Research on the mechanism of EGFR/Foxo3a/Snail1 pathway in bleomycin-induced pulmonary fibrosis in mice_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

YANG Weiyuan ^1,2 , LI Li ² , ZHENG Linxin ² ,  LI Weifeng ²

1. Guangzhou University of Traditional Chinese Medicine, Guangzhou, Guangdong 510405, P. R. China;
2. General Hospital of Guangzhou Theatre Command, Guangzhou, Guangdong 510010, P. R. China;

Corresponding?author：

LI Weifeng, Email: lwf980622@126.com

Keywords：

Forkhead box O3a; Pulmonary fibrosis; Epithelial mesenchymal transition; Bleomycin; Gefitinib

DOI：

10.7507/1671-6205.201904028

Video：

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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 O3a (Foxo3a) in C57BL/6 mice who are induced to pulmonary fibrosis with bleomycin, as so to illuminate the possible mechanism of Foxo3a in epithelial-mesenchymal transition (EMT) of pulmonary fibrosis.Methods Thirty C57BL/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 of 3 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 mRNA levels of α-smooth muscle actin (α-SMA), E-cadherin, high mobility group protein box 1 (HMGB1), Foxo3a, FoxM1 and Snail1 in the lung tissues were detected by RT-PCR. The protein expressions of α-SMA, E-cadherin, HMGB1, phospho-Foxo3a (p-Foxo3a), Foxo3a, FoxM1 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 mRNA level of α-SMA, Snail1 (P<0.01) and HMGB1 (P<0.05) were declined, but mRNA level of E-cadherin (P<0.01), Foxo3a and FoxM1 (P>0.05) were ascendant in the gefitinib group. Meanwhile, western blot analysis showed reduced protein expressions of α-SMA (P<0.05), Snail1(P<0.01), HMGB1 (P<0.05) and p-Foxo3a/Foxo3a (P<0.01) in lung tissues, while expressions of E-cadherin (P<0.05), Foxo3a and FoxM1 proteins (P>0.05) were increased in the gefitinib group.Conclusions Increased activity of Foxo3a can down-regulate Snail1, which decreases the expression of α-SMA and increases the expression of E-cadherin, thereby attenuating bleomycin-induced pulmonary fibrosis in mice.

Citation： YANG Weiyuan, LI Li, ZHENG Linxin, LI Weifeng. Research on the mechanism of EGFR/Foxo3a/Snail1 pathway in bleomycin-induced pulmonary fibrosis in mice. Chinese Journal of Respiratory and Critical Care Medicine, 2020, 19(4): 371-378. doi: 10.7507/1671-6205.201904028 Copy

1.	葛均波, 徐永健, 王辰, 主編. 內科學. 第 9 版. 北京: 人民衛生出版社, 2018, 87.
2.	Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A, 2006, 103(35): 13180-13185.
3.	Margetts PJ, Bonniaud P, Liu L, et al. Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol, 2005, 16: 425-436.
4.	Fang L, Wang H, Zhou L, et al. Akt-FoxO3a signaling axis dysregulation in human oral squamous cell carcinoma and potent efficacy of FoxO3a-targeted gene therapy. Oral Oncol, 2011, 47(1): 16-21.
5.	蔣宗勝. 基于轉錄因子 FOXM1 調控下的抗非小細胞肺癌上皮間質轉化的機制研究[D]. 2018, 福建醫科大學. 48-49.
6.	Ishii Y, Fujimoto S, Fukuda T. Gefitinib prevents bleomycin-induced lung fibrosis in mice. Am J Resp Crit Care Med, 2006, 174: 550-556.
7.	Le Cras TD, Korfhagen TR, Davidson C, et al. Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Pathol, 2010, 176(2): 679-686.
8.	Wang XT, McCullough KD, TF Franke, et al. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem, 2000, 275(19): 14624-14631.
9.	Gomes AR, Zhao F, Lam EWF. Role and regulation of the forkhead transcription factors FOXO3a and FOXM1 in carcinogenesis and drug resistance. Chin J Cancer, 2013, 32(7): 365-370.
10.	李偉峰, 李理, 袁偉鋒, 等. 吉非替尼抑制博萊霉素誘導的小鼠肺纖維化. 中國病理生理雜志, 2010, 26(8): 1565-1569.
11.	李理, 李偉峰, 蔡琳, 等. 吉非替尼對博萊霉素致肺纖維化小鼠 α 平滑肌肌動蛋白的抑制作用. 南方醫科大學學報, 2010, 30(12): 2675-2678.
12.	彭婷, 杜海堅, 李理, 等. 吉非替尼通過增加 Foxo3a 活性抑制肺纖維化小鼠上皮–間質轉分化. 中國病理生理雜志, 2014, 30(3): 444-448.
13.	熊俊. 腎間質纖維化中的上皮–間充質轉型及 Snail1 的作用研究[D]. 2011, 第二軍醫大學. 12.
14.	Mikawa K, Nishina K, Takao Y, et al. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg, 2003, 97(6): 1751-1755.
15.	Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol, 1998, 41(4): 467-470.
16.	趙永強, 楚海燕, 膝禮, 等. 博萊霉素誘導肺纖維化小鼠模型的建立及特征. 疑難病雜志, 2013, 12(9): 705-709.
17.	李偉峰, 胡玉潔, 袁偉鋒, 等. 氣管內滴入與霧化博萊霉素致小鼠肺纖維化模型的比較研究. 南方醫科大學學報, 2012, 32(2): 221-225.
18.	齊曼古麗吾守爾, 夏宇, 巴哈爾古麗米吉提, 等. 博萊霉素致大鼠肺纖維化模型的建立方法及比較. 新疆醫科大學學報, 2005, 28(6): 495-498.
19.	劉祉寧. 上皮–間充質轉化與肺纖維化的研究進展. 齊齊哈爾醫學院學報, 2018, 39(8): 950-953.
20.	胡青青, 周恩敏, 金克, 等. 褪黑素對博來霉素誘導肺纖維化小鼠 α-SMA 和 E-cadherin 表達的影響. 溫州醫科大學學報, 2018, 48(8): 577-581.
21.	蔣宗勝, 阮君山, 王少明. FOX 家族介導的上皮間質轉化在腫瘤轉移中的研究進展. 中國藥理學通報, 2016, 32(8): 1053-1058.
22.	張郡, 李長天, 趙騰蛟. FOXM1、FOXO3a 在肺腺癌中的表達及臨床意義. 甘肅科技, 2018, 34(5): 124-127.
23.	McGovern UB, Francis RE, Peck B, et al. Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer. Mol Cancer Ther, 2009, 8(3): 582-591.
24.	張均. Snail 及 α-SMA 在 IgA 腎病中的表達及意義[D]. 2015, 重慶醫科大學. 21.
25.	蔡琳, 李理, 徐軍. 高遷移率族蛋白 B1 和 α 平滑肌肌動蛋白在博萊霉素誘導肺纖維化中的表達. 中國呼吸與危重監護雜志, 2009, 8(2): 181-185.
26.	鄭林鑫, 陳燦, 麥玉梅, 等. 兩種表皮生長因子抑制劑下調腫瘤抑制素 M 抑制博來霉素誘導的小鼠肺纖維化. 中國呼吸與危重監護雜志, 2018, 17(4): 400-406.
27.	吳海蘭, 戴偉, 趙明, 等. 高遷移率族蛋白 B1 在急性肺損傷中的研究進展. 臨床肺科雜志, 2019, 24(12): 2276-2280.
28.	Voutsadakis IA. The ubiquitin-proteasome system and signal transduction pathways regulating epithelial mesenchymal transition of cancer. J Biomed Sci, 2012, 19(1): 67.
29.	Nho RS, Hergert P, Kahm J, et al. Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type I collagen matrix. Am J Pathol, 2011, 179(5): 2420-2430.
30.	姚怡, 張建林, 趙虹, 等. 叉頭框蛋白 O3a 表達下調對人腎小管上皮細胞 EMT 的影響. 中國醫學創新, 2017, 12(13): 33-36.
31.	劉伏友, 孫巖, 孫林, 等. 去甲斑蝥素對 TGF-β1 誘導的人近端腎小管細胞上皮間質轉分化以及轉錄因子 Snail1 表達的影響. 中國中西醫結合腎病雜志, 2009, 10(11): 952-957.
32.	Balli D, Ustiyan V, Zhang YF, et al. FoxM1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO J, 2013, 32(2): 231-244.
33.	金成, 方鈿德, 詹杰鋒, 等. FoxM1 與 EMT 相關蛋白 E-Cadherin 在結腸癌中的表達及其相關性研究. 實用預防醫學, 2018, 25(5): 594-596.
34.	于水, 于春鵬, 石磊, 等. FoxM1 調控 Snail1 促進肝癌上皮–間充質轉換的作用. 青島大學醫學院學報, 2016, 52(6): 645-654.
35.	Ye H, Kelly TF, Samadani U, et al. Hepatocyte nuclear factor 3/Forkhead homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol, 1997, 17(3): 1626-1641.
36.	Korver W, Roose J, Wilson A, et al. The winged-helix transcription factor trident is expressed in cycling cells. Nucleic Acids Res, 1997, 25(9): 1715-1724.
37.	譚擁軍, 陳含笑. 轉錄因子 FoxM1 剪接異構體在乳腺癌 EMT 過程中的初步研究. 湖南大學學報, 2015, 42(12): 100-106.

1. 葛均波, 徐永健, 王辰, 主編. 內科學. 第 9 版. 北京: 人民衛生出版社, 2018, 87.
2. Kim KK, Kugler MC, Wolters PJ, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A, 2006, 103(35): 13180-13185.
3. Margetts PJ, Bonniaud P, Liu L, et al. Transient overexpression of TGF-β1 induces epithelial mesenchymal transition in the rodent peritoneum. J Am Soc Nephrol, 2005, 16: 425-436.
4. Fang L, Wang H, Zhou L, et al. Akt-FoxO3a signaling axis dysregulation in human oral squamous cell carcinoma and potent efficacy of FoxO3a-targeted gene therapy. Oral Oncol, 2011, 47(1): 16-21.
5. 蔣宗勝. 基于轉錄因子 FOXM1 調控下的抗非小細胞肺癌上皮間質轉化的機制研究[D]. 2018, 福建醫科大學. 48-49.
6. Ishii Y, Fujimoto S, Fukuda T. Gefitinib prevents bleomycin-induced lung fibrosis in mice. Am J Resp Crit Care Med, 2006, 174: 550-556.
7. Le Cras TD, Korfhagen TR, Davidson C, et al. Inhibition of PI3K by PX-866 prevents transforming growth factor-alpha-induced pulmonary fibrosis. Am J Pathol, 2010, 176(2): 679-686.
8. Wang XT, McCullough KD, TF Franke, et al. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem, 2000, 275(19): 14624-14631.
9. Gomes AR, Zhao F, Lam EWF. Role and regulation of the forkhead transcription factors FOXO3a and FOXM1 in carcinogenesis and drug resistance. Chin J Cancer, 2013, 32(7): 365-370.
10. 李偉峰, 李理, 袁偉鋒, 等. 吉非替尼抑制博萊霉素誘導的小鼠肺纖維化. 中國病理生理雜志, 2010, 26(8): 1565-1569.
11. 李理, 李偉峰, 蔡琳, 等. 吉非替尼對博萊霉素致肺纖維化小鼠 α 平滑肌肌動蛋白的抑制作用. 南方醫科大學學報, 2010, 30(12): 2675-2678.
12. 彭婷, 杜海堅, 李理, 等. 吉非替尼通過增加 Foxo3a 活性抑制肺纖維化小鼠上皮–間質轉分化. 中國病理生理雜志, 2014, 30(3): 444-448.
13. 熊俊. 腎間質纖維化中的上皮–間充質轉型及 Snail1 的作用研究[D]. 2011, 第二軍醫大學. 12.
14. Mikawa K, Nishina K, Takao Y, et al. ONO-1714, a nitric oxide synthase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits. Anesth Analg, 2003, 97(6): 1751-1755.
15. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol, 1998, 41(4): 467-470.
16. 趙永強, 楚海燕, 膝禮, 等. 博萊霉素誘導肺纖維化小鼠模型的建立及特征. 疑難病雜志, 2013, 12(9): 705-709.
17. 李偉峰, 胡玉潔, 袁偉鋒, 等. 氣管內滴入與霧化博萊霉素致小鼠肺纖維化模型的比較研究. 南方醫科大學學報, 2012, 32(2): 221-225.
18. 齊曼古麗吾守爾, 夏宇, 巴哈爾古麗米吉提, 等. 博萊霉素致大鼠肺纖維化模型的建立方法及比較. 新疆醫科大學學報, 2005, 28(6): 495-498.
19. 劉祉寧. 上皮–間充質轉化與肺纖維化的研究進展. 齊齊哈爾醫學院學報, 2018, 39(8): 950-953.
20. 胡青青, 周恩敏, 金克, 等. 褪黑素對博來霉素誘導肺纖維化小鼠 α-SMA 和 E-cadherin 表達的影響. 溫州醫科大學學報, 2018, 48(8): 577-581.
21. 蔣宗勝, 阮君山, 王少明. FOX 家族介導的上皮間質轉化在腫瘤轉移中的研究進展. 中國藥理學通報, 2016, 32(8): 1053-1058.
22. 張郡, 李長天, 趙騰蛟. FOXM1、FOXO3a 在肺腺癌中的表達及臨床意義. 甘肅科技, 2018, 34(5): 124-127.
23. McGovern UB, Francis RE, Peck B, et al. Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer. Mol Cancer Ther, 2009, 8(3): 582-591.
24. 張均. Snail 及 α-SMA 在 IgA 腎病中的表達及意義[D]. 2015, 重慶醫科大學. 21.
25. 蔡琳, 李理, 徐軍. 高遷移率族蛋白 B1 和 α 平滑肌肌動蛋白在博萊霉素誘導肺纖維化中的表達. 中國呼吸與危重監護雜志, 2009, 8(2): 181-185.
26. 鄭林鑫, 陳燦, 麥玉梅, 等. 兩種表皮生長因子抑制劑下調腫瘤抑制素 M 抑制博來霉素誘導的小鼠肺纖維化. 中國呼吸與危重監護雜志, 2018, 17(4): 400-406.
27. 吳海蘭, 戴偉, 趙明, 等. 高遷移率族蛋白 B1 在急性肺損傷中的研究進展. 臨床肺科雜志, 2019, 24(12): 2276-2280.
28. Voutsadakis IA. The ubiquitin-proteasome system and signal transduction pathways regulating epithelial mesenchymal transition of cancer. J Biomed Sci, 2012, 19(1): 67.
29. Nho RS, Hergert P, Kahm J, et al. Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type I collagen matrix. Am J Pathol, 2011, 179(5): 2420-2430.
30. 姚怡, 張建林, 趙虹, 等. 叉頭框蛋白 O3a 表達下調對人腎小管上皮細胞 EMT 的影響. 中國醫學創新, 2017, 12(13): 33-36.
31. 劉伏友, 孫巖, 孫林, 等. 去甲斑蝥素對 TGF-β1 誘導的人近端腎小管細胞上皮間質轉分化以及轉錄因子 Snail1 表達的影響. 中國中西醫結合腎病雜志, 2009, 10(11): 952-957.
32. Balli D, Ustiyan V, Zhang YF, et al. FoxM1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition. EMBO J, 2013, 32(2): 231-244.
33. 金成, 方鈿德, 詹杰鋒, 等. FoxM1 與 EMT 相關蛋白 E-Cadherin 在結腸癌中的表達及其相關性研究. 實用預防醫學, 2018, 25(5): 594-596.
34. 于水, 于春鵬, 石磊, 等. FoxM1 調控 Snail1 促進肝癌上皮–間充質轉換的作用. 青島大學醫學院學報, 2016, 52(6): 645-654.
35. Ye H, Kelly TF, Samadani U, et al. Hepatocyte nuclear factor 3/Forkhead homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol, 1997, 17(3): 1626-1641.
36. Korver W, Roose J, Wilson A, et al. The winged-helix transcription factor trident is expressed in cycling cells. Nucleic Acids Res, 1997, 25(9): 1715-1724.
37. 譚擁軍, 陳含笑. 轉錄因子 FoxM1 剪接異構體在乳腺癌 EMT 過程中的初步研究. 湖南大學學報, 2015, 42(12): 100-106.

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

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