An integrative model of cirrhosis-associated hepatocarcinogenesis in rat: histological, biochemical and molecular approaches

The present study aimed at proposing a novel chemically-induced cirrhosis-associated rat hepatocarcinogenesis model, involving the characterization of histological, biochemical and molecular features. Male Wistar rats received a single dose of diethylnitrosamine (DEN, 200 mg/Kg body weight [b.wt.]), and were submitted to several cycles of thioacetamide (TAA, 200 mg/Kg b.wt.), during 23 weeks. Blood and liver were collected from untreated and DEN/TAA-treated groups. Liver samples were processed for global gene expression (cDNA microarray), histopathological (HE) and collagen content (picrosirius red) evaluations, immunohistochemical (Ki-67, GST-P and α-SMA), biochemical (catalase, glutathione peroxidase and glutathione-S-transferase) and gelatin zymography (MMP-2 and 9) analysis. Using a very stringent analysis (FDR<0.01 and fold change>3), gene expression array evidenced 359 differentially expressed genes upon DEN/TAA regimen. Gene Ontology and functional analyses showed several upregulated genes involved in extracellular matrix organization, mainly collagen type I α1 and 2 (Col1α1, Col1α2) and tissue inhibitor of metalloproteinase 1 and 2 (Timp1 and Timp2) genes. In addition, glutathione S-transferase, pi 1 and 2 (Gstp1 and Gstp2) genes were markedly upregulated. In contrast, functional analyses also revealed the downregulation of antioxidant response genes, as catalase, glutathione peroxidase 1 and glutathione S-transferase mu type 3 (Cat, Gpx1 and Gstm3). In agreement with gene expression data, our model presented extensive liver cirrhosis with increased α-SMA expression and collagen deposition, as well as marked development of preneoplastic GST-P positive hyperplastic lesions and some neoplasms. Besides, we observed a decrease in total glutathione peroxidase, total glutatione-S-tranferase and catalase activities. The characterization of a suitable cirrhosis-associated hepatocarcinogenesis model could provide insights into molecular characteristics of the human disease and be applied to evaluate potential preventive and therapeutic approaches.本研究旨在构建一种新型化学诱导的肝硬化相关性大鼠肝癌发生模型，并对其组织学、生化及分子特征进行全面表征。雄性Wistar大鼠单次给予二乙基亚硝胺（diethylnitrosamine, DEN，200 mg/kg体质量），随后在23周内接受多轮硫代乙酰胺（thioacetamide, TAA，200 mg/kg体质量）给药周期处理。采集未处理对照组与DEN/TAA处理组大鼠的血液及肝脏组织。对肝脏样本开展全基因组基因表达分析（cDNA微阵列，cDNA microarray）、组织病理学（HE，苏木精-伊红）染色检测、胶原含量测定（天狼星红染色，picrosirius red）、免疫组织化学（immunohistochemical）检测Ki-67、谷胱甘肽S-转移酶P（GST-P）与α-平滑肌肌动蛋白（α-SMA）的表达、生化指标检测（过氧化氢酶（catalase）、谷胱甘肽过氧化物酶（glutathione peroxidase）及谷胱甘肽S-转移酶（glutathione-S-transferase）），以及明胶酶谱法（gelatin zymography）分析基质金属蛋白酶2和9（MMP-2、MMP-9）的活性。采用严格的统计筛选标准（错误发现率FDR<0.01且倍数变化fold change>3），基因表达芯片结果显示，经DEN/TAA造模方案处理后，共有359个差异表达基因。基因本体（Gene Ontology, GO）富集与功能分析结果显示，多个上调基因参与细胞外基质（extracellular matrix）组织过程，其中主要包括I型胶原蛋白α1、α2（Col1α1、Col1α2）以及金属蛋白酶组织抑制剂1和2（Timp1、Timp2）基因。此外，谷胱甘肽S-转移酶π1、π2（Gstp1、Gstp2）基因的表达水平显著上调。与之相对，功能分析同时发现抗氧化应答（antioxidant response）相关基因的表达出现下调，包括过氧化氢酶（Cat）、谷胱甘肽过氧化物酶1（Gpx1）及谷胱甘肽S-转移酶μ3（Gstm3）。与基因表达数据一致，本模型大鼠肝脏出现广泛肝硬化病变，伴随α-SMA表达升高与胶原沉积，同时可见明显的癌前（preneoplastic）GST-P阳性增生性病变及部分肿瘤（neoplasms）组织。此外，我们检测到总谷胱甘肽过氧化物酶、总谷胱甘肽S-转移酶及过氧化氢酶的活性均有所下降。该适配性良好的肝硬化相关性肝癌发生模型的构建与表征，可为人类相关疾病的分子特征研究提供参考依据，并可用于评估潜在的预防与治疗策略。