鞣花酸经活性氧与血管再生途径对四氯化碳诱导肝硬化的保护作用研究

The protective effects of ellagic acid on carbon tetrachloride-induced cirrhosis through reactive oxygen species and angiogenesis ways

目的研究鞣花酸(EA)对四氯化碳(CCl4)诱导的肝硬化小鼠模型的保护作用,并探讨其作用机制。方法将40只小鼠随机分为对照组(n=10,予0.9%氯化钠溶液)和肝硬化模型组(n=30,由橄榄油稀释10%CCl4诱导建模),模型组又随机分为EA未干预组(n=10)、7.5 mg/kg EA组(n=10)、15 mg/kg EA组(n=10)。检测所有小鼠血清谷氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)和白蛋白(ALB)水平,苏木精-伊红(HE)染色观察肝细胞形态。采用逆转录-聚合酶链反应(RT-PCR)检测肝细胞Ⅰ型胶原蛋白m RNA水平,酶联免疫吸附试验(ELISA)检测谷胱甘肽过氧化物酶(GSH-PX)、谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA)及半胱氨酸天冬氨酸特异性蛋白酶(caspase)-3活性,蛋白印迹检测诱导型一氧化氮合酶(i NOS)、血管内皮细胞生长因子(VEGF)、VEGF受体(VEGFR)-2表达水平。结果根据肝细胞ALT、AST、ALB含量检测及HE染色细胞形态观察结果,推测EA对CCl4诱导的肝硬化小鼠具有保护作用。EA能显著抑制Ⅰ型胶原和i NOS表达,氧化应激及活性氧(ROS)形成显著降低,EA干预组VEGF、VEGFR-2表达及caspase-3活性显著提高。结论 EA通过ROS及血管再生途径对CCl4诱导的肝硬化有保护作用。

Objective To investigate the protective effect of ellagic acid （EA） on carbon tetrachloride （CCL） induced cirrhosis in mouse models, and to discuss its mechanism. Methods A total of 40 mice were randomly divided into the control group （n=10, receiving 0.9% sodium chloride solution） and the cirrhosis model group （n=30, cirrhosis modeling was induced by olive oil-diluted 10% CC14）. The cirrhosis model group was further randomly divided into EA non-intervention group （n=10）, 7.5 mg/kg EA group （n=10） and 15 mg/kg EA group （n=10）. The serum levels of ALT, AST and ALB were checked for all experimental mice. Hematoxylin eosin （HE） staining was adopted to observe the morphology of hepatocytes. Using reverse transcriptase polymerase chain reaction （RT-PCR） technique to detect the mRNA level of type I collagen of liver cells, enzyme linked immunosorbent assay （ELISA） was adopted to test the activities of the glutathione peroxidase （GSH-PX）, glutathione （GSH）, superoxide dismutase （SOD）, malonaldehyde （MDA） and caspase- 3. Western blot method was employed to measure the inducible nitric oxide synthase （iNOS）, the protein expression levels of vascular endothelial cell growth factor （VEGF） and VEGF receptor-2 （VEGFR-2）. Results According to the results of content determination of ALT, AST and ALB and the findings of HE- staining cell morphology observation, it could be presumably judged that EA had protective effects on CCl4-induced cirrhosis mice. EA could significantly inhibit the expressions of type I collagen and iNOS, the formation of ROS was strikingly decreased, and the expressions of VEGF, YEGFR- 2 and the activity of caspase-3 were remarkably improved. Conclusion Through ROS and vascular regeneration ways, EA exerts its protective effects on CCl4-induced cirrhosis in experimental mice.