肝纤维化过程中calpain2与caspase-3表达变化及意义

Changes of calpain 2,caspase-3 expression and their effects on liver fibrosis in rats

目的探讨大鼠肝纤维化过程中钙激活中性蛋白酶2(calpain 2)与半胱氨酸天冬氨酸酶3(caspase-3)表达变化及其在肝纤维化发生发展中作用。方法雄性Wistar大鼠40只,随机分为对照4、8周组和肝纤维化模型4、8周组,每组各10只;肝纤维化组按0.3 mL/100g皮下注射40%CCl4植物油溶液,每隔3 d注射1次,对照组给予等量植物油;测定肝脏指数、血清谷丙转氨酶(ALT)及谷草转氨酶(AST)含量,苏木素伊红(HE)染色观察肝组织病理学改变;免疫组织化学法及western blot检测肝组织中calpain 2与活化的caspase-3蛋白表达含量;聚合酶链反应检测肝组织中calpain 2 mRNA表达。结果肝纤维化4、8周组大鼠血清ALT、AST分别为(727.00±249.70)、(1 260.00±579.25)和(616.00±209.98)、(1 167.00±318.18)U/L,均高于对照组(P<0.01);肝纤维化组大鼠肝组织发生明显的纤维化改变,其中以8周组更为明显;与对照组比较,肝纤维化8周组大鼠肝组织中calpain 2蛋白(23.1±3.77)和mRNA(167.93±50.39)表达均升高(P<0.01),而在肝纤维化4、8周组活性caspase-3蛋白表达[(18.76±3.54)、(25.46±4.77)]均增加(P<0.01)。结论 calpain 2与caspase-3在蛋白及基因水平表达变化,可能参与了肝纤维化的发生发展过程。

Objective To observe the changes of calpain 2, caspase-3 expression in liver fibrosis and to explore their effects on liver fibrosis in rats. Methods Forty male Wistar rats were randomly divided into four groups （4 weeks nor mal control group,8 weeks normal control group,4 weeks liver fibrosis model group and 8 weeks liver fibrosis model group, 10 in each group）. The model groups were treated with hypodermic injection of 40% carbon tetrachloride （CCI4 ） every 3 day for 4 weeks and 8 weeks, respectively, at the dosage of 0. 3 ml/100 g body weight. The liver index, blood ala nine aminotransfersase （ALT）and aspartate amiontransferase （AST）were analyzed. The liver fibrosis was observed with microscope. Additionally,the expression of calpain 2 and activated caspase-3 protein were determined by immunohisto chemistry and western blot;mRNA expression of calpaln 2 was determined with real-time PCR. Results The level of ALT and AST in 4 weeks model group （727. 00±249. 70 and 1 260. 00 ±579. 25 U/L） and in 8 weeks model group （616. 00±209.98 and 1167. 00 ±318.18 U/L） were obviously higher than those of normal control groups（P 〈0. 01 ）. The protein（23.1 ± 3.77 ）and mRNA （ 167.93 ± 50. 39）expression of calpain 2 in liver tissue of 8 weeks fibrosis model group were obviously higher than those in normal age-matched control group （ P 〈 0. 01 ）. Compared with the control groups, the protein of activated caspase-3 （ 18.76 ± 3.54 and 25.46 ± 4. 77 ） in 4 weeks and 8 weeks liver fibrosis model group also elevated（P 〈 0. 01 ）. Conclusion In the process of liver fibrosis induced by CC14, the expression of calpain 2 and caspase-3 increase obviously, suggesting that calpain 2 and caspase-3 may play important roles in liver fibrosis.