Hunan Province Key Laboratory of Tumor Cellular and Molecular Pathology, Cancer Institute, School of Medicine, University of South China, Hengyang, Hunan 421001, P.R. China.
Department of Respiratory and Critical Care Medicine, Zhangjiajie City Hospital, Zhangjiajie, Hunan 427000, P.R. China.
Mol Med Rep. 2017 Nov;16(5):6946-6952. doi: 10.3892/mmr.2017.7413. Epub 2017 Aug 31.
Asthma is a complicated systemic disease of the airways, which is characterized by variable symptoms, including bronchial hyper‑responsive-ness, inflammation and airflow obstruction. The prevalence of asthma has increased 2‑3‑fold over recent decades in developed countries; however, the molecular mechanism of asthma remains unclear. In the current study, the expression of recombinant protein Dermatophagoides farinaeI (Derf I) was induced by isopropyl β‑D‑1‑thiogalactoside (IPTG) and purified using Ni‑NTA. Derf I, an important antigen of asthma, was used to establish the animal model of asthma. Airway hyper‑responsiveness was mea-sured using unrestrained whole‑body plethysmography with a four‑chamber system. Immunoglobulin (Ig)E, IgG and IgG2a were analyzed using indirect enzyme‑linked immunosorbent assay (ELISA). Proteomic technology was applied to detect the difference between the normal lung tissue and asthma lung tissue samples of the asthma model. Cytokines in bronchoalveolar lavage fluid and the splenocyte culture medium were measured by ELISA and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to detect the mRNA expression of ATP synthase, H+ transporting, mitochondrial F1 complex, β polypeptide (ATP5b). In addition, cell growth of arterial smooth muscle cells (ASMCs) was evaluated by MTT assay. In the current study, Derf I was successfully used to construct the animal model of asthma. Out of 23 proteins that exhibit 3‑fold upregulation or downregulation, ATP5b was chosen for further investigation. The data indicated that ATP5b was overexpressed in the asthma lung tissue when compared with the normal lung tissue. However, when ATP5b was knocked down, cell growth decreased. Therefore, overexpressed ATP5b leads to airway smooth muscle cell (ASMC) proliferation and finally to ASM thickening. Thus, to the best of our knowledge, this is the first study to report that the expression level of ATP5b was markedly increased in lung tissue samples of an asthma model compared with the tissue samples from normal lungs, which promoted ASMC proliferation and contributed to airway remodeling.
哮喘是一种气道的复杂系统性疾病，其特征在于包括支气管高反应性、炎症和气流阻塞在内的可变症状。在过去几十年中，哮喘在发达国家的发病率增加了 2-3 倍；然而，哮喘的分子机制仍不清楚。在本研究中，通过异丙基 β-D-1-硫代半乳糖苷（IPTG）诱导重组蛋白屋尘螨 I（Derf I）的表达，并使用 Ni-NTA 进行纯化。Derf I 是哮喘的重要抗原，用于建立哮喘动物模型。使用四室系统的无约束全身 plethysmography 测量气道高反应性。间接酶联免疫吸附试验（ELISA）分析免疫球蛋白（Ig）E、IgG 和 IgG2a。应用蛋白质组学技术检测正常肺组织和哮喘模型肺组织样本之间的差异。通过 ELISA 测量支气管肺泡灌洗液和脾细胞培养液中的细胞因子，并通过逆转录-定量聚合酶链反应（RT-qPCR）检测三磷酸腺苷合酶、H+转运、线粒体 F1 复合物、β 多肽（ATP5b）的 mRNA 表达。此外，通过 MTT 测定评估动脉平滑肌细胞（ASMC）的细胞生长。在本研究中，成功地使用 Derf I 构建了哮喘动物模型。在 23 个表达上调或下调 3 倍的蛋白质中，选择 ATP5b 进行进一步研究。数据表明，与正常肺组织相比，哮喘肺组织中 ATP5b 过度表达。然而，当 ATP5b 被敲低时，细胞生长减少。因此，过度表达的 ATP5b 导致气道平滑肌细胞（ASMC）增殖，最终导致 ASM 增厚。因此，据我们所知，这是第一项报道哮喘模型肺组织中 ATP5b 的表达水平明显高于正常肺组织的研究，促进了 ASMC 增殖并导致气道重塑。
