Nan Yue-min, Wu Wen-juan, Yao Xi-xian, Wang Lei
Traditional and Western Medical Department of Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang 050051, China.
Zhonghua Gan Zang Bing Za Zhi. 2007 Jan;15(1):41-6.
To study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH).
An experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit.
In MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05).
In our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.
研究细胞凋亡以及凋亡相关基因Fas配体（FasL）、Fas、半胱天冬酶-3（caspase-3）和半胱天冬酶-8在非酒精性脂肪性肝炎（NASH）动物模型中的作用。
通过给雄性C57BL6/J小鼠喂食高脂肪、蛋氨酸-胆碱缺乏（MCD-）饮食2天、5天、10天、3周和8周，建立实验性进行性NASH模型。对照小鼠喂食补充蛋氨酸-胆碱（MCD+）的饮食。通过检查其苏木精和伊红染色的肝脏切片对肝脂肪变性、炎症和纤维化进行分级。采用TUNEL法检测肝细胞凋亡。通过定量实时逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测FasL、Fas和半胱天冬酶-8的mRNA和蛋白质表达。使用ApoAlert半胱天冬酶-3检测试剂盒进行半胱天冬酶-3活性检测。
在MCD-小鼠中，第5天观察到轻度肝脂肪变性，到第10天，发现伴有炎症浸润的轻度脂肪变性。第3周出现严重脂肪性肝炎，第8周出现纤维化。TUNEL法显示，MCD-组在第3周（15.59%±4.87%对5.17%±3.19%，P<0.05）和第8周（11.29%±3.22%对5.41%±1.54%，P<0.05）的凋亡指数高于MCD+组。与MCD+组相比，MCD-小鼠在第10天和第3周FasL的mRNA和蛋白质水平均显著升高（P<0.05和P<0.01）。MCD-组在第3周和第8周Fas mRNA表达上调（P<0.01），在第8周Fas蛋白质水平更高（P<0.01）。MCD-组在第3周和第8周mRNA水平半胱天冬酶-8表达显著增加（分别为P<0.01和P<0.05），在第8周蛋白质水平显著增加（P<0.05）。除第5天外，在所有时间点，MCD-组半胱天冬酶-3活性均比MCD+组显著增强（P<0.05）。
在我们的实验性NASH模型中经常检测到肝细胞凋亡。凋亡增加可能归因于凋亡相关基因如FasL/Fas系统的上调以及半胱天冬酶途径的激活。这些变化可能引发肝细胞凋亡以及炎症和纤维化的发展。
