EGFR/Foxo3a/Snail1通路在博来霉素肺纤维化小鼠中的作用机制探讨

Research on the mechanism of EGFR/Foxo3a/Snail1 pathway in bleomycin-induced pulmonary fibrosis in mice

目的以表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)吉非替尼对博来霉素所致小鼠肺纤维化进行干预,探讨转录因子叉头盒O3a(Foxo3a)的相关下游信号通路,阐明Foxo3a在肺纤维化中可能的作用机制。方法将30只6周龄SPF级C57BL/6小鼠(雌雄各半)随机平均分为3组:对照组(气管内滴入0.9%的氯化钠注射液)、博来霉素组(气管内滴入博来霉素3 mg/kg)和吉非替尼组(气管内滴入博来霉素3 mg/kg后以吉非替尼20 mg·kg^–1·d^–1灌胃)。14 d后收集小鼠肺组织,左肺行HE及Masson染色,取右肺以逆转录–聚合酶链反应法与免疫印迹法分别检测α-平滑肌肌动蛋白(α-SMA)、上皮钙黏素(E-cadherin)、高迁移率族蛋白B1(HMGB1)、Foxo3a、叉头盒M1(Fox M1)、Snail1 m RNA与蛋白表达水平。结果吉非替尼处理后,肺组织病理损伤及纤维化程度评分较博来霉素组明显降低(均P<0.01),与博来霉素组比较,α-SMA、Snail1(均P<0.01)、HMGB1(P<0.05)m RNA表达水平下调,E-cadherin(P<0.01)、Foxo3a、Fox M1(均P>0.05)m RNA表达水平上调;α-SMA(P<0.05)、Snail1(P<0.01)、HMGB1蛋白水平(P<0.05)以及Foxo3a蛋白磷酸化水平(P<0.01)表达下调,E-cadherin(P<0.05)、Foxo3a及Fox M1蛋白表达上调(均P>0.05)。结论Foxo3a活性增加可下调Snail1,使α-SMA表达降低、E-cadherin表达升高,从而减轻博来霉素诱导的小鼠肺纤维化。

Objective By intervening with gefitinib,an epidermal growth factor receptor tyrosine kinase inhibitor,to explore the downstream signaling pathway of the transcription factor forkhead box O3 a(Foxo3 a)in C57 BL/6 mice who are induced to pulmonary fibrosis with bleomycin,as so to illuminate the possible mechanism of Foxo3 a in epithelial-mesenchymal transition(EMT)of pulmonary fibrosis.Methods Thirty C57 BL/6 mice aged 6 weeks in half genders were randomly divided into a control group,a bleomycin group and a gefitinib group.The mice in the control group were injected with saline via trachea.The mice in the bleomycin group were injected with bleomycin at a dose of3 mg/kg via trachea.The mice in the gefitinib group were injected with bleomycin at a dose of 3 mg/kg via trachea and then gastrically perfused with gefitinib(20 mg·kg^–1·d^–1).14 days after the treatment,all mice were killed and lung tissue specimens were collected for further detection.Lung tissue sections were stained with hematoxylin eosin and Masson’s trichrome.The m RNA levels ofα-smooth muscle actin(α-SMA),E-cadherin,high mobility group protein box 1(HMGB1),Foxo3 a,Fox M1 and Snail1 in the lung tissues were detected by RT-PCR.The protein expressions ofα-SMA,E-cadherin,HMGB1,phospho-Foxo3 a(p-Foxo3 a),Foxo3 a,Fox M1 and Snail1 in the lung tissues were determined by western blot.Results The scores of lung inflammation and fibrosis were evidently decreased in the gefitinib group compared with that in the bleomycin group(P<0.01).Compared with bleomycin group,the m RNA level ofα-SMA,Snail1(P<0.01)and HMGB1(P<0.05)were declined,but m RNA level of E-cadherin(P<0.01),Foxo3 a and Fox M1(P>0.05)were ascendant in the gefitinib group.Meanwhile,western blot analysis showed reduced protein expressions ofα-SMA(P<0.05),Snail1(P<0.01),HMGB1(P<0.05)and p-Foxo3 a/Foxo3 a(P<0.01)in lung tissues,while expressions of E-cadherin(P<0.05),Foxo3 a and Fox M1 proteins(P>0.05)were increased in the gefitinib group.Conclusions Increased activity of Foxo3 a can down-regulate Snail1,which decreases the expression ofα-SMA and increases the expression of E-cadherin,thereby attenuating bleomycin-induced pulmonary fibrosis in mice.