补中益气汤灌胃大鼠血清对TGF-β1介导A549细胞MRP1表达影响随机平行对照研究

Effects of Serum of Rats with Intragastric Administration of Buzhong Yiqi Decoction(补中益气汤) on MRP1 Expression in A549 Cells Mediated by TGF-β1 a Randomized Parallel Controlled Study

[目的]观测补中益气汤灌胃大鼠血清对TGF-β1介导A549细胞MRP1表达影响。[方法]血清药理学法,制备灌胃大鼠血清:使用随机平行对照方法,将20只SPF级SD大鼠,按随机数字表法分为2组,10只/组,补中益气汤组以2m L生药量5.67g/kg灌胃,空白血清组等量生理盐水,1次/d,连续3d,末次灌胃1h后,腹主动脉取血,提取上清。血清干预:取人肺腺癌A549细胞,接种于培养瓶中,37℃,5%CO2培养箱中培养;细胞密度达70%,饥饿12h,空白组(A组)加空白血清;TGF-β1组(B组)加空白血清及TGF-β1;补中益气汤+TGF-β1组(C组)加补中益气汤血清及TGF-β1,培养48h。观测MRP1蛋白表达(免疫细胞化学法和Western blot法)、MRP1基因表达(Realtime PCR法)。[结果]MRP1主要在细胞膜表达,各组细胞中均可见棕黄色阳性产物表达,TGF-β1组(B组)和补中益气汤+TGF-β1组(C组)棕黄色颗粒阳性表达量均高于空白组(P<0.05);补中益气汤+TGF-β1组(C组)低于TGF-β1组(B组)(P<0.05);Western blot结果与组化结果一致。Realtime PCR结果为TGF-β1组(B组)和补中益气汤+TGF-β1组(C组)基因表达水平均高于空白组(P<0.05);补中益气汤+TGF-β1组(C组)低于TGF-β1组(B组)(P<0.05)。[结论]补中益气汤血清干预TGF-β1诱导A549细胞EMT过程,细胞中MRP1在蛋白和基因水平均降低。

[Objective] To observe the effect of Buzhong Yiqi Decoction serum on the expression of MRP1 in epithelial mesenchymal transformation process induced by TGF-β 1 in the human lung cancer cell A549. [Methods] Serum pharmacological method was used to prepare the serum of rats in gastric lavage： using randomized controlled methods, 20 male SPF SD rats were randomly divided into 2 groups, 10 rats in each group, Buzhong Yiqi Decoction group with 2mL crude drug 5.67 g/kg gavage, blank serum group, normal saline, 1 time/d, 3d, continuous, final gavage after lh, blood from the abdominal aorta, extraction supernatant. Serum intervention： The human lung carcinoma A549 cells were seeded in culture flasks, 37 ℃, 5%CO2incubator; when the cells grow to 70% density, hunger 12h, blank group （A group） with blank serum medium; TGF- β 1 group （B group） with blank serum medium and TGF- β 1; Buzhong Yiqi Decoction＋TGF-β1 group （C group） with Buzhong Yiqi Decoction and TGF-β 1, 48h. MRP1 protein expression （immunocytochemistry and Western blot） and MRP1 gene expression （Realtime PCR） were observed. [Results] MRP1 is mainly expressed in the cell membrane, the expression of these cells were visible brown positive product of TGF- β 1 group （group B） and Buzhong Yiqi Decoction＋TGF- β 1group （group C） brown granules of positive expression were higher than the blank group （P〈0.05）; Buzhong Yiqi Decoction＋TGF- β1 group （C group） is lower than TGF-β 1 group （B group） （P〈0.05）; The results of Western blot were consistent with the immunoeytochemistry results.The results of Realtime PCR showed that the levels of gene expression in TGF- β 1 group （group B） and Buzhong Yiqi Decoction＋TGF- β lgroup （C group） were higher than those in the blank group （P〈0.05）; Buzhong Yiqi Decoction＋TGF-β1 group（C group） was lower than TGF- β 1 group （group B） （P〈0.05）. [Conclusion] Buzhong Yiqi Decoction containing serum can inhibit the protein expression of MRP1 mediated epithelial mesenchymal transition in A549 cells.