Department of Pulmonary Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, NO.241, West HuaiHai Road, Shanghai 200030, PR China; Department of Respiratory and Critical Care, Anhui Geriatric Institute, The First Affiliated Hospital of Anhui Medical University, NO.218, Jixi Road, Hefei, Anhui, 230022, .PR China.
Department of Respiratory and Critical Care, Anhui Geriatric Institute, The First Affiliated Hospital of Anhui Medical University, NO.218, Jixi Road, Hefei, Anhui, 230022, .PR China.
Free Radic Biol Med. 2019 Apr;134:229-238. doi: 10.1016/j.freeradbiomed.2019.01.004. Epub 2019 Jan 9.
Transient receptor potential protein (TRP) ion channels TRPA1 and TRPV1 may be important in mediating airway tissue injury and inflammation. This study was designed to clarify the role of TRPA1 and TRPV1 channels in cigarette smoke extract (CSE)-induced damage to bronchial and alveolar epithelial cells. Alveolar epithelial (A549) cells and bronchial epithelial (Beas-2B) cells were treated with CSE in the presence and absence of a TRPA1 inhibitor (100 μM, A967079), a TRPV1 inhibitor (100 μM, AMG9810) or both. DCFH-DA and MitoSOX Red probes were used to assay intracellular and mitochondrial oxidative stress, respectively. The mRNA levels of inflammatory mediators (IL-1β, IL-8, IL-18, IL-33) and antioxidants (HO-1, NQO1, MnSOD, catalase) and the protein expression levels of mitochondrial and inflammasome factors (MFN2, OPA1, DRP1, MFF, NLRP3,caspase-1) were respectively detected by RT-PCR and Western Blot. The results were validated in TRPA1 shRNA and TRPV1 shRNA cells. In both cell types, 10% CSE increased intracellular and mitochondrial oxidative stress, induced Ca influx, increased inflammatory gene expression, reduced antioxidant gene expression and inhibited the activities of mitochondrial respiratory chain (MRC) complexes. 10% CSE increased the expression of mitochondrial fission proteins (MFF and DRP1), Caspase-1 and NLRP3 protein expression and decreased that of mitochondrial fusion proteins (MFN2 and OPA1). Both inhibitors and gene-knockout of TRPA1 and TRPV1 reduced oxidative stress, blocked Ca influx, and inhibited inflammatory and increased antioxidant gene expression. They also prevented the changes in mitochondrial fission and fusion proteins and in MRC complexes activities induced by CSE. Both TRPA1 and TRPV1 mediate CSE-induced damage of bronchial and alveolar epithelial cells via modulation of oxidative stress, inflammation and mitochondrial damage and their inhibition should be considered as potential therapy for COPD.
瞬时受体电位蛋白（TRP）离子通道 TRPA1 和 TRPV1 可能在介导气道组织损伤和炎症中起重要作用。本研究旨在阐明 TRPA1 和 TRPV1 通道在香烟烟雾提取物（CSE）诱导的支气管和肺泡上皮细胞损伤中的作用。用 CSE 处理肺泡上皮（A549）细胞和支气管上皮（Beas-2B）细胞，同时存在或不存在 TRPA1 抑制剂（100µM，A967079）、TRPV1 抑制剂（100µM，AMG9810）或两者。使用 DCFH-DA 和 MitoSOX Red 探针分别测定细胞内和线粒体氧化应激。通过 RT-PCR 和 Western Blot 分别检测炎症介质（IL-1β、IL-8、IL-18、IL-33）和抗氧化剂（HO-1、NQO1、MnSOD、过氧化氢酶）的 mRNA 水平以及线粒体和炎性体因子（MFN2、OPA1、DRP1、MFF、NLRP3、caspase-1）的蛋白表达水平。在 TRPA1 shRNA 和 TRPV1 shRNA 细胞中验证了这些结果。在这两种细胞类型中，10% CSE 增加了细胞内和线粒体氧化应激，诱导了 Ca2+内流，增加了炎症基因表达，降低了抗氧化基因表达，并抑制了线粒体呼吸链（MRC）复合物的活性。10% CSE 增加了线粒体分裂蛋白（MFF 和 DRP1）、Caspase-1 和 NLRP3 蛋白的表达，降低了线粒体融合蛋白（MFN2 和 OPA1）的表达。TRPA1 和 TRPV1 的抑制剂和基因敲除均降低了氧化应激，阻断了 Ca2+内流，抑制了炎症并增加了抗氧化基因的表达。它们还防止了 CSE 诱导的线粒体分裂和融合蛋白以及 MRC 复合物活性的变化。TRPA1 和 TRPV1 均通过调节氧化应激、炎症和线粒体损伤介导 CSE 诱导的支气管和肺泡上皮细胞损伤，其抑制作用应被视为 COPD 的潜在治疗方法。
