大黄(庶虫)虫超微粉剂对肝纤维化大鼠肝组织蛋白表达的影响

目的:利用荧光差异凝胶电泳技术(2D DIGE)研究大黄蜜虫超微粉剂对大鼠肝纤维化组织蛋白表达的影响.方法:采用超微粉碎技术对大黄(蜜)虫丸原药材进行剂型改进,用猪血清复制大鼠免疫性肝纤维化模型.实验动物分为正常组、模型组、给药组.正常组ip等渗生理盐水0.5 mL,其余组ip猪血清0.5 mL,每周2次,连续12周.药物组于造模之日起ig给予大黄蜜虫超微粉水溶液(剂量0.27 g·kg-1),正常组与模型组ig等容积生理盐水,每天1次,连续12周.第12周末处死大鼠,取血与肝组织.检测指标包括肝组织病理学变化:HE染色与Masson染色观察肝纤维化程度；血清纤维化指标:透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)；血清肝功能指标:丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST).同时利用2D DIGE和质谱分析技术比较模型组与给药组大鼠肝组织蛋白表达量的差异,用免疫印迹法及免疫组化法对差异蛋白进行验证.结果:模型组肝组织假小叶形成,肝组织胶原纤维含量与血清ALT,AST及HA,LN,PCⅢ,Ⅳ-C指标显著高于正常组(P＜0.01).给药组肝脏无假小叶形成,肝组织胶原纤维含量与血清ALT,AST及HA,LN,PCⅢ,Ⅳ-C指标显著低于模型组(P＜0.01).2D DIGE结果显示有58个表达量差异大于2倍的蛋白质点,初步鉴定了12个,并验证了Regucalcin,ERp57,CAⅢ蛋白在各组大鼠肝组织的表达,与2D DIGE结果一致.结论:大黄蜜虫超微粉剂能显著降低大鼠肝组织纤维化程度、降低血清纤维化及肝功能指标,提示其对实验性免疫性肝纤维化有较好防治作用；它能调节大鼠肝纤维化组织Regucalcin,ERp57,CAⅢ蛋白表达量,这些蛋白可能通过细胞钙稳态失调、过氧化应激、炎症反应、免疫反应、纤维组织癌变等机制参与肝纤维化的病理过程.

Effects and Mechanisms of Dahuang Zhechong Recipe on Immunity Hepatic Fibrosis in Rats

Objective:Dahuang Zhechong recipe(DHZC) is a traditional Chinese formula for treatment of cirrhosis.The present study was conducted to investigate the effect of ultramicro-powder of DHZC on rats liver fibrosis and its possible mechanisms by Two-dimensional differential in-gel electrophoresis(2D DIGE)based proteomics analysis.Method: SD rats were randomly divided into 3 groups: normal group,model group and DHZC-treated group.Except in normol group,liver fibrosis in rat was produced in each group.The rats were injected intraperitoneally with 0.5 mL sterile porcine serum twice a week for 12 weeks.In addition,rats of normal group treated with physiological saline in the same way.At the same day,aqueous solution of ultramicro-powder of DHZC was given to the DHZC-treated group(0.27 g · kg-1,ig) for 12 weeks.Rats of three groups were given equal volume of physiological saline.The rats were killed after 12 weeks of porcine serum treatment.Blood samples of all groups were drawn from the abdominal aorta.Livers were stained with HE and Masson to observe the histopathological chang.The lever of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),hyaluronic acid(HA),laminin(LN),procollagen type III(PC-III) and collagen type IV(IV-C) were measured.The difference gel electrophorisis(2-D DIGE) combined MALDI-TOF MS/MS technique was used to analyze differentially expressed proteins of rat livers from model group and DHZC-treated group.Result: Rats of model group showed significantly increased serum ALT,AST,HA,LN,PCⅢ and IV-C levels compared with rats of normal group(P<0.01).The histopathological analysis suggested that the severe liver fibrosis happened in model group compared with normal group.Administration with ultramicro-powder of DHZC obviously alleviated the degree of liver fibrosis based on HE stained and Masson stain tissue sections.Fifty-eight spots representing significant changes in protein expression were detected utilizing DIGE.Twelve of these proteins were send to identify by MALDI-TOF MS/MS.Two proteins were confirmed by Western blot and immunohistochemistry.The contents of ALT,AST,HA,LN,PCⅢ and IV-C in model rats were increased significantly,while DHZC could decrease those significantly.The histopathological analysis suggested that DHZC obviously alleviated the degree of liver brosis induced by porcine serum.Conclusion:DHZC can effectively prevent hepatic fibrosis and improve liver function in rats.The regulation on the expressions of some proteins may be one of the pathways for DHZC to exert its anti-hepatic fibrosis effect.