The Role of TNF in Hepatotoxicity

肿瘤坏死因子α(TNFpha)是内毒素休克的主要介质。肿瘤坏死因子诱导的损伤机制之一是通过激活核因子-kB和产生活性氧。我们推测，2，3，7，8-四氯二苯并二恶英(TCDD)诱导的内毒素超敏反应和随后的凋亡诱导可能是通过调节TNFpha信号通路而发生的。使用内毒素超敏的体内模型来研究肿瘤坏死因子α信号与细胞凋亡的关系的研究正在进行中。我们通过评估B6C3F1小鼠在存在或不存在内毒素的情况下暴露于TCDD的关键时间点的血清酶水平、量化凋亡细胞和测量基因表达的变化来表征TCDD诱导的肝脏损伤的动力学。TCDD调节内毒素介导的Fas早期表达，上调caspase3(第10天)、NFkappaB(第14天)和TNFpha(第10天和第14天)的表达。此外，TCDD/内毒素联合应用改变了TCDD诱导的肝毒性动力学，血清峰值提前4天。在本研究条件下，TCDD对TNFR1和TNFR2基因表达、IkappaBalpha和IkappaBβ蛋白表达以及NFkappaB DNA结合活性似乎没有调节作用。以前的研究表明，在内毒素暴露之前用TCDD治疗啮齿动物会显著增加毒性，并且在这个模型中，放线菌亚胺抑制蛋白质合成阻断了TCDD诱导的对肿瘤坏死因子的敏感性。已有研究表明，caspase的过表达可诱导细胞凋亡，而caspase的激活似乎是形成细胞凋亡表型所必需的。放线菌酮的保护作用及对肿瘤坏死因子α和NFkappaB基因表达的影响提示，TCDD可能通过TNFα/TNFR2/TRAF通路改变肿瘤坏死因子诱导的NFkappaB的激活。Fas和Caspase 3基因表达的改变表明细胞凋亡的启动，提示参与了Fas配体/Fas/Caspase途径。为了探讨TCDD改变NFkappaB表达的机制，我们目前正在研究TCDD是否在较低剂量的内毒素作用下改变IkappaB/NFkappaB复合体的降解。此外，我们试图通过分离和定量处理和未处理的动物中的活性和前活性形式的蛋白水解酶来阐明Caspase1和3的作用。

Tumor necrosis factor alpha (TNFalpha) has been demonstrated to be a primary mediator of endotoxin shock. One mechanism of TNF-induced injury is via the activation of NF-kB and the generation of reactive oxygen species. We have hypothesized that 2,3,7,8-tetrachlorodibenzodioxin (TCDD) -induced endotoxin hypersensitivity and subsequent induction of apoptosis may occur through modulation of TNFalpha signaling pathways. Studies using in vivo models of endotoxin hypersensitivity to examine the relationship between TNFalpha signaling and apoptosis are ongoing. We have characterized the kinetics of TCDD-induced hepatic damage in the liver by evaluating serum enzyme levels, quantifying apoptotic cells, and measuring alterations in gene expression at critical time points in B6C3F1 mice exposed to TCDD in the presence or absence of endotoxin. TCDD modulated endotoxin-mediated early expression of Fas, and upregulated expression of caspase 3 (day 10), NFkappaB (day 14), and TNFalpha (days 10 and 14). In addition, combined TCDD/endotoxin altered the kinetics of TCDD-induced hepatoxicity, with peak serum levels occurring 4 days earlier. TNFR1 and TNFR2 gene expression, IkappaBalpha and IkappaBbeta protein expression, and NFkappaB DNA-binding activity did not appear to be modulated by TCDD under the conditions of the study. Previous studies had shown that when rodents are treated with TCDD prior to endotoxin exposure a significant increase in toxicity occurs, and that inhibition of protein synthesis with cycloheximide blocks the TCDD-induced sensitivity to TNF in this model. Overexpression of caspases has been shown to induce apoptosis, and activation of the caspases appears to be necessary for development of the apoptotic phenotype. The protective effects of cycloheximide and alterations in TNFalpha, and NFkappaB gene expression suggest that TCDD treatment may result in alterations in TNF-induced activation of NFkappaB via the TNFalpha/TNFR2/TRAF pathway. The alterations in Fas and Caspase 3 gene expression indicate initiation of apoptosis and suggest involvement of the Fas Ligand/Fas/Caspase pathway.To investigate the mechanism by which TCDD alters NFkappaB expression we are currently examining whether TCDD alters the degradation of the IkappaB/NFkappaB complex at lower doses of endotoxin. In addition, we are attempting to clarify the role of Caspases 1 and 3 by separating and quantifying active and pro-active forms of the protease in treated and untreated animals.