Institute of Biomedicine and Molecular Immunology, National Research Council, Via Ugo La Malfa, 153, 90146 Palermo, Italy.
Toxicol Lett. 2013 Nov 25;223(2):198-204. doi: 10.1016/j.toxlet.2013.09.013. Epub 2013 Sep 25.
Cigarette smoke represents the major risk factor for chronic obstructive pulmonary disease (COPD). Cigarette smoke extracts (CSE) alter TLR4 expression and activation in bronchial epithelial cells. Carbocysteine, an anti-oxidant and mucolytic agent, is effective in reducing the severity and the rate of exacerbations in COPD patients. The effects of carbocysteine on TLR4 expression and on the TLR4 activation downstream events are largely unknown. This study was aimed to explore whether carbocysteine, in a human bronchial epithelial cell line (16-HBE), counteracted some pro-inflammatory CSE-mediated effects. In particular, TLR4 expression, LPS binding, p21 (a senescence marker), IL-8 mRNA and release in CSE-stimulated 16-HBE as well as actin reorganization in neutrophils cultured with supernatants from bronchial epithelial cells which were stimulated with CSE and/or carbocysteine were assessed. TLR4 expression, LPS binding, and p21 expression were assessed by flow cytometry, IL-8 mRNA by Real Time PCR and IL-8 release by ELISA. Actin reorganization, a prerequisite for cell migration, was determined using Atto 488 phalloidin in neutrophils by flow cytometry and fluorescence microscopy. CSE increased: (1) TLR4, LPS binding and p21 expression; (2) IL-8 mRNA and IL-8 release due to IL-1 stimulation; (3) neutrophil migration. Carbocysteine in CSE stimulated bronchial epithelial cells, reduced: (1) TLR4, LPS binding and p21; (2) IL-8 mRNA and IL-8 release due to IL-1 stimulation; (3) neutrophil chemotactic migration. In conclusion, the present study provides compelling evidences that carbocysteine may contribute to control the inflammatory and senescence processes present in smokers.
香烟烟雾代表慢性阻塞性肺疾病（COPD）的主要危险因素。香烟烟雾提取物（CSE）改变了支气管上皮细胞中 TLR4 的表达和激活。半胱氨酸羧甲基醚，一种抗氧化和黏液溶解剂，在减少 COPD 患者的严重程度和恶化率方面有效。半胱氨酸羧甲基醚对 TLR4 表达及其下游 TLR4 激活事件的影响在很大程度上尚不清楚。本研究旨在探讨半胱氨酸羧甲基醚是否在人支气管上皮细胞系（16-HBE）中抵消了一些由 CSE 介导的促炎作用。特别是，在 CSE 刺激的 16-HBE 中评估 TLR4 表达、LPS 结合、p21（衰老标志物）、IL-8mRNA 和释放，以及用 CSE 和/或半胱氨酸羧甲基醚刺激的支气管上皮细胞的上清液培养的中性粒细胞中的肌动蛋白重组。通过流式细胞术评估 TLR4 表达、LPS 结合和 p21 表达，通过实时 PCR 评估 IL-8mRNA，通过 ELISA 评估 IL-8 释放。通过流式细胞术和荧光显微镜在中性粒细胞中用 Atto 488 鬼笔环肽测定肌动蛋白重组，这是细胞迁移的先决条件。CSE 增加：（1）TLR4、LPS 结合和 p21 表达；（2）IL-1 刺激引起的 IL-8mRNA 和 IL-8 释放；（3）中性粒细胞迁移。半胱氨酸羧甲基醚在 CSE 刺激的支气管上皮细胞中，减少：（1）TLR4、LPS 结合和 p21；（2）IL-1 刺激引起的 IL-8mRNA 和 IL-8 释放；（3）中性粒细胞趋化性迁移。总之，本研究提供了令人信服的证据，表明半胱氨酸羧甲基醚可能有助于控制吸烟者体内的炎症和衰老过程。
