FXR激动剂奥贝胆酸抑制吉非替尼所致的肺纤维化机制的研究

Inhibition of gefitinib-induced pulmonary fibrosis by FXR agonist olbecholic acid

目的 探讨FXR激动剂奥贝胆酸抑制吉非替尼诱导的肺纤维化的机制,为其临床应用提供理论依据。方法 细胞组实验选择人肺成纤维细胞HFL1细胞作为研究对象。分为3组:对照组、吉非替尼组、奥贝胆酸+吉非替尼组。将吉非替尼2.5μmol/L、奥贝胆酸5 mg/L各3 ml分组与HFL1细胞共培养,采用ELISA方法对转化生长因子β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)及Smad2/3、Smad7蛋白的表达进行检测。动物组实验选用健康清洁级SD雄性大鼠60只,随机分为4组:对照组、博来霉素组、吉非替尼+博来霉素组、吉非替尼+博来霉素+奥贝胆酸组。采用ELISA方法对1、3、7、14、21 d大鼠血浆中TGF-β1、α-SMA及Smad2/3、Smad7蛋白的表达进行检测。采用Western blot法对21 d大鼠肺组织中TGF-β1、α-SMA及Smad2/3、Smad7蛋白的表达进行检测。HE染色观察1、3、7、14、21 d大鼠肺组织病理变化。结果 细胞组实验中吉非替尼组及奥贝胆酸+吉非替尼组均随着时间延长,TGF-β1、α-SMA及Smad2/3蛋白的表达逐渐升高,各时点均明显高于对照组,Smad7蛋白的表达逐渐下降,各时点均明显低于对照组,差异有统计学意义(P<0.01)。奥贝胆酸+吉非替尼组TGF-β1、α-SMA及Smad2/3蛋白的表达各时点均明显低于吉非替尼组,但仍高于对照组,Smad7蛋白的表达显著高于吉非替尼组,但仍低于对照组,差异有统计学意义(P<0.01)。动物组实验中博来霉素组、吉非替尼+博来霉素组、吉非替尼+博来霉素+奥贝胆酸组均随着时间延长,TGF-β1、α-SMA及Smad2/3蛋白的表达逐渐升高,各时点均明显高于对照组,Smad7蛋白的表达逐渐下降,各时点均明显低于对照组,差异有统计学意义(P<0.01)。吉非替尼+博来霉素+奥贝胆酸组TGF-β1、α-SMA及Smad2/3蛋白的表达各时点均明显低于吉非替尼+博来霉素组及博来霉素组,但仍高于对照组,Smad7蛋白的表达显著高于吉非替尼+博来霉素组及博来霉素组,但仍低于对照组,差异有统计学意义(P<0.01)。结论 奥贝胆酸通过调控TGF-β1、α-SMA及Smad2/3、Smad7蛋白的表达来抑制吉非替尼诱导的肺纤维化。

Objective To investigate the possible mechanism of farnesoid X receptor(FXR)agonist olbecholic acid inhibiting gefitinib-induced pulmonary fibrosis and provide a theoretical basis for its clinical application.Methods Human lung fibroblast HFL1 cells were selected as the research object in the cell group,divided into blank control group,gefitinib group,olbecholic acid+gefitinib group.The gefitinib 2.5μmol/L,olbecholic acid 5 mg/L each 3 ml group was co-cultured with HFL1 cells,and the expression of transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),Smad3 and Smad2 protein was detected by ELISA.In the animal experiment,60 healthy and clean SD rats were randomly divided into 4 groups:saline control group,bleomycin group,gefitinib+bleomycin group,gefitinib+bleomycin+obecholic acid group.The expression of TGF-β1,α-SMA,Smad2/3 and Smad7 protein in lung tissues of rats at 1,3,7,14 and 21 days was detected by ELISA method.The expression of TGF-β1,α-SMA,Smad2/3 and Smad7 protein in lung tissue of 21-day rats was detected by Western blot.HE staining was used to observe the pathological changes of lung tissue in rats at 1,3,7,14 and 21 d.Results In the cell group,the expression of TGF-β1,α-SMA and Smad2/3 protein increased gradually with gefitinib group and olfinic acid+gefitinib group,and the time was significantly higher.In the blank group,the expression of Smad7 protein gradually decreased,and the time points were significantly lower than the blank group.The difference was statistically significant(P<0.01).The expressions of TGF-β1,α-SMA and Smad2/3 in the oleic acid+gefitinib group were significantly lower than those in the gefitinib group,but still higher than the blank group.The expression of Smad7 protein was significantly higher than that in the gefitinib group.The fentanyl group was still lower than the blank group,and the difference was statistically significant(P<0.01).In the animal group,the bleomycin group,gefitinib+bleomycin group,gefitinib+bleomycin+oleic acid group were prolonged with time,TGF-β1,α-SMA and Smad2 The expression of Smad2/3 protein was gradually increased,and the expression of Smad7 protein was gradually decreased at each time point,and the expression of Smad7 protein was gradually lower than that of the blank group.The difference was statistically significant(P<0.01).The expressions of TGF-β1,α-SMA and Smad2/3 protein in gefitinib+bleomycin+oleic acid group were significantly lower than those in gefitinib+bleomycin group,but still higher than that in blank group,the expression of Smad7 protein was significantly higher than that of gefitinib+bleomycin group,but it was still lower than the blank group,and the difference was statistically significant(P<0.01).Conclusion Oribolic acid inhibits gefitinib-induced pulmonary fibrosis by regulating the expression of TGF-β1,α-SMA and Smad2/3,Smad7 proteins.