桃红四物汤对肝纤维化模型大鼠肝脏组织uPA/PAI-1mRNA表达的影响

Effects of Taohongsiwu decoction on the expression of uPA/PAI-1 mRNA in the liver tissues of hepatic fibrosis model rat

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 effects of Taohongsiwu decoction on the matrix metalloproteinases and the expression of urokinase type plasminogen activator / Plasminogen activator inhibitor 1 mRNA. To explore the modern scientific connotation of Taohongsiwu decoction on treatment of liver fibrosis from the fibrinolytic system- closely related to liver fibrosis. Methods： Prepared the Taohongsiwu decoction according to traditional decocting method,then used the HPLC method to determine the content of ferulic acid and hydroxy safflower in the preparation.The subcutaneous injection of 50% CCl4（ twice a week） for 12 weeks method was used to make the hepatic fibrosis model. The model rats were randomly divided into model group,positive group（ colchicine 2mg / kg）,Taohongsiwu decoction dose groups（ 4. 25,8. 50,17. 00 g / kg）,10 rats in each group,10 normal rats were taken as normal group. Starting from the seventh week the rats were administered by dose（ 10 ml /kg weight）,normal group and model group were given equal volume of distilled water,once a day for 6 weeks. After last administration,the rats were fasted for 12 hours and sacrificed by 3% chloral hydrate,the blood was taken from the abdominal aorta and the liver tissue was removed. The serum AST,ALT activity were determined by automatic biochemical analyzer,the hydroxyproline content in liver tissue was measured by alkaline hydrolysis. The content of MMP-1 and TIMP-1 were determined by Western blotting method,and the expression of u PA /PAI-1 mRNA were detected by fluorescence quantitative PCR method. Parts of liver tissue were fixed by 10% formalin,HE staining method was used to examine the pathological changes. Results： 12 weeks after modeling,model rat serum AST,ALT activity and liver tissue Hyp content increased significantly,the content of MMP-1 and the expression of u PA significantly decreased and TIMP-1 and PAI-1 obviously increased,there were significant difference compared with normal group（ P 〈 0. 05）. After 6 weeks of treatment,Taohongsiwu decoction could increase the activity the of MMP-1 and decrease the expression of TIMP-1,the effect of Taohongsiwu high group was obviously,there were significant difference compared with model group（ P 〈 0. 05）. Groups of Taohongsiwu decoction could significantly increase the u PA activity and reduce PAI-1 mRNA expression,there were significant difference compared with model group（ P 〈 0. 05）. HE staining showed： In normal group,the hepatic Cord was ordered,the structure was clear,liver cells were radial distribution to the central vein as the center; Model group rats liver tissue structure of hepatic lobule was destroyed,liver cords were disorder,the hepatocyte showed extensive vacuolar degeneration and necrosis,liver leaflets were separated by fibrous tissue into different sizes of liver cells. In Taohongsiwu decoction groups the damage degree of hepatic tissue was decreased,and the degeneration of hepatic cells was decreased. Conclusion： Taohongsiwu decoction antifibrotic mechanism may be activated by u PA plasminogen system and increase the activity of MMPs and inhibit collagen synthesis,maintain the balance of extracellular matrix.