补阳还五汤大鼠含药血清对肺成纤维细胞TGF-β1及其基因表达的影响

Effect of Rats Serum Containing Buyang Huanwu Decoction on Expression of TGF-β1 and Gene Expression in Lung Fibroblasts

目的：观察补阳还五汤大鼠含药血清对博莱霉素诱导的肺纤维化大鼠肺成纤维细胞中TGF-β1及其基因表达的影响。方法：含药血清的制备：24只SPF级SD大鼠,雄性,随机分为3组,即补阳还五汤水煎液组（简称补阳还五汤组,1 g生药/m L）,强的松水溶液组（简称阳性组,5 mg/m L）和生理盐水组（简称模型组）,每组8只,补阳还五汤组以9.24 g/（kg·d）（临床等效剂量）进行灌胃,阳性组及模型组以1 m L/100 g灌服相应溶液,1次/d,连续灌服7 d,采血前禁食12 h,于末次灌胃1 h后,腹主动脉取血,离心后分离血清,将收集到的补阳还五汤含药血清配制成含药血清浓度分别为5%,10%,20%的培养液备用;体外培养的肺成纤维细胞,分别加入含5%,10%,20%浓度的补阳还五汤药物血清干预,72 h后收集细胞及上清,采用酶联免疫吸附法（ELISA）检测上清中TGF-βl的表达,采用实时荧光定量PCR（Real-Time PCR）检测细胞中的TGF-βl蛋白基因的表达。结果：与模型组比较,阳性组和20%含药血清组,10%含药血清组肺纤维化大鼠成纤维细胞中TGF-β1的表达均有显著减少（P〈0.01）,5%含药血清组细胞中TGF-β1的表达有减少趋势,3组不同浓度的补阳还五汤含药血清作用效果表现出一定的量效关系;与模型组比较,阳性组和20%含药血清组肺纤维化大鼠成纤维细胞中TGF-β1蛋白基因的表达均有显著减少（P〈0.01）;10%含药血清组TGF-β1蛋白基因的表达减少（P〈0.05）,5%含药血清组TGF-β1蛋白基因的表达有减少趋势,三组不同浓度的补阳还五汤含药血清作用效果表现出一定的量效关系。结论：补阳还五汤含药血清能够有效抑制TGF-βl及TGF-βl蛋白基因的表达,这可能是其防治肺纤维化作用的机制之一。

Objective： To investigate the possible mechanism of rats serum contained Buyang Huanwu Decoction（ BYHWD） on the expression of transforming growth factor-β1（ transforming growth factor-β1,TGF-β1） and gene expression on bleomycin induced lung fibroblast. Method： Twenty-four rats were divided into 3 groups in random： the BYHWD group,the positive group and the model group,with 8 rats in each group. The BYHWD group was fed with BYHWD at dosage of 9. 24 g/（ kg· d）（ clinically equivalent dose）. The positive group and the model group were fed with corresponding solution 1 m L/100 g once a day,continuously for 7 d. After fasting blood for 12 h,the blood sample was taken out and 1 h after the last fed,abdominal aortic blood was taken out and then the serum was obtained after centrifugation.The collected serum containing BYHWD was formulated to the serum containing drugs which concentrations were 5%,10% and 20%. Vitro cultured lung fibroblasts were added with 5%,10% and 20% concentrations of serum containing BYHWD. The cells and supernatants were collected after 72 h,using ELISA to detect the expression of TGF-βl in the supernatants and using Real-Time PCR method to detect the expression of TGF-βl gene expression in cells. Result：Compared with the model group,the expression of TGF-β1 in the pulmonary fibrosis fibroblasts was significantly reduced（ P 0. 01） in the positive group,20% serum containing group and 10% serum containing group and the 5% serum containing group＇s cells expression of TGF-β1 had a decreasing trend. The three different concentrations of serum containing BYHWD showed some effects of dose-effect relationship. The expression of TGF-β1 gene expression in the pulmonary fibrosis fibroblasts was significantly reduced（ P 0. 01） in the positive group and 20% serum containing group and the expression of TGF-β1 gene expression in the pulmonary fibrosis fibroblasts was reduced（ P 0. 05） in the 10%serum containing group. The 5% serum containing group＇s cells TGF-β1 gene expression had a decreasing trend and three different concentrations of serum containing BYHWD showed some effects of dose-effect relationship. Conclusion：BYHWD can effectively inhibit the overexpressing of the TGF-β1 and its gene expression,which may be the mechanism of anti-pulmonary fibrosis.