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目的:观察桃红四物汤对肝纤维化模型大鼠肝脏组织基质金属蛋白酶(MMPs)以及尿激酶型纤溶酶原激活物/纤溶酶原激活物抑制因子1(u PA/PAI-1)mRNA表达的影响,从纤溶系统-肝纤维化密切关联角度探讨该方药治疗肝纤维化的现代科学内涵。方法:按传统水煎法制备桃红四物汤制剂,采用HPLC法测定制剂中阿魏酸和羟基红花黄素含量以控制质量。采用皮下注射50%CCl4连续12周(每周2次)的方法制备肝纤维化模型大鼠,将模型动物随机分成模型组、秋水仙碱组(2mg/kg)、桃红四物汤4.25、8.50、17.00g/kg剂量组,每组10只,另取10只正常大鼠为正常组。从造模第7周开始,各组动物按剂量灌胃给药,10ml/kg体重,正常组和模型组给予等容量蒸馏水,每天1次,连续给药6周。末次给药后,禁食不禁水12h,3%水合氯醛麻醉处死动物,腹主动脉取血,分离血清,采用全自动生化分析仪测定AST、ALT活性;摘取部分肝脏,采用碱水解法测定羟脯氨酸(Hyp)含量;采用Westblot法测定MMP-1、TIMP-1的表达;采用荧光定量PCR法检测u PA、PAI-1mRNA的表达。另取部分肝脏组织,10%的福尔马林固定,HE染色进行病理学检查。结果:造模12周后,模型大鼠血清AST、ALT活性及肝脏组织Hyp含量显著增加,肝脏组织MMP-1、u PA表达显著降低、TIMP-1和PAI-1活性明显增加;连续给药6周后,桃红四物汤8.5、17g/kg剂量组能明显降低AST、ALT活性及肝脏组织Hyp含量;桃红四物汤各组均能增加MMP-1活性、降低TIMP-1的表达,其中桃红四物汤17g/kg剂量组作用明显;桃红四物汤各组均能明显增加u PA的活性,显著降低PAI-1mRNA的表达。HE染色显示:正常组大鼠肝组织肝索排列规则有序,结构清晰,肝细胞以中央静脉为中心呈放射状分布;模型组大鼠肝脏组织肝小叶结构破坏,肝索排列紊乱,肝细胞广泛性空泡样变性、坏死,汇管区扩大、并见大量纤维组织增生,将肝小叶分隔成大小不等的肝细胞团;桃红四物汤各组肝小叶结构破坏程度降低,肝细胞脂肪空泡变性减少。结论:桃红四物汤抗肝纤维化作用可能是通过激活u PA纤溶酶系统,增加MMPs活性,抑制胶原异常增生,维持细胞外基质平衡。
Objective: To observe the effect of Taohong Siwu decoction on the expression of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator / plasminogen activator inhibitor 1 (u PA / PAI-1) in hepatic fibrosis model rats, mRNA expression of fibrinolytic system from the perspective of the close association of liver fibrosis to explore the treatment of liver fibrosis of the modern scientific connotation. Methods: According to the traditional decoction method, the preparation of Taohong Siwu Decoction was prepared. The content of ferulic acid and hydroxy-canthaxanthin in preparation was determined by HPLC to control the quality. The model rats were randomly divided into model group, colchicine group (2mg / kg), Taohongsiwutang 4.25 and 8.50, which were injected subcutaneously with 50% CCl4 for 12 weeks (twice a week) , 17.00g / kg dose group, each group of 10, another 10 normal rats for the normal group. From the beginning of the 7th week of model establishment, each group of animals was dosed by gavage with 10ml / kg body weight. The normal group and the model group were given distilled water of equal volume once a day for 6 weeks. After the last administration, fasting can not help but water 12h, 3% chloral hydrate anesthesia killed animals, abdominal aorta blood, serum was separated, using automatic biochemical analyzer AST, ALT activity; part of the liver was removed by alkaline hydrolysis The content of hydroxyproline (Hyp) was measured. The expressions of MMP-1 and TIMP-1 were detected by Westblot method. The expressions of u PA and PAI-1 mRNA were detected by real-time quantitative PCR. Another part of the liver tissue, 10% formalin fixed, HE staining for pathological examination. Results: After 12 weeks of modeling, serum AST, ALT activity and Hyp content in liver tissue of model rats were significantly increased, the expression of MMP-1 and u PA in liver tissue was significantly decreased, and the activities of TIMP-1 and PAI-1 were significantly increased. After 6 weeks, the dosage of 8.5,17g / kg of Taohong Siwu Decoction could significantly reduce the activity of AST and ALT and the content of Hyp in liver tissue. Each group of Taohong Siwu Decoction could increase the activity of MMP-1 and decrease the expression of TIMP-1 The effect of Taohong Siwu Decoction at 17g / kg dose group was obvious. Each group of Taohong Siwu Decoction could obviously increase the activity of u PA and significantly reduce the expression of PAI-1 mRNA. Hematoxylin-eosin staining showed that the arrangement of hepatic cords in the normal group was orderly and the structure was clear. The hepatocytes distributed radially around the central vein. The structure of the hepatic lobules in the model group was disorganized, the hepatic cord was disordered and the hepatocytes were extensive Vacuolar degeneration, necrosis and portal area enlargement, and see a large number of fibrous tissue hyperplasia, the hepatic lobule is divided into groups of hepatocytes with varying sizes; the lesser degree of destruction of hepatic lobule structure in each group of Taohong Siwu Decoction, Degeneration reduced. Conclusion: The anti-hepatic fibrosis effect of Taohong Siwu decoction may be through the activation of u PA plasmin system, increasing the activity of MMPs, inhibiting collagen abnormal proliferation and maintaining the balance of extracellular matrix.