Chu Shuyuan, Zhang Xiufeng, Sun Yabing, Yu Yuanyuan, Liang Yaxi, Jiang Ming, Huang Jianwei, Ma Libing
Department of Respiratory Medicine, The Affiliated Hospital of Guilin Medical University, Guilin 541001, Guangxi, China; Institute of Respiratory Diseases, Guilin Medical University, Guilin 541001, Guangxi, China; Guangxi Colleges and Universities Key Laboratory of Respiratory Disease, Guilin 541001, Guangxi, China.
Department of Respiratory Medicine, The Second Affiliated Hospital of University of South China, Hengyang 421000, Hunan, China.
Peptides. 2017 Apr;90:1-9. doi: 10.1016/j.peptides.2017.02.002. Epub 2017 Feb 13.
Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-β1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA expressions were increased after induction by TGF-β1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and α-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased α-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with α-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-β1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-β1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.
心房利钠肽（ANP）在气道中的表达逐渐增加，并抑制肺动脉重塑。然而，ANP在呼吸系统重塑中的作用仍不清楚。本研究探讨了ANP在气道重塑中的作用及其可能机制。用转化生长因子-β1（TGF-β1）、ANP、环磷酸鸟苷（cGMP）抑制剂、蛋白激酶G（PKG）抑制剂和cGMP类似物刺激人支气管上皮16HBE-14o细胞和肺泡上皮A549细胞。通过定量实时聚合酶链反应（qRT-PCR）和蛋白质印迹法评估上皮标志物、间充质标志物和Smad3的表达。当16HBE-14o或A549细胞中的Smad3被小干扰RNA（siRNA）沉默后，采用免疫组织化学染色评估Smad3的表达。我们的结果显示，在16HBE-14o和A549细胞中，TGF-β1诱导后E-钙黏蛋白的mRNA和蛋白表达降低，而α-平滑肌肌动蛋白（α-SMA）表达增加。ANP刺激后，E-钙黏蛋白表达增加，α-SMA表达降低。抑制cGMP或PKG会降低E-钙黏蛋白表达，但增加α-SMA表达，而cGMP类似物可逆转这种情况。此外，磷酸化Smad3的表达与α-SMA表达一致。在16HBE-14o或A549细胞上皮-间质转化（EMT）过程中，Smad3被沉默后，其主要表达于细胞质而非细胞核，与未沉默细胞不同。总之，ANP通过cGMP/PKG信号通路抑制TGF-β1诱导的16HBE-14o和A549细胞EMT，通过减弱Smad3磷酸化靶向TGF-β1/Smad3。这些发现提示ANP在治疗气道重塑相关肺部疾病方面具有潜力。
