二乙基亚硝胺诱导小鼠肝纤维化动态模型的建立及病理机制初探

摘要:目的 为 探 究 肝 纤 维 化 的 发 生 发 展 机 制 及 病 理 病 因 的 特 点, 本 研 究 通 过 建 立 二 乙 基 亚 硝 胺( diethylnitrosamine,DEN)诱导的肝纤维化动态小鼠模型,动态监测上述小鼠模型的血清生化指标,推断肝纤维化阶段。 方法 雄性 C57BL / 6 小鼠按梯度剂量每周 2 次腹腔注射 DEN,连续 8 周。 分别在第 2、4、6 和 8 周收集小鼠的血清和肝组织,通过检测相应血清生化指标、肝组织羟脯氨酸含量以及组织病理学检查监测和评估小鼠肝纤维化的发病进程。 采用 Western blot 检测 TGF-β1、α-SMA 和 TLR4 / MyD88 / NF-κB 信号通路相关蛋白以探究肝纤维化的发病机制。 结果 从第 4 周开始,与 对 照 组 相 比, DEN 诱 导 小 鼠 的 肝 功 能 相 关 血 清 生 化 指 标 ( AST、 ALT、 ALP、T-BIL、A / G 等)出现显著变化,且体质量出现明显下降。 肝脏指数出现明显下降,组织中羟脯氨酸含量均显著升高(P<0. 01) ,组织病理学结果显示 DEN 诱导组小鼠肝组织开始出现轻微胶原沉积。 Western blot 结果表明模型组小鼠肝组织中TGF-β1 和 α-SMA 表达显著上调,TLR4、MyD88、TRAF6 及细胞核 NF-κB p65 蛋白表达水平显著升高,细胞质 p65 NF-κB 表达显著下调。 结论 研究结果表明,肝纤维化的发生机制与肝损伤后激活 TLR4 / MyD88 / NF-κB 信号通路相关,而 TLR4 炎症通路的激活又进一步促进分泌 TGF-β1 并激活肝星状细胞从而导致大量细胞外基质沉积。 本研究表明,通过 DEN 诱导的小鼠肝纤维化发病机制可能与炎症相关,且可通过结合多个血清生化指标初步判断肝纤维化阶段。

To Establish a Dynamic Model of Hepatic Fibrosis Induced by Diethylnitrosamine and Explore its Pathological Mechanism in Mice

Abstract: Objective A dynamic mouse model of liver fibrosis was established induced by diethylnitrosamine ( DEN) to investigate the pathogenesis and pathologic etiology of liver fibrosis and to explore the possibility of predicting the stage of liver fibrosis by dynamically monitoring serum biochemical indexes. Method Male C57BL / 6 mice, were intraperitoneally injected with DEN twice a week at a gradient dose for 8 weeks. During the whole experiment, serum and liver tissues of mice were collected on the 2nd, 4th , 6th and 8th weeks respectively, and the progression of progressive liver fibrosis in mice was monitored and evaluated by detecting the corresponding serum biochemical indicators, the content of hydroxyproline in liver tissues, and the preparation observation. To explore the pathogenesis of hepatic fibrosis, western blotting was used to detect proteins involved in the TLR4 / MyD88 / NF-κB signaling pathway, and proteins associated with fibrosis including TGF-β1 and α-SMA. Result Compared with the control group, the serum biochemical indexes ( AST, ALT, ALP, T-BIL, A / G, etc. ) related to liver functions in DEN treated mice showed significant difference from the 4th week, and the body weight decreased after treated with DEN significantly, liver index was significantly decreased and hydroxyproline content in the liver tissues remained at a higher level from the 4th week ( P<0. 01) . The histopathological result indicated that DEN treatment induced hepatic fibrosis in mice on the 4th week. Western blotting analysis showed that the liver expressions of TGF- β1 and α-SMA in DEN treated mice significantly increased from the 6th week, compared with the control group, TLR4 / MyD88 / NF-κB signaling pathway was activated during the process of fibrosis, as shown by increased TLR4, MyD88, TRAF6 and p65 NF-κB in nuclear, accompanying with decreased p65 NF-κB in cytoplasm.Conclusion The result suggests that the mechanism of hepatic fibrosis may be related to the activation of TLR4 / MyD88 / NF-κB signaling pathway, which further promotes the secretion of TGF-β1 and activates hepatic stellate cells resulting in a large amount of extracellular matrix deposition. This study suggests that the pathogenesis of liver fibrosis in mice induced by DEN may be related to inflammation and indicates that to preliminarily judge the stage of liver fibrosis through multiple serum biochemical indexes.