柴胡对胰腺纤维化大鼠TIMP-1及TGF-β1增效作用的研究

Synergistic effects of Chaihu( Bupleuri Radix) on TIMP-1 and TGF-β1 in rats with pancreatic fibrosis

目的探讨引经药柴胡对胰腺纤维化动物模型TIMP-1及TGF-β1增效作用及部分机制。方法将50只SD大鼠随机分成模型组、假手术组、柴胡组、大黄丹参组和柴胡大黄丹参组。假手术组开腹后直接关腹,其余各组均通过结扎胰胆管联合腹腔注射雨蛙素复制胰腺纤维化模型;用药组均分别于术前、后3天用相应药物灌胃(剂量1.38 g·kg^(-1)·d^(-1)、3.04 g·kg^(-1)·d^(-1)及4.11 g·kg^(-1)·d^(-1))。术后第4天取材。采用ELISA测血清TIMP-1表达,免疫组化测胰腺TGF-β1表达,Real-time PCR和Western blot分别测胰腺TGF-β1mRNA和蛋白表达。结果与模型组比较,用药组均可降低血清TIMP-1含量(P<0.01);与柴胡组比较,柴胡大黄丹参组TIMP-1含量降低(P<0.01);与大黄丹参组比较,柴胡大黄丹参组TIMP-1含量下降(P<0.01)。与模型组比较,用药组TGF-β1 mRNA及蛋白均表达下降(P<0.01);与柴胡组比较,柴胡大黄丹参组TGF-β1 mRNA及蛋白表达下降(P<0.01),大黄丹参组TGF-β1蛋白表达下降(P<0.01);与大黄丹参组比较,柴胡大黄丹参组TGF-β1蛋白表达下降(P<0.01)。结论 "下调TGF-β1表达—抑制PSC活化—减少ECM合成"和"下调TGF-β1表达—抑制TIMP分泌—促进ECM降解"可能是柴胡、大黄丹参以及柴胡大黄丹参抗胰腺纤维化的两条作用主线。三者对TGF-β1的调控在ECM合成和降解中发挥双重作用。柴胡对大黄丹参在抗胰腺纤维化方面存在一定的增效作用,主要表现在抑制TIMP分泌和下调TGF-β1蛋白方面。

Objective To discuss the synergistic effects and mechanism of channel ushering drug Chaihu( Bupleuri Radix) on tissue inhibitor of metalloproteinases-1( TIMP-1) and transforming growth factor-β1( TGF-β1) in rats with pancreatic fibrosis. Methods 50 SD rats were randomly divided into the model group,sham operation group,Chaihu group,Dahuang( Rhei Radix et Rhizoma)-Danshen( Salviae Miltiorrhizae Radix et Rhizoma) group and ChaihuDahuang-Danshen group. In sham operation group,the rats were performed laparotomy and then the abdominal cavities was closed without any operation,while in the model group and three drug treatment groups,the rat model of pancreatic fibrosis was established by pancreatic duct ligation combined with cerulein by intraperitoneal injection. Before and after operation,three drug treatment groups were treated with Chaihu,Dahuang-Danshen and Chaihu-Dahuang-Danshen by intragastric administration at doses of 1.38 g·kg^(-1) d^(-1),3.04 g·kg^(-1) d^(-1) and 4.11 g·kg^(-1) d^(-1),respectively,with a course of 3 days. Rats were sacrificed and the samples were collected on the 4 th day after operation. The expression of serum TIMP-1 was measured by ELISA and the expression of TGF-β1 in pancreas was measured by immunohistochemistry. The mRNA and protein expression levels of TGF-β1 in pancreas were detected by Real-time PCR and Western blot,respectively. Results Compared with the model group,the level of serum TIMP-1 was decreased in three drug treatment groups( P < 0.01). Compared with Chaihu group,the level of TIMP-1 was decreased in Chaihu-Danhuang-Dansheng group( P<0.01). Compared with Dahuang-Danshen group,the level of TIMP-1 was decreased in Chaihu-Danhuang-Dansheng group( P<0.01). Compared with the model group,the mRNA and protein expressions of TGF-β1 were decreased in three drug treatment groups( P<0.01). Compared with Chaihu group,the mRNA and protein expressions of TGF-β1 were decreased in Chaihu-Dahuang-Danshen group( P<0.01),and the protein expression of TGF-β1 was decreased in Dahuang-Danshen group( P<0.01). The protein expression of TGF-β1 was decreased in Chaihu-Dahuang-Danshen group compared with Dahuang-Danshen group( P < 0.01). Conclusion "Down-regulating TGF-β1 expression-inhibiting PSC activation-reducing ECM synthesis"and"down-regulating TGF-β1 expression-inhibiting TIMP secretion-promoting ECM degradation"may be two main lines in anti-pancreatic fibrosis effects of Chaihu,Dahuang-Danshen and Chaihu-Dahuang-Danshen. The regulation effects of these three drugs on TGF-β1 play a dual role in ECM synthesis and degradation. Chaihu shows some synergism on the anti-pancreatic fibrosis effects of Dahuang-Danshen,which mainly displays in inhibiting TIMP-1 secretion and down-regulating protein expression of TGF-β1.