强肝软坚丸对肝纤维化大鼠肝组织中MMP、TIMP表达的影响

Effect of Qianggan Ruanjian Pill on Expressions of MMP and TIMP in Rat Liver Fibrosis

目的:观察强肝软坚丸对肝纤维化的干预和治疗作用,探讨其作用机理。方法:120只SPF级雄性SD大鼠按照随机数字表法将其分为正常对照组、模型组、秋水仙碱治疗组、秋水仙碱干预组、强肝软坚丸治疗组、强肝软坚丸干预组6组。除正常对照组外,各组均采用未灭活猪血清腹腔注射造模,每只每次0.5 m L,每周两次,正常对照组给予同剂量生理盐水腹腔注射。各干预组造模同时每日1次灌胃给药:秋水仙碱溶液0.1 g·kg-1(秋水仙碱质量浓度为0.01 g·m L^(-1))、强肝软坚丸溶液2 g·kg-1(药物质量浓度为0.2 g·m L^(-1)),正常组、模型组和各治疗组每天同剂量灭菌水灌胃。于造模第5周末各组均随机抽取5%大鼠断头处死,肝组织病理提示造模成功,第6周初开始分别给予各治疗组药物灌胃,方法和剂量同各干预组。于造模10周结束后禁食12 h,各组均随机取10只大鼠断头处死,采集肝组织标本,通过常规HE染色和Masson染色观察各组大鼠肝组织病理学变化并进行Masson染色肝纤维化半定量分析;通过免疫组化染色,用图像分析法检测各组肝组织基质金属蛋白酶(matrix metalloproteinase,MMP)-1、MMP-2和MMP抑制因子(tissue inhibitor of matrix metalloproteinase,TIMP)-1、TIMP-2的表达。结果:与模型组比较,秋水仙碱组、强肝软坚丸组MMP-1表达均升高,差异有统计学意义(P<0.05);提示强肝软坚丸可提高肝纤维化大鼠肝脏组织MMP-1的表达。与模型组比较,秋水仙碱干预组、强肝软坚丸治疗组和强肝软坚丸干预组MMP-2表达均升高,差异有统计学意义(P<0.05)。与模型组比较,秋水仙碱组、强肝软坚丸组TIMP-1表达均下降,差异有统计学意义(P<0.05);提示强肝软坚丸可降低肝纤维化大鼠肝脏组织TIMP-1的表达。与正常对照组比较,其余各组TIMP-2表达明显升高,差异有统计学意义(P<0.05);与模型组比较,秋水仙碱组、强肝软坚丸组TIMP-2表达均下降,差异有统计学意义(P<0.05)。结论:强肝软坚丸不仅可改善免疫损伤性肝纤维化大鼠一般情况和肝纤维化程度,也可使免疫损伤性肝纤维化大鼠肝组织中MMP-1、MMP-2的表达升高,TIMP-1、TIMP-2的表达降低。

Objective：To research Qianggan Ruanjian Pill for intervention and treatment of liver fibrosis, and to explore its action mechanism. Methods： 120 SD male rats were randomly divided into 6 groups ：normal control group,model control group,Colchicine intervention group, Colchicine treatment group, Qianggan Ruanjian Pill intervention group, Qianggan Ruanjian Pill treatment group. Except the normal control group, each group used inactivated porcine serum intraperitoneal injection to manufacturing models, two times a week, each time each one 0.5mL, 10 weeks. Normal control group with the same method, the same dose, with intra- peritoneal injection of normal saline. The intervention group was intragastricing administration at the same time intraperitoneally in- jecting, one time a day, with colchicine solution ：0.1 g/Kg （ colchicine concentration 0.01 g · mL -1 ）, Qianggan Ruanjian Pill solu- tion 2 g · kg- 1 （ drug concentration 0.2 g · mL- 1 ）. The normal group, the model group and the treatment group with the same dose of sterile water gavage every day. At the end of the fifth week, each group will select 5% rats randomly and kill them by decapita- tion method, then liver pathology will prove that we make model success. At the beginning of sixth week, each treatment group will be given intragastric administration of drug,and the same dose in each intervention group. Each group selected ten rats randomly and decapitated at the end of the tenth week. The pathological changes of liver tissues in rats were observed by routine HE staining and Masson staining and semi quantitative analysis was performed by Masson staining;liver tissue matrix metalloproteinase MMP- 1 and MMP-2 in each group and its inhibitor TIMP-1 and TIMP-2 were detected by image analysis, using immunohistochemical staining. Results ： Compared with normal control group, increased Qianggan Ruanjian pill group the content of MMP-1, the differ- ence was statistically significant （ P 〈 0.05 ） ; control group and model group, colchicine, Qianggan Ruanjian pill group the expres- sion of MMP-1 was increased, the difference was statistically significant （P 〈 0.05 ） ;prompt Qianggan Ruanjian pill can improve the expression of liver fibrosis of rat liver MMP-1. Compared with the model group, colchicine group, Ruanjian pill treatment group and Qianggan Ruanjian pill liver MMP-2 expression in the intervention group were increased, the difference was statistically signifi- cant （ P 〈 0.05 ） ; Qianggan Ruanjian pill group the expression of TIMP-1 was decreased, the difference was statistically significant （ P 〈 0.05 ） ; prompt Qianggan Ruanjian pill can decrease the expression of hepatic fibrosis of rat liver TIMP-1 ; compared with nor- mal control group, Qianggan Ruanjian pill group TIMP-2 expression was significantly increased, the difference was statistically sig- nificant （P 〈 0.05 ） ; and compared to the model group, Qianggan Ruanjian pill treatment group and The expression of TIMP-2 in the intervention group decreased, the difference was statistically significant （ P 〈 0.05 ）. Conclusion ： Qianggan Ruanjian Pill can not only improve the immune injury of liver fibrosis in rats general situation and the degree of liver fibrosis. Also can make the MMP-1 rat liver fibrosis,increased the expression of MMP-2 TIMP-1 decreased the expression of TIMP-2.