丹芍化纤胶囊通过上调ALK2、p-Smad1/5/8改善四氯化碳所致大鼠肝纤维化

Danshaohuaxian capsule attenuates hepatic fibrosis induced by CCl_4 in rats by up-regulating expression of ALK2 and p-Smad1/5/8

目的观察丹芍化纤胶囊(DSHX)对四氯化碳(CCl4)所致肝纤维化大鼠肝脏骨形态发生蛋白Ⅰ型受体(BMPR-Ⅰ)中的激活素受体样激酶2(ALK2)、磷酸化Smad1/5/8(p-Smad1/5/8)与DNA结合(分化)抑制因子2(Id2)表达的影响,探讨该药治疗肝纤维化的可能机制。方法雄性Wistar大鼠50只分为对照组(A组)、肝纤维化模型组(B组)、自然恢复组(C组)、DSHX低剂量治疗组(D组)、DSHX高剂量治疗组(E),每组10只。除A组外,其余各组用CCl4复合因素复制大鼠肝纤维化模型,共8周。D、E组分别用0.5、1.0g/kg DSHX灌胃8周,1次/日。实验结束后分别采用酶联免疫吸附试验测定大鼠肝匀浆透明质酸(HA)、羟脯氨酸(Hyp)水平,Masson染色法观察肝组织内胶原纤维沉积程度,实时荧光定量聚合酶链式反应(RT-PCR)检测肝组织ALK2、Id2mRNA水平,蛋白质印迹法(Western blotting)分析肝组织ALK2、Id2及p-Smad1/5/8蛋白的表达。结果 B组肝匀浆HA、Hyp水平高于A组(P<0.01),肝组织ALK2、Id2mRNA和蛋白的表达及p-Smad1/5/8蛋白表达均低于A组(P<0.01);D、E组大鼠肝纤维化程度较B组和C组明显改善,肝匀浆HA、Hyp水平均低于B组和C组(P<0.01),肝组织ALK2mRNA和蛋白及p-Smad1/5/8蛋白表达均高于B组和C组(P<0.01),肝组织Id2mRNA的表达高于B组和C组(P<0.01),但Id2蛋白表达与B组、C组比较,差异均无统计学意义(P>0.05)。结论 DSHX对大鼠肝纤维化的治疗作用机制可能与上调ALK2、p-Smad1/5/8的表达有关。

Objective To investigate the effect of Danshaohuaxian capsule（DSHX） on expressions of bone morphogenetic protein receptor I （BMPR- I ,ALK2） ,phosphorylated Smadl/5/8 （p-Smad1/5/8） and inhibitor of DNA binding/differentiation 2 （Id2） in rat liver of hepatic fibrosis induced by carbon tetrachloride（CC14 ）,and to explore its possible mechanism for treating liver fibrosis. Methods A total of 50 male Wistar rats were divided into control group（A） ,CCL-induce hepatic fibrosis group（13）, untreated model group（C）, low-dose-DSHX treated group（D） ,high-dose-DSHX treated group（E）, 10 rats in each group. Fibrous liver models of rats were induced by subcutaneous injection of CC14 and high-lipid/low-protein diet for 8 weeks except for control group. Then the two DSHX treated groups were treated respectively with low dose（0.5 g/kg） and high dose（1.0 g/kg） DSHX capsule once per day for 8 weeks. By the end of the experiment,the level of hyaluronic acid（HA） and hydroxyproline（Hyp） in the liver homogenate were determined by using enzyme-linked immunosorbent assay, the mRNA expressions of ALK2 and Id2 were determined by using RT-PCR, the protein expressions of ALK2, Id2 and p-Smad 1/5/8 were determined by using Western blotting, respectively. Results Compared with group A,the concentration of HA and Hyp increased in group B（P〈0.01） ,the mRNA and protein expression of ALK2 and Id2, the protein expression of p-Smad 1/5/8 decreased in group B（P〈0.01）. Compared with groups B and C, the degree of hepatic fibrosis was significantly improved, the level of HA and H yp decreased, and the mRNA and protein expression of ALK2,the protein expression of p-Smad 1/5/8, the mRNA expression of Id2 were significantly higher in the two DSHXtreated groups respectively（P〈0.01）. However, there was no significant difference in the expression of Id2 protein between the two DSHX-treated groups and groups B and C（P〉0.05）. Conclusion The therapeutic mechanism of DSHX for hepatic fibrosis in rats may be associated with up-regulation of the expression of ALK2 and p-Smad 1/5/8.