蛋氨酸-胆碱缺乏饮食诱导大鼠脂肪性肝纤维化肝细胞再生障碍的机制

Mechanism of hepatocyte regeneration inhibition in rats with fiver fibrosis induced by fipogenic methionine-choline-deficient diet

目的获取通过改变饮食习惯可逆转脂肪性肝纤维化的证据；明确非酒精性脂肪性肝纤维化肝细胞增殖障碍的发生机制。方法（1）蛋氨酸-胆碱缺乏饮食（MCDD）喂养10周诱导非酒精性脂肪性肝纤维化大鼠模型，恢复组于第9周开始将MCDD转变为蛋氨酸-胆碱对照饮食（MCCD）喂养2周。（2）纤维化和炎症程度采用肝组织病理染色方法评价，肝星状细胞、Kupffer细胞活化采用免疫组化、蛋白质印迹或实时PCR方法评价。（3）肝细胞凋亡和增殖分别采用原位缺口末端标记法（TUNEL）染色法和BrdU染色法评价，肝细胞再生相关细胞因子白细胞介素（IL）-6、抗信号转导与转录激活因子3（STAT3）、多克隆抗体抗c-JunN端激酶1（JNK-1）、c-Jun、多克隆抗体抗CCAAT／增强子结合蛋白α（C／EBPα）、p21、多克隆抗体抗肝细胞核因子6（HNF6）、肝细胞生长因子（HGF）-d表达分别采用实时PCR及蛋白质印迹方法评价。结果（1）MCDD转变为MCCD后组织病理学显示肝细胞脂肪沉积及炎症反应明显减轻。（2）伴随着活化的肝星状细胞和Kupffer细胞减少，肝纤维化程度明显减轻。（3）模型组肝细胞凋亡数量显著高于正常组和恢复组（68±16比40±8、48±6，均P〈0．05）；肝细胞增殖／凋亡比值显著低于正常组和恢复组（0．10±0．03比0．19±0．03、0．41±0．09，均P〈0．01）；恢复组增殖数量显著高于模型组（17．2±4．4比7．5±3．0，P〈0．01）。（4）模型组IL-6、JNK-1、c-Jun、C／EBPα、p21、HNF6mRNA表达均显著低于正常组（0．34±0．18、0．41±0．11、0．19±0．03、1．94±0．64、0．34±0．20、0．47±0．21比1．33±0．44、0．83±0．26、1．53±1．20、4．51±1．15、1．30±0．75、0．92±0．16，P〈0．05或P〈0．01）；且模型组上述指标及STAT3蛋白表达亦明显低于正常组（P〈0．05或P〈0．01）；恢复组仅IL-6、JNK-1和p21mRNA和蛋白表达显著高于模型组（P〈0．05或P〈0．01）。结论饮食结构调整对于脂肪性肝纤维化的组织学改善具有重要的治疗意义；MCDD脂肪性肝纤维化肝细胞再生抑制与Gn至S期进入障碍有关，促进IL-6、JNK-1和p21的表达可能对于脂肪性肝纤维化恢复具有重要的治疗意义。

Objective To obtain the evidence of fibrotic resolution in fatty liver by changing the diet and to clarify the mechanism of hepatoeyte proliferation inhibition in rat with fatty liver fibrosis. Methods （1） Nonalcoholic steatohepatitis with advanced fibrosis was induced in rats by giving them a methionine-choline-deficient diet （MCDD） for 10 weeks （group M）. A methionine-choline-control diet （MCCD） instead of MCDD was given for the last 2 weeks to the experimental group（group R）. （2） Fibrosis and inflammation were determined by tissue staining. The activation of hepatic stellate cells and Kupffer cells were determined by immunostaining, immunoblot or quantitative RT-PCR respectively. （3） Hepatocytic apoptosis and proliferation were determined by TUNEL and BrdU staining respectively. Expressions of IL-6, STAT3, JNK-1, e-Jun, p21, C/EBPct, HNF6 and HGF-alpha were evaluated by quantitative RT-PCR and immanoblot to clarify the mechanism of hepatocytic proliferation inhibition. Results （ 1 ） Changing the diet from MCDD to MCCD triggered the reduction of fat in hepatocytes and a decrease in inflammatory response. （2） The regression of fibrosis was accompanied by the disappearance of activated stellate cells and macrophages. （3） Compared with control group （group C ）, hepatocytic apoptotic number increased significantly in group M （68 ± 16 vs 40 ± 8 ,P 〈 0.05 ） and the ratio of hepatocytic proliferation/apoptosis decreased markedly in group M （ 0. 10 ± 0. 03 vs 0. 19 ± 0. 03, P 〈 0. 01 ） ; compared to group M, hepatocytic apoptotic number decreased significantly in group R （ 48 ± 6, P 〈 0. 05 ） and hepatocytic proliferation number and the ratio of hepatocytic proliferation/apoptosis increased markedly in group R （ 17.2 ±4. 4 vs 7.5 ±3.0, 0. dl ±0. 09 vs 0. 10 ±0. 03 respectively, P 〈0. 01 ）. （4） Compared with group C, the mRNA level of IL-6, JNK-1, c-Jun, C/EBPα, p21 and HNF6 mRNA decreased significantly （0. 34 ± 0. 18 vs 1.33 ±0.44, 0.41 ±0. 11 vs 0.83 ±0.26, 0. 19 ±0.03 vs 1.53 ±1.2, 1.94 ±0.64 vs 4.51 ±1.15, 0.54±0.20 vs 1.30±0.75, 0.47 ±0.21 vs 0.92 ±0. 16 respectively, P 〈0.05 or P 〈0.01）, and protein level of IL-6, STA33, JNK-1, c-Jun, C/EBPα, P21 and HNF6 also decreased significantly in liver fibrotic stage （ P 〈 0. 05 or P 〈 0. 01 ） while only IL-6, JNK-1 and p21 recovered immediately after a changed diet from MCDD to MCCD （P 〈 0. 05 or P 〈 0. 01 ）. Conclusion Food intake is a very important factor for controlling the fatty status and pathology of liver. Hepatocytic proliferation inhibition is associated with the arrested G0-S phasic transition in fatty liver fibrosis and the up-regulated expression of IL-6, JNK-1 and p21. These factors play a very important role in the recovery of fatty liver fibrosis.