Department of Surgery, Division of Pediatric Surgery, New York University School of Medicine, New York, New York.
J Surg Res. 2010 Apr;159(2):611-7. doi: 10.1016/j.jss.2009.10.038. Epub 2009 Nov 20.
Hepatic fibrosis leading to cirrhosis is the major morbidity in patients with biliary atresia (BA). This fibrosis is due to an imbalance in extracellular matrix (ECM) breakdown and deposition. We have previously demonstrated increased mRNA expression for inhibitors of ECM breakdown without increased expression for mediators of ECM deposition in our animal model of BA by d 14. However, only a mild degree of hepatic fibrosis was seen at this time. We hypothesized that expression patterns for these proteins may change once more significant fibrosis had been established, and added resuscitation to the model to improve survival. Interestingly, we found that some mice spontaneously recovered at later time points with resuscitation, and thus compared expression for inhibitors of ECM breakdown and deposition in sick and recovered mice to determine the differences.
Newborn Balb/c mice received an intraperitoneal injection 1.0 x 10(6) fluorescence forming units of rhesus rotavirus 24h after birth. Mice were monitored daily for weight gain, development of jaundice, acholic stools, and bilirubinuria. Fifty muL/g of 5% dextrose in normal saline were subcutaneously injected daily to each mouse starting on d 7 until sacrifice. Mice that survived past d 14 were sacrificed at d 21 after saline or RRV infection. Livers were then harvested post-injection d 21 for histologic and immunohistochemical analysis. RNA expression of known mediators of fibrosis was evaluated using quantitative real-time PCR. Protein expression was assessed using ELISA. Weights and normally distributed data were compared using Student's t test. Histologic findings were compared using Fisher's exact test. Comparisons of gene expression and skewed data were performed by the Mann-Whitney U test. Statistical significance was assigned to any P value less than 0.05.
Daily resuscitation resulted in a 35% (24/68) survival rate to d 21 in our model. Mice that recovered were significantly heavier than those that remained ill on d 14 (6.15 +/- 1.16 versus 4.94 +/- 0.82, P = 0.02) and 21 (7.31 +/- 1.41 versus 4.14 +/- 0.53, P < 0.001) despite the fact that there was no difference between the groups with respect to weight on d 7 (4.29 +/- 0.90 versus 3.89 +/- 0.81, P = 0.32). We found that all (10/10) animals that displayed clinical signs of biliary atresia on d 21 had moderate or severe histologic findings, while only one (1/9) of the recovered animals had liver abnormalities at sacrifice (P < 0.001 versus sick group). We also found that the sick mice had statistically significant median fold-increases of mRNA expression for TIMP-1 (31.9 versus 9.1, P = 0.041), TIMP-4 (88.1 versus 1.8, P = 0.022), and MMP-7 (51.8 versus 11.9, P = 0.006) compared with those that recovered. There was a trend toward decreased mRNA expression for PAI-1, which did not reach statistical significance (median 27.7 versus 2.19, P = 0.066). Increased protein expression for TIMP-1 and PAI-1 were also found in the sick group. The mRNA expression for the fibrillar collagens, fibronectin-1, connective tissue growth factor, snail-1, TIMP-2 and -3, and MMP-2 and MMP-9 was not different in the sick and recovered groups 21 d after RRV infection, and was not elevated from baseline gene expression.
With resuscitation added to the animal model of BA, some mice spontaneously recover while others progress to more significant hepatic fibrosis. Mice with hepatic fibrosis have a continued increase in mRNA expression of TIMP-1, TIMP-4, and MMP-7, with a trend toward increased mRNA expression of PAI-1 on d 21. Protein levels for TIMP-1 and PAI-1 were also increased in the sick mice. Recovered mice display mild to no hepatic parenchymal disease and a normal pattern of mRNA expression for the mediators of fibrosis tested. No increase in mRNA expression for the mediators of ECM deposition was found in either group. These data further support the notion that inhibition of ECM breakdown alone is sufficient to induce hepatic fibrosis. Modulation of this process may be a putative target for preventing liver injury in patients with BA.
胆道闭锁（BA）患者的肝纤维化导致肝硬化是主要的发病率。这种纤维化是由于细胞外基质（ECM）分解和沉积的失衡所致。我们之前通过动物模型研究发现，在 BA 第 14 天时，ECM 分解抑制剂的 mRNA 表达增加，而 ECM 沉积的介质表达没有增加。然而，此时只观察到轻度的肝纤维化。我们假设，一旦建立更显著的纤维化，这些蛋白的表达模式可能会发生变化，并且通过复苏模型来提高存活率。有趣的是，我们发现一些老鼠在复苏后会在后期自行恢复，因此我们比较了患病和恢复的老鼠中 ECM 分解和沉积抑制剂的表达，以确定差异。
新生 Balb/c 小鼠在出生后 24 小时内接受 1.0 x 10(6)个荧光形成单位的恒河猴轮状病毒的腹腔内注射。每天监测每只老鼠的体重增加、黄疸、白便和胆红素尿情况。从第 7 天开始，每天给每只老鼠皮下注射 5%葡萄糖生理盐水 50μL/g，直至处死。在第 14 天之后幸存的老鼠在感染后的第 21 天处死。然后在注射后第 21 天采集肝脏进行组织学和免疫组织化学分析。使用实时定量 PCR 评估已知纤维化介质的 RNA 表达。使用 ELISA 评估蛋白表达。使用 Student's t 检验比较体重和正态分布数据。使用 Fisher's 精确检验比较组织学发现。使用 Mann-Whitney U 检验比较基因表达和偏态数据。任何 P 值小于 0.05 的都被认为具有统计学意义。
在我们的模型中，每日复苏使 35%（24/68）的老鼠在第 21 天存活。在第 14 天仍患病的老鼠中，体重明显更重（6.15 +/- 1.16 与 4.94 +/- 0.82，P = 0.02）和第 21 天（7.31 +/- 1.41 与 4.14 +/- 0.53，P < 0.001），尽管两组在第 7 天的体重没有差异（4.29 +/- 0.90 与 3.89 +/- 0.81，P = 0.32）。我们发现，在第 21 天出现胆道闭锁临床症状的所有（10/10）动物都有中度或重度组织学发现，而只有一只（1/9）恢复的动物在处死时肝脏异常（P < 0.001 与患病组）。我们还发现，患病老鼠的 TIMP-1（31.9 与 9.1，P = 0.041）、TIMP-4（88.1 与 1.8，P = 0.022）和 MMP-7（51.8 与 11.9，P = 0.006）的中位折叠增加具有统计学意义。PAI-1 的 mRNA 表达呈下降趋势，但没有达到统计学意义（中位数 27.7 与 2.19，P = 0.066）。TIMP-1 和 PAI-1 的蛋白表达也在患病组中增加。RRV 感染后第 21 天，患病和恢复组的纤维胶原、纤维连接蛋白-1、结缔组织生长因子、蜗牛-1、TIMP-2 和 -3 以及 MMP-2 和 MMP-9 的 mRNA 表达没有差异，并且与基线基因表达相比没有升高。
在 BA 的动物模型中加入复苏后，一些老鼠会自行恢复，而另一些老鼠则会发展为更严重的肝纤维化。患有肝纤维化的老鼠 ECM 分解抑制剂的 mRNA 表达持续增加，MMP-7 在第 21 天有增加趋势，而 PAI-1 的 mRNA 表达呈增加趋势。患病老鼠的 TIMP-1 和 PAI-1 蛋白水平也增加。恢复的老鼠表现出轻微至无肝实质疾病和测试的纤维化介质的正常 mRNA 表达模式。在任何一组中都没有发现 ECM 沉积介质的 mRNA 表达增加。这些数据进一步支持了这样一种观点，即单独抑制 ECM 分解就足以诱导肝纤维化。调节这一过程可能是预防胆道闭锁患者肝损伤的潜在靶点。
