Erickson Nissa, Mohanty Sujit Kumar, Shivakumar Pranavkumar, Sabla Gregg, Chakraborty Ranajit, Bezerra Jorge A
Cincinnati Children's Hospital Medical Center and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229-3039, USA.
Hepatology. 2008 May;47(5):1567-77. doi: 10.1002/hep.22229.
Biliary atresia is a fibro-inflammatory cholangiopathy that obstructs the extrahepatic bile ducts in young infants. Although the pathogenesis of the disease is undefined, studies in livers from affected children and neonatal mice with experimental biliary atresia have shown increased expression of proapoptosis molecules. Therefore, we hypothesized that apoptosis is a significant mechanism of injury to duct epithelium. To test this hypothesis, we quantified apoptosis using terminal transferase dUTP nick end labeling and active caspase-3 staining in livers and extrahepatic bile ducts from Balb/c mice infected with Rhesus rotavirus (RRV) within 24 hours of birth. RRV induced a significant increase in labeled cells in the portal tracts and in epithelial and subepithelial compartments of extrahepatic bile ducts, with onset within 3 days and peaks at 5-10 days. Exploring mechanisms of injury, we found increased biliary expression of caspases 1 and 4 and of interferon-gamma (IFNgamma)-related and tumor necrosis factor-alpha (TNFalpha)-related genes. Using a cholangiocyte cell line, we found that neither IFNgamma nor TNFalpha alone affected cell viability; however, simultaneous exposure to IFNgamma and TNFalpha activated caspase-3 and decreased cell viability. Inhibition of caspase activity blocked apoptosis and restored viability to cultured cholangiocytes. In vivo, administration of the caspase inhibitor IDN-8050 decreased apoptosis in the duct epithelium and the extent of epithelial injury after RRV challenge.
The biliary epithelium undergoes early activation of apoptosis in a mouse model of biliary atresia. The synergistic role of IFNgamma and TNFalpha in activating caspase-3 in cholangiocytes and the decreased apoptosis following pharmacologic inhibition of caspases support a prominent role for apoptosis in the pathogenesis of experimental biliary atresia.
胆道闭锁是一种纤维炎性胆管病，可阻塞幼儿的肝外胆管。尽管该疾病的发病机制尚不清楚，但对患病儿童肝脏和患有实验性胆道闭锁的新生小鼠的研究表明，促凋亡分子的表达增加。因此，我们推测凋亡是胆管上皮损伤的重要机制。为了验证这一假设，我们使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法和活性半胱天冬酶-3染色，对出生后24小时内感染恒河猴轮状病毒（RRV）的Balb/c小鼠的肝脏和肝外胆管中的凋亡进行了定量分析。RRV诱导门管区以及肝外胆管上皮和上皮下区标记细胞显著增加，在3天内开始出现，5-10天达到峰值。在探究损伤机制时，我们发现半胱天冬酶1和4以及干扰素-γ（IFNγ）相关和肿瘤坏死因子-α（TNFα）相关基因的胆管表达增加。使用胆管上皮细胞系，我们发现单独的IFNγ或TNFα均不影响细胞活力；然而，同时暴露于IFNγ和TNFα会激活半胱天冬酶-3并降低细胞活力。抑制半胱天冬酶活性可阻断凋亡并恢复培养的胆管上皮细胞的活力。在体内，给予半胱天冬酶抑制剂IDN-8050可减少RRV攻击后胆管上皮细胞的凋亡以及上皮损伤程度。
在胆道闭锁的小鼠模型中，胆管上皮细胞发生早期凋亡激活。IFNγ和TNFα在激活胆管上皮细胞中的半胱天冬酶-3方面的协同作用以及药物抑制半胱天冬酶后凋亡减少，支持了凋亡在实验性胆道闭锁发病机制中的重要作用。
