中药双甲五灵冲剂对免疫性肝纤维化大鼠抗肝纤维化的分子机制研究

目的探讨中药双甲五灵冲剂对免疫诱导型肝纤维化大鼠的治疗作用及机制。方法90只雌性Wistar大鼠。10只大鼠为正常对照组，80只大鼠予尾静脉注射人血白蛋白，建立免疫损伤性肝纤维化大鼠模型，分为5组：预防组、治疗1组、治疗2组、秋水仙碱组、观察组。用光镜观察肝脏HE染色、VanGieson（VG）胶原染色，采用原位杂交及免疫组织化学染色技术检测大鼠肝脏组织中TIMP-1、TIMP-2、Ⅰ型胶原、Ⅲ型胶原mRNA及蛋白的表达强度；用电镜观察肝脏超微结构的变化。结果免疫诱导型肝纤维化大鼠在造模结束后肝组织学改变呈进行性加重，以造模结束后3个月为著；TIMP-1、TIMP-2、Ⅰ型胶原、Ⅲ型胶原mRNA及蛋白呈强阳性表达，电镜见观察组有较多激活的HSC及大量胶原纤维沉积在肝窦间隙及肝细胞间，经双甲五灵冲剂治疗后的大鼠与观察组大鼠相比，肝组织结构明显好转，纤维组织减少，未见到激活的HSC、TIMP-1和TIMP-2mRNA及蛋白表达与观察组相比均明显降低，Ⅰ、Ⅲ型胶原蛋白及基因表达明显降低（P〈0．05），并且其效果为预防组效果最好，治疗1组较治疗2组效果好，三组均明显优于秋水仙碱组。结论双甲五灵冲剂可以逆转肝纤维化，抗肝纤维化机制可能是：①抑制肝星状细胞活化，减少ECM的生成及TIMP分泌；②直接抑制TIMP—1、TIMP-2mRNA及蛋白的表达；二者共同作用使得TIMP分泌减少，从而降低对MMPs的抑制，有利于MMPs对ECM的降解；③具有加速Ⅰ、Ⅲ型胶原的降解，抑制其生成及沉积的作用。

Investigation on molecular mechanism of curing liver fibrosis with Chinese medicine Shuangjiawuling in immune induced liver fibrosis rat

Objective To investigate the therapeutic effect and mechanism of Chinese traditionary medicine Shuangjiawuling using rat immune induced liver fibrosis model. Methods Rat liver fibrosis was induced by the administration of 200 g/L human serum albumin （HSA） intravenously through the tail vein. Ninety rats were divided into 6 groups. Normal control group is untreatment control. In prevention group, Chinese medicine Shuangjiawuling was taken orally at the same time with the administration of HSA. In treatment 1 group, Chinese medicine Shuangjiawuling was taken orally immediately after the administration of HSA. In treatment 2 group, Chinese medicine Shuangjiawuling was taken orally after 3 months of the administration of HSA. In eolchicine group, colchicine tablets was taken orally at the same time with the administration of HAS. In observation group, HSA was administrated without orally treatment. The rat livers were harvestd and the changes of HE, VG staining were observed with optical microscope. The expression of mRNA and proteins of Ⅰ, Ⅲ collage were determined by in situ hybridization and immunohistochemisty analysis. Moreover, the ultrastructure was observed with electron microscope. Results In the immune induced liver fibrosis rat model, the fibrosis was most serious at 3 months after the administration of HAS and the expression of Ⅰ, Ⅲ collagen was the strongest at the same time. The liver fibrosis and expression of Ⅰ, Ⅲ collagen were reduced significantly in the Shuangjiawuling treatment rats （P 〈 0.05）. In the Shuangjiawuling treatment rats, treatment 1 group was better than treatment 2 group. Shuangjiawuling treatment was superior to colehieine tablets treatment. Conclusion Animal study has confirmed that Shuangjiawuling decoction/capsule has the ability to prevent and cure liver fibrosis. The possible mechanism may be ： ① Shuangjiawuling inhibits the formation of TIMP and decreasing the secretion of ECM by inhibiting the activation of HSC ; ② Shuangjiawuling reduces the inhibi