hepatocyte apoptosis in mice

The major heat shock protein, HSP70, plays a critical role in cell survival in response to stress, possibly by inhibiting a number of antisurvival pathways. However, heat stress (HS) and HSPs also sensitize cells to certain apoptotic stimuli, such as TNF-alpha. To clarify the relations between HS and apoptosis, we examined the differential effects of the intensity of HS on liver injury and apoptosis induced by TNF-alpha in mice. TNF-alpha was injected into D-galactosamine (GalN)-sensitized mice that were pretreated with or without HS. Liver injury was assessed biochemically and histologically. In GalN-sensitized mice, application of HS for 7 days led to significant enhancement of TNF-alpha-induced hepatotoxicity, despite upregulation of HSP70 in the liver. In contrast, application of HS for 1 day led to attenuation of TNF-alpha-induced liver injury. Repeated HS decreased the levels of the FLICE inhibitory protein short (FLIP(S)) and activated caspase-8 in the liver. The caspase-8 inhibitor Z-IETD-FMK effectively protected both the nontreated and HS-pretreated mice from the hepatotoxicity induced by GalN/TNF-alpha. HS shows dual effects on TNF-alpha-induced hepatocyte apoptosis. Exposure to repeated HS, but not to single HS, leads to enhancement of TNF-alpha-induced hepatocyte apoptosis via the interaction of FLIP and caspase-8.     

Apoptotic cell death in the response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.

The apoptotic cell death induced in D-galactosamine-sensitized mice by administration of lipopolysaccharide was characterized. Administration of lipopolysaccharide caused apoptotic cell death in livers of D-galactosamine-sensitized mice. Apoptotic cells were also detected in the kidney, thymus, spleen, and lymph node. Severe hepatic apoptosis in D-galactosamine-sensitized mice was reproduced by transfer of the sera from mice injected with D-galactosamine and lipopolysaccharide. The hepatocyte apoptosis induced by lipopolysaccharide was completely prevented by an anti-tumor necrosis factor alpha antibody but not by an anti-gamma interferon antibody. Administration of recombinant tumor necrosis factor into D-galactosamine-sensitized mice also caused hepatocyte apoptosis. Lipopolysaccharide-induced hepatocyte apoptosis in D-galactosamine-sensitized mice did not seem to be mediated by Fas antigen. It was suggested that lipopolysaccharide- induced hepatic injury and failure in D-galactosamine-sensitized mice was due to the apoptotic cell death of hepatocytes caused by tumor necrosis factor alpha released in the circulation.     

Necrostatin-1 Protects Against <Emphasis Type="SmallCaps">d</Emphasis>-Galactosamine and Lipopolysaccharide-Induced Hepatic Injury by Preventing TLR4 and RAGE Signaling

Fulminant hepatic failure (FHF) is a life-threatening clinical syndrome results in massive inflammation and hepatocyte death. Necroptosis is a regulated form of necrotic cell death that is emerging as a crucial control point for inflammatory diseases. The kinases receptor interacting protein (RIP) 1 and RIP3 are known as key modulators of necroptosis. In this study, we investigated the impact of necroptosis in the pathogenesis of FHF and molecular mechanisms, particularly its linkage to damage-associated molecular pattern (DAMP)-mediated pattern recognition receptor (PRR) signaling pathways. Male C57BL/6 mice were given an intraperitoneal injection of necrostatin-1 (Nec-1, RIP1 inhibitor; 1.8 mg/kg; dissolved in 2% dimethyl sulfoxide in phosphate-buffered saline) 1 h before receiving d-galactosamine (GalN; 800 mg/kg)/lipopolysaccharide (LPS; 40 μg/kg). Hepatic RIP1, RIP3 protein expression, their phosphorylation, and RIP1/RIP3 complex formation upregulated in the GalN/LPS group were attenuated by Nec-1. Nec-1 markedly reduced the increases in mortality and serum alanine aminotransferase activity induced by GalN/LPS. Increased serum high mobility group box 1 (HMGB1) and interleukin (IL)-33 release, HMGB1-toll-like receptor 4 and HMGB1-receptor for advanced glycation end products (RAGE) interaction, and nuclear protein expressions of NF-κB and early growth response protein-1 (egr-1) were attenuated by Nec-1. Our finding suggests that necroptosis is responsible for GalN/LPS-induced liver injury through DAMP-activated PRR signaling.     

淫羊藿苷对ConA诱导的小鼠肝脏损伤保护机理的研究

目的:利用刀豆蛋白A(ConA)诱导建立小鼠肝炎模型,观察淫羊藿苷对该损伤模型保护作用的细胞免疫学和分子免疫学机制。方法:采用C57BL/6雄性小鼠,随机分为淫羊藿苷+ConA组、生理盐水+ConA组、淫羊藿苷+生理盐水组。小鼠尾静脉注射ConA,建立T细胞介导的免疫性肝脏损伤模型;采用转氨酶试剂盒测定各组小鼠血液中转氨酶含量;组织病理学观察实验小鼠肝脏组织和肝细胞坏死变化;采用ELISA试剂盒测定血清中炎性细胞因子IFN-γ、TNF-α含量;流式细胞术观察注射ConA后肝脏淋巴细胞的活化变化。结果:与ConA对照组相比,淫羊藿苷干预组小鼠血液中ALT和AST水平明显降低,P<0.01;IFN-γ、TNF-α含量明显降低,P<0.05;H&E染色可见淫羊藿苷干预组小鼠肝细胞核完整,未见炎性细胞的浸润,而ConA对照组小鼠肝细胞核固缩,部分核膜破裂,肝组织内有炎症细胞和红细胞浸润;流式细胞技术发现淫羊藿苷可明显延缓由ConA引起的肝脏NKT细胞的活化,减弱T细胞的浸润。结论:淫羊藿苷对ConA诱导的小鼠肝脏损伤有明显的保护作用,其机理可能与淫羊藿苷降低血液中IFN-γ、TNF-α表达及影响肝脏NKT细胞的活化有关。     

依赖天冬氨酸特异性半胱氨酸蛋白酶-3的肝细胞凋亡在肝硬化大鼠肝缺血再灌注损伤中的作用

目的:探讨肝硬化大鼠肝缺血/再灌注(I/R)损伤是否与肝细胞凋亡相关及天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)活性变化与肝细胞凋亡的关系。方法:Pringle法复制肝I/R模型,将肝硬化大鼠随机分为2组:A组:单纯肝门阻断;B组:血流阻断+抑制剂:N-苯甲基氧化碳酰-缬氨酸-丙氨酸-天冬氨酸-氟化丙酮(ZVAD-fmk)15mg/kg;取无肝硬化大鼠,作单纯肝门阻断为C组。各组肝门阻断时间均为30min,再灌注72h。比较3组的血清天冬氨酸转氨酶(AST)、肝组织的caspase-3活性和肝细胞凋亡数。结果:A组大鼠肝组织caspase-3活性、肝细胞凋亡数在再灌注后6h达高峰,分别为(18.1±1.8)μmolAMC·h^-1·g^-1(tissue)和20.9%±4.9%,与I/R前的(6.6±2.0)μmolAMC·h^-1·g^-1(tissue)和0.5%±0.3%相比,P＜0.01。肝细胞凋亡数、caspase-3的活性随灌注时间的延长而减低,两者随时间的变化一致。3组中A组肝损伤最严重,表现为再灌注后6h血清AST最高,与B、C组比较有显著差异,大鼠7d生存率只为62.5%。进一步研究表明,再灌注后6h,A组的肝组织caspase-3活性、肝细胞凋亡数亦明显比B、C组高。结论:肝细胞凋亡是肝硬化大鼠肝I/R损伤的主要病理改变。肝细胞凋亡的发生可能主要依赖于肝组织caspase-3活性的改变,抑制caspase-3能明显减轻肝I/R损伤。肝硬化肝脏比无硬化肝脏对缺血损伤敏感性高的病理机制与依赖caspase-3的肝细胞凋亡密切相关。     

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80⁺ Kupffer cells, as well as in isolated nonparenchymal cells. MLK3^−/− mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.Key words: Alcoholic liver disease (ALD), Kupffer cells, Necroptosis, Toll-like receptor 4 (TLR4), Cytokines     

Prevention of hepatic apoptosis and embryonic lethality in RelA/TNFR-1 double knockout mice

Mice deficient in the nuclear factor κB (NF-κB)-transactivating gene RelA (p65) die at embryonic days 14–15 with massive liver apoptosis. In the adult liver, activation of the NF-κB heterodimer RelA/p50 can cause hepatocyte proliferation, apoptosis, or the induction of acute-phase response genes. We examined, during wild-type fetal liver development, the expression of the Rel family member proteins, as well as other proteins known to be important for NF-κB activation. We found these proteins and active NF-κB complexes in the developing liver from at least 2 days before the onset of lethality observed in RelA knockouts. This suggests that the timing of NF-κB activation is not related to the timing of lethality. We therefore hypothesized that, in the absence of RelA, embryos were sensitized to tumor necrosis factor (TNF) receptor 1 (TNFR-1)-mediated apoptosis. Thus, we generated mice that were deficient in both RelA and TNFR-1 to determine whether apoptotic signaling through TNFR-1 was responsible for the lethal phenotype. RelA/TNFR-1 double knockout mice survived embryonic development and were born with normal livers without evidence of increased hepatocyte apoptosis. These animals became runted shortly after birth and survived an average of 10 days, dying from acute hepatitis with an extensive hepatic infiltration of immature neutrophils. We conclude that neither RelA nor TNFR-1 is required for liver development and that RelA protects the embryonic liver from TNFR-1-mediated apoptotic signals. However, the absence of both TNFR-1 signaling and RelA activity in newborn mice makes these animals susceptible to endogenous hepatic infection.     

刀豆蛋白A诱导急性肝损伤的病理学观察

目的：探讨刀豆蛋白Ａ（ＣｏｎＡ）诱导的小鼠急性肝损伤的病理机制，方法：应用ＣｏｎＡ诱导建立小鼠实验性急性肝损伤模型，并进行光镜和电镜观察。结果：小鼠早期肝损在出表现为肝细胞凋亡，结论，凋亡可能为刀豆蛋白Ａ诱导的肝细胞死亡的主要机制，ＣｏｎＡ性肝损伤为研究肝炎等发病机制和病理生理提供了一种较理想的实验动物模型。     

Hepatopoietin Cn reduces ethanol‐induced hepatoxicity via sphingosine kinase 1 and sphingosine 1‐phosphate receptors

The hepatic growth factor hepatopoietin Cn (HPPCn) prevents liver injury induced by carbon tetrachloride in rats. Sphingosine 1‐phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK). S1P and S1P receptors (S1PRs) are involved in liver fibrogenesis and oxidative injury. This work sought to understand the mechanism by which SphK/S1P/S1PRs are involved in the protective effects of HPPCn on ethanol‐induced liver injury and fibrosis. Transgenic mice with liver‐specific overexpression of HPPCn (HPPCn^liver+/+) were generated. Two ethanol feeding protocols were used to assess the protective effect of HPPCn on acute and chronic liver injury in mice. Specific inhibitors of S1PR1, S1PR2 and S1PR3 and siRNA were used to examine the roles of S1PRs in hepatic stellate cell (HSC) activation and hepatocyte apoptosis. Increased HPPCn expression in transgenic mice attenuated fibrosis induced by ethanol and carbon tetrachloride (CCl₄). Treatment with recombinant human HPPCn prevented human hepatocyte apoptosis and HSC activation. JTE‐013 or S1PR2‐siRNA attenuated the effect of HPPCn on HSC activation induced by tumour necrosis factor‐α (TNF‐α). Consistent with the effect of N,N‐dimethylsphingosine (DMS), suramin or S1PR3‐siRNA treatment blocked HPPCn‐induced Erk1/2 phosphorylation in human hepatocytes. This study demonstrated that HPPCn attenuated oxidative injury and fibrosis induced by ethanol feeding and that the SphK1/S1P/S1PRs signalling pathway contributes to the protective effect of HPPCn on hepatocyte apoptosis and HSC activation. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

REV-ERBα Agonist GSK4112 attenuates Fas-induced Acute Hepatic Damage in Mice

Fas-induced apoptosis is a central mechanism of hepatocyte damage during acute and chronic hepatic disorders. Increasing evidence suggests that circadian clock plays critical roles in the regulation of cell fates. In the present study, the potential significance of REV-ERBα, a core ingredient of circadian clock, in Fas-induced acute liver injury has been investigated. The anti-Fas antibody Jo2 was injected intraperitoneally in mice to induce acute liver injury and the REV-ERBα agonist GSK4112 was administered. The results indicated that treatment of GSK4112 decreased the level of plasma ALT and AST, attenuated the liver histological changes, and promoted the survival rate in Jo2-insulted mice. Treatment with GSK4112 also downregulated the activities of caspase-3 and caspase-8, suppressed hepatocyte apoptosis. In addition, treatment with GSK4112 decreased the level of Fas and enhanced the phosphorylation of Akt. In conclusion, treatment with GSK4112 alleviated Fas-induced apoptotic liver damage in mice, suggesting that REV-ERBα agonist might have potential value in pharmacological intervention of Fas-associated liver injury.     

c-Myc partially mediates IFNgamma-induced apoptosis in the primary hepatocyte

Interferon-gamma (IFNgamma) is a central component of the complex cytokine and inflammatory response that contributes to liver cell injury in hepatitis. We report that in the primary hepatocyte IFNgamma synergizes with the mechanistically distinct apoptotic stimuli CD95, tumour necrosis factor-alpha (TNFalpha) and UV-irradiation. For the first time in primary hepatocytes, we show that IFNgamma-mediated apoptotic signalling requires the cell surface interaction of CD95 and its ligand, and we demonstrate that IFNgamma induces soluble CD95 ligand release from hepatocyte monolayers. Utilizing c-myc phosphorothioate antisense fragments, we suppresses hepatocyte apoptosis induced by IFNgamma. In summary, we identify apoptotic pathways that contribute to IFNgamma-mediated cell death. The hepatocellular response to IFNgamma signalling can be modulated by cytokines and by the interruption of CD95 interaction with its ligand. We present evidence to suggest that c-myc contributes to IFNgamma signalling.     

Growth inhibition and apoptosis in liver myofibroblasts promoted by hepatocyte growth factor leads to resolution from liver cirrhosis

Liver cirrhosis is characterized by hepatic dysfunction with extensive accumulation of fibrous tissue in the liver. In response to chronic hepatic injury, hepatic portal myofibroblasts and activated hepatic stellate cells (HSCs) play a role in liver fibrosis. Although administration or gene expression of hepatocyte growth factor (HGF) leads to improvement in hepatic fibrosis/cirrhosis, the related mechanisms are not fully understood. We investigated mechanisms involved in resolution from liver cirrhosis by HGF, focusing on growth regulation and apoptosis in portal myofibroblasts. Cultured rat HSCs could not proliferate, were withdrawn after passage, and were replaced by proliferating portal myofibroblasts during the passages. In quiescent HSCs, c-Met receptor expression was undetected whereas c-Met receptor expression was detected in activated HSCs and liver myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA), suggesting that activated HSCs and portal myofibroblasts are targets of HGF. For cultured rat portal myofibroblasts, HGF counteracted phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and mitogenic stimulus induced by platelet-derived growth factor, induced c-jun N-terminal kinase (JNK) 1 phosphorylation, and promoted apoptotic cell death. In the dimethylnitrosamine rat model of liver cirrhosis, administration of HGF suppressed proliferation while promoting apoptosis of alpha-SMA-positive cells in the liver, events that were associated with reduced hepatic expressions of alpha-SMA and histological resolution from liver cirrhosis. Growth inhibition and enhanced apoptosis in portal myofibroblasts by HGF are newly identified mechanisms aiding resolution from liver fibrosis/cirrhosis by HGF.     

Studies of the protective effect and antioxidant mechanism of blueberry anthocyanins in a CC14-induced liver injury model in mice

This study investigated the protective effects and antioxidant mechanisms of blueberry anthocyanin extract (BBAE) in vivo and in vitro using the model of acute CC1₄-induced liver injury in mice. It was found that BBAE significantly inhibited CC1₄-induced liver injury and inhibited acute ethanol-induced increases in alanine aminotransferase, aspartate aminotransferase and malondialdehyde in a dose-dependent manner (P<0.05). The activities of superoxide dismutase, catalase and glycogen and glutathione reductase were markedly increased in the anthocyanin group (P<0.01). BBAE also significantly suppressed cellular injury and apoptotic cell death. In summary, BBAE possesses marked protective effects against CC1₄-induced liver injury, related to its ability to reduce human embryonic-liver cell (L-02) apoptosis and damage induced by oxidative stress. These findings indicate that BBAE may be of value in protecting against acute liver injury, providing pharmacological evidence supporting its clinical application.     

Depletion of hepatic glutathione prevents death receptor-dependent apoptotic and necrotic liver injury in mice

The activation of the death receptors, tumor necrosis factor-receptor-1 (TNF-R1) or CD95, is a hallmark of inflammatory or viral liver disease. In different murine in vivo models, we found that livers depleted of gamma-glutamyl-cysteinyl-glycine (GSH) by endogenous enzymatic conjugation after phorone treatment were resistant against death receptor-elicited injury as assessed by transaminase release and histopathology. In apoptotic models initiated by engagement of CD95, or by injection of TNF or lipopolysaccharide into galactosamine-sensitized mice, hepatic caspase-3-like proteases were not activated in the GSH-depleted state. Under GSH depletion, also caspase-independent, TNF-R1-mediated injury (high-dose actinomycin D or alpha-amanitin), as well as necrotic hepatotoxicity (high-dose lipopolysaccharide) were entirely blocked. In the T-cell-dependent model of concanavalin A-induced hepatotoxicity, GSH depletion resulted in a suppression of interferon-gamma release, delay of systemic TNF release, hepatic nuclear factor-kappaB activation, and an abrogation of sinusoidal endothelial cell detachment as assessed by electron microscopy. When GSH depletion was initiated 3 hours after concanavalin A injection, ie, after the peak of early pro-inflammatory cytokines, livers were still protected. We conclude that sufficient hepatic GSH levels are a prerequisite for the execution of death receptor-mediated hepatocyte demise.     

Menadione biphasically controls JNK-linked cell death in leukemia Jurkat T cells

Signals for cell-death induction by menadione were studied in Jurkat T cells. Low concentrations of menadione (10-20 microM) and H(2)O(2) (10-50 microM) induced cell death accompanying low (menadione: <5%) or moderate (H(2)O(2): 10-15%) levels of DNA fragmentation in Jurkat cells. These concentrations of menadione (10 microM) and H(2)O(2) also caused membrane (necrotic) cell death at unproportionally high (80%) and proportional (10-30%) levels, respectively. Higher concentrations (100-5,000 microM) of H(2)O(2) exclusively induced membrane cell death. Unexpectedly, 30-300 microM menadione induced ever-decreasing levels of necrotic cell death in a concentration-dependent manner. An in vitro kinase assay showed that 20-50 microM, but not >100 microM, menadione induced activation of c-Jun NH(2)-terminal kinase (JNK), whereas a striking activation of JNK was induced by 500-5,000 microM H(2)O(2). Induction of cell death by a low concentration of menadione was partially inhibited in dominant negative JNK gene-transfected Jurkat/VPF cells. A high concentration (300 microM) of menadione was found to inhibit cell-death induction by high concentrations (200-5,000 microM) of H(2)O(2). The JNK inhibitory activity of menadione was also demonstrated in a cell-free system. However, menadione did not activate JNK in vitro. These results suggest that JNK is required for induction of not only apoptotic cell death, but also necrotic cell death in Jurkat T cells and that menadione biphasically controls this JNK-linked signal for inducing cell death.     

IKKbeta is required for prevention of apoptosis mediated by cell-bound but not by circulating TNFalpha

IkappaB kinase beta (IKKbeta) is required for NF-kappaB activation and suppression of TNFalpha-mediated liver apoptosis. To investigate how IKKbeta suppresses apoptosis, we generated hepatocyte-specific Ikkbeta knockout mice, Ikkbeta(Deltahep), which exhibit little residual NF- kappaB activity but are healthy with normal liver function. Unexpectedly, Ikkbeta(Deltahep) mice are slightly more sensitive than controls to LPS-induced liver apoptosis but are highly susceptible to liver destruction following concanavalin A (ConA)-induced T cell activation. Unlike LPS, a potent inducer of circulating TNFalpha, ConA exerts cytotoxic effects through cell-bound TNFalpha, which activates type 1 and 2 TNF receptors (TNFR). While TNFR2 does not contribute to NF-kappaB activation, it is important for ConA-induced JNK activation, which is augmented by the absence of IKKbeta. Using JNK-deficient mice we show that JNK is required for ConA-induced liver damage. Thus, the antiapoptotic function of IKKbeta, which is most critical in situations that involve cell-bound TNFalpha, is mediated partially through attenuation of JNK activity.     

Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications.

Tumor necrosis factor-alpha (TNF-alpha) contributes to liver injury by inducing hepatocyte apoptosis. Recent evidence suggests that cathepsin B (cat B) contributes to TNF-alpha-induced apoptosis in vitro. The aim of the present study was to determine whether cat B contributes to TNF-alpha-induced hepatocyte apoptosis and liver injury in vivo. Cat B knockout (catB(-/-)) and wild-type (catB(+/+)) mice were first infected with the adenovirus Ad5I kappa B expressing the I kappa B superrepressor to inhibit nuclear factor-kappa B-induced survival signals and then treated with murine recombinant TNF-alpha. Massive hepatocyte apoptosis with mitochondrial release of cytochrome c and activation of caspases 9 and 3 was detected in catB(+/+) mice 2 hours after the injection of TNF-alpha. In contrast, significantly less hepatocyte apoptosis and no detectable release of cytochrome c or caspase activation occurred in the livers of catB(-/-) mice. By 4 hours after TNF-alpha injection, only 20% of the catB(+/+) mice were alive as compared to 85% of catB(-/-) mice. Pharmacological inhibition of cat B in catB(+/+) mice with L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline (CA-074 Me) also reduced TNF-alpha-induced liver damage. The present data demonstrate that a cat B-mitochondrial apoptotic pathway plays a pivotal role in TNF-alpha-induced hepatocyte apoptosis and liver injury.