血管紧张素转换酶2在四氯化碳诱导的大鼠肝纤维化中的表达及其相关性

Expression and correlation of angiotensin-converting enzyme 2 in CClcinduced rat Hver fibrosis

目的运用CCl4肝纤维化模型研究血管紧张素转换酶（ACE）2在肝纤维化形成中的表达及其与纤维化的关系。方法将64只雄陛SD大鼠随机分为2组。模型组按3ml／kg皮下注射40％CCl4，对照组按3ml／kg皮下注射橄榄油，每周2次。分别在注射前（0周）及注射后第2、4、6周取肝组织及血清。实时荧光定量PCR检测ACE2和ACE的mRNA相对表达量，wescemblot检测ACE2蛋白相对表达量，酶联免疫吸附法检测血清中ACE2浓度。两组间差异比较用f检验，多组样本的比较用单因素方差分析或KnmkalWallis检验，等级相关分析用Spmmmn等级相关检验。结果造模0、2、4、6周组的ACE2mRNA相对表达量分别为0．991±0．079、3．055±1．034、3．545±1．947、6．448±1．835，差异有统计学意义（H=23．224，P〈0．01）；ACE2mRNA的表达在造模2、4、6周逐步升高，且均明显高于对照组（t值分别为-5．760、-3．578和-8．453，P值均〈0．01）。造模0、2、4、5周组肝组织ACEmRNA相对表达量分别为1．005±0．193、3．956±0．934、4．974±2．004、6。265±2．718，差异有统计学意义（F=12．982，P〈0．01）lACEmRNA的表达在造模2、4、6周逐步升高，且均明显高于对照组（f值分别为-8．790、-5．544和-5．431，P值均〈0．01）。ACE2蛋白在正常组低表达，随着造模时间的延长，其表达逐渐升高，造模0、2、4、5周组的ACE2蛋白相对表达量分别为0．034、0．097、0．355、0．512，造模第5周表达最高。ACE2mRNA的表达与肝纤维化等级评分及ALT、AST变化具有良好的相关陛（r值分别为0．850、0．669和0．815，P值均〈0．01），外周血中游离ACE2的含量与肝纤维化等级评分具有良好相关陛（r=0．730，P〈0．01）。结论ACE2参与肝纤维化的形成，在肝纤维化形成过程中的表达明显E调，且其表达量与肝纤维化程度明显相关。

Objective To investigate the expression and pathogenic relevance of angiotensin- converting enzyme 2 （ACE2） in liver fibrosis by using the rat model of CC14-induced liver fibrosis. Methods The liver fibrosis model was generated by delivering subcutaneous injections of CCI4 （dissolved in olive oil at a 2：3 ratio; injection dose： 3 ml/kg） every three days for six weeks into male Sprague-Dawley rats. Another group of rats that received simultaneous injections of olive oil alone （3 ml/kg） were used as controls. At week 0, 2, 4, or 6 after the first injection, a subset of rats from each group was sacrificed to obtain liver tissues and serum samples. Pathological analyses were carded out to detect the presence and extent of liver cell degeneration, necrosis, inflammatory cell infiltration, and collagen deposition. ACE2 and ACE gene and protein expressions were measured by real-time PCR and Western blotting, respectively. The significance of differential expression between groups and time points was assessed by t-test and one-way ANOVA or IOrnskal-Wallis tests, and correlation with fibrosis was assessed by Spearman＇s rank correlation coefficient. Results CCI4 administration led to significantly up-regulated ACE2 mRNA levels at week 2 （3.055 ± 1.034）, 4 （3.545 ± 1.947）, and 6 （6.448 ± 1.836） （vs. conlrols; H= 23.224, P〈0.001）. Similarly, hepatic ACE mRNA was significantly increased after the CC14 injections （week 2：3.055 ± 1.034, week 4：3.545 ± 1.947, week 6： 6.448± 1.836; vs. controls： F= 12.982, P〈0.001）. There was a significant correlation between the ACE and ACE2 gale expression levels （r=0.750,P〈0.001）. Protein levels of ACE2 also showed an increasing trend following CC14 administration （week 0： 0.034, week 2： 0.097, week 4： 0.356, week 6： 0.512）. The hepatic ACE2 gene expression strongly correlated with levels of alanine aminotransferase （r=0.669, P〈0.0001） and aspartate aminoUansferase （r=0.815, P〈0.0001）, and with the Ishak fibrosis score （r=0.850, P〈0.001）. Finally, there was a significant correlation between circulating ACE2 and the Ishak fibrosis score （r=0.730, P〈 0.001）. Conelnsion A significant relationship exists between ACE2 gene expression and extent of liver fibrosis. ACE2 may play a crucial role in liver fibrogenesis.