Department of Pulmonary Medicine, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China.
Cell Biochem Funct. 2013 Aug;31(6):496-503. doi: 10.1002/cbf.2926. Epub 2012 Nov 27.
This study aimed to identify the role and regulation of thymic stromal lymphopoietin (TSLP) in asthmatic airway remodelling. To identify the expression of TSLP, α smooth muscle actin (α-SMA) and collagen I in bronchial tissues, bronchial biopsy specimens were collected from patients with asthma and healthy controls and stained with specific antibodies, respectively. To characterize the signalling pathways regulated by TSLP, we silenced or overexpressed TSLP in human lung fibroblast (HLF-1) cells by shRNA approaches or transfection and detected the expression of TSLP receptor (TSLPR) by enzyme-linked immunosorbent assay and Western blot analysis. In TSLP signalling pathway, the protein expression of total signal transducer and activator of transcription 3 (STAT3), STAT5, the phosphorylation of STAT3 (pSTAT3) and STAT5 (pSTAT5), TSLP, α-SMA and collagen I were also detected by Western blotting. In addition, the α-SMA, collagen I and mRNA expression were determined by real-time reverse-transcription. To further confirm the TSLP-STAT3 signalling pathway in HLF-1 cells, we inhibited STAT3 activity by targeted small molecules and then detected TSLP-induced expression of α-SMA and collagen I in both mRNA and protein levels by quantitative real-time reverse-transcription and Western blotting, respectively. First, overexpression of TSLP, α-SMA and collagen I was detected in epithelium collected from patients with asthma. Second, STAT3 activity and the expression of α-SMA and collagen I were controlled, regulated by TSLP. Specifically, the pSTAT3, α-SMA and collagen I were induced by the introduction of TSLP in HLF-1 cells, and the repression of α-SMA and collagen I was detected after TSLP silencing. Third, no changes of pSTAT5 were found in the presence of the STAT3 inhibitor, and TSLP-induced α-SMA and collagen I upregulation is in a STAT3 dependent manner. If we inhibit STAT3 activity by STAT3 targeted small molecules, TSLP-induced α-SMA and collagen I upregulation cannot be detected. The functions of TSLP in asthmatic airway remodelling were performed through STAT3 signalling pathway.
本研究旨在确定胸腺基质淋巴细胞生成素 (TSLP) 在哮喘气道重塑中的作用和调节机制。为了确定 TSLP、α 平滑肌肌动蛋白 (α-SMA) 和胶原 I 在支气管组织中的表达，我们分别收集了哮喘患者和健康对照者的支气管活检标本，并使用特异性抗体进行染色。为了描述 TSLP 调节的信号通路，我们通过 shRNA 方法或转染沉默或过表达人肺成纤维细胞 (HLF-1) 中的 TSLP，并通过酶联免疫吸附试验和 Western blot 分析检测 TSLP 受体 (TSLPR) 的表达。在 TSLP 信号通路中，还通过 Western blot 检测总信号转导和转录激活因子 3 (STAT3)、STAT5 的蛋白表达、STAT3(pSTAT3)和 STAT5(pSTAT5)的磷酸化、TSLP、α-SMA 和胶原 I 的蛋白表达。此外，通过实时逆转录测定α-SMA、胶原 I 和 mRNA 的表达。为了进一步证实 HLF-1 细胞中的 TSLP-STAT3 信号通路，我们通过靶向小分子抑制 STAT3 活性，然后分别通过定量实时逆转录和 Western blot 检测 TSLP 诱导的 α-SMA 和胶原 I 在 mRNA 和蛋白水平上的表达。首先，在哮喘患者的上皮细胞中检测到 TSLP、α-SMA 和胶原 I 的过表达。其次，TSLP 控制和调节 STAT3 活性以及 α-SMA 和胶原 I 的表达。具体而言，在 HLF-1 细胞中引入 TSLP 后诱导 pSTAT3、α-SMA 和胶原 I，在 TSLP 沉默后检测到 α-SMA 和胶原 I 的抑制。第三，在存在 STAT3 抑制剂的情况下未发现 pSTAT5 的变化，并且 TSLP 诱导的 α-SMA 和胶原 I 的上调依赖于 STAT3。如果我们通过 STAT3 靶向小分子抑制 STAT3 活性，则无法检测到 TSLP 诱导的 α-SMA 和胶原 I 的上调。TSLP 通过 STAT3 信号通路在哮喘气道重塑中发挥作用。
