Respiratory Medicine, Hunan Provincial People's Hospital, No. 61 Jiefang Xi Road, Changsha, 410219, Hunan, PR China.
State Key Lab of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University, No. 151 Yanjiang Road, Guangzhou, 510120, Guangdong, PR China.
Biochimie. 2019 Mar;158:139-148. doi: 10.1016/j.biochi.2018.12.004. Epub 2018 Dec 11.
The present study was designed to explore the role of transient receptor potential canonical 3 (TRPC3) in nicotine-induced chronic obstructive pulmonary disease (COPD) and its underlying mechanism. In this study, the expression and localization of α5 nicotinic acetylcholine receptor (α5-nAchR) in lung tissues were determined by western blotting and immunohistochemistry. The quantitative real-time PCR (qRT-PCR) analysis was performed to examine the mRNA expression levels of α5-nAchR and TRPC3 in human airway smooth muscle cells (HASMCs). Cell viability was assessed by CCK-8 assay. Proliferation was detected by cell counting and EdU immunofluorescent staining. Fluorescence calcium imaging was carried out to measure cytosolic Ca ([Ca]cyt) concentration. The results showed that the α5-nAchR and TRPC3 expressions were significantly up-regulated in lung tissues of COPD smokers. Nicotine promoted HASMC proliferation, which was accompanied by elevated α5-nAchR and TRPC3 expressions, basal [Ca]cyt, store-operated calcium entry (SOCE) and the rate of Mn quenching in HASMCs. Further investigation indicated that nicotine-induced Ca response and TRPC3 up-regulation was reversibly blocked by small interfering RNA (siRNA) suppression of α5-nAChR. The knockdown of TRPC3 blunted Ca response and HASMC proliferation induced by nicotine. In conclusion, nicotine-induced HASMC proliferation was mediated by TRPC3-dependent calcium entry via α5-nAchR, which provided a potential target for treatment of COPD.
本研究旨在探讨瞬时受体电位经典型 3（TRPC3）在尼古丁诱导的慢性阻塞性肺疾病（COPD）中的作用及其潜在机制。在这项研究中，通过 Western blot 和免疫组织化学测定了肺组织中α5 烟碱型乙酰胆碱受体（α5-nAchR）的表达和定位。通过定量实时 PCR（qRT-PCR）分析检测了人气道平滑肌细胞（HASMCs）中α5-nAchR 和 TRPC3 的 mRNA 表达水平。通过 CCK-8 测定评估细胞活力。通过细胞计数和 EdU 免疫荧光染色检测增殖。通过荧光钙成像测量胞质 Ca([Ca]cyt)浓度。结果表明，COPD 吸烟者肺组织中α5-nAchR 和 TRPC3 的表达明显上调。尼古丁促进 HASMC 增殖，伴随着α5-nAchR 和 TRPC3 表达升高、基础[Ca]cyt、储存操纵钙内流（SOCE）和 HASMC 中 Mn 猝灭率升高。进一步的研究表明，α5-nAChR 的小干扰 RNA（siRNA）抑制可逆转尼古丁诱导的 Ca 反应和 TRPC3 上调。TRPC3 的敲低减弱了尼古丁诱导的 Ca 反应和 HASMC 增殖。总之，尼古丁诱导的 HASMC 增殖是通过α5-nAchR 依赖性钙内流介导的 TRPC3 介导的，这为 COPD 的治疗提供了一个潜在的靶点。
