己酮可可碱对小鼠酒精性肝病酒精代谢酶和核受体PPAR-α的影响

Effects of pentoxifylline on ethanol metabolic enzymes and peroxisome proliferator-activated receptor alpha in mice with alcoholic liver disease

目的研究己酮可可碱(PTX)对酒精性肝病小鼠酒精代谢酶和过氧化物酶增殖物激活受体(PPAR-α)的影响。方法将64只C57BL/6小鼠随机分为模型组、治疗组和对照组,用50%酒精灌胃建立急性肝损伤模型,以20%酒精连续灌胃6周建立慢性酒精性肝病模型;采用比色法检测各组小鼠血清乙醇脱氢酶(ADH)和细胞色素P4502E1(CYP2E1)活性;采用RT-PCR法检测肝组织ADH、CYP2E1和PPAR-αmRNA水平;采用免疫组化法检测肝组织CYP2E1和PPAR-α蛋白表达。结果急性和慢性酒精性肝损伤模型小鼠血清ADH活性分别为(11.2±1.6)U/ml和(5.8±1.4)U/ml,与相应对照组比无显著性差异[分别为(12.5±1.2)U/ml和(4.3±0.6)U/ml];急性和慢性酒精性肝损伤小鼠CYP2E1活性分别为(12.2±1.8)U/ml和(11.8±1.7)U/ml,均显著高于对照组[(7.9±1.4)U/ml和(6.5±1.2)U/ml,P<0.01)]和治疗组[(8.1±1.5)U/ml和(7.8±1.5)U/ml,P<0.01];急性和慢性酒精性肝损伤小鼠肝组织CYP2E1阳性细胞相对表达强度为(765±21)和(682±25),均显著高于对照组[分别为(308±12)和(305±18),P<0.01)]和大剂量PTX治疗组[分别为(521±18)和(418±12),P<0.01];急性和慢性酒精性肝损伤小鼠肝组织ADH mRNA水平与对照组比无显著性差异,但肝组织CYP2E1 mRNA相对水平分别为(1.47±0.32)和(1.13±0.52),显著高于对照组[(0.89±0.23)和(0.45±0.28),P<0.01)]及大剂量PTX治疗组[分别为(0.92±0.27)和(0.48±0.32),P<0.01)];急性酒精性肝病动物肝组织PPAR-αmRNA水平与对照组或PTX治疗组比无统计学差异,但慢性酒精性肝损伤小鼠肝组织PPAR-αmRNA相对水平[(0.45±0.31)]显著低于对照组[(0.85±0.21),(P<0.05)];急性和慢性酒精性肝损伤小鼠肝组织PPAR-α阳性相对表达强度为(322±15)和(262±23),均显著低于对照组[分别为(721±18)和(689±14),(P<0.01)]和大剂量PTX治疗组[分别为(548±20)和(725±19),P<0.01)]。结论 PTX能够减轻急慢性酒精性肝损伤,可能与其上调酒精代谢酶CYP2E1和下调PPAR-α表达有关,而与ADH无关。

Objective To investigate the effect of pentoxifylline （PTX） on ethanol metabolic enzymes and peroxisome proliferator-activated receptor alpha （PPAR-α） in C57BL/6 mice with alcoholic liver disease. Meth-ods Sixty -four mice were randomly divided into alcoholic liver disease model, PTX intervention and control group; Acute alcoholic liver injury was induced in mice by intragastric administration with 50% alcohol,and chronic alcoholic liver injury was induced by intragastric administration with 20% alcohol daily for six weeks;Serum alcohol dehydrogenase （ADH） and cytochrome P4502E1 （CYP2E1） activity was measured by colorimetric method;The mRNA levels of ADH,CYP2E1 and PPAR-α were measured by PT-PCR;The protein expression of CYP2E1 and PPAR-α was determined by immunohistochemistry. Results The serum activity of ADH did not differ among mice with acute [（11.2±1.6）U/ml] or chronic [（5.8±1.4） U/ml] alcoholic liver injury and controls [（12.5±1.2）U/ml and（4.3±0.6）U/ml,respectively];In mice with acute and chronic liver injury,the CYP2E1 activ-ity was（12.2±1.8）U/ml and（11.8±1.7） U/ml,respectively,significantly higher than those in normal controls[（7.9±1.4） U/ml and （6.5±1.2） U/ml,P〈0.01）] and those in PTX-intervented mice [（8.1±1.5） U/ml and （7.8±1.5）U/ml, P〈0.01];The relative CYP2E1 positive cells in liver tissues in mice with acute and chronic alcoholic liver injury were （765±21） and （682±25）,respectively,significantly higher than those in normal controls [（308±12） and （305±18）,P〈0.01）] and in mice with high-dosage of PTX intervention [（521±18） and （418±12）,respectively,P〈0.01];The mRNA levels of ADH in liver tissues of mice with acute and chronic alcoholic liver injury were simi-lar to that in the controls, however, the relative CYP2E1 mRNA levels[（1.47±0.32） and （1.13±0.52）] were sig-nificantly higher than those in normal controls [（0.89±0.23） and （0.45±0.28）,respectively,P〈0.01）] and in mice with high-dose of PTX[（0.92±0.27） and（0.48±0.32）,respectively,P〈0.01）];PPAR-α mRNA levels in liver tissues did not differ among mice with acute liver injury and normal controls,however,it was significantly lower than that in normal controls （0.85±0.21） in liver of mice with chronic alcoholic liver injury [（0.45±0.31）,P〈0.05];The PPAR-α positive cells in mice of acute and chronic alcohol injury were（322±15） and（262±23）,respectively,sig-nificantly higher than those in normal controls [（721±18）and （689±14）,P〈0.01] and in mice with high-dose of PTX intervention[（548±20） and （725±19）,P〈0.01]. Conclusion PTX attenuates acute or chronic alcoholic liver injury,probably by through up-regulation of CYP2E1 and down-regulation of PPAR-α.