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  

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

作　　者：王明亮 彭蕴茹[1] WANG Mingliang;PENG Yunru(Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Jiangsu Province Academy of Traditional Chinese Medicine,Nanjing 210028,China)

机构地区：[1]南京中医药大学附属中西医结合医院,江苏省中医药研究院,南京210028

出　　处：《实验动物科学》2021年第3期1-9,共9页Laboratory Animal Science

基　　金：国家自然科学基金资助项目(81774178,81373888)。

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。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 C57 BL/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 2^(nd),4^(th),6^(th) and 8^(th) 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 andα-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 4^(th) 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 4^(th) week(P<0.01).The histopathological result indicated that DEN treatment induced hepatic fibrosis in mice on the 4^(th) week.Western blotting analysis showed that the liver expressions of TGF-β1 andα-SMA in DEN treated mice significantly increased from the 6^(th)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

关 键 词：肝纤维化 二乙基亚硝胺 炎症 TLR4 动态模型 

分 类 号：S852.3[农业科学—基础兽医学]