Gene expression profile in the liver of BALB/c mice infected with Fasciola hepatica

Background: Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with Fasciola hepatica metacercariae using a microarray-based methodology. Methodology: A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). Results: We found that Fasciola hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. Conclusion: The present study provides us insights at the molecular level about the underlying mechanisms used by Fasciola hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis. We used three experimental groups each containing 3 mice. Group 1 remains untreated and served as control. Group 2 was infected with Fasciola hepatica metacercariae on day 0 and humanely necropsied at 7 days post-infection. Group 3 was infected with Fasciola hepatica metacercariae on day 0 and humanely necropsied at 21 days post- infection. At the time of necropsy, liver of each mice were removed and the RNA was isolated. We compared the gene expression profile in the liver of mice infected with Fasciola hepatica.

**背景**：肝片形吸虫（Fasciola hepatica）感染仍是被忽视的热带病（Neglected Tropical Diseases, NTDs）之一。该寄生虫具有广泛的全球分布范围，主要感染牛类，偶尔也会侵袭人类。现有研究已报道了蠕虫感染及肝片形吸虫感染诱导的免疫应答，但针对其靶器官——肝脏——的基因表达谱却鲜有研究；肝脏是成虫定植并长期寄生的部位，也是该寄生虫引发特征性病理损伤的核心场所。本研究采用基于微阵列的实验方法，对感染肝片形吸虫囊蚴的小鼠肝脏的早期与晚期基因表达谱进行了分析。

**方法学**：本实验共使用9只6周龄雌性BALB/c小鼠（Charles River Laboratories，西班牙巴塞罗那），体重区间为20~35g。将小鼠分为三组：两组经口感染7枚肝片形吸虫囊蚴，另一组不予任何处理作为空白对照。分别在感染当日（t0）、感染后7天（t7）及感染后21天（t21）对小鼠实施安乐死并进行剖检，采集肝脏样本，依次开展肝脏损伤组织学评估、RNA提取、微阵列设计及基因表达分析。

**结果**：本研究发现，肝片形吸虫感染可在感染早期诱导小鼠肝脏内128个基因出现差异表达，感染晚期则有308个基因发生差异表达，且绝大多数差异基因呈现上调趋势。Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）结果显示，与代谢、生物合成及信号转导相关的通路，以及参与诱导肝毒性、肝损伤及肝细胞死亡的基因均发生了显著变化。

**结论**：本研究从分子层面揭示了肝片形吸虫诱导肝脏损伤及其后续病理生理过程的潜在机制。本研究得到的基因表达模式，还可用于解释肝片形吸虫感染与胆管癌之间缺乏关联的现象，但该假说仍需更多后续研究加以验证。

本研究设置三个实验组，每组各包含3只小鼠：第1组不予任何处理，作为空白对照；第2组于第0日经口感染肝片形吸虫囊蚴，并于感染后7天实施安乐死剖检；第3组于第0日经口感染肝片形吸虫囊蚴，并于感染后21天实施安乐死剖检。剖检时采集每只小鼠的肝脏组织并提取RNA，最终对比感染肝片形吸虫的小鼠肝脏的基因表达谱。